THE EFFECTS OF ɤ-GLUTAMYL MODULATION ON CANCER TREATMENT AND DNA METHYLATION IN HUMAN CANCER CELLS

by

Sung-Eun Kim

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Nutritional Sciences University of Toronto

© Copyright by Sung-Eun Kim 2013

THE EFFECTS OF ɤ-GLUTAMYL HYDROLASE MODULATION ON CANCER TREATMENT AND DNA METHYLATION IN HUMAN CANCER CELLS

Sung-Eun Kim

Doctor of Philosophy

Graduate Department of Nutritional Sciences University of Toronto

2013 Abstract

Folate and are retained in cells by polyglutamylation mediated by folylpolyglutamate synthase (FPGS), and are exported from cells after hydrolysis to monoglutamates by γ-glutamyl hydrolase (GGH). Polyglutamylated (anti) are retained in cells longer and are better substrates than their monoglutamate counterparts for intracellular -dependent .

GGH modulation may therefore affect chemosensitivity of cancer cells to antifolates and 5- by altering polyglutamylation of antifolates and a specific target intracellular folate for 5-fluorouracil (5,10-methylenetetrahydrofolate), respectively.

We generated an in vitro model of GGH modulation in HCT116 and MDA-MB-435 cells with predictable functional consequences and investigated chemosensitivity to 5-fluorouracil and antifolates. Overall, GGH overexpression decreased chemosensitivity to 5-fluorouracil+ leucovorin and , while GGH inhibition increased chemosensitivity to 5-fluorouracil

+leucovorin. However, these effects appeared to depend not only on the GGH modulation- induced changes in polyglutamylation of 5,10-methylenetetrahydrofolate and antifolates but also on intracellular folate levels as well as adaptive and compensatory changes in other enzymes

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involved in intracellular folate and accumulation and in response to GGH modulation.

Polyglutamylation is also important in DNA methylation as polyglutamylated folates are better substrates for methylenetetrahydrofolate and involved in the generation of S-adenosylmethionine, which is a substrate for DNA methylation. We hypothesized that GGH and FPGS modulation may affect DNA methylation at global and - specific levels with consequent functional ramifications. GGH and FPGS modulation demonstrated the inverse relationship between GGH activity and global DNA methylation as well as was associated with differential and altered CpG promoter DNA methylation involved in important biological pathways. Some of the observed altered gene expression appear to be regulated by DNA methylation. Whether or not GGH modulation may be an important determinant of chemosensitivity to 5-fluorouracil- and antifolate-based , and the potential roles of GGH and FPGS modulation in DNA methylation need further exploration.

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Acknowledgments

First and foremost, I would like to express my sincere gratitude to my supervisor, Dr.

Young-In Kim for the opportunity to complete a Ph.D. degree under his supervision. I truly appreciate his encouragement, guidance and invaluable assistance throughout the project. His knowledge and enthusiasm towards my research have been a true inspiration. I would also like to thank my committee members, Dr. Robert Bruce, Dr. Deborah O’Connor and Dr. Andrew

Bognar for their expert knowledge and guidance throughout the course of my research.

I would like to extend my gratitude to the collaborators - Dr. Toshinori Hinoue for the bioinformatic analysis of our microarray data, Dr. Daniel Weisenberger and Dr. Peter Laird for advice on the methylation analysis, and Ruth Croxford for the detailed explanations and guidance on the statistics behind the data. I am also grateful to all the past and present lab members for their support and for all the memories we have shared. I am thankful to my friends from the Department of Nutritional Sciences for their support and encouragement throughout my doctorate degree.

My deepest thanks are reserved to people dearest and nearest to me. I would like to thank

Dr. Mi-Kyung Sung for her continued moral support towards pursuing a graduate degree. I am grateful to all my friends in Toronto and Korea for their attention, encouragement, and support.

Special thanks to my family for their love and inspiration throughout my studies. Lastly, to my parents, words cannot describe the gratitude you deserve. I will always appreciate your unwavering love, understanding, and support that have made me who I am today. I would also like to express my gratitude to my grandparents who were always by my side and wished my success. Again, my sincere and lasting gratitude to my father and mother, and it is to them that I dedicate this thesis.

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Table of Contents

ACKNOWLEDGMENTS ...... IV TABLE OF CONTENTS ...... V LIST OF ABBREVIATIONS ...... VII LIST OF TABLES ...... X LIST OF FIGURES ...... XV LIST OF APPENDICES ...... XIX CHAPTER 1: INTRODUCTION ...... 1 CHAPTER 2: LITERATURE REVIEW ...... 5 2.1 Folate ...... 6 2.1.1 Chemistry of Folate ...... 6 2.1.2 Folate Metabolism and Biochemical Function ...... 7 2.1.3 Intracellular Homeostasis of Folate ...... 16 2.1.4 Folate and Health ...... 27 2.2 Folate and Cancer Risk ...... 30 2.3 Folate and Cancer Treatment ...... 31 2.3.1 Folate and Chemotherapeutic Agents ...... 31 2.3.2 5-Fluorouracil ...... 34 2.3.3 Methotrexate ...... 37 2.3.4 ...... 39 2.3.5 Trimetrexate ...... 42 2.3.6 Drug Resistance ...... 44 2.4 and Cancer Treatment ...... 46 2.4.1 Epigenetics ...... 46 2.4.2 DNA Methylation and Cancer ...... 47 2.4.3 Inhibition of DNA Methylation as Chemotherapeutic Strategy ...... 52 2.4.4 Epigenetic Silencing and Response to Chemotherapeutic Agents ...... 53 2.4.5 Analysis of DNA methylation ...... 54 2.4.6 Folate and Epigenetics ...... 56 CHAPTER 3: RATIONALE, HYPOTHESES, AND OBJECTIVES ...... 58 CHAPTER 4: STUDY 1 – THE DEVELOPMENT AND VALIDATION OF AN IN VITRO MODEL OF GGH MODULATION AND INVESTIGATION OF THE EFFECT OF GGH

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MODULATION ON CHEMOSENSITIVITY OF HUMAN COLON AND BREAST CANCER CELLS TO CHEMOTHERAPEUTICS (PROOF-OF-PRINCIPLE) ...... 63 4.1 Abstract ...... 64 4.2 Introduction ...... 65 4.3 Materials and Methods ...... 67 4.4 Results...... 73 4.5 Discussion ...... 87 CHAPTER 5: STUDY 2 – THE EFFECTS OF GGH MODULATION AND FOLATE ON CHEMOSENSITIVITY OF HUMAN COLON AND BREAST CANCER CELLS TO CHEMOTHERAPEUTICS IN IN VITRO AND IN VIVO MODELS ...... 93 5.1 Abstract ...... 94 5.2 Introduction ...... 95 5.3 Materials and Methods ...... 98 5.4 Results ...... 103 5.5 Discussion ...... 123 CHAPTER 6: STUDY 3 – THE EFFECT OF GGH MODULATION ON GLOBAL AND GENE-SPECIFIC DNA METHYLATION AND GENE EXPRESSION IN HUMAN COLON AND BREAST CANCER CELLS ...... 130 6.1 Abstract ...... 131 6.2 Introduction ...... 132 6.3 Materials and Methods ...... 134 6.4 Results ...... 142 6.5 Discussion ...... 195 CHAPTER 7: STUDY 4 – THE EFFECT OF FPGS MODULATION ON GLOBAL AND GENE-SPECIFIC DNA METHYLATION AND GENE EXPRESSION IN HUMAN COLON AND BREAST CANCER CELLS ...... 202 7.1 Abstract ...... 203 7.2 Introduction ...... 204 7.3 Materials and Methods ...... 206 7.4 Results ...... 210 7.5 Discussion ...... 266 CHAPTER 8: OVERALL DISCUSSION AND FUTURE DIRECTIONS ...... 276 8.1 Overall Discussion ...... 277 8.2 Future Directions ...... 281 REFERENCES ...... 284

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List of Abbreviations

ABC transporter ATP-binding-cassette transporter AICARFT aminoimidazole-carboxomide ribonucleotide transformylase ALL acute lymphoblastic leukemia 5-aza-CR 5-azacytidine 5-aza-CdR 5-aza-2’-deoxycytidine BCRP breast-cancer-resistance protein BDR basal dietary requirement BHMT betaine- S- CBS cystathionine-β-synthase ChIP chromatin immunoprecipitation CIMP+ CpG island methylator phenotype CpG cytosine-guanine dinucleotide sequence CRC colorectal cancer CTH γ-cystathionase DDATHF 5,10-dideazatetrahydrofolate DFEs dietary folate equivalents DHF dihydrofolate DHFR dihydrofolate reductase DHFU dihydrofluorouracil DMG dimethylglycine DNMT DNA methyltransferase DPD dihydropyrimidine dehydrogenase dTMP deoxythymidine-5-monophosphate; thymidylate dUMP deoxyuridine-5-monophosphate FA folic acid FBP folate-binding protein FBS fetal bovine serum 5FdUMP 5-fluoro-deoxyuridine-monophosphate FPGS folylpolyglutamate synthase FR folate receptor

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5FU 5-fluorouracil 5FUMP 5-fluorouridine-monophosphate GARFT glycinamide ribonucleotide transformylase GCP II glutamate II GGH γ-glutamyl hydrolase Hcy homocysteine HDAC histone deacetylase LV leucovorin MAT methionine adenosyl trasnferase MBD2 methyl-CpG binding domain protein 2, DNA demethylase MFR mitochondrial folate transporter MGMT O-6-methylguanine-DNA methyltransferase MRP multidrug-resistance-associated protein MS methionine synthase MSR methionine synthase reductase MTA pemetrexed; multi-targeted antifolate 5-MTHF 5-methyltetrahydrofolate MTHFCH 5,10-methenylTHF cyclohydrolase MTHFD 5,10-methyleneTHF dehydrogenase MTHFR methylenetetrahydrofolate reductase MTX methotrexate NAALADase N-acetyl-α-linked acidic NHANES National Health and Nutrition Examination Survey NTD neural tube defect OPRT orotate phosphoribosyl PABA para-aminobenzoic acid PCFT proton-coupled folate transporter PRPP phosphoribosyl pyrophosphate PSMA prostate specific membrane antigen RA rheumatoid arthritis RBC red blood cells

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RFC reduced folate carrier RT-PCR reverse transcriptase-polymerase chain reaction SAH S-adenosylhomocysteine SAM S-adenosylmethionine SEM standard error of mean SD standard deviation SHMT serine hydroxymethyltransferase siRNA small-interfering RNA SNP single- polymorphism THF tetrahydrofolate TK thymidine kinase TMTX trimetrexate TP TS UMFA unmetabolized folic acid

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List of Tables

Table 2. 1 Dual modulatory role of folate in carcinogenesis ...... 31

Table 4. 1 Summary of phenotypic and molecular characteristics of HCT116 and MDA-MB- 435 cell lines ...... 68

Table 4. 2 IC50 values of 5FU and MTX in the GGH-modulated HCT116 colon and MDA- MB-435 breast cancer cells ...... 86

Table 5. 1 Intracellular folate concentrations of the GGH-overexpressed HCT116 and MDA- MB-435 cells at different concentrations of media ...... 104 Table 5. 2 Intracellular folate concentrations of the GGH-inhibited HCT116 and MDA-MB- 435 cells at different ...... 105

Table 5. 3 IC50 values of 5FU and MTX in HCT116 colon cancer cells transfected with the sense GGH and GGH-targeted siRNA in comparison with corresponding control cells expressing endogenous GGH at different concentrations of media ...... 114

Table 5. 4 IC50 values of 5FU and MTX in MDA-MB-435 breast cancer cells transfected with the sense GGH and GGH-targeted siRNA in comparison with corresponding control cells expressing endogenous GGH at different concentrations of media ...... 115 Table 5. 5 Summary of the in vitro chemosensitivity of the GGH-modulated HCT116 and MDA-MB-435 cells at different folate levels (Chapters 4 and 5) ...... 129

Table 6. 1 Primer sequences for selected for qRT-PCR ...... 141 Table 6. 2 Summary of number of genes differentially methylated in the GGH-modulated HCT116 and MDA-MB-435 cell lines ...... 150 Table 6. 3 The top molecular and cellular functions associated with differentially methylated genes in the GGH-modulated HCT116 colon cancer cells ...... 151 Table 6. 4 The top networks matched by the genes differentially methylated in the GGH- modulated HCT116 colon cancer cells ...... 152 Table 6. 5 The top molecular and cellular functions associated with differentially methylated genes in the GGH-modulated MDA-MB-435 breast cancer cells ...... 153 Table 6. 6 The top networks matched by the genes differentially methylated in the GGH- modulated MDA-MB-435 breast cancer cells ...... 154 Table 6. 7 The top molecular and cellular functions associated with genes with commonly differentially methylated in response to GGH modulation in both the HCT116 and MDA- MB-435 cells ...... 157 Table 6. 8 The top networks matched by the genes differentially methylated in both the GGH- overexpressed HCT116 and MDA-MB-435 cells ...... 158 Table 6. 9 The top networks matched by the genes differentially methylated in both the GGH- inhibited HCT116 and MDA-MB-435 cells ...... 158

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Table 6. 10 Summary of number of genes differentially expressed in the GGH-modulated HCT116 and MDA-MB-435 cell lines ...... 159 Table 6. 11 The top molecular and cellular functions associated with differentially expressed genes in the GGH-modulated HCT116 colon cancer cells ...... 162 Table 6. 12 List of the top differentially expressed genes in the GGH-overexpressed HCT116 colon cancer cells ...... 163 Table 6. 13 List of the top differentially expressed genes in the GGH-inhibited HCT116 colon cancer cells ...... 164 Table 6. 14 The top networks matched by the genes differentially expressed in the GGH- modulated HCT116 colon cancer cells ...... 165 Table 6. 15 The top molecular and cellular functions associated with differentially expressed genes in the GGH-modulated MDA-MB-435 breast cancer cells ...... 166 Table 6. 16 List of the top differentially expressed genes in the GGH-overexpressed MDA- MB-435 breast cancer cells ...... 167 Table 6. 17 List of the top differentially expressed genes in the GGH-inhibited MDA-MB-435 breast cancer cells ...... 168 Table 6. 18 The top networks matched by the genes differentially expressed in the GGH- modulated MDA-MB-435 breast cancer cells ...... 169 Table 6. 19 The top molecular and cellular functions associated with genes that are commonly differentially expressed in the GGH-modulated HCT116 and MDA-MB-435 cells ...... 171 Table 6. 20 The top networks matched by the genes differentially expressed in both the GGH- overexpressed HCT116 and MDA-MB-435 cells ...... 172 Table 6. 21 The top networks matched by the genes differentially expressed in both the GGH- inhibited HCT116 and MDA-MB-435 cells ...... 172 Table 6. 22 Summary of number of genes with altered expression and promoter DNA methylation in the GGH-modulated HCT116 and MDA-MB-435 cell lines ...... 175 Table 6. 23 The top molecular and cellular functions associated with genes with altered expression and promoter methylation in the GGH-modulated HCT116 colon cancer cells ...... 176 Table 6. 24 List of the top genes with altered promoter methylation and expression in the GGH-overexpressed HCT116 colon cancer cells ...... 176 Table 6. 25 List of the top genes with altered promoter methylation and expression in the GGH-inhibited HCT116 colon cancer cells ...... 177 Table 6. 26 The top networks matched by the genes with altered expression and promoter methylation in the GGH-modulated HCT116 colon cancer cells ...... 178 Table 6. 27 The top molecular and cellular functions associated with genes with altered expression and promoter methylation in the GGH-modulated MDA-MB-435 breast cancer cells ...... 179

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Table 6. 28 List of the top genes with altered promoter methylation and expression in the GGH-overexpressed MDA-MB-435 breast cancer cells ...... 180 Table 6. 29 List of the top genes with altered promoter methylation and expression in the GGH-inhibited MDA-MB-435 breast cancer cell ...... 181 Table 6. 30 The top networks matched by the genes with altered expression and promoter methylation in the GGH-modulated MDA-MB-435 breast cancer cells ...... 182 Table 6. 31 Comparison of fold changes in gene expression detected by Illumina HumanHT- 12 v4.0 BeadChip and qRT-PCR analyses in the GGH-modulated HCT116 and MDA-MB- 435 cell lines ...... 184 Table 6. 32 List of differentially expressed genes involved in folate biosynthesis and one- carbon pool by folate pathways in the GGH-modulated HCT116 and MDA-MB-435 cells ...... 186 Table 6. 33 List of differentially expressed genes involved in the cell cycle pathway in the GGH-modulated HCT116 and MDA-MB-435 cells ...... 187 Table 6. 34 List of differentially expressed genes involved in the apoptosis pathway in the GGH-modulated HCT116 and MDA-MB-435 cells ...... 188 Table 6. 35 The top molecular and cellular functions associated with the GGH-specific gene expression in the GGH-modulated HCT116 colon cancer cells ...... 190 Table 6. 36 The top networks matched by the genes with the GGH-specific altered expression in the GGH-modulated HCT116 colon cancer cells ...... 190 Table 6. 37 List of the top genes associated with the GGH-specific altered expression in the GGH-modulated HCT116 colon cancer cells ...... 191 Table 6. 38 The top molecular and cellular functions associated with the GGH-specific gene expression in the GGH-modulated MDA-MB-435 breast cancer cells ...... 193 Table 6. 39 The top networks matched by the genes with the GGH-specific altered expression in the GGH-modulated MDA-MB-435 breast cancer cells ...... 193 Table 6. 40 List of the top genes associated with the GGH-specific altered expression in the GGH-modulated MDA-MB-435 breast cancer cells ...... 194

Table 7. 1 Primer sequences for genes selected for qRT-PCR ...... 209 Table 7. 2 Summary of number of genes differentially methylated in the FPGS-modulated HCT116 and MDA-MB-435 cell lines ...... 218 Table 7. 3 The top molecular and cellular functions associated with differentially methylated genes in the FPGS-modulated HCT116 colon cancer cells ...... 219 Table 7. 4 The top networks matched by the genes differentially methylated in the FPGS- modulated HCT116 colon cancer cells ...... 220 Table 7. 5 The top molecular and cellular functions associated with differentially methylated genes in the FPGS-modulated MDA-MB-435 breast cancer cells ...... 221

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Table 7. 6 The top networks matched by the genes differentially methylated in the FPGS- modulated MDA-MB-435 breast cancer cells ...... 222 Table 7. 7 The top molecular and cellular functions associated with genes with commonly differentially methylated in response to FPGS modulation in both the HCT116 and MDA- MB-435 cells ...... 225 Table 7. 8 The top networks matched by the genes differentially methylated in both the FPGS- overexpressed HCT116 and MDA-MB-435 cells ...... 226 Table 7. 9 The top networks matched by the genes differentially methylated in both the FPGS- inhibited HCT116 and MDA-MB-435 cells ...... 226 Table 7. 10 Summary of number of genes differentially expressed in the FPGS-modulated HCT116 and MDA-MB-435 cell lines ...... 227 Table 7. 11 The top molecular and cellular functions associated with differentially expressed genes in the FPGS-modulated HCT116 colon cancer cells ...... 230 Table 7. 12 List of the top differentially expressed genes in the FPGS-overexpressed HCT116 colon cancer cells ...... 231 Table 7. 13 List of the top differentially expressed genes in the FPGS-inhibited HCT116 colon cancer cells ...... 232 Table 7. 14 The top networks matched by the genes differentially expressed in the FPGS- modulated HCT116 colon cancer cells ...... 233 Table 7. 15 The top molecular and cellular functions associated with differentially expressed genes in the FPGS-modulated MDA-MB-435 breast cancer cells ...... 234 Table 7. 16 List of the top differentially expressed genes in the FPGS-overexpressed MDA- MB-435 breast cancer cells ...... 235 Table 7. 17 List of the top differentially expressed genes in the FPGS-inhibited MDA-MB-435 breast cancer cells ...... 236 Table 7. 18 The top networks matched by the genes differentially expressed in the FPGS- modulated MDA-MB-435 breast cancer cells ...... 237 Table 7. 19 The top molecular and cellular functions associated with genes that are commonly differentially expressed in the FPGS-modulated HCT116 and MDA-MB-435 cells ...... 239 Table 7. 20 The top networks matched by the genes differentially expressed in both the FPGS- overexpressed HCT116 and MDA-MB-435 cells ...... 240 Table 7. 21 The top networks matched by the genes differentially expressed in both the FPGS- inhibited HCT116 and MDA-MB-435 cells ...... 240 Table 7. 22 Summary of number of genes with altered expression and promoter DNA methylation in the FPGS-modulated HCT116 and MDA-MB-435 cell lines ...... 241 Table 7. 23 The top molecular and cellular functions associated with genes with altered expression and promoter methylation in the FPGS-modulated HCT116 colon cancer cells ...... 243

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Table 7. 24 List of the top genes with altered promoter methylation and expression in the FPGS-overexpressed HCT116 colon cancer cells ...... 244 Table 7. 25 List of the top genes with altered promoter methylation and expression in the FPGS-inhibited HCT116 colon cancer cells ...... 245 Table 7. 26 The top networks matched by the genes with altered expression and promoter methylation in the FPGS-modulated HCT116 colon cancer cells ...... 246 Table 7. 27 The top molecular and cellular functions associated with genes with altered expression and promoter methylation in the FPGS-modulated MDA-MB-435 breast cancer cells ...... 247 Table 7. 28 List of the top genes with altered promoter methylation and expression in the FPGS-overexpressed MDA-MB-435 breast cancer cells ...... 248 Table 7. 29 List of the top genes with altered promoter methylation and expression in the FPGS-inhibited MDA-MB-435 breast cancer cells ...... 249 Table 7. 30 The top networks matched by the genes with altered expression and promoter methylation in the FPGS-modulated MDA-MB-435 breast cancer cells ...... 250 Table 7. 31 Comparison of fold changes in gene expression detected by Illumina HumanHT- 12 v4.0 BeadChip and qRT-PCR analyses in the FPGS-modulated HCT116 and MDA-MB- 435 cell lines ...... 252 Table 7. 32 List of differentially expressed genes involved in folate biosynthesis and one- carbon pool by folate pathways in the FPGS-modulated HCT116 and MDA-MB-435 cells ...... 254 Table 7. 33 List of differentially expressed genes involved in the cell cycle pathway in the FPGS-modulated HCT116 and MDA-MB-435 cells ...... 256 Table 7. 34 List of differentially expressed genes involved in the apoptosis pathway in the FPGS-modulated HCT116 and MDA-MB-435 cells ...... 259 Table 7. 35 The top molecular and cellular functions associated with the FPGS-specific gene expression in the FPGS-modulated HCT116 colon cancer cells ...... 261 Table 7. 36 The top networks matched by the genes with the FPGS-specific altered expression in the FPGS-modulated HCT116 colon cancer cells ...... 261 Table 7. 37 List of the top genes associated with the FPGS-specific altered expression in the FPGS-modulated HCT116 colon cancer cells ...... 262 Table 7. 38 The top molecular and cellular functions associated with the FPGS-specific gene expression in the FPGS-modulated MDA-MB-435 breast cancer cells ...... 264 Table 7. 39 The top networks matched by the genes with the FPGS-specific altered expression in the FPGS-modulated MDA-MB-435 breast cancer cells ...... 264 Table 7. 40 List of the top genes associated with the FPGS-specific altered expression in the FPGS-modulated MDA-MB-435 breast cancer cells ...... 265

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List of Figures

Figure 2. 1 Chemical structures of folic acid (A) and folate (B) ...... 7 Figure 2. 2 Simplified biochemical pathway of folate metabolism and reactions ...... 8 Figure 2. 3 Summary of chemotherapeutic agents used in the current research on the folate pathway ...... 33 Figure 2. 4 Chemical structure of 5-fluorouracil ...... 34 Figure 2. 5 Metabolic pathway of 5-fluorouracil ...... 35 Figure 2. 6 Chemical structure of methotrexate ...... 37 Figure 2. 7 Chemical structure of pemetrexed ...... 39 Figure 2. 8 Chemical structure of trimetrexate ...... 42 Figure 2. 9 A proposed model of the modulation of MRPs and BCRP by folate supplementation (A) and folate depletion (B) ...... 45 Figure 2. 10 Distribution of CpG dinucleotides in the and CpG methylation patterns in normal and tumor cells ...... 48 Figure 2. 11 Effects of DNA methylation and chromatin structure on gene transcription in normal and tumor cells ...... 50

Figure 4. 1 Maps of pIRESneo (A) and pSilencer neo (B) expression vectors ...... 70 Figure 4. 2 ɤ-Glutamyl hydrolase (GGH) protein expression and GGH activity in the GGH-modulated HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cells ...... 74 Figure 4. 3 Concentrations of intracellular total folate (A) and long-chain length polyglutamates (B) in the GGH-modulated HCT116 colon cancer cells ...... 76 Figure 4. 4 Concentrations of intracellular total folate (A) and long-chain length polyglutamates (B) in the GGH-modulated MDA-MB-435 breast cancer cells ...... 77 Figure 4. 5 Thymidylate synthase (TS) catalytic activity in the GGH-modulated HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cells ...... 78 Figure 4. 6 Dihydrofolate reductase (DHFR) protein expression and DHFR enzyme activity in the GGH-overexpressed (A) and GGH-inhibited (B) HCT116 colon cancer cells ...... 79 Figure 4. 7 Dihydrofolate reductase (DHFR) protein expression and DHFR enzyme activity in the GGH-overexpressed (A) and GGH-inhibited (B) MDA-MB-435 breast cancer cells ... 80 Figure 4. 8 Doubling time of the GGH-modulated HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cells ...... 81 Figure 4. 9 In vitro chemosensitivity of HCT116 colon cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to pemetrexed (A, F; MTA, positive control), trimetrexate (B, G; TMTX, negative control), 5-fluorouracil plus leucovorin (C, H; 5FU+LV), 5-fluorouracil alone (D, I; 5FU), or methotrexate (E, J; MTX) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH) ...... 84

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Figure 4. 10 In vitro chemosensitivity of MDA-MB-435 breast cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to pemetrexed (A, F; MTA, positive control), trimetrexate (B, G; TMTX, negative control), 5-fluorouracil plus leucovorin (C, H; 5FU+LV), 5-fluorouracil alone (D, I; 5FU), or methotrexate (E, J; MTX) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH) ...... 85

Figure 5. 1 Experimental design for in vivo chemosensitivity study ...... 99 Figure 5. 2 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to pemetrexed (MTA; positive control) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH) ...... 109 Figure 5. 3 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to trimetrexate (TMTX; negative control) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH) ...... 110 Figure 5. 4 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to methotrexate (MTX) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH) ...... 111 Figure 5. 5 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to 5-fluorouracil (5FU) plus leucovorin (LV) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH) ...... 112 Figure 5. 6 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to 5-fluorouracil (5FU) alone in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH) ...... 113 Figure 5. 7 Food intake (A) and body weight (B) of mice from four different diet and treatment groups ...... 116 Figure 5. 8 Plasma folate (A) and homocysteine (B) concentrations of mice at different folic acid diets ...... 117 Figure 5. 9 Relative tumor volume of HCT116 colon cancer xenografts expressing either GGH overexpression (Sense) or endogenous GGH (Control-S) treated with saline or 5FU+LV at 2 mg FA/kg control (A) and 8 mg FA/kg supplemented (B) diets ...... 118 Figure 5. 10 Representative images of Ki-67-stained xenografts (A) and percentage of Ki-67- positive cells (B) of HCT116 colon cancer xenografts expressing either GGH overexpression (Sense) or endogenous GGH (Control-S) ...... 121 Figure 5. 11 Representative images of xenografts apoptosis as determined by TUNEL assay (A) and percentage of TUNEL-positive cells (B, C) of HCT116 colon cancer xenografts expressing either GGH overexpression (Sense) or endogenous GGH (Control-S) ...... 122

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Figure 6. 1 Global DNA methylation in the GGH-modulated HCT116 colon (A, B) and MDA- MB-435 breast (C, D) cancer cells ...... 143 Figure 6. 2 DNA methyltransferase enzyme activity in the GGH-modulated HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cells ...... 144 Figure 6. 3 Scatter plots of DNA methylation β-value of GGH overexpression and inhibition in HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cell lines ...... 145 Figure 6. 4 Number of CpG sites methylated at different β-values in the GGH-modulated HCT116 colon cancer cells ...... 146 Figure 6. 5 Number of CpG sites methylated at different β-values in the GGH-modulated MDA-MB-435 breast cancer cells ...... 147 Figure 6. 6 Two-dimensional hierarchical clustering of DNA methylation β-value in the GGH- modulated HCT116 cell line ...... 148 Figure 6. 7 Two-dimensional hierarchical clustering of DNA methylation β-value in the GGH- modulated MDA-MB-435 cell line ...... 149 Figure 6. 8 Number of genes commonly differentially methylated in HCT116 and MDA-MB- 435 cells and distribution of differentially methylated CpG loci in response to GGH overexpression ...... 155 Figure 6. 9 Number of genes commonly differentially methylated in HCT116 and MDA-MB- 435 cells and distribution of differentially methylated CpG loci in response to GGH inhibition ...... 156 Figure 6. 10 Two-dimensional hierarchical clustering of gene expression fold change in the GGH-modulated HCT116 cell line ...... 160 Figure 6. 11 Two-dimensional hierarchical clustering of gene expression fold change in the GGH-modulated MDA-MB-435 cell line ...... 161 Figure 6. 12 Number of genes commonly differentially expressed in both the HCT116 and MDA-MB-435 cells in response to GGH overexpression (A) and GGH inhibition (B) .... 170 Figure 6. 13 Integrated analysis of gene expression and promoter DNA methylation changes between Sense and Control-S (A, C) and between siRNA and Control-si (B, D) in the GGH- modulated HCT116 (A, B) and MDA-MB-435 cells (C, D) ...... 174 Figure 6. 14 Number of genes differentially expressed in the opposite direction between GGH overexpression and inhibition in the GGH-modulated HCT116 colon cancer cells ...... 189 Figure 6. 15 Number of genes differentially expressed in the opposite direction between GGH overexpression and inhibition in the GGH-modulated MDA-MB-435 breast cancer cells 192

Figure 7. 1 Global DNA methylation in the FPGS-modulated HCT116 colon (A) and MDA- MB-435 breast (B, C) cancer cells ...... 211 Figure 7. 2 DNA methyltransferase enzyme activity in the FPGS-modulated HCT116 colon (A) and MDA-MB-435 breast (B, C) cancer cells ...... 212 Figure 7. 3 Scatter plots of DNA methylation β-value of FPGS overexpression and inhibition in HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cell lines ...... 213

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Figure 7. 4 Number of CpG sites methylated at different β-values in the FPGS-modulated HCT116 colon cancer cells ...... 214 Figure 7. 5 Number of CpG sites methylated at different β-values in the FPGS-modulated MDA-MB-435 breast cancer cells ...... 215 Figure 7. 6 Two-dimensional hierarchical clustering of DNA methylation β-value in the FPGS-modulated HCT116 cell line ...... 216 Figure 7. 7 Two-dimensional hierarchical clustering of DNA methylation β-value in the FPGS-modulated MDA-MB-435 cell line ...... 217 Figure 7. 8 Number of genes commonly differentially methylated in HCT116 and MDA-MB- 435 cells and distribution of differentially methylated CpG loci in response to FPGS overexpression ...... 223 Figure 7. 9 Number of genes commonly differentially methylated in HCT116 and MDA-MB- 435 cells and distribution of differentially methylated CpG loci in response to FPGS inhibition ...... 224 Figure 7. 10 Two-dimensional hierarchical clustering of gene expression fold change in the FPGS-modulated HCT116 cell line ...... 228 Figure 7. 11 Two-dimensional hierarchical clustering of gene expression fold change in the FPGS-modulated MDA-MB-435 cell line ...... 229 Figure 7. 12 Number of genes commonly differentially expressed in both the HCT116 and MDA-MB-435 cells in FPGS overexpression (A) and FPGS inhibition (B) ...... 238 Figure 7. 13 Integrated analysis of gene expression and promoter DNA methylation changes between Sense and Control(-S) (A, C), between Antisense and Control (B), and between siRNA and Control-si (D) in the FPGS-modulated HCT116 (A, B) and MDA-MB-435 cells (C, D) ...... 242 Figure 7. 14 Number of genes differentially expressed in the opposite direction between FPGS overexpression and inhibition in the FPGS-modulated HCT116 colon cancer cells ...... 260 Figure 7. 15 Number of genes differentially expressed in the opposite direction between FPGS overexpression and inhibition in the FPGS-modulated MDA-MB-435 breast cancer cells 263

Figure 8. 1 Number of genes whose altered expression was affected by both GGH and FPGS in HCT116 (A) and MDA-MB-435 (B) cells ...... 280

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List of Appendices

Appendix 1 Cell survival …………………………………………………………………… 327

Appendix 2 Diet composition………………………………………………...……………… 329

Appendix 3 Gene-specific promoter CpG island methylation analysis……………………… 332

Appendix 4 Gene expression analysis ……………………………………………………… 334

Appendix 5 The top 50 genes with most differentially altered expression in response to GGH modulation ………………………………………………………………………… 337 Appendix 6 The list of differentially expressed genes associated with the top functions in response to GGH modulation ………………………………………………………… 347 Appendix 7 The list of differentially expressed genes associated with the top networks in response to GGH modulation ………………………………………………………… 412 Appendix 8 Genes with altered promoter methylation and expression in response to GGH modulation ………………………………………………………………………… 416 Appendix 9 The list of differentially methylated and expressed genes associated with the top networks in response to GGH modulation ……………………………………… 427 Appendix 10 The list of genes associated with the GGH-specific gene expression analysis ... 431

Appendix 11 The top 50 genes with most differentially altered expression in response to FPGS modulation………………………………………………………………………… 440 Appendix 12 The list of differentially expressed genes associated with the top functions in response to FPGS modulation ………………………………………………………… 451 Appendix 13 The list of differentially expressed genes associated with the top networks in response to FPGS modulation ………………………………………………………… 537 Appendix 14 Genes with altered promoter methylation and expression in response to FPGS modulation………………………………………………………………………… 541 Appendix 15 The list of differentially methylated and expressed genes associated with the top networks in response to FPGS modulation ……………………………………… 555 Appendix 16 The list of genes associated with the FPGS-specific gene expression analysis… 560

Appendix 17 Genes with altered expression associated with both GGH and FPGS ………… 574

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CHAPTER 1: INTRODUCTION

1

2

Folate, a water-soluble B and an important mediator of one-carbon transfer, plays an essential role in DNA synthesis and methylation (1). Intracellular folate homeostasis is maintained by folylpolyglutamate synthase (FPGS) that facilitates intracellular retention of folate by polyglutamylation and by γ-glutamyl hydrolase (GGH) that catalyzes the hydrolysis of polyglutamylated folate into monoglutamates, thereby facilitating the export of folate out of the cell (1). Polyglutamylated folates are better substrates for folate-dependent enzymes and are better retained within cells (2). Antifolates, such as methotrexate (MTX), are metabolized in a similar way to folate (2, 3). As with folate, polyglutamylated antifolates generally have a higher affinity for, and thus inhibit their target folate-dependent enzymes in de novo synthesis of thymidylate and to a greater extent compared with the monoglutamate form (2, 3). FPGS and GGH may affect chemosensitivity of cancer cells to antifolates and 5-fluorouracil (5FU) by changing intracellular retention of antifolates and by changing intracellular retention of a folate cofactor (5,10-methylenetetrahydrofolate) necessary for the cytotoxic effects of 5FU, respectively. In addition to polyglutamylation of folate and antifolates, FPGS and GGH induce changes in intracellular folate concentrations (4, 5). Intracellular folate status is a critical determinant of chemosensitivity of cancer cells to chemotherapeutic agents designed to interrupt intracellular folate metabolism and DNA synthesis (4). As such, FPGS and GGH are important enzymes for the maintenance of intracellular homeostasis of folates and antifolates for optimal folate-dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects, respectively.

Folate deficiency is essentially nonexistent (< 1%) in Canada and the United States (6-8) owing to mandatory folic acid (FA) supplementation and widespread supplemental use of FA. Also, up to 50% of cancer patients and long-term survivors have reported the use of FA containing supplements (9, 10). While FA supplementation is beneficial to prevent and reduce side effects and toxicities of antifolates and 5FU, a growing body of evidence suggests that FA supplementation may interfere with the sensitivity of chemotherapeutic drugs and cause drug resistance (11-14).

There is accumulating evidence demonstrating that FPGS modulation significantly affects sensitivity of tumor cells to chemotherapeutic agents. High FPGS expression/activity is shown to increase polyglutamylation and chemosensitivity of antifolates and 5FU (4, 15-17), whereas

3 reduced FPGS expression/activity is associated with drug resistance (4, 17-26). However, it is largely unknown whether GGH modulation can affect chemosensitivity of cancer cells to chemotherapeutic agents.

Folate also mediates the transfer of one-carbon units for the generation of S-adenosylmethionine, the primary methyl group donor for most biological methylation reactions including DNA methylation, which is catalyzed by DNA methyltransferase (DNMT) (1, 27). Polyglutamylation is also important in DNA methylation as polyglutamylated folates are better substrates for folate- dependent enzymes such as methylenetetrahydrofolate reductase and methionine synthase that are involved in the generation of S-adenosylmethionine, which is a substrate for DNA methylation mediated by DNMT (2, 28). And hence, FPGS and GGH may affect DNA methylation at global and gene-specific levels with consequent functional ramifications. Both global DNA hypomethylation and gene-specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis (29). DNA methylation and DNMT are also potential therapeutic targets and may modify the effect of specific chemotherapeutic agents (30), suggesting that the FPGS- and GGH-modulated DNA methylation changes might influence chemosensitivity to chemotherapeutic agents. In addition, a number of recent studies suggest that aberrant DNA methylation might affect chemosensitivity of cancers by altering expression of genes critical to the drug response (31-34).

Given these considerations, the overarching goal of my research was to investigate the pharmacogenetic role of GGH in modulating chemosensitivity of human HCT116 colon and MDA-MB-435 breast cancer cell lines to chemotherapeutic agents using in vitro and in vivo systems and the effects of GGH and FPGS modulation on DNA methylation and its functional ramifications. This thesis consists of two main research questions: the first research question was whether chemosensitivity of antifolates and 5FU would be altered by GGH modulation (Study 1 and Study 2); and the second research question was whether DNA methylation and gene expression would be altered by GGH and FPGS modulation (Study 3 and Study 4). The objective of Study 1 was to characterize the role of GGH in modulating chemosensitivity of human cancer cells to antifolates and 5FU, and the aim of Study 2 was to determine whether exogenous folate concentrations would further influence the effect of GGH modulation on chemosensitivity of human cancer cells to antifolates and 5FU. The objective of Study 3 was to investigate the effect of GGH modulation on global and gene-specific DNA methylation and gene expression, and the

4 purpose of Study 4 was to determine whether FPGS modulation would affect global and gene- specific DNA methylation and gene expression.

CHAPTER 2: LITERATURE REVIEW

5

6

2.1 Folate

Folate is a water-soluble B vitamin that is naturally present in many foods, including green leafy vegetables, asparagus, broccoli, Brussels sprouts, citrus fruit, legumes, dry cereals, whole grain, yeast, lima beans, liver, and other organ meats (1). Naturally occurring folates are highly unstable, rapidly lose their activity in foods, and are easily oxidized under low pH (1). Folate bioavailability varies widely depending on the food source and preparation method (1). Approximately 50-75% of the original folate values are lost through food harvesting, storage, processing, and preparation (1). In contrast, folic acid (FA), also known as pteroyl(mono)glutamate, is the most stable and the fully oxidized monoglutamyl synthetic form of folate that is commercially used in supplements and in fortified foods (1). Since the mandatory fortification of white wheat flour, cereal, and enriched pastas with FA in 1998, grain products have become a major source of folate in Canada and the United States (35).

2.1.1 Chemistry of Folate

FA consists of three units: a 2-amino-4-hydroxy-pteridine (pterin) ring linked via a methylene bridge to para-aminobenzoic acid (PABA), which in turn is bound to glutamate via a bond (Figure 2.1A) (1). Folate, on the other hand, is the generic term referring to compounds that have similar chemical structures and nutritional properties, and folates found in food differ from the oxidized FA because they are in the reduced state in their pteridine ring (Figure 2.1B). As shown in Figure 2.1B, one-carbon units (R) can be linked to tetrahydrofolate (THF) at the N- 5 and N-10 positions, which confers folate the role of mediating the transfer of one-carbon units. The most reduced form of folate is 5-methylTHF (5-MTHF) and the most oxidized forms are 5- and 10-formylTHF (Figure 2.1B). In addition, multiple glutamate residues of varying numbers (up to nine) can be added via a γ-peptide linkage (Figure 2.1B).

Although have the ability to synthesize all of the component parts of folate, they lack the enzyme required for coupling the pteridine ring to PABA and as a result, cannot synthesize folate de novo (1). Thus, with the exception of the incorporation of folate synthesized by intestinal flora, mammals must obtain this vitamin from dietary and supplemental sources (36).

7

R One-carbon N5 N10 moiety Reduced -CH3 -H 5-methyl- -CH2- 5,10-methylene- Level of =CH= 5,10-methenyl- Oxidation -CHNH -H 5-formimino- -CHO -H 5-formyl- Oxidized -H -CHO 10-formyl-

Figure 2. 1 Chemical structures of folic acid (A) and folate (B). Adapted and modified by permission from the publisher (John Wiley and Sons) (29).

2.1.2 Folate Metabolism and Biochemical Function

Naturally occurring folate exists mainly as polyglutamylated derivatives of 5-MTHF and 10- formylTHF, with 5-MTHF as the predominant food folate. 5-MTHF is readily oxidized to 5- methyl-5,6-dihydrofolate, and this oxidized form is rapidly degraded under the mildly acidic conditions which prevail in the postprandial gastric environment (36). Under the same conditions, 5-MTHF is relatively stable. Once ingested, the stomach optimizes bioavailability of folate by secreting ascorbic acid into the gastric lumen, reducing 5-methyl-5,6-dihydrofolate back to 5- MTHF (36). By contrast, FA is rapidly absorbed and becomes metabolically active upon reduction, first to dihydrofolate (DHF) and then to THF by DHF reductase (DHFR), and methylated to 5-MTHF in the liver and, to a lesser degree, in the intestine (37), before it can enter the folate cycle (Figure 2.2). Since the capacity to reduce folate (e.g., DHFR) is limited, high intakes of FA result in its appearance of being unaltered in circulation (38).

8

Figure 2. 2 Simplified biochemical pathway of folate metabolism and reactions. GCP II (glutamate carboxypeptidase II) hydrolyzes intraluminal folate; FR (folate receptor), PCFT (proton- coupled folate transporter) and RFC (reduced folate carrier) are involved in intracellular folate uptake while MRP (multidrug resistant protein) is associated with folate efflux; FPGS (folylpolyglutamate synthase) is involved in intracellular folate retention and GGH (γ-glutamyl hydrolase) is involved in folate efflux; DHFR (dihydrofolate reductase) and SHMT (serine hydroxymethyltransferase) are involved in maintenance of the intracellular folate pool; TS (thymidylate synthase) is involved in nucleotide biosynthesis. Aminoimidazole-carboxomide ribonucleotide (AICAR) transformylase (AICARFT) and glycinamide ribonucleotide (GAR) transformylase (GARFT) each catalyze the acceptance of a one-carbon unit in AICAR and GAR from 10-formylTHF during the biosynthesis of the ring; MS (methionine synthase), MSR (methionine synthase reductase) and MTHFR (methylenetetrahydrofolate reductase) are involved in the methionine cycle; DNMT1, 3a, 3b are CpG involved in DNA methylation while MBD2 (methyl-CpG binding domain protein 2, DNA demethylase) is involved in DNA demethylation. Filled circle represents a pteridine ring conjugated to PABA. Each filled triangle represents a glutamate, which is linked via a peptide bond to form various chain lengths of polyglutamylated folate. AHCY, S-adenosylhomocysteine hydrolase; BHMT, betaine-homocysteine S-methyltransferase; CBS, cystathionine-β-synthase; CTH, γ-cystathionase; CpG, cytosine-guanine dinucleotide sequence; dTMP, deoxythymidine-5-monophosphate (thymidylate); dUMP, deoxyuridine-5-monophosphate; FAD, flavin adenine dinucleotide; FADH2, 1,5-dihydro-flavin adenine dinucleotide; FAICAR, formylaminoimidazole- carboxomide ribonucleotide; FGAR, formylglycinamide ribonucleotide; GNMT, glycine N- methyltransferase; IMP, inosine 5’-monophosphate; MAT, methionine adenosyltransferase; MTFMT, mitochondrial methionyl-tRNA formyltransferase; MTHFCH, methenyltetrahydrofolate cyclohydrolase; MTHFD1, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1; SAH, S- adenosylhomocysteine; SAM, S-adenosylmethionine; THF, tetrahydrofolate. Adapted and modified by permission from the publisher (John Wiley and Sons) (29).

9

2.1.2.1 Intestinal Absorption

Folate absorption can occur along the entire small and large intestine, and is most efficient in the jejunum (35). Prior to absorption into enterocytes, polyglutamylated forms of folate found in dietary sources need to be hydrolyzed by the intestinal apical brush border enzyme glutamate carboxypeptidase II (GCP II) to their monoglutamylated forms in the lumen of the intestine since glutamate tails with three or more residues cannot cross the intestinal cell membrane (1, 39). Monoglutamylated folates are then transported across the enterocyte brush border membrane through folate transporters including an anion exchange mechanism driven by the transmembrane pH gradient (Figure 2.2) (36).

2.1.2.2 Folate Transport Systems

There are four transporters of folates and antifolates in the body: reduced folate carrier, proton- coupled folate transporter, folate receptor, and ATP-binding-cassette transporter (Figure 2.2). Due to the highly lipophobic bivalent property of folate, there is minimal passive diffusion across cell membranes only when pharmacological doses of FA are consumed (40).

Reduced Folate Carrier

Reduced folate carrier (RFC; SLC19A1) is a saturable anion dependent membrane carrier with a greater affinity for reduced folates (Km = 1-5 μM) and hydrophilic antifolates such as methotrexate (MTX; Km = 5-10 μM) than FA (Km = 100-200 μM) (1). The affinity for pemetrexed (MTA) is 2-fold higher than that for MTX (41, 42). RFC is a member of the superfamily of solute carriers, and utilizes the high transmembrane anion gradient, particularly, the organic phosphate gradient which is modulated by energy status of the cell in order to achieve uphill folate transport into cells (40, 43). The human RFC gene is ubiquitously and differentially expressed in normal human tissues as well as neoplastic tissues (44). Expression of human RFC transcripts is highest in the placenta and lowest in skeletal muscle. RFC can also be detected in the liver, leukocytes, kidney, lung, bone marrow, small intestine, regions of the central nervous system, and brain (44).

RFC functions as a bidirectional transporter for monoglutamylated reduced folates and classical antifolates, with maximal absorption occurring at an intestinal pH of 5.0 to 6.0 (45). The

10 transport of cytotoxic antifolates by RFC is not tumor specific and hence causes toxicity to normal tissue since RFC is ubiquitously expressed and exhibits a high level of activity at neutral pHs, which are characteristic of most normal tissues (45). For chemotherapeutic antifolates such as MTX or MTA, synthesis of mutant RFCs or loss of RFC transcripts and proteins results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and insufficient substrate for polyglutamate synthesis (45). It has been known that is associated with overexpression of folate influx systems including RFC (46, 47), and downregulation of ATP-dependent folate exporters (48). However, Ifergan and colleagues also found that RFC mRNA levels were decreased by around 2.5-fold in MCF7 breast cancer and CEM T-cell leukemia cell lines that were accompanied by a consistent fall in MTX influx under 3-7 days of folate deprivation (49). As a result of this observation along with that of a mathematical biomodeling, they suggest that upon severe short term (up to 7 days) folate deprivation, RFC transport activity becomes reduced since RFC, but not ATP-driven folate exporters, efficiently exports folate monoglutamates out of the cell, indicating that the downregulation of RFC may serve as a novel adaptive response to severe folate deficiency (49).

Proton-Coupled Folate Transporter

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate-H+ symporter that functions most efficiently in an acidic extracellular environment; the downhill flow of protons via PCFT is coupled to the uphill flow of folates into cells (40, 50). The transport mediated by PCFT is greatest at low pH, but transport is also detected at pH 7.4 (51, 52). Human PCFT has a high affinity for FA, 5-MTHF, and 5-formylTHF at pH 5.5 (Km = 1-5 μM), and the highest affinity for MTA (Km = 0.2-0.8 μM) among folate analogs (41, 53). At pH 5.5, PCFT has a ~20-fold higher affinity for MTA than MTX (41). There are high levels of PCFT mRNA in the small intestine, kidney, liver, placenta, retina, and brain in human and murine tissues. The highest PCFT is found in the proximal jejunum and duodenum (50, 54).

The important difference between RFC and PCFT is the optimum pH that impacts their affinities for transport substrates. At pH 7.4, the transport of antifolates is mediated predominantly by RFC as RFC activity is optimal and the activity of PCFT is minimized. Alternatively, PCFT activity is more prominent as pH is decreased (40). In addition to the role of PCFT in mediating intestinal folate absorption and uptake into tissues in which there is an acidic extracellular environment

11 such as solid tumors or, less efficiently, transport into tissues in a neutral pH environment, PCFT may play a role in folate receptor-mediated endocytosis (55).

Folate Receptor

Folate receptors (FRα and FRβ) are high-affinity folate-binding proteins that are anchored in the cell membrane by a glycosylphosphoinositol domain (56). FRs have a higher affinity for FA (Km < 1 nM), to a lesser extent have an affinity for 5-MTHF (Km = 1-10 nM), and the lowest affinity for other reduced fo1ates (Km = 10-300 nM) (1, 57). FRα has a low affinity for MTX (Km = ~300 nM) but has an affinity for MTA higher than for FA (Km < 1 nM) (42). FRs transport folates into cells via an endocytic mechanism. Once in the cytoplasm, when the vesicle acidifies to a pH of 6.0-6.5, folate is released from the receptor, and is exported from the endosome by a mechanism proposed to be mediated at least in part by PCFT (40, 58). Although FRα has a higher affinity for its preferred substrates than RFC, its transport into cells requires a series of steps such as binding, invagination, vesicle formation and translocation, acidification, and export of substrate from the vesicle into cytoplasm. Due to these multiple steps required for the transport into cells, the maximum rate of FR-mediated transport into the cell is 1/100 the rate mediated by RFC (59).

FRα is known to be expressed on the membrane of epithelial cells of the kidney, choroid plexus, and retina, and a high degree of expression is found in several carcinomas, particularly in uterine and ovarian cancer cells (60). In normal and tumour cells, FRα expression has been shown to increase in response to folate deficiency and decrease with increased intracellular folate concentration, suggesting that FRα is important in regulating intracellular folate pools (61, 62). FRβ is expressed in placenta and hematopoietic malignancies such as acute and chronic myelogenous leukemia (63) and on activated macrophages and tumor-infiltrating macrophages (64, 65) whereas FRγ is a secretory protein localized to hematopoietic tissues (66). Generally, the affinity of 5-MTHF for FRα is higher than that of FRβ (67).

ATP-Binding-Cassette Transporter

Recent studies reported that several of the ATP-binding-cassette (ABC) transporters are low- affinity, high-capacity ATP-dependent efflux pumps of folates and antifolates (Km = 0.2-2 mM for folate and antifolates); these include the multidrug-resistance-associated proteins, MRP1-

12

MRP5 (ABCC1-ABCC5) and the breast-cancer-resistance protein, BCRP (ABCG2) (11, 68, 69). In particular, it has been known that BCRP is associated with the efflux of mono-, di-, and tri- glutamate conjugates of FA and antifolates such as MTX, while MRP1-4 export monoglutamates of folates and MTX (11, 14, 70, 71). MRP5 is known to export both mono- and diglutamate forms (11, 14).

Members of this family are widely expressed in mammalian cells and suppress the concentrations of folates or antifolates accumulated in cells grown in vitro (70). MRP1-MRP4 and BCRP are expressed in human jejunum, but their relative levels have not been established (72, 73). MRP1 and MRP4 are known to locate at the basolateral membrane of some tissues, but their localization in human jejunum has not been reported (70). In animal studies, MRP2 is expressed at the apical membrane of rat jejunum, which would oppose absorption mediated by PCFT (74), while MRP3 is expressed at the basolateral membrane of rat jejunum (75). MRP1, MRP3, and MRP5 are expressed in the basolateral membrane of mice duodenum and jejunum, a location that should facilitate vectorial transport across these epithelia (76-78). Conversely, MRP2 and BCRP are located at the apical membrane of these epithelia opposing intestinal folate reabsorption and reabsorption of folate from the glomerular filtrate (74, 76, 79). The overexpressed ABC transporters in vitro decrease cellular folate concentrations and increase folate growth requirements (48).

2.1.2.3 Transport

Once absorbed by enterocytes, folate is either polyglutamylated and retained within cells or released into portal circulation for various compartments for metabolism, storage, or enterohepatic recirculation (80). The liver takes up the majority of folate that enters portal circulation, while the remaining folate passes through the liver, enters the general circulation and is taken up by other tissues where it is converted into its polyglutamylated form for intracellular storage (1).

Most folate circulating in the blood (monoglutamylated 5-MTHF) is bound nonspecifically to albumin, α-2macroglobulin, and transferrin, although about one third circulates unbound. Only a small fraction of serum folate is bound by specific folate-binding proteins (FBPs), several of which appear to be derived from folate receptors of cell membranes (81, 82). Folate is also

13 reabsorbed in the proximal tubule of nephrons by a mechanism involving FBPs and contributes to the availability of circulating folate (36). Folate concentrations found in serum and red blood cells (RBC) vary greatly. Serum folate concentrations of 13.5-45.3 nM are considered normal levels (83). The WHO has currently recommended deficiency cutoffs of < 10 nM for serum and < 340 nM for RBC folate based on metabolic indicators (84). RBC folate, mainly 5-MTHF and 10-formylTHF, is used as an indicator of tissue folate status because it is not affected by recent dietary folate intake (36). The majority of folate found in RBC is polyglutamylated, with penta- and hexaglutamates predominating, and RBC folate acts as a long-term storage reservoir and buffer for maintaining folate homeostasis (85). Perhaps as much as 100 μg of folate undergoes enterohepatic recycling daily, with biliary excretion followed by reabsorption (1). Most of the estimated 200 μg of folate is excreted daily in feces, and only a few micrograms of intact folate is eliminated in the urine (86).

2.1.2.4 Intracellular Folate Metabolism

The major function of folate is to mediate the transfer of one-carbon units involved in nucleotide biosynthesis, the remethylation of homocysteine (Hcy), and biological methylation reactions (29). As an essential cofactor for the de novo biosynthesis of nucleotide (1, 36), folate plays an important role in DNA synthesis, stability and integrity, and repair. Folate also provides the primary methyl group donor for the methylation of DNA (regulates gene expression), proteins (important post-translational modifications), and lipids (important in their synthesis, for example, phosphatidylcholine) (87).

Folate-mediated one-carbon metabolism is highly compartmentalized between the cytoplasm, mitochondria, and nucleus (88). In general, it is believed that cytosolic folates are mainly associated with purine and thymidylate synthesis and biological methylation reactions while mitochondria folates (30-50% of cellular folates) are primarily related with the serine/glycine cycle (89). The major folate metabolites found in the cytosol are THF and 5-MTHF, whereas THF and 10-formylTHF are found in the mitochondria (89).

Cytoplasmic Pathways

Prior to transferring one-carbon units, folate needs to be reduced to THF by DHFR, a NADPH requiring enzyme (80) while FA undergoes a two-step reduction, first being reduced to DHF and

14 then to THF, the last reaction being mediated by DHFR. There is evidence suggesting that DHFR activity in human mucosa and liver is much lower than in rodents (37). In most organisms, serine derived from glycolysis provides one-carbon units to THF, a reaction catalyzed by serine hydroxymethyltransferase (SHMT), generating 5,10-methyleneTHF and glycine. 5,10- methyleneTHF is required for de novo thymidylate synthesis catalyzed by thymidylate synthase (TS), and its reduction mediated by methylenetetrahydrofolate reductase (MTHFR) leads to 5- MTHF, which is involved in remethylation of Hcy. In addition, 5,10-methyleneTHF is oxidized to 10-formylTHF, necessary for purine biosynthesis, requiring two moles of 10-formylTHF per mole of purine ring. The conversion of 5,10-methyleneTHF to 10-formylTHF is achieved by the sequential enzymes 5,10-methyleneTHF dehydrogenase (MTHFD) and 5,10-methenylTHF cyclohydrolase (MTHFCH) (Figure 2.2) (88).

In the methionine cycle, 5-MTHF transfers single methyl groups to Hcy, catalyzed by cobalamin

(vitamin B12)-dependent methionine synthase (MS) (90, 91), to synthesize methionine. Methionine can then be activated by ATP and methionine adenosyl trasnferase (MAT) to yield S-adenosylmethionine (SAM), which is the primary methyl group donor for most biological methylation reactions including that of DNA, a reaction that is catalyzed by DNA methyltransferases (DNMTs) (27, 92). During the process, SAM is converted to S- adenosylhomocysteine (SAH), which is then hydrolyzed back to Hcy to recommence the remethylation cycle (93). Meanwhile, Hcy transsulphuration involves the condensation of thiol with serine to form cystathionine, a vitamin B6-dependent step activated by cystathionine-β- synthase (CBS). Cystathionine is then hydrolyzed to cysteine and α-ketobutyrate mediated by γ- cystathionase (CTH). As such, SAM regulates the remethylation and transsulphuration pathways, which coordinates the the utilization of Hcy (94). 5-MTHF regulates the methionine cycle by controlling the SAM/SAH ratio and the availability of dietary methionine by donating a methyl group to Hcy (95). 5-MTHF is converted to THF by MS and subsequently to 5,10- methyleneTHF by the vitamin B6-dependent SHMT (Figure 2.2) (96, 97).

Nucleotide biosynthesis is another important role of folate cofactors. In purine biosynthesis, aminoimidazole carboxamide ribonucleotide (AICAR) and glycinamide ribonucleotide (GAR) each receive a one-carbon unit from 10-formylTHF through the action of AICAR transformylase (AICARFT) and GAR transformylase (GARFT), respectively, yielding THF and intermediates in the formation of purines (36). The synthesis of thymidylate requires 5,10-methyleneTHF to

15 donate a methyl group to deoxyuridylate monophosphate (dUMP) (Figure 2.2). The reduction of TS is the rate-limiting step in DNA synthesis (36). The expression of TS and DHFR peaks during the S phase of the cell cycle and is related to the replication rate (36). In actively proliferating cells, increased TS activity may lead to the elevation of the steady state DHF levels, and inhibition of MTHFR activity, providing a regulatory mechanism for ensuring that priority is given to synthesis over methionine formation (98). Since the synthesis of purines and thymidylate is essential for DNA replication and cell proliferation, alterations such as decreased folate-dependent enzyme activity and binding will affect these cellular processes.

Mitochondrial Pathways

Studies have suggested that a reduced, monoglutamylated form of cytoplasmic folate (probably THF or 5-formylTHF) is transported into the mitochondria by the mitochondrial folate transporter (MFR), followed by mitochondrial FPGS induced polyglutamylation resulting in mitochondrial folate accumulation (99). Lin and Shane showed that mitochondrial polyglutamates can exit from the mitochondria without prior polyglutamate removal although polyglutamates could not enter the mitochondria in cultured CHO cells (100). RFC is also found in the mitochondrial membrane, as demonstrated in one study using the human CCRF-CEM T- cell lymphoblastic leukemia cell line (101).

Although the exchange between the cytoplasmic and mitochondrial pools of THF is limited, the transport of one-carbon units between two compartments occurs by serine, glycine, and formate. In mammals, excess choline is oxidized to betaine, a reaction catalyzed by the cytoplasmic betaine-homocysteine S-methyltransferase (BHMT), which donates a methyl group to Hcy, forming methionine in the liver (88). Dimethylglycine (DMG, the other product of the BHMT reaction) and sarcosine can also serve as one-carbon donors in the mitochondria via DMG and reactions. SAM is also known to be transported to the mitochondria in a passive carrier-mediated process (102).

Folate-mediated one-carbon metabolism in the mitochondria consists of three functions: (1) the initiation of mitochondrial protein synthesis; (2) glycine synthesis catalyzed by SHMT; and (3) serine cleavage to form 5,10-methyleneTHF. Serine formed by cytosolic glycolysis enters the

16 mitochondria and transfers a one-carbon group (CH2) to THF, forming glycine and 5,10- methyleneTHF. Formate is reversibly converted to 10-formylTHF to regenerate THF (89).

Nuclear One-Carbon Metabolism

The evidence of folate-mediated dTMP (deoxythymidine-5-monophosphate; thymidylate) synthesis in the nucleus is presented in a recent study. Stover and colleagues observed that cytoplasmic SHMT, TS, and DHFR are all translocated into the nucleus during S and G2/M cell cycle phases following modification by the small ubiquitin-like modifier in HeLa and MCF-7 cells (103, 104). Nuclear dTMP synthesis may not occur in all cells since the tissue-specific expression of cytoplasmic SHMT is more restricted than that of TS and DHFR (88).

2.1.3 Intracellular Homeostasis of Folate

While monoglutamates are the only circulating forms of folate in the blood and the only form of folate that is transported across the cell membrane, once taken up into cells, intracellular folate exists primarily as polyglutamates (1). Intracellular folate is converted to polyglutamates by FPGS, whereas GGH removes the terminal glutamates, thereby facilitating the export of folate out of the cell (1). Polyglutamylated folates are better retained in cells and are better substrates for intracellular folate dependent enzymes compared with monoglutamates (2).

Similar to folates, antifolates such as MTX are retained in tumor and normal cells by the FPGS- induced polyglutamylation and are exported from cells after hydrolysis to monoglutamates by GGH (2, 3). As with folate, polyglutamylated antifolates are retained in cells longer, thereby increasing their cytotoxicity by extending the length of exposure (2, 3). Polyglutamylated antifolates generally have a higher affinity for and, hence, inhibit their target folate-dependent enzymes in thymidylate and purine biosynthesis to a greater extent than the monoglutamate forms (Figure 2.2) (2, 3). As such, FPGS and GGH are important enzymes for the maintenance of intracellular homeostasis of folates and antifolates for optimal folate-dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects.

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2.1.3.1 Folylpolyglutamate synthase

Folylpolyglutamate synthase (FPGS, EC 6.3.2.17) is an ATP-dependent that catalyzes the formation of polyglutamate derivatives of folates and antifolates (105). The chemical reaction catalyzed by FPGS involves simple amide bond formation via a γ-glutamyl phosphate intermediate (106). The reaction requires three substrates, folate or antifolate, ATP, and glutamate, and yields three products, a γ-glutamyl peptide metabolite of folate or antifolate, ADP, and inorganic phosphate (Pi). Generally, the folate products of this reaction can function as FPGS substrates again (105). The substrates bind in the order of ATP, folate/antifolate, and glutamate and the products are released in the order of ADP, folate/antifolate-Glu, and Pi (107, 108).

Tissue Distribution

In general, FPGS activity in mammalian cells is very low compared with bacterial sources (109). The steady state level of human FPGS mRNA expression was found to be highest in the liver, pancreas, and kidney, but can be distributed in most mammalian tissues (110). Human heart and skeletal muscle have very high levels of FPGS-specific mRNA, whereas FPGS activity is negligible in mouse muscle tissue (110). However, FPGS mRNA levels were not predictive of FPGS activity in primary human leukemia blasts and normal hematopoietic progenitors (111). It appears that FPGS expression might be controlled by tissue/lineage-specific mechanisms and linked to proliferation (111). Distributions of polyglutamate chain lengths differ in tissues within and between (112). For example, the pentaglutamylate form predominates in rat liver, while the hexaglutamylate form primarily exists in pig and mouse liver (109). Furthermore, FPGS activity is high in actively proliferating cells and drops in cells undergoing differentiation or entering an extended G0 phase (113).

FPGS activity has been observed to be higher in tumor samples and cancer cell lines compared with normal tissue. FPGS activity of head and neck tumor biopsies varied from 25 to 1827 pmol/hr/mg in tumor tissue compared to 7 to 297 pmol/hr/mg in normal tissue, and head and neck cell lines showed 335 to 1305 pmol/hr/mg protein of FPGS activity (114). In addition, FPGS activity in human colon tumors or metastases varied greatly from 531 to 6315 pmol/hr/mg protein, and 2- to 3- fold increased FPGS activity was observed in cells grown in reduced folate

18 medium (115).

Cellular Location

Most FPGS activity is present in the cytoplasm of cells, while the remaining activity occurs in the mitochondria. Mitochondrial and cytosolic forms of FPGS are derived from a single gene, arising from the use of two different translation initiation codons, and their translation products differ by the presence of a 42-residue amino-terminal mitochondrial leader peptide (116). Two thirds of the FPGS transcripts in leukemia cells correspond to mRNA encoding mitochondrial enzyme while the remaining third corresponds to mRNA encoding cytosolic enzyme (116). Mitochondrial FPGS activity is required for mitochondrial folate accumulation used in glycine metabolism (117). Cells lacking mitochondrial FPGS are auxotrophic for glycine, and cytosolic FPGS overexpression does not correct for low or absent mitochondrial FPGS activity (118). The folate pools found in mitochondria and cytoplasm are not in equilibrium, and mitochondrial polyglutamylate derivatives are longer compared to cytosolic derivatives (119).

Polyglutamylation by FPGS

During FPGS catalyzed reactions, the carboxylate groups of glutamic acid residues carry a negative charge that increases with each additional glutamate molecule (120). The negatively charged polyglutamates cannot cross the cell membrane, and thus, are retained and concentrated intracellularly. Polyglutamylated folates also have a higher affinity for folate-dependent enzymes in pig and rabbit liver (109, 121) and human colon adenocarcinoma xenograft (122).

The rate and extent of polyglutamylation in cells is determined by several factors. Monoglutamylated folates taken up into cells are metabolized by FPGS into polyglutamylated forms, which then provide feedback to inhibit FPGS activity (123, 124). Eventually, intracellular polyglutamylate levels will reach a concentration that suppresses the entry of monoglutamylates into the cell. At this point, the rate of monoglutamyl glutamylation is equal to the rate of hydrolysis mediated by GGH and the low leakage rates from cells, reaching THF-cofactor steady state (123, 124). The steady-state level differs among various tissues and the THF-cofactor pool is also an important determinant for the polyglutamylation of antifolates, limiting the rate and extent of formation of these derivatives. In addition, Tomsho and colleagues found FPGS catalyzed multiple ligations of glutamic acid to antifolate in a processive manner but the degree

19 of processivity was dependent on the concentration of antifolate substrate (125). They suggested that the concentration dependence of processivity in polyglutamate formation may have evolved as a mechanism for regulation of folate homeostasis: low intracellular concentrations of folate would encourage the formation of long-chain polyglutamate metabolites, thereby increasing their retention within the cell and efficacy as cofactors, whereas, at high folate concentrations, cellular retention is not required and long-chain polyglutamate metabolite formation is reduced, thus allowing efflux of folate from the cell while minimizing unnecessary consumption of ATP (125).

Effect of Folate on FPGS Activity

Previous findings on the effect of folate on FPGS activity are inconclusive, but recent investigations into reactions catalyzed by FPGS support the inverse association between folate and FPGS activity (125). It has been shown that a low folate diet induced an increase in FPGS activity in murine tissues and human pancreatic carcinoma xenograft, suggesting that the tissue distribution and cytotoxicities of antifolates requiring polyglutamylation for activation and cellular retention would be influenced by folate status (126). Similarly, a low folate diet led to the upregulation of FR, elevated FPGS activity, and longer polyglutamate forms (octa- and hepta-) in mouse liver compared to the standard diet (127). In another study using Chinese hamster ovary AUXB1 cells transfected with human FPGS, the effects of FPGS levels on folate accumulation in different medium folate levels (2 nM-20 μM) were investigated. At low medium folate levels, folate accumulation was limited by influx and was independent of FPGS activity except in cells expressing extremely low levels of FPGS. As the medium folate concentration increased from physiological to pharmacological levels, cellular folate accumulation became proportional to FPGS activity. At high medium folate concentrations, competition between substrates for FPGS limited the extent of polyglutamylation and less than 5% of transported folate was retained by the cells (128).

Other Functions of FPGS

Altered FPGS expression was found to be associated with the outcome of colorectal cancer (CRC) patients. Odin and colleagues reported that the expression of RFC, FPGS, GGH, and TS were significantly higher in CRC tumor biopsies than the adjacent non-neoplastic mucosa (129). The same group also demonstrated that low expression of RFC and FPGS correlated with lack of

20 a putative tumor suppressor gene Deleted in Colorectal Carcinoma (DCC) mRNA splice variant in normal-appearing mucosa of CRC patients (130). In contrast, the expression of FPGS mRNA in peripheral mononuclear cells was shown to be predictive of a poor response to MTX therapy in rheumatoid arthritis (RA) patients (131). Although several single-nucleotide polymorphisms (SNPs) in the FPGS gene are reported, less is known regarding functionality or frequencies (132, 133). The mutant Cys346Phe FPGS is known to interfere with L-glutamate or ATP binding thereby presumably disrupting FPGS activity (19). Recently, Leclerc and colleagues proposed that FPGS expression is epigenetically regulated by the binding of selected ALL fusions to a multiprotein complex, which also controls the cell cycle dependence of FPGS expression. As part of a multiprotein complex, NFY and Sp1 recruit histone deacetylase (HDAC) 1 to the FPGS promoter region to regulate its expression. In cells expressing TEL-AML1, HDAC1 interacts with TEL-AML1 and possibly other regulatory factors such as NCoR, mSin3A, and Rb to downregulate FPGS mRNA expression (134). On the other hand, HDAC inhibitors induced FPGS mRNA expression and intracellular accumulation of long-chain MTX polyglutamates in childhood ALL cell lines, which was potentiated by downregulated DHFR and TS probably resulting from cell cycle arrest induced by HDAC inhibitors (135).

Effect of FPGS Modulation on Chemosensitivity

Generally, it has been established that the FPGS-induced polyglutamylation may affect the sensitivity of tumor cells to chemotherapeutic agents such as antifolates and 5-fluorouracil (5FU). of FPGS cDNA has been shown to increase sensitivity to MTX in variant hamster cells that lack endogenous FPGS activity (16). FPGS gene transfer into rat and human glioma, gliosarcoma, and glioblastoma cell lines already expressing FPGS significantly enhanced sensitivity to MTX and other antifolates (15). However, FPGS downregulation appears to be a mechanism of resistance to MTX and other antifolates in human and murine leukemia cell lines (19-22, 24, 26). Decreased FPGS activity has previously been shown to confer resistance to 5FU in some human cancer cell lines (18, 23, 25).

We have previously reported that FPGS overexpression increases, whereas FPGS inhibition decreases, chemosensitivity of human HCT116 colon cancer cells to 5FU (17). We have also demonstrated that at supraphysiologial folate medium concentrations, FPGS overexpression increases chemosensitivity of human MDA-MB-435 breast cancer cells to MTX, while FPGS

21 inhibition decreases 5FU-induced chemosensitivity in MDA-MB-435 cells (4). Furthermore, FPGS modulation affects polyglutamylation of not only antifolates and specific target intracellular folate cofactors (e.g., 5,10-methyleneTHF for 5FU), but also of all other intracellular folate cofactors, which are important determinants of the cytotoxic effects of antifolates and 5FU (3, 4, 17, 46, 136). Therefore, the FPGS modulation-induced changes in intracellular folate concentrations and folylpolyglutamylation may counterbalance the effects of the FPGS modulation-induced changes in polyglutamylation of antifolates and specific 5,10- methyleneTHF. As such, FPGS seems to play an important role in the sensitivity of cancer cells to antifolates and 5FU, and thus, FPGS modulation might be a potential therapeutic target for increasing sensitivity of cancer cells to these chemotherapeutic agents.

2.1.3.2 γ-Glutamyl hydrolase

γ-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes the hydrolysis of γ-polyglutamate tails that attached to folates and antifolates after they enter the cell in order to yield their monoglutamate form, which can then be released from the cell (137). GGH is a secretory lysosomal enzyme with an acidic pH 4.5-6 optimum (138) that has exo- and activity depending on the species and tissue of origin (139, 140). While rat GGH acts as an endopeptidase on the innermost γ-glutamate bond, human GGH catalyzes the hydrolysis of the ultimate or penultimate γ- glutamate bond with the sequential release of either monoglutamate or diglutamate (138, 141).

One study investigating site-directed mutagenesis indicated that Cys-110 might be an essential factor for GGH activity (142), and His-220, Glu-222, and His-171 of human GGH were also important in GGH activity (143). Furthermore, the three-dimensional structure of human GGH suggestd that Tyr-36 functioned as a catalytic residue in GGH activity (144). The proposed catalytic mechanism of GGH involves attack by the active-site Cys-110 nucleophile on a γ- carbonyl of a Glu-γ-Glu bond to yield a thioacyl enzyme intermediate, followed by release of an amine component (145). GGH requires the presence of reduced sulfhydryls for maximum catalytic activity (146). Human GGH exhibits greater preference for longer chain polyglutamylates (Glu4 vs. Glu1 derivatives, Km 17- and 15-fold lower for folate and MTX, respectively) (138). GGH mRNA levels and activity were decreased in folate-deprived MCF7 breast cancer cells with moderate RFC levels, suggesting downregulation of GGH may serve as an adaptive response to severe folate deficiency (49).

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Tissue Distribution

GGH is present in serum, bile, and pancreatic secretions and also secreted by rat and human normal and tumor cells in vitro (139, 140, 147). Human GGH mRNA expression is highest in the liver and kidney, moderate in the placenta, brain, and colon, and low in the thymus, spleen, small intestine, lung, and peripheral blood leukocytes (148). GGH mRNA expression was examined in various cancer cell lines and seven of the eight cell lines examined showed extremely high expression (148). Furthermore, in an examination of 12 normal breast tissues and 12 breast cancer tissues, GGH mRNA expression was relatively homogenous in the normal breast tissue whereas expression was more heterogenous among the breast cancer tissues (148).

Cellular Location

GGH is a lysosomal enzyme, which allows the cell to separate the anabolic process by FPGS from catabolic process by GGH and presumably enables the tight control necessary to maintain the proper intracellular homeostasis of folate and antifolate (144). Interestingly, a large portion, if not the majority, of GGH is secreted into the medium in tissue culture (138-140), which probably corresponds to GGH found in serum (149). The role of the secreted form of GGH is still unknown, nor is it known whether the secreted GGH is catalytically active outside the lysosome (144).

A facilitative transport system for MTX polyglutamates was found in sarcoma cell lysosomes with a higher affinity for longer chain length polyglutamates (150-152). The limiting feature of MTX polyglutamate hydrolysis was related with the rate of transport into lysosome and not the catalytic capacity of GGH (153).

Role as a Tumor Marker

GGH has been suggested as a useful diagnostic biomarker for various cancers. Plasma GGH activity was significantly higher in patients with metastatic breast cancer compared to control subjects without disease and women whose cancer was in remission (154). In addition, infiltrating carcinoma tissue showed higher GGH activity than normal adjacent tissue. This observation indicated that increased GGH levels may occur due to hormonal stimulation and increased cellular proliferation since the mammary gland is a target tissue for estrogen. This

23 study, therefore, suggests that plasma GGH might quantitatively reflect the tumor burden. However, it is not clear whether higher activity observed in this study was caused by increased secretion of the lysosomal GGH or increased expression of the plasma membrane GCP II. Higher GGH expression in a malignant and metastatic breast cell line compared with a normal, premalignant breast cell line was also demonstrated by mass spectrometry-protein identification, confirming that GGH could be a potential biomarker of breast cancer (155).

GGH expression in pulmonary neuroendocrine tumors has been found to be associated with poor prognosis (156). The patients with GGH-positive tumors showed 28% survival probability at 9 years, while 83% of those with GGH-negative tumors survived for 9 years. In addition, recent data also implicate GGH as a novel biomarker for bladder cancer (157). Furthermore, GGH expression was increased in CRC compared with adjacent normal colonic mucosa (129, 158). GGH mRNA expression was also increased by 13.9-fold in the bladder cancer tissues compared with normal tissues, and was significantly higher in muscle-invasive than noninvasive urothelial cancer (157).

Single Nucleotide Polymorphisms of GGH

Several SNPs identified in the GGH gene at bases -401C>T, -354G>T, -124T>G, +16T>C, +452C>T, and +1102A>G have been shown to affect both the promoter and the coding region of the gene (159). These promoter polymorphisms showed increased GGH expression in HepG2 cells. Furthermore, -401C>T and -124T>G SNPs were associated with enhanced GGH expression in MCF7 cells, suggesting that these polymorphisms in the GGH gene promoter may increase GGH expression (159). One study in patients with RA reported the TT genotype of - 401C>T was associated with higher GGH activity than patients with CC or CT genotypes, indicating the TT genotype might be related to a poor response to chemotherapeutic agents such as antifolates (160). However, a poor response to platinum-based neoadjuvant chemotherapy was associated with CC genotype of -401C>T in Korean cervical cancer patients with radical hysterectomy, which might be due to the possibility that CC genotypes of both XRCC1 A194T and GGH -401C>T could negate the positive effects of each SNP in cervical cancer (161). In addition, the -124 G allele was associated with significant stepwise elevations in DNA uracil content for each additional G allele (162). This observation might be explained by the fact that reduced availability of polyglutamylated 5,10-methyleneTHF by increased GGH activity in -

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124T>G (159) would lead to a cellular accumulation of uracil with the subsequent incorporation into DNA, which is associated with increased DNA breakage (163) and ultimately a high risk of carcinogenesis (162). On the other hand, it has been shown that +452C>T is associated with low GGH activity and high MTX polyglutamate accumulation in acute lymphoblastic leukemia (ALL) blasts of patients treated with a high dose of MTX (164). In summary, it appears that at least some of the SNPs identified in the GGH gene have functional and pharmacological consequences.

In addition to SNPs, epigenetic regulation alters GGH activity and MTX polyglutamate accumulation in human leukemia cells by CpG island methylation in the human GGH promoter region (165). Two CpG islands (CpG1 and CpG2) were identified in the region extending from the GGH promoter through the first exon and into intron 1, and methylation of both CpG islands in the GGH promoter was associated with significantly reduced GGH mRNA expression and activity and with a higher accumulation of MTX polyglutamates in ALL cells (165). Furthermore, CpG1 methylation was leukemia cell-specific while CpG2 methylation was common in leukemia cells and normal leukocytes, and methylation of CpG1 had a much greater effect on GGH expression than methylation of CpG2.

Other Enzymes with γ-Glutamyl Hydrolase Activity

In addition to GGH, there is another enzyme with γ-glutamyl hydrolase activity called glutamate carboxypeptidase II (GCP II, EC 3.4.17.21, FOLH1), which resides in the cell membrane. The catalytic mechanism of GCP II is distinct from that of GGH although they have their common catalytic activity, and they represent separate enzyme families. While GGH belongs to the peptidase family C26, GCP II belongs to the peptidase family M28 (144). GCP II is a co- catalytic zinc peptidase, and it utilizes a protein-coordinated zinc ion to polarize a water molecule for attack on the susceptible bond, whereas GGH involves the nucleophilic attack of the Cys on the carbonyl of the Glu-γ-Glu bond (166).

GCP II is also synonymous with intestinal folate conjugase, prostate specific membrane antigen (PSMA), and N-acetyl-α-linked acidic dipeptidase (NAALADase). Intestinal folate conjugase, also known as pteroylpoly-γ-glutamate carboxypeptidase, is a GCP II enzyme on the surface of intestinal jejunum brush border cells that hydrolyzes pteroylpoly-γ-glutamates to pteroyl-mono-

25 glutamates, which are then available for absorption by intestinal cells and delivery into the bloodstream (144). PSMA is highly expressed in prostate cancer cells, and has been proposed as a biomarker for prostate cancer. NAALADase is primarily found on neuronal cells, and catalyzes the hydrolysis of N-acetyl-aspartylglutamate to yield N-acetyl-aspartate and the important neurotransmitter glutamic acid (167). It also hydrolyzes the γ-glutamyl linkages of pteroylpoly-γ- glutamates (168). It has been suggested that these three enzymes are expression of the same FOLH1 gene with different functions for the protein product (169).

Effect of GGH on Chemosensitivity

As mentioned earlier, both FPGS and GGH are important enzymes for the maintenance of intracellular homeostasis of folates and antifolates for optimal folate-dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects, respectively. It has been shown that the ratio of FPGS/GGH was a good predictor of the relative amounts of long-chain MTX polyglutamates in blast cells from acute leukemia patients (170). In this study, the linear regression curve relating the relative levels of long-chain polyglutamates/total polyglutamates with the ratio of GGH/FPGS showed an r value of 0.81 (P < 0.001). Similary, the ratio of

FPGS/GGH was positively associated with the accumulations of total MTX and MTX-Glu4-6 in childhood leukemia patients (171).

In general, lower FPGS and/or higher GGH activity has been associated with reduced antifolate polyglutamylation that is associated with drug resistance (144). Increased GGH activity was identified as the mechanism of antifolates resistance in H35 rat hepatoma cells and HT-1080 human sarcoma cells (172, 173). Compared to parental H35 cell line, approximately 7-fold increased GGH activity and no change in FPGS activity was observed in the another antifolate 5,10-dideazatetrahydrofolate (DDATHF)-resistant H35D rat hepatoma cells (172). The same group also reported that these two H35 cell lines had nearly equal intracellular folate concentration, but the length of polyglutamate chains consisted of predominantly Glu3 and Glu4 in H35D cells whereas the H35 cell line consisted mainly of Glu5 and Glu6. Furthermore, the accumulation of folate and antifolates were reduced in H35D cells with increased GGH activity (147). Similary, increased GGH activity was identified as the mechanism of resistance in intrinsically MTX-resistant HT-1080 human sarcoma cells (173), and higher GGH activity also found in DDATHF-resistant CCRF-CEM human leukemia cells (174).

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In addition, the RA patients who had the TT genotype of -401C>T exhibited increased GGH activity and lower levels of long-chain MTX polyglutamates (160), and in Japanese patients with RA, patients having the RFC80A SNP, demonstrated higher folate polyglutamate levels in blood cells (175) and a better response to MTX (176). Finally, RA patients with the GGH -401T alleles were less responsive to MTX than those with RFC80A and without GGH -401T alleles (177).

On the other hand, there are a few studies showing that decreased GGH activity is associated with an increased accumulation of long-chain MTX polyglutamates. In H35 rat hepatoma cells, cellular and secreted GGH was reduced by 60% in the presence of (140). Given that the presence of insulin caused MTX polyglutamylation to be increased by 3-fold in intact H35 cells (178, 179), O’Connor and colleagues suggested that enhanced polyglutamylation by insulin observed in previous studies might be related with the reduction in GGH (140). This explanation was also confirmed by another study which showed that insulin caused decreased GGH activity that was inversely related to the changes in cellular MTX polyglutamate synthesis and accumulation (180). Insulin-induced enhanced polyglutamylation was observed, without change in FPGS activity, ATP, and glutamate, suggesting that the effect of insulin may be indirect and/or related to the metabolic state of the cells (181). Furthermore, in ALL blasts of patients treated with a high dose of MTX, the inverse relationship between GGH activity and the accumulation of total and long-chain (Glu4-7) MTX polyglutamates was observed, and specifically the ALL patients with +452C>T SNP was associated with low GGH activity and high MTX polyglutamate accumulation (164).

There are few studies examining the effect of GGH on the sensitivity of antifolates and 5FU. In H35D cells, which is a cell line resistant to antifolates mainly by increased GGH activity, intracellular MTA polyglutamate accumulation was markedly reduced and IC50s of MTA and

MTX were greater compared to H35 rat hepatoma cell line (182). MTA Glu3 and Glu5 are good substrates for GGH, having higher rates compared with MTX Glu3 and Glu5 (182). A recent study performed by a Japanese group demonstrated that decreased GGH expression in DLD-1 colon cancer cells induced by small-interfering RNA (siRNA) resulted in increased sensitivity to 5FU with leucovorin (LV) as well as 5FU alone, presumably due to the better retention of 5,10- methyleneTHF (5). This study also found that siRNA- induced FPGS downregulation reduced the basal levels of reduced folates, lowered the accumulation and retention of folate in DLD-1 colon cancer cells treated with LV, and abolished the increase in 5FU cytotoxicity in the

27 presence of LV. Thus, the authors suggest that tumors expressing high levels of FPGS and low levels of GGH are expected to be most sensitive to 5FU-induced chemosensitivity in the presence of LV (5). In addition, low GGH and TS expression as well as high RFC, FPGS, and O- 6-methylguanine-DNA methyltransferase (MGMT) were reported recently to correlate with positive responses to 5FU-based therapy in a study of metastatic CRC (183). It has been shown that the CpG island methylator phenotype (CIMP+) CRC, accounting for approximately 17% of CRC cases and occuring predominantly in the proximal colon of aging patients (184, 185), is associated with low GGH expression and high 5,10-methyleneTHF levels compared to CIMP- tumors. CIMP+ CRC exhibits a higher response to 5FU compared with CIMP- CRC (186, 187). FPGS levels were not significantly different, however, between CIMP+ and CIMP− tumors (187), suggesting that FPGS has less of an influence than GGH in modulating the chemosensitivity of CRC to 5FU. On the other hand, Cole and colleagues reported that GGH overexpression alone may be insufficient to produce resistance to MTX at least for HT-1080 and MCF7 cell lines although intracellular folate pools were reduced in the GGH-overexpressed HT- 1080 cells and accumulations of MTX and 5-MTHF were decreased in the GGH-overexpressed MCF7 cells (188).

2.1.4 Folate and Health

Folate has an important role in human health and disease. Folate deficiency has been associated with the development of anemia, atherosclerosis, neural tube defects (NTDs) and congenital disorders, adverse pregnancy outcomes, neuropsychiatric disorders, and cognitive impairment (189). In addition, a substantial amount of epidemiological evidence suggests an inverse relationship between folate status, assessed by dietary folate intake or by the measurement of blood folate levels, and the risk of several malignancies, including cancer of the lungs, oropharynx, esophagus, stomach, colorectum, pancreas, cervix, ovary, prostate, and breast and the risk of neuroblastoma and leukemia (29, 189-191).

The Recommended Dietary Allowance (RDA) for adults in North America is 400 μg/day of Dietary Folate Equivalents (DFEs) with the upper limit set at 1 mg FA per day (192). DFE is a term used in the Institute of Medicine’s Dietary Reference Intakes and accounts for the lower

28 bioavailability of natural folate compared to FA: 1 μg DFE = 1 μg of food folate = 0.6 μg of FA from fortified food or as a supplement taken with meals = 0.5 μg of a supplement taken on an empty stomach (193).

The Canadian and United States governments implemented mandatory fortification with FA in 1998 based on the overwhelming body of evidence that FA supplementation reduces the risk of NTDs. Recent studies from Canada, the United States and Chile demonstrated that mandatory FA fortification is associated with a significant reduction in NTDs prevalence by 40-50% (194- 196). A recent population-based study reported that less than 1% of the Canadian population is folate deficient (RBC folate < 305 nmol/L) and 40% have the RBC folate level in what's considered to be a very high range (> 1360 nmol/L) (6). This observation is consistent with trends observed in the United States as reported in the National Health and Nutrition Examination Survey (NHANES) (8) from prefortification (1988-1994) to postfortification (1999- 2010), the postfortification prevalence of low serum (< 10 nmol/L) or RBC (< 340 nmol/L) folate levels were ≤ 1%.

2.1.4.1 Use of Folic Acid Supplement

A recent study using the Canadian Community Health Survey Cycle 2.2 (n = 34,381) reported that the overall prevalence of vitamin/mineral supplement consumption in the population was 40% including 25% who consumed an FA-containing supplement (197). Among individuals ≥ 14 y who consumed vitamin/mineral supplements, 9-14% were above the UL for FA (197). A study using NHANES 2003-2006 data (n = 11,462) demonstrated that 53% of the United States population used dietary supplements and 34.5% used dietary supplements that contained FA (198). The use of dietary supplements that contained FA was highest among men and women between 51-71 years of age who reported a mean daily intake of 436 ± 21.4 μg FA from supplements. In this age group, 5% were above the UL from dietary supplements alone (198). In an analysis of NHANES 2001-2002 data (≥ 60 y, n = 1,121, 58% women), 47% of the population reported using FA-containing supplements, and unmetabolized folic acid (UMFA) was detected in 38% of the population in a fasted state, with a mean concentration of 4.4 ± 0.6 nmol/L (median: 1.2 ± 0.2 nmol/L) (199). Serum folate and vitamin B12 concentrations were significantly higher in the UMFA+ group compared to UMFA- group (51 ± 2.6 vs. 37 ± 0.9 nmol/L for folate and 432 ± 12 vs. 384 ± 17 pmol/L for vitamin B12, P ≤ 0.006). The detectable

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UMFA is caused by the relatively low activity of DHFR in human liver (37, 38). It appears that daily ingestion of more than 400 μg of FA saturates not only hepatic DHFR activity, but also cellular uptake and renal clearance mechanisms (200, 201). At present, there is no conclusive evidence that exposure to UMFA causes adverse health effects. However, potential concerns include negative effects on vitamin B12 deficiency, cancer development, immune function, and epigenetic regulation (202).

Supplement use among cancer patients and long-term survivors is common. In studies combining different cancer sites, 64-81% of cancer survivors reported using single vitamin or mineral supplements and 26-77% reported using a multivitamin (10). This systematic review reported that between 14% and 32% of cancer survivors initiated supplement use after diagnosis, and use differed by cancer site (10). The use of FA supplements in cancer survivors has been mostly studied in CRC although the highest prevalence of vitamin and/or mineral use (75-87%) was reported in breast cancer survivors (10). A substantial increase in the use of FA-containing supplements from 35.4% to 55.1% after diagnosis with CRC has been noted, with use or initiation more likely among women, caucasians, U.S. residents, and those consuming less meat, more fruits and vegetables, and possibly non-smokers (9). Multivitamin use ranged from 38% to 43%, and FA use ranged from 1% to 12% in CRC survivors (203-205). A study of 924 CRC survivors in Western Washington State found that 12% of CRC survivors supplemented daily with FA at doses above the amount typically found in a multivitamin (203). The Colorectal Adenoma Prevention Study reported a 49% prevalence of vitamin and 1% of specifically FA use (204). FA or FA-containing supplement use was higher among female CRC survivors (9, 204, 205).

2.1.4.2 Potential Adverse Effect of Excess Folic Acid Supplementation

There have been some concerns, however, regarding potential adverse effects of too much FA and folate. The most commonly addressed consequence of a high folate status is the inability to detect the symptoms and signs of vitamin B12 deficiency, resulting in a missed diagnosis and subsequent progression of B12 deficiency-induced neurological sequela (206). UMFA has been associated with lower cognitive test scores in seniors who have an inadequate vitamin B12 status (< 148 pmol/L), indicating that high FA might harm the nervous system via a mechanism that involves UMFA (207). In theory, high FA combined with low vitamin B12 status could bypass

30 the metabolic block in the nucleotide synthesis, thus allowing the continuous process of cell division to occur in the bone marrow, perpetuating a masked anemic state (208). As a result, the growing cells deplete the pool of methyl groups with their increased demands and further compromise the methionine cycle, which is already undermined due to an inadequate vitamin B12 status. Other potentially harmful effects of excess FA include an increased risk for the progression of established neoplastic lesions (29), decreased natural killer cell cytotoxicity (209), genetic selection of disease alleles (202), increased risk of insulin resistance and (210), and asthma (211) in children born to mothers supplemented with high levels of FA. High FA supplementation has also been associated with resistance or tolerance to antifolate drugs used to treat arthritis (212, 213) and cancer (214, 215). It is likely that FA fortification and supplementation may benefit some with certain diseases and conditions, but may be detrimental to others.

2.2 Folate and Cancer Risk

Epidemiological studies collectively suggest an inverse association (in some cases dose- dependent) between folate status (measured by either folate intake [dietary and supplemental] or blood levels of folate) and the risk of several malignancies including cancer of the colorectum, oropharynx, esophagus, stomach, pancreas, lungs, cervix, ovary, and breast and neuroblastoma and leukemia (29, 190, 191, 216-218). However, data from human clinical trials and animal studies suggest that folate possesses a dual modulatory effect on cancer development and progression, a dose-dependent effect which also depends on the stage of cell transformation at the time of folate intervention (29). The dual effects of folate on cancer development and progression are related to its biochemical function of mediating the transfer of one-carbon moities involved in DNA synthesis and repair, and DNA methylation. Folate deficiency in normal tissues predisposes them to neoplastic transformation, and modest supplemental levels suppress, whereas supraphysiologic supplemental doses enhance, the development of tumors in normal tissues. In contrast, folate deficiency has an inhibitory effect, whereas folate supplementation has a promoting effect on the progression of established neoplasms. The dual modulatory role of folate in carcinogenesis is summarized in Table 2.1.

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Table 2. 1 Dual modulatory role of folate in carcinogenesis

Normal Cancer

Folate deficiency Tumor promoting mechanism: Tumor inhibitory mechanism: 1. DNA strand breaks 1. Ineffective DNA synthesis 2. Impaired DNA repair 2. Reversal of promoter CpG 3. Increased mutagenesis islands hypermethylation 4. Genomic DNA hypomethylation

Folate supplementation Tumor inhibitory mechanism: Tumor promoting mechanism: 1. DNA stability and integrity 1. Provision of for 2. Optimal DNA repair proliferation and growth 3. Decreased mutagenesis 2. De novo methylation of 4. Prevention of aberrant DNA promoter CpG islands methylation 3. Hypermutability of CH3-CpG

Adapted and modified by permission from the publisher (John Wiley and Sons) (29).

2.3 Folate and Cancer Treatment

2.3.1 Folate and Chemotherapeutic Agents

Folate is an essential cofactor for the de novo biosynthesis of purines and thymidylate, and in this role, folate plays an important role in DNA synthesis and replication (190, 216). In neoplastic cells where DNA replication and cell division are occurring at an accelerated rate, interruption of folate metabolism causes ineffective DNA synthesis, resulting in inhibition of tumor growth (190, 216). Indeed, this has been the basis for cancer chemotherapy using a number of antifolate agents and 5FU (190, 216). Furthermore, folate deficiency has been shown to induce regression and suppress progression of pre-existing neoplasms in experimental models (219, 220).

It was initially thought that acute leukemia might result from folate deficiency, but FA treatment increased the number of leukemia cells while folate deficiency caused a decrease in the leukemia cell count (221). It has been proposed that a folate analogue might be a useful cytotoxic agent by interfering with the normal metabolism of folate or a folate-dependent reaction (3). For example, , the first folate analogue, was developed in 1947 (222). Farber and colleagues

32 reported its ability to induce temporary remissions in children with ALL (223), leading to significant interest in antifolates for cancer treatment.

The mechanism of action for antimetabolites, a class of chemotherapeutic agents including antifolates, is to interrupt normal cellular metabolism and thus inhibit cell growth or induce cell death. Antifolates are structurally similar to folate and act by either binding or inhibiting intracellular folate enzymes. In addition, antifolates are typically described as classic or non- classic: classic antifolates such as MTX and MTA refer to antifolates that are transported by folate transporters (e.g., RFC) and require polyglutamylation for cytotoxicity, whereas non- classic antifolates such as trimetrexate (TMTX) do not require active transport or polyglutamylation for their cytotoxicity upon entry into the cell (224). As mentioned in Sections 2.1.2 and 2.1.3, antifolates are transported by RFC, FR or PCFT and retained in tumor and normal cells by the FPGS-induced polyglutamylation and then are exported from cells after hydrolysis to monoglutamates by GGH (2, 3). As with folate, polyglutamylated antifolates are retained in cells longer, thereby increasing their cytotoxicity by extending the length of exposure (2, 3). Polyglutamylated antifolates also generally have a higher affinity for and, hence, inhibit their target folate-dependent enzymes involved in thymidylate and purine biosynthesis to a greater extent than the monoglutamate forms (2, 3).

There are four chemotherapeutic agents of interest for the current research: fluorinated pyrimidines and antifolates (Figure 2.3).

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Figure 2. 3 Summary of chemotherapeutic agents used in the current research on the folate pathway. 5FdUMP, 5-fluoro-deoxyuridine-monophosphate; 5FU, 5-fluorouracil; MTA, pemetrexed; MTX, methotrexate; TMTX, trimetrexate. The box with straight line indicates the primary inhibitory effect of drug. The box with dotted line indicates the inhibitory effect of drug. Adapted and modified by permission from the publisher (Future Medicine) (225).

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2.3.2 5-Fluorouracil

Figure 2. 4 Chemical structure of 5-fluorouracil (http://www.sigmaaldrich.com/catalog/product/sigma/f6627?lang=en®ion=CA)

5-Fluorouracil (5FU) is widely used for the treatment of colon, breast and head and neck cancer (Figure 2.4) (226). 5FU is a prototype of pyrimidine antagonists that structurally resemble naturally occurring nucleotides. Cytotoxic nucleotides can be formed by three routes: (1) conversion of 5FU to 5-fluorouridine-monophosphate (5FUMP) by orotate phosphoribosyl transferase (OPRT) with phosphoribosyl pyrophosphate (PRPP); (2) sequential conversion of 5FU to 5FUMP by uridine phosphorylase and uridine kinase; and (3) sequential conversion of 5FU to 5FdUMP by thymidine phosphorylase (TP) and thymidine kinase (TK) (Figure 2.5) (227, 228). Only a small amount of the 5FU is activated via these routes; in humans 80-90% of the administered dose is degraded to 5,6-dihydrofluorouracil (DHFU) by dihydropyrimidine dehydrogenase (DPD) (Figure 2.5) (227, 228). DHFU can be further degraded to fluoro-β- ureidopropionate by and subsequently to fluoro-β-alanine by β- ureidopropionase (227, 228). 5FU degradation occurs in all tissues, including tumor tissues, but to the greatest extent in the liver (228).

5FU has several potential mechanisms for its cytotoxicity although, in general terms, it is considered to be a folate antimetabolite. 5-Fluoro-deoxyuridine-monophosphate (5FdUMP), a metabolite of 5FU, can form a ternary complex with TS and 5,10-methyleneTHF (Figure 2.5) (227). This ternary complex inhibits TS activity, which subsequently depletes intracellular thymidylate levels and, ultimately, suppresses DNA synthesis (Figure 2.5) (227). The dissociation of 5FdUMP from the ternary complex with TS and 5,10-methyleneTHF is suppressed when levels of 5, 10-methyleneTHF are increased (229). Leucovorin (LV; also known as ) or 5-formylTHF, a precursor of 5,10-methyleneTHF, potentiates the cytotoxic effect of 5FU by stabilizing the inhibitory 5,10-methyleneTHF-TS-5FdUMP ternary complex (Figure 2.5) (227). Furthermore, two metabolites of 5FU, 5FdUTP and 5FUTP, can be

35 incorporated into DNA and RNA, respectively, resulting in DNA instability and interfering with RNA processing and function (227). 5FU is considered to be an S phase-active chemotherapeutic agent (230), and treatment of cells with 5FU induces DNA damage during the S phase due to the misincorporation of FdUTP into DNA (231, 232).

Figure 2. 5 Metabolic pathway of 5-fluorouracil. TMP, (thymidylate) ; dUMP, deoxyuridine monophosphate; 5FdUDP, 5-fluoro-deoxyuridine- diphosphate; 5FdUMP, 5-fluoro-deoxyuridine-monophosphate; 5FdUR, 5-fluoro-deoxyuridine; 5FdUTP, 5-fluoro-deoxyuridine-triphosphate; 5FU, 5-fluorouracil; 5FUDP, 5-fluorouridine- diphosphate; 5FUMP, 5-fluorouridine-monophosphate; 5FUR, 5-fluorouridine;5FUTP, 5- fluorouridine-triphosphate; DHFU, 5,6-dihydrofluorouracil; TS, thymidylate synthase. Adapted and modified by permission from the publisher (Future Medicine) (225).

The expression and/or activity of TS and DPD, the rate-liiting enzymes involved in 5FU metabolism, are major determinants of 5FU efficacy (233, 234). TS overexpression has been shown to be associated with 5FU resistance in CRC (235, 236). Similary, DPD overexpression in cancer cell lines is associated with 5FU resistance (237), and high DPD mRNA expression in colorectal tumors has been shown to correlate with resistance to 5FU (238). In contrast, in addition to low GGH expression and high 5,10 methyleneTHF in CIMP+ tumors, methylation- induced silencing of DPD may also be associated with an improved response of CIMP+ to 5FU (186).

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The 5FU resistant HCT116 cell line is associated with cellular phenotypes such as reduced apoptosis and more aggressive growth as well as upregulation of TS compared with the parental HCT116 cell line (239). Based on microarray gene expression analysis, this study also suggested that altered regulation of nucleotide metabolism, metabolism, cytoskeleton organization, transport, and metabolism may underlie the differential resistance to 5FU observed in these cell lines (236). Furthermore, it is known that pretreatment with MTX before 5FU administration enhance 5FU efficacy by promoting the conversion of 5FU to 5FUMP through increased PRPP levels (240), which is correlated with increased formation of 5FU ribonucleotides and increased 5FU incorporation into RNA (241).

Effect of the MTHFR Polymorphism on 5FU Chemosensitivity

MTHFR is a critical enzyme that catalyzes the irreversible conversion of 5,10-methyleneTHF to 5-MTHF as previously mentioned (Figure 2.2). The MTHFR C677T polymorphism, a in the MTHFR gene, is associated with reduced MTHFR activity (242), leading to decreased 5- MTHF and the accumulation of 5,10-methyleneTHF (243). Increased intracellular levels of 5,10- methyleneTHF may enhance 5FU-induced chemosensitivity by augmenting the formation and stabilization of the 5,10-methyleneTHF-TS-5FdUMP ternary structure (225). Human colon and breast cancer cells expressing the mutant 677T MTHFR were associated with decreased MTHFR activity, decreased 5-MTHF, increased formylTHF/methenylTHF (and thus increased methyleneTHF), and increased TS activity compared with cells expressing the wild-type MTHFR (244). The MTHFR 677T mutation increased sensitivity of colon and breast cancer cells to 5FU alone, and to 5FU+LV in vitro (244). HCT116 colon cancer cells xenografts expressing the mutant 677T MTHFR showed higher sensitivity to 5FU+LV than xenografts expressing the wild-type MTHFR (244). In addition, antisense inhibition of MTHFR has been related with increased sensitivity of several human cancer cell lines to 5FU in vitro and in nude mice (244- 246). Overall, these findings suggest that MTHFR inhibition might be a potential therapeutic target for 5FU-based chemotherapy, although the results of clinical studies have been inconclusive possibly due to the limitations of these studies such as the small sample size, small number of subjects carrying the homozygous variant MTHFR genotypes, and nonuniform treatment protocols (225). Other genetic variants or SNPs of folate metabolic pathway may also affect chemosensitivity of cancer cells to 5FU. The effects of FPGS and GGH modulations on 5FU-based chemotherapy have been discussed previously in Sections 2.1.3.1 and 2.1.3.2.

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2.3.3 Methotrexate

Figure 2. 6 Chemical structure of methotrexate. Adapted and reprinted by permission from the publisher (Elsevier) (11).

Methotrexate (MTX), a 4-aminofolate analog, is a classic antifolate used to treat various human malignancies including leukemia, osteosarcoma, breast cancer, and head and neck cancer and RA (Figure 2.6) (124). MTX inhibits DHFR and decreases the intracellular folate pool required for thymidylate and purine biosynthesis (Figure 2.3) (120, 247). Inhibition of DHFR prevents the reduction of DHF to THF, leading to the accumulation of DHF in the cell. MTX and accumulated DHF are also capable of directly inhibiting folate-dependent enzymes in the thymidylate (TS) and purine (GARFT and AICARFT) biosynthetic pathways (Figure 2.3) (120, 247).

MTX enters cells mainly through RFC and is retained in cells by the FPGS-induced polyglutamylation, and polyglutamylated MTX is exported from cells after hydrolysis to monoglutamates by GGH (3). Polyglutamylated MTX is retained in cells longer, thereby increasing their cytotoxicity by extending the length of exposure (248, 249). Polyglutamylated MTX generally has a higher affinity for and, hence, inhibits their target folate-dependent enzymes in the thymidylate and purine biosynthesis pathways to a greater extent than the monoglutamate form (250-253). Compared to natural folates and MTA, MTX is a relatively ineffective substrate for FPGS, and forms polyglutamate derivatives slowly in cells (3, 124).

FA Supplementation During MTX Treatment

Previous evidence suggests that FA supplementation during MTX treatment might be beneficial to reduce toxicity although the mechanisms have not been entirely elucidated. An animal study

38 using mice inoculated with L1210 mouse lymphoid leukemia found that those receiving FA supplementation (25 mg/kg/day) 1 hour before or at the same time as MTX administration had a significantly longer median survival time than mice receiving either LV (20 mg/kg/day) or no supplementation (254). Similarly, in a study of mice inoculated with P388 mouse lymphocytic leukemia cells, the concomitant use of FA (62.5 mg/kg) with MTX (8.7 mg/kg) increased median survival time compared to mice treated with MTX alone and compared to mice receiving FA either 12 or 24 hours before MTX treatment (255). A cross-sectional observational study of children with ALL receiving MTX treatment investigated the effect of FA supplementation before and during MTX administration (256). This study found that there was no significant difference in erythrocyte MTX levels between children receiving FA supplementation (75-200 μg/day) before and during MTX treatment and those who did not, suggesting that FA supplementation may not compete with MTX for uptake into the cell. It is recommended that if LV rescue is deemed necessary, typically 15 mg/m2 LV is administered intravenously or orally every 6 hours beginning 24 hours after MTX treatment begins and continued until signs of MTX toxicity have resolved (215), whereas recommendations from the European rheumatology community include 5 mg/day FA orally with MTX treatment of RA (257).

Effect of Folate on MTX Chemosensitivity

As mentioned earlier, previous studies suggest that FA supplementation may increase MTX efficacy as well as reduce its toxicity. However, more recent clinical studies show that concomitant FA also reduces the efficacy of MTX (258, 259). Previous research findings suggest that cells containing less folate are potentially more sensitive to MTX cytotoxicity since more MTX polyglutamates can accumulate which can lengthen the half-life of the drug in the cell (260-262). This inverse relationship between intracellular folate levels and MTX activity is also observed in other antifolates, and it will be discussed in more detail in Sections 2.3.4 and 2.3.5. The accumulation of intracellular 5,10-methyleneTHF caused by the variant MTHFR 677T allele would compromise MTX-induced cytotoxicity since this would counteract the mode of MTX action, which depletes intracellular 5,10-methyleneTHF for thymidylate and purines biosynthesis. In MDA-MB-435 cells transfected with the mutagenized 677T human MTHFR cDNA, sensitivity to MTX was decreased compared with cells expressing the wild-type MTHFR (244). In contrast, the MTHFR 677T mutation had no significant effect on the sensitivity of HCT116 colon cancer cells to MTX, and antisense inhibition of MTHFR showed significantly decreased

39 chemosensitivity of HCT116 to MTX (244). This study suggested the possibility that greater MTHFR inhibition might affect chemosensitivity of cancer cells to MTX, although clinical observations investigating the effect of the MTHFR C677T polymorphism on MTX sensitivity have been inconsistent (225). Other genetic variants or SNPs of folate metabolic pathway may affect chemosensitivity of cancer cells to MTX. The effects of FPGS and GGH modulations on MTX-based chemotherapy have been discussed previously in Sections 2.1.3.1 and 2.1.3.2.

2.3.4 Pemetrexed

Figure 2. 7 Chemical structure of pemetrexed. Adapted and reprinted by permission from the publisher (Elsevier) (11).

Pemetrexed (a multi-targeted antifolate, MTA) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate that has shown clinical activity in a number of solid tumors such as non-small cell lung, breast, mesothelioma, colorectal, pancreatic, gastric, bladder, cervix, and head and neck cancers (263), and has recently been approved for the treatment of mesothelioma and non-small cell lung cancer (264, 265). MTA primarily inhibits TS that results in the suppression of the generation of DHF, and to a lesser extent, potentially suppresses GARFT (Figure 2.3) (266, 267). MTA also targets DHFR, but its affinity for DHFR is 1/1,000 that of MTX (Figure 2.3) (266, 267). MTA is an excellent substrate for FPGS resulting in very rapid and efficient conversion to active polyglutamate derivatives in contrast with the gradual polyglutamylation profile observed with MTX (267, 268). The active MTA polyglutamate derivatives accumulate rapidly in cells (269), resulting in both prolonged and marked suppression of TS, the primary target enzyme (266). The

MTA Glu5 (Ki = 1.3 nM) was an ~100-fold better inhibitor of TS than the MTA Glu1 (Ki = 109

40 nM), and similarly, the MTA Glu5 (Ki = 65 nM) showed enhanced activity against GARFT compared to the MTA Glu1 (Ki = 9,300 nM) (266, 267).

Furthermore, MTA has a high affinity for transporters of folate and antifolate such as RFC, FR, and PCFT as explained in Section 2.1.2.3. Its affinity for RFC is twice that of MTX (41, 42) and its affinity for FRα is 2 orders greater than that of MTX and comparable to that of FA (42). Recent studies suggest that PCFT plays an important role in the delivery of MTA into the cell when RFC activity is impaired or lost (270).

In a randomized, double-blind, phase II study conducted to evaluate the efficacy and safety of MTA in patients with newly diagnosed metastatic breast cancer or locally recurrent breast cancer, high expression of FPGS and TP was associated with better MTA response rates and longer median time to tumor progression compared with low expression of these genes (271). TP catalyzes the reversible phosphorolysis of thymidine to , and is involved in the activation of fluoropyrimidine. It has been shown that high TP expression in cancer cells is associated with an increased response to TS inhibitors (272). This study also reported that high GGH expression was unexpectedly associated with greater risk for grade 3/4 MTA toxicity (271).

FA Supplementation During MTA Treatment

There are a number of studies investigating the effect of FA supplementation on the efficacy and toxicity of MTA. Worzalla and colleagues found decreased MTA toxicity without compromising treatment effectiveness in mice receiving FA supplementation compared with mice on a low- folate diet during MTA treatment (273). A retrospective analysis of phase I and phase II clinical trial findings related to MTA toxicity (exclusive of patients who had received FA supplementation during treatment) suggested that decreasing Hcy levels in response to vitamin supplementation led to a better safety profile of MTA with an improved efficacy (274). Bajetta and colleagues found that in advanced gastric cancer patients, all MTA responding patients were in the FA supplemented group (5 mg/day on days -2 to +2 of every cycle), and patients receiving FA supplementation did not discontinue MTA treatment due to toxicity (275). Similarly, malignant pleural mesothelioma patients treated with MTA and supplemented with FA (350- 1,000 μg) and vitamin B12 (1,000 μg) had less toxicity, tolerated more cycles of treatment, and a 5-month greater median overall survival compared with nonsupplemented patients (276). Based

41 on reports from a number of studies, current clinical regimens require the routine administration of 350-1000 μg FA and 1,000 μg of vitamin B12 intramuscularly at least 1 week prior to, and concurrent with MTA treatment every 9 weeks (214).

Effect of Folate on MTA Chemosensitivity

In clinical regimens, MTA is administered with FA to minimize its toxicity. However, it has been known that MTA has a marked sensitivity to physiological intracellular folate levels (270), and excessive folate supplementation may decrease MTA sensitivity. The increased extracellular LV was associated with reduction in the formation of MTA polyglutamates in L1210 murine leukemia cells (136). Also, as extracellular LV levels were increased over the range of normal blood levels, MTA activity was substantially decreased in HCT-15 human colon adenocarcinoma, A549 human lung cancer, and NCI-H2052 and NCI-H2373 human mesothelioma cell lines (214).

In contrast, as intracellular folates decreased due to decreased extracellular reduced folate, or loss of RFC function in HeLa cells, MTA inhibition of GARFT was increased (277). Similarly, loss of RFC function in HCT-15 human colon cancer cells was associated with increased sensitivity to MTA, likely caused by cellular folate pool contraction (278). In addition, low folate levels were associated with higher sensitivity to MTA in four colon cancer and three squamous cell carcinoma of the head and neck cell lines (46), and mice bearing a colon tumor showed increased sensitivity to MTA on a folate-depleted diet compared to standard diet (279). Taken together, intracellular folate concentration is an important determinant of chemosensitivity of MTA.

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2.3.5 Trimetrexate

Figure 2. 8 Chemical structure of trimetrexate. Adapted and reprinted by permission from the publisher (Elsevier) (11).

Trimetrexate (TMTX), a 2,4-diaminoquinazoline, is a non-classical lipophilic antifolate that directly inhibits DHFR (Figures 2.3 and 2.8) (280). It has been used in treating pneumocystis pneumonia and a variety of tumors including leiomyosarcoma and skin lymphoma (281). In addition, TMTX plays a role as a biochemical modulator of 5FU and LV (282), which results from inhibition of DHFR and from the increase of intracellular concentrations of 5- phosphoribosyl-1-pyrophosphate via inhibition of purine synthesis. This leads to an increased conversion of 5FU to its active metabolite, FdUMP (283). The synergistic effect of TMTX in combination with 5FU and LV is explained by the fact that TMTX does not compete with LV for cellular uptake and metabolism (284). TMTX does not require active transport but enters the cell by passive diffusion, and does not need to be polyglutamylated for its cytotoxicity (285). There are several studies regarding TMTX activity in CRC. The treatment with TMTX in combination with 5FU and LV showed increased cytotoxicity in HCT-8 human colon cancer cells (286). In two phase II studies in previously untreated patients with advanced CRC, TMTX/5FU/LV treatment was associated with 50% and 36% response rates, respectively (287, 288). Furthermore, a phase III randomized European study with 365 advanced CRC patients reported that the addition of TMTX to a weekly regimen of 5FU/LV showed a small but significant improvement in progression-free survival without adding toxicity or worsening quality of life (289).

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Folate Supplementation During TMTX Treatment

Current treatment guidelines for the use of TMTX in cancer patients have not been established yet. One animal study using Wistar rats found that TMTX-related toxicities and mortality could be completely prevented with < 5 mg/kg LV for TMTX doses of up to 45 mg/kg (290). In a study of 11 pediatric and young-adult patients with a variety of cancers refractory to conventional therapy who received 28 day cycles of high-dose TMTX (45 mg/m2 every 12 hours for 14 days, then 14 days of rest), Hum and colleagues determined that 2.5 mg LV/m2 (which is close to the estimated average requirement for folate for this age group [250-330 μg/day]), was the smallest dose needed to prevent TMTX toxicity (291).

Effect of Folate on TMTX Chemosensitivity

TMTX is also very sensitive to physiological intracellular folate levels due to the interconversion of THF cofactors to DHF and due to the competition between DHF and TMTX at the level of DHFR (136, 292). It has been shown that there is an inverse relationship between intracellular folate levels and TMTX activity. As extracellular LV levels were increased within physiological blood levels, TMTX activity was substantially decreased in human colon adenocarcinoma, lung cancer, and mesothelioma cell lines (214). By contrast, HeLa cells with loss of RFC function, showed increased TMTX sensitivity compared with wild-type HeLa cells; the magnitude between these two cell lines was greater in the presence of lower extracellular folate concentrations (278, 293, 294). Similarly, loss of RFC function in HCT-15 human colon cancer cells was associated with increased sensitivity to TMTX, likely due to cellular folate pool contraction (278). The uptake of TMTX is not affected by changes in the activities of folate transporters since it is transported by passive diffusion (280).

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2.3.6 Drug Resistance

In general, the mechanisms of resistance to antifolates are shown to correlate with (1) impaired drug uptake due to loss of RFC function, (2) increased drugs efflux due to overexpression of MRPs and BCRP, (3) decreased affinity of DHFR and/or TS for drugs due to overexpression and of these enzymes, (4) decreased polyglutamylation due to low FPGS and/or high GGH expression, and (5) increased intracellular THF cofactor pools (13, 14).

As mentioned earlier, loss of RFC led to a resistance to MTX and MTA due to an incomplete inhibition of target enzymes and insufficient substrates for polyglutamate synthesis (45). Folate deficiency was associated with increased RFC expression and increased sensitivity to MTA in human colon cancer and head and neck squamous cell carcinoma cell lines (46). The enhanced inhibitory effect of 5FU by LV was more effective in folate-depleted cells (46) and in mice fed a folate-deficient diet compared to a normal diet (5). In addition, Sakamoto and colleagues demonstrated that siRNA-induced FPGS downregulation decreased the basal level of reduced folate, intracellular folate level after LV treatment (10 μM overnight), and LV efficacy in enhancing the inhibitory effect of 5FdUR in DLD-1 human colon cancer cells, whereas siRNA- induced GGH downregulation was associated with an increased intracellular folate level after LV treatment and enhanced chemosensitivity to 5FdUR+LV (5).

Consistent with this observation, FPGS expression was also positively related with reduced folate levels in CRC tissues from patients who received LV, while GGH expression was negatively associated with reduced folate levels in tissues from CRC patients after LV administration (295). These results suggest that the expression levels of FPGS and GGH in tumor tissues might affect the efficacy of LV in CRC patients. In addition to high FPGS expression and low GGH expression, low MRP1 expression in CRC tissues could predict the level of reduced folate after LV administration (295). MRP1 might regulate the concentrations of reduced folate in tumor tissue after LV treatment by exporting monoglutamate or short-chain polyglutamate forms of reduced folate, including LV (295).

MRP 1-4 efflux MTX preferentially in its monoglutamate whereas MRP5 can efflux mono- and diglutamate conjugates form, indicating MRP5 is associated more with resistant to MTX after short-term MTX exposure (14, 68). BCRP can also export both mono-, di- and triglutamate

45 forms of MTX, suggesting it may be significantly related with MTX resistance (12). It has been shown that MRP1-5 and MRP8 is associated with antifolates resistance, and MRP5 and MRP8 are also involved in 5FU resistance (12). However, MRP5 is only capable of transporting 5FdUMP, a metabolite of 5FU (296). Whether MRPs and BCRP contribute to drug resistance or increase drug efficacy depends on polyglutamylation of antifolates and intracellular folate concentrations. Recently, Porcelli and colleagues proposed a model for the modulation of MRPs and BCRP by cellular folate status that also takes into account the subcellular localization of these transporters (13). When MRPs/BCRP are located at the plasma membrane, folate supplementation would increase the expression and/or activity of these transporters, while folate depletion would decrease their expression and/or activity (Figure 2.9). In contrast, when MRPs/BCRP are located intracellularly, folate supplementation would decrease the expression and/or activity of MRPs/BCRP, whereas folate depletion would increase their expression and/or activity (13). This proposal suggests that folate supplementation during cancer chemotherapy may be a double-edged sword, since it might improve drug efficacy or contribute to drug resistance.

A B

Figure 2. 9 A proposed model of the modulation of MRPs and BCRP by folate supplementation (A) and folate depletion (B). When MRPs/BCRP are located at the plasma membrane, folate supplementation would increase the expression and/or activity of these transporters (A), while folate depletion would decrease their expression and/or activity (B). Conversely, when MRPs/BCRP are located intracellularly, folate supplementation would decrease the expression and/or activity of MRPs/BCRP, whereas folate depletion would increase their expression and/or activity. The yellow circles represent folate molecules. Adapted and modified by permission from the publisher (Bentham Science) (13).

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2.4 Epigenetics and Cancer Treatment

2.4.1 Epigenetics

The current definition of epigenetics is ‘the study of heritable changes in gene expression that occur independent of changes in the primary DNA sequence’ (297, 298). Most of these stable heritable changes are established during differentiation and are maintained through multiple cycles of cell division, enabling cells to have distinct identities while containing the same genetic information. Chromatin is made of repeating units of nucleosomes, which consist of ~146 base pairs of DNA wrapped around an octamer of four core histone proteins (H3, H4, H2A and H2B) (299). Epigenetic mechanisms that modify chromatin structure can be divided into four main categories: DNA methylation, covalent histone modifications, noncovalent mechanisms such as chromatin remodeling and non-coding RNAs including miRNAs. These modifications work together to regulate the functioning of the genome by altering the local structural dynamics of chromatin, primarily regulating its accessibility and compactness.

DNA methylation occurs in the context of chemical modification of histone proteins (300). The N-terminal tails of histones can undergo a variety of posttranslational covalent modifications including methylation, acetylation, ubiquitylation, sumoylation and phosphorylation on specific residues (301). These modifications affect gene transcription, replication and repair (301). In contrast to DNA methylation, the patterns of histone modifications are more labile. Furthermore, histone modifications and DNA methylation interact with each other at multiple levels to determine gene expression status, chromatin organization and cellular identity as well as performing their individual roles (302).

Nucleosomes not only serve as the basic modules for DNA packaging within a cell, but also regulate gene expression by altering the accessibility of regulatory DNA sequences to transcription factors (303). The interaction of nucleosome remodeling machinery with DNA methylation and histone modifications plays an important role in establishing global gene expression patterns and chromatin architecture (304, 305). In addition, miRNAs are short, ~22- nucleotide, non-coding RNAs that regulate gene expression by posttranscriptional silencing of target genes and control biological processes including cell proliferation, apoptosis and differentiation. miRNAs expression can be regulated by epigenetic mechanisms (306), and

47 miRNAs can modulate epigenetic regulatory mechanisms inside a cell by targeting enzymes responsible for DNA methylation (DNMT3a and DNMT3b) and histone modifications (EZH2) (307, 308). These interactions described above highlight the integrated nature of epigenetic mechanisms involved in the maintenance of global gene expression patterns.

The interplay of these modifications creates an ‘epigenetic landscape’ that regulates the way the mammalian genome manifests itself in different cell types, developmental stages and disease states, including cancer (301, 303, 309-312). In contrast to genetic changes in human diseases, epigenetic changes are gradual in onset and progressive, their effects are dose-dependent, and are potentially reversible by dietary and pharmacologic manipulations (92, 313). Of the various epigenetic mechanisms, we focus on DNA methylation and gene expression in the present study.

2.4.2 DNA Methylation and Cancer

2.4.2.1 DNA Methylation

DNA methylation is heritable, tissue- and species-specific, and an important epigenetic inverse determinant in gene expression (298, 313). Methylation also plays a critical role in the maintenance of DNA integrity and stability, and in chromatin modifications (30, 298). DNA methylation primarily occurs by covalent addition of a methyl group at the 5’ carbon of the cytosine ring located within the cytosine-guanine (CpG) sequences (314). About 80% of all CpG sites in human DNA are methylated in normal cells (298, 315). However, this global DNA methylation occurs primarily in the bulk of the genome where CpG density is low, including exons, noncoding regions, and repeat DNA sites, and allows correct organization of chromatin in active and inactive states (Figure 2.10) (33). By contrast, most CpG rich areas clustered in small stretches of DNA, termed CpG islands, span the 5’ end of approximately half of all transcribed human genes including the promoter, untranslated region, and exon 1. These areas are generally unmethylated in normal cells, thereby allowing transcription to occur (Figure 2.10) (298, 315, 316). Most definitions of CpG islands include a minimum length (e.g., 200 or 500 base pairs), a minimum observed:expected CpG ratio (e.g., > 0.6 or 0.65), and a minimum GC content (e.g., 50% or 55%) (317). According to Takai and Jones, CpG islands are defined as regions of DNA > 500 base pairs with a GC content ≥ 55%, and a ratio of observed and expected CpG > 0.65 (318).

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DNA methylation is critically involved in regulating many developmental and cellular processes, including embryonic development, transcription, chromatin structure, X inactivation, , and chromosome stability (319). Aberrant patterns and dysregulation of DNA methylation are mechanistically related to the development of several human diseases, including cancer (298, 315).

Figure 2. 10 Distribution of CpG dinucleotides in the human genome and CpG methylation patterns in normal and tumor cells. Normal cells contain methylated CpG sites in the CpG-poor bulk of the genome and unmethylated CpG islands. On the contrary, cancer cells simultaneously harbor widespread loss of methylation in the CpG depleted regions, where most CpG dinucleotides should be methylated, and are also characterized by methylated CpG islands in gene promoter regions. Open circles represent unmethylated CpG sites, whereas filled circles are methylated CpG sites. Boxes 1, 2, and 3 represent exons, and line between exons are introns. X at the transcription start site represents transcriptional silencing. Adapted and reprinted by permission from the publisher (John Wiley and Sons) (29).

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2.4.2.2 Regulation of DNA Methylation

DNA methylation is a dynamic process between active methylation, mediated by CpG DNA methyltransferases (DNMT1, 3a, 3b) using SAM as the methyl donor, and active and/or passive demethylation (320). Active DNA demethylation removes methyl groups from 5-methylcytosine residues by a purported demethylase (MBD2; methyl-CpG binding domain protein 2), and can occur independently of DNA replication; Passive DNA demethylation occurs when maintenance methyltransferases (DNMT1) are inactive during the cell cycle following DNA replication, resulting in a retention of the unmethylated state of the newly synthesized strand (321). DNA methylation patterns are reprogrammed during embryogenesis by genome-wide demethylation after fertilization. Active and passive demethylation of the paternal and maternal methylation patterns, respectively, occurs, which erases significant parts of the parental DNA methylation patterns. This is followed by de novo methylation, which establishes a new DNA methylation pattern soon after implantation, with methylation limited to non-CpG island areas, except for the rare genes silenced in normal cells (320, 322). The maintenance of CpG DNA methyltransferase (DNMT1) uses hemimethylated sites to ensure DNA methylation patterns, whereas de novo CpG DNA methyltransferases (DNMT3a, 3b) do not require pre-existing methylation and, therefore, establish a new DNA methylation pattern (320).

The methylation of promoter region CpG islands causes stable, heritable transcriptional silencing (298, 315). DNMTs may regulate gene silencing accompanying promoter DNA methylation by recruiting HDACs and other chromatin-binding proteins to promoter sites to maintain histone deacetylation (323). Furthermore, both nucleosome structure and histone modification affect chromatin structure and regulate gene transcription. In CpG islands that are unmethylated, the nucleosomes are in a more open configuration and histones acetylation is required to maintain chromatin in an open and transcriptionally active state. This allows binding of transcriptional factors, histone acetyltransferases, and other regulatory coactivators that promote gene expression (Figure 2.11) (323). In contrast, when CpG islands are hypermethylated, the nucleosomes become more tightly compacted and histone deacetylation suppresses transcription by allowing tighter nucleosomal packaging, thus rendering an inactive chromatin conformation (314, 324). Binding of HDACs to hypermethylated chromatin is directed by DNMTs as well as methyl-CpG binding domain proteins, which form a complex with other regulatory proteins to block access of the transcriptional machinery to the promoter (Figure 2.11) (314, 323, 324).

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Normal

Cancer

Figure 2. 11 Effects of DNA methylation and chromatin structure on gene transcription in normal and tumor cells. Ovals with bold asterisks (purple) represent nucleosomes with histone deacetylation. Ovals with light asterisks (green) represent nucleosomes with histone acetylation. Open circles represent unmethylated CpG sites, whereas filled circles are methylated CpG sites. X indicates transcriptional silencing. Curved arrows indicate transcriptional activity. CA, transcriptional coactivators; CR, transcriptional corepressor; DNMTs, DNA methyltransferases; HAT, histone acetyltransferase; HDAC, histone deacetylase; MBPs, methyl- CpG binding domain proteins; TF, primary transcription factors. Adapted and reprinted by permission from the publisher (Nature Publishing Group) (323).

While the role of promoter CpG island methylation in gene silencing is well established, much less is known about the role of methylation of non-CpG island promoters. Recent studies have shown that DNA methylation is also important for the regulation of non-CpG island promoters (30). The epigenetic signatures of DNA methylation, histone marks and nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis (325).

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2.4.2.3 DNA Methylation in Cancer

Neoplastic cells simultaneously harbor widespread global hypomethylation and more specific regional areas of hypermethylation (298, 326). Global hypomethylation is an early, and consistent, event in carcinogenesis (298, 326). Global hypomethylation is known to occur at various genomic sequences including repetitive elements, retrotransposons, CpG poor promoters, introns and gene deserts (327) and is associated with genomic instability (328) and increased mutations (329).

In addition to global hypomethylation, site-specific hypermethylation at promoter CpG islands of tumor suppressor and mismatch repair genes is an important mechanism in gene silencing in carcinogenesis (32, 330). As shown in Figure 2.10, in contrast to methylated CpG sites in the CpG-poor bulk region of the genome and unmethylated CpG islands in normal cells, cancer cells simultaneously harbor widespread loss of methylation in the CpG depleted regions, where most CpG dinucleotides should be methylated and gains in the methylation of CpG islands in gene promoter regions (92, 331). Since the initial finding of promoter CpG island hypermethylation of Rb, a tumor suppressor gene associated with retinoblastoma (332), other tumor suppressor genes including p16, MLH1 and BRCA1 have also been found to be associated with tumor-specific silencing by hypermethylation (298, 309, 323). These genes are involved in cellular processes, which are essential to cancer development and progression, including DNA repair, cell cycle, cell adhesion, apoptosis and angiogenesis.

DNA hypermethylation can also indirectly inactivate additional classes of genes by silencing transcription factors and DNA repair genes. Promoter hypermethylation-induced silencing of transcription factors, such as RUNX3 in esophageal cancer (333) and GATA-4 and GATA-5 in colorectal and gastric cancers (334), leads to inactivation of their downstream targets. Silencing of DNA repair genes such as MLH1 and BRCA1 enables cells to accumulate further genetic lesions leading to the rapid progression of cancer (30). Furthermore, DNA methylation has been associated with alterations in signaling pathways in cancer. For example, epigenetic inactivation of SFRP1, SFRP2, SFRP5, DKK1, DKK2, and DKK3, six negative regulators of WNT signaling, contributes to the full activation of T cell Factor (TCF) β-catenin activity in CRC (335, 336), while epigenetic inactivation of RASSF1 and RASSF2, negative regulators of the Ras signaling pathway, contributes to full activation of oncogenic Ras signaling (337, 338).

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2.4.3 Inhibition of DNA Methylation as Chemotherapeutic Strategy

The reversible nature of epigenetic alterations has led to the development of therapeutic strategies targeting various epigenetic components. The aim of this epigenetic therapy is to reverse the causal epigenetic aberrations that occur in cancer, leading to the restoration of a normal epigenome (30). Many epigenetic drugs have been discovered in recent years that can effectively reverse DNA methylation and histone modification aberrations that occur in cancer (339). Among them, DNA methylation inhibitors such as 5-azacytidine (5-aza-CR) and 5-aza-2’- deoxycytidine (5-aza-CdR) were the first epigenetic drugs proposed for use as cancer therapeutics. These two DNA methylation inhibitors have hypomethylating activity after incorporation into the DNA of actively replicating tumor cells (340, 341). This drug-induced reduction of DNA methylation causes growth inhibition in cancer cells by activating tumor suppressor genes which are aberrantly silenced in cancer (339).

In addition to nucleoside analogs, non-nucleoside compounds such as SGI-1027, RG108 and MG98 have been developed to inhibit DNA methylation by either blocking catalytic/cofactor- binding sites of DNMTs or by targeting their regulatory messenger RNA sequences (342, 343). HDAC inhibitors are a family of epigenetic drugs that increase acetylation of histone proteins and cytoplasmic proteins such as p53. Since reversing epigenetically silenced genes is coordinated by increasing promoter histone acetylation levels and DNA demethylation, combination treatment strategies such as DNA methylation and HDAC inhibitors together have been found to be more synergistically effective than individual treatment approaches (30).

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2.4.4 Epigenetic Silencing and Response to Chemotherapeutic Agents

It has been shown that genomic instability caused by impairment or inactivation of DNA repair systems could represent a molecular target of cancer therapy. Initially, evidence regarding the positive relationship between MGMT gene methylation and sensitivity to alkylating agents was reported (344). This study suggested that methylation of the MGMT promoter is predictive of a good outcome in patients with malignant glioma treated with alkylating agents. Alkylating agents irreversibly bind to DNA, cause DNA strand breaks, and ultimately prevent DNA replication, while MGMT is able to repair DNA error induced by alkylating agents. As a result, the efficacy of alkylating agents is diminished in tumors expressing MGMT (345). Moreover, Hegi and colleagues found that inactivation of MGMT by promoter hypermethylation was associated with longer survival in a prospective clinical trial (346).

Approximately 15% of CRC cases exhibit microsatellite instability due to methylation of the mismatch repair gene hMLH1 (347). It has been demonstrated that CRC with hMLH1 methylation expresses high levels of TS (348). Moreover, CRC cell lines displaying microsatellite instability are resistant to 5FU due to methylation of hMLH1, but they become susceptible to treatment upon exposure to 5-aza-CdR (349). Therefore, methylation of hMLH1 appears to be a predictive molecular marker of the sensitivity of CRC to 5FU. Simiarly, inactivation of hMLH1 by methylation in some ovarian tumors results in loss of cisplatin sensitivity (350, 351), whereas hMLH1 expression and sensitivity to cisplatin were restored after treatment with the demethylating agent 5-aza-CdR (352).

Based on the comparison of DNA methylation profiles for 32 genes with drug sensitivity in NCI- 60 cell lines, methylation of p73 could be a predictive marker of sensitivity to alkylating agents (353). p73 is a member of the p53 family and, like other p53 family members, it is involved in cell-cycle checkpoint function, apoptosis, DNA repair, and cellular differentiation (354). p73 overexpression has been observed in cancers of the bladder, lung, and ovary, and is often associated with resistance to treatment with DNA damaging agents (355-357). In addition, siRNA-induced p73 inhibition was associated with reduced cellular viability after treatment with BCNU and cisplatin. However, the molecular mechanism by which silencing of p73 sensitizes cancer cells to alkylating agents remains unknown.

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2.4.5 Analysis of DNA methylation

The DNA methylation profile across the entire genome is important to understand the influence of epigenetics. There has been a revolution in technology of DNA methylation analysis. Analyses were previously restricted to specific loci, but now can be performed on a genome- wide scale and on entire methylomes at single-base-pair resolution (317). In contrast to the initial analysis using two-dimensional gel electrophoresis, recent studies use microarray hybridization techniques from gene expression and genomic fields to the profiling of histone modifications and DNA methylation patterns. The current revolution in sequencing technology has recently approached single-base-pair resolution genome-wide DNA methylation analysis (317, 358).

DNA methylation measurements can be made either of the pattern of methylated target sequences along individual DNA molecules or as an average methylation level at a single genomic across many DNA molecules (359). Almost all sequence-specific DNA methylation analysis methods require methylation-dependent pretreatments of DNA before amplification or hybridization in order to reveal the presence or absence of the methyl group at cytosine residues (317, 359). There are three main methods for a methylation-dependent pretreatment of DNA: (1) endonuclease digestion, (2) affinity enrichment, and (3) bisulphite conversion. Pretreatment of DNA with one of the methylation-dependent steps is followed by various techniques including probe hybridization and sequencing to identify the location of the 5meC residues (317).

Sequence-specific restriction endonuclease has an accompanying DNMT that protects the endogenous DNA from the restriction defense system by methylating bases in the recognition site. The initial locus-specific DNA methylation analyses were determined by the use of methylation-sensitive restriction enzymes followed by gel electrophoresis and Southern blotting. Methylation-sensitive restriction digestion also can be followed by PCR across the restriction site, which is a very sensitive method that is still being used. It is, however, prone to false-positive results that occur because of incomplete digestion (317, 359).

Chromatin immunoprecipitation (ChIP), a technique used to identify the location of DNA- binding proteins and epigenetic marks in the genome, followed by microarray hybridization (ChIP-chip) or next-generation sequencing (ChIP-seq) has proven to be a useful method for

55 genome-wide studies of histone modifications. Likewise, affinity enrichment of methylated regions using antibodies specific for 5meC (in the context of denatured DNA) or using methyl- binding proteins with an affinity for methylated native genomic DNA, are powerful tools for comprehensive profiling of DNA methylation in complex genomes. Affinity-based methods perform rapid and efficient genome-wide assessment of DNA methylation, but they do not produce information on individual CpG dinucleotides and require substantial experimental or bioinformatic adjustment for varying CpG density at different regions of the genome (317).

Lastly, cytosine methylation status of individual CpG sequences can be determined using techniques based on the conversion of unmethylated but not methylated cytosines to uracil by bisulfite treatment. Illumina has adapted the bisulfite conversion method to Infinium platform for DNA methylation analysis (360), which is used in this study. The Infinium platform incorporates a whole-genome amplification step after bisulfite conversion, followed by fragmentation and hybridization of samples to methylation-specific DNA oligomers that are linked to individual bead types. Each bead type corresponds to a specific DNA CpG site and methylation state (317). More detailed information regarding Illumina Infinium HumanMethylation27 BeadChip will be described in Appendix 3.

The choice of method will be influenced by the number of samples and the quality and quantity of DNA, as well as the desired coverage and resolution. Furthermore, it is necessary to consider the organism that is being studied: array-based analysis requires that a suitable array for the species of interest is available, whereas sequence-based analyses are generally applicable to any species for which a reference genome exists (317).

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2.4.6 Folate and Epigenetics

A recent review extensively discussed the effects of folate status on DNA methylation (331). Evidence from in vitro, animal, and human studies suggest that the epigenetic effects of folate on DNA methylation are highly complex. Overall, the results from animal studies suggest that DNA methylation patterns are gene- and site-specific and depend on cell type, target organ, and stage of transformation as well as on the timing, degree, and duration of folate intervention (331). The majority of observational studies have described a direct relationship between dietary and blood levels of folate and genomic DNA methylation in both lymphocytes and colonic tissues such that a low folate status is associated with genomic hypomethylation. This positive association is more consistent in individuals with colorectal adenomas, adenocarcinomas, or previously resected neoplastic tumors as well as in those at a greater risk of health complications compared with normal subjects (331). For gene-specific DNA methylation, the direction and magnitude of effect due to dietary and blood folate concentrations on gene-specific methylation remain unclear (331).

Recently, the possibility of an inverse relationship between FA supplementation and DNA methylation status has been raised. Among reproductive-aged women not previously exposed to FA in China, FA supplementation (0.1, 0.4, and 4 mg/day) for 1 month significantly decreased genomic methylation by 13% and continued to remain significantly lower with subsequent interventions (361). This seemingly paradoxical effect of FA supplementation on global DNA methylation may be partly explained by the preferential shuttling of the flux of one-carbon units to the nucleotide synthesis pathway over the methionine cycle necessary for biological methylation reactions in response to FA supplementation (331). Although FA is an inhibitor of DHFR (362), in certain situations, it may upregulate DHFR (363), and this upregulation may increase TS activity, because the transcription of these genes is co-regulated by several transcription factors (364, 365). A mathematical modeling framework has indicated that this would increase thymidylate production, thereby increasing cellular proliferation, at the expense of biological methylation reactions (366).

Folate may affect the post-translational modifications of histones which cause alterations to the chromatin structure and thus influence transcription. Since histone modifications are more diverse than DNA, more complicated and diverse possibilities in affecting histone marks may exist. Overall there are two major ways in which these histone marks can be altered: altering the

57 abundance and/or efficacy of the enzymes responsible for the modification and altering the availability of the enzyme substrate (367). Less is known about the effects of dietary factors on activities of histone methyltransferase and histone demethylase. A recent study revealed a new relationship between folate and histone methylation, as folate was found to be an enzymatic cofactor for the -specific histone demethylase 1A (KDM1A) (368). Specifically, THF can serve as an acceptor for formaldehyde which is generated during the oxidative demethylation of histone tails. Such a reaction would serve not only to trap and convert formaldehyde, which is a toxic compound, but also to recharge THF with a single-carbon moiety that can then be used for future methylation reactions (368). As histone modifications and DNA methylation have a combined role in transcriptional regulation, dietary influences, including folate, alter one of these epigenetic marks and may also affect one another. For example, a localized increase in DNA methylation in response to increased dietary methyl donors may attract HDAC enzymes to that genomic site, leading to secondary histone deacetylation (367).

CHAPTER 3: RATIONALE, HYPOTHESES, AND OBJECTIVES

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Rationale: Folate mediates the transfer of one-carbon units necessary for thymidylate and purine biosynthesis, and hence is an essential factor for DNA synthesis (1). In neoplastic cells, folate depletion and disrupted folate metabolism cause ineffective DNA synthesis, leading to inhibition of tumor growth (3). This has been the basis for cancer chemotherapy using antifolates and 5FU (3). While monoglutamates are the only circulating forms of folate in blood, once taken up into cells, intracellular folate exists primarily as polyglutamates (1). Intracellular folate is converted to polyglutamates by FPGS within the cytosol, while the lysosomal enzyme GGH removes the terminal glutamates, thereby facilitating export of folate (1). Polyglutamylated folates are better retained intracellularly and are better substrates for intracellular folate-dependent enzymes than monoglutamates (2).

Similar to folate, antifolates, such as MTX and MTA, are retained in cells by the FPGS-induced polyglutamylation, and are exported from cells after hydrolysis to monoglutamates by GGH (2, 3). As with folate, polyglutamylated antifolates are retained in cells longer, thereby increasing their cytotoxicity by extending the length of exposure (2, 3). Polyglutamylated antifolates generally have a higher affinity for, and hence, inhibit their target folate-dependent enzymes in thymidylate and purine biosynthesis to a greater extent than the monoglutamate form (2, 3). One of mechanisms of 5FU cytotoxicity is the formation of a ternary complex involving 5FdUMP, TS and 5,10-methyleneTHF, thereby inhibiting TS activity with consequent suppression of DNA synthesis (227). 5,10-MethyleneTHF with long-chain length polyglutamates is better retained in cells and is more efficient in the formation and stabilization of this inhibitory ternary complex compared with shorter chain polyglutamates (122). Accordingly, modulation of 5,10- methyleneTHF polyglutamylation may affect chemosensitivity of cancer cells to 5FU.

FPGS and GGH may affect chemosensitivity of cancer cells to antifolates by inducing changes in the intracellular retention of antifolates and to 5FU by inducing changes in the intracellular retention of specific folate cofactor such as 5,10-methyleneTHF, respectively. It has been shown that there is an inverse relationship between the ratio of GGH and FPGS and levels of long-chain MTX polyglutamates (170, 171). Generally, low FPGS and/or high GGH expression and activity have been associated with reduced antifolates polyglutamylation that is associated with drug resistance (144). The effects of the differences in FPGS activity and of specific FPGS

60 modulation on drug resistance of and chemosensitivity to antifolates and 5FU have been well investigated. Generally, high FPGS activity or upregulation appears to enhance chemosensitivity of cancer cells to antifolates and 5FU, whereas low FPGS activity or downregulation seems to be a mechanism of resistance to antifolates as well as 5FU. In contrast, the role of GGH modulation on chemosensitivity of cancer cells to antifolate and 5FU has not yet been clearly established (144). FPGS and GGH also modulate intracellular folate concentrations, which might be a critical determinant of chemosensitivity of cancer cells to chemotherapeutic agents designed to interrupt folate metabolism and DNA synthesis (4, 5). The GGH modulation-induced changes in intracellular folate concentrations and folylpolyglutamylation may counterbalance the effects of the GGH modulation-induced changes in polyglutamylation of antifolates and 5,10- methyleneTHF. We have previously demonstrated this complex interaction in an in vitro model of FPGS overexpression and inhibition (4, 17). In the present study, we generated an in vitro model of GGH modulation in human HCT116 colon and MDA-MB-435 breast cancer cell lines and determined the effects of GGH modulation on chemosensitivity of these cells to 5FU and antifolates (Chapter 4).

FA-containing supplement use among cancer patients and survivors is common (9, 10), and FA and LV are administered with most antifolates and 5FU to reduce toxicity and increase efficacy (215, 369). However, increased folate levels may interfere with the sensitivity of chemotherapeutic drugs and cause drug resistance (11-14). Thus, we investigated the effect of different physiological levels of folate on the GGH-modulated chemosensitivity of HCT116 and MDA-MB-435 cell lines to antifolates and 5FU using in vitro and in vivo systems (Chapter 5).

Furthermore, folate mediates the transfer of one-carbon units for the generation of SAM, the primary methyl group donor for most biological methylation reactions including DNA methylation, which is catalyzed by DNMT (1, 27). Polyglutamylation is also important in DNA methylation as polyglutamylated folates are better substrates for folate-dependent enzymes such as MTHFR and MS that are involved in the generation of SAM, which is a substrate for DNA methylation mediated by DNMT (2, 28). And hence, FPGS and GGH may affect DNA methylation at global and gene-specific levels with consequent functional ramifications. Both genomic DNA hypomethylation and gene-specific promoter CpG island hypermethylation are

61 important epigenetic mechanisms of carcinogenesis (29). DNA methylation and DNMT are also potential therapeutic targets and may modify the effect of specific chemotherapeutic agents (30), suggesting that the FPGS- and GGH-modulated DNA methylation changes might influence chemosensitivity to chemotherapeutic agents. In addition, a number of recent studies suggest that aberrant DNA methylation might affect chemosensitivity of cancers by altering expression of genes critical to the drug response. For example, hypermethylation of DNA repair genes, such as MGMT, was associated with increased sensitivity to alkylating agents in glioma patients, and hypermethylation of p73, a homolog of the p53 tumor supressor gene, was also associated with increased sensitivity to cisplatin in NCI-60 cells. In contrast, hypermethylation of hMLH1 was associated with decreased sensitivity to cisplatin in ovarian cancer cells.(31-34, 353). Given these special considerations, we investigated the effects of GGH and FPGS modulation on global and gene-specific DNA methylation and gene expression in HCT116 and MDA-MB-435 cell lines (Chapters 6 and 7).

Research Question 1

Objective: To investigate the role of GGH in modulating chemosensitivity of human HCT116 and MDA-MB-435 cancer cells to antifolates and 5FU using in vitro and in vivo systems.

Hypothesis: We hypothesized that GGH modulation would affect chemosensitivity of HCT116 and MDA-MB-435 cancer cells to antifolates and 5FU by changing the intracellular retention of antifolates and changing the intracellular retention of a folate cofactor necessary for the formation of the inhibitory ternary complex involving TS, respectively. Moreover, we hypothesized that total intracellular folate concentrations and degree of polyglutamylation may further modify the effects of the GGH-modulated chemosensitivity.

Thesis Organization:

Chapter 4 – Study 1 - The development and validation of an in vitro model of GGH modulation and investigation of the effect of GGH modulation on chemosensitivity of human colon and breast cancer cells to chemotherapeutics (proof-of-principle)

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Chapter 5 – Study 2 -The effects of GGH modulation and folate on chemosensitivity of human colon and breast cancer cells to chemotherapeutics in in vitro and in vivo models

Research Question 2

Objective: To investigate the effects of GGH and FPGS modulation on global and gene-specific DNA methylation and gene expression.

Hypothesis: We hypothesized that GGH overexpression and FPGS inhibition would decrease global DNA methylation and DNMT activity, whereas GGH inhibition and FPGS overexpression would increase global DNA methylation and DNMT activity. In addition, GGH and FPGS modulation may further affect the degree of promoter CpG island methylation and the expression of genes.

Thesis Organization:

Chapter 6 – Study 3 -The effect of GGH modulation on global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

Chapter 7 – Study 4 -The effect of FPGS modulation on global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

CHAPTER 4: STUDY 1 – THE DEVELOPMENT AND VALIDATION OF AN IN VITRO MODEL OF GGH MODULATION AND INVESTIGATION OF THE EFFECT OF GGH MODULATION ON CHEMOSENSITIVITY OF HUMAN COLON AND BREAST CANCER CELLS TO CHEMOTHERAPEUTICS (PROOF-OF-PRINCIPLE)

Modified from: Sung-Eun Kim, Peter D. Cole, Robert C. Cho, Anna Ly, Lisa Ishiguro, Kyoung-Jin Sohn, Ruth Croxford, Barton A. Kamen, and Young-In Kim. Effects of folate and γ-glutamyl hydrolase modulation on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and antifolates in vitro and in vivo. Submitted to British Journal of Cancer (under review)

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4.1 Abstract

Backgroud: Folate and antifolates are retained in cells by polyglutamylation mediated by FPGS, and are exported from cells after hydrolysis to monoglutamates by GGH. Polyglutamylated folates and antifolates are retained in cells longer and are better substrates than their monoglutamate counterparts for folate dependent enzymes involved in thymidylate and purine biosynthesis. GGH modulation may therefore affect chemosensitivity of cancer cells to antifolates and 5FU by altering polyglutamylation of antifolates and a specific target intracellular folate cofactor for 5FU (5,10-methyleneTHF), respectively.

Methods: We generated an in vitro model of GGH overexpression and inhibition in human HCT116 colon and MDA-MB-435 breast cancer cells with predictable functional consequences, and investigated the effects of GGH modulation on the chemosensitivity to 5FU and MTX.

Results: Functionally significant GGH overexpression and inhibition were confirmed by GGH protein expression/activity, total and long-chain polyglutamylated intracellular folate concentrations, TS activity, and DHFR protein expression/activity in both cell lines. GGH overexpression decreased chemosensitivity to 5FU with LV and MTX, while GGH inhibition increased chemosensitivity to 5FU with LV. The GGH modulation-induced changes in the chemosensitivity of colon and breast cancer cells to 5FU were in the expected direction when a precursor for 5,10-methyleneTHF (i.e., LV) was supplied exogenously. The GGH modulation- induced changes in MTX-polyglutamylation appeared to be the primary determinant of chemosensitivity of colon and breast cancer cells to MTX in the GGH overexpression system but not in the GGH inhibition system.

Conclusions: As proof-of-principle, we provide functional evidence that GGH status alters chemosensitivity of colon and breast cancer cells to 5FU and MTX. However, these effects appeared to depend not only on the GGH modulation-induced changes in polyglutamylation of 5,10-methyleneTHF and MTX but also on adaptive and compensatory changes in other enzymes involved in intracellular folate and antifolate accumulation and metabolism in response to GGH modulation.

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4.2 Introduction

Folate mediates the transfer of one-carbon units necessary for thymidylate and purine biosynthesis, and hence is an essential factor for DNA synthesis (370). In neoplastic cells, folate depletion and disrupted folate metabolism cause ineffective DNA synthesis, resulting in inhibition of tumor growth (3). This has been the basis for cancer chemotherapy using antifolates and 5FU (3). While monoglutamates are the only circulating forms of folate in blood and the only form of folate that is transported across the cell membrane, once taken up into cells, intracellular folate exists primarily as polyglutamates (370). Intracellular folate is converted to polyglutamates by FPGS, while GGH removes the terminal glutamates (370). Polyglutamylated folates are better retained in cells and are better substrates than monoglutamates for intracellular folate-dependent enzymes (2).

Similarly, antifolates (e.g., MTX) are retained in cells by the FPGS-induced polyglutamylation, and are exported from cells after hydrolysis to monoglutamates by GGH (2, 3). As with folate, polyglutamylated antifolates are retained in cells longer, thereby increasing their cytotoxicity by extending the length of exposure (2, 3). Polyglutamylated antifolates generally have a higher affinity for, and hence inhibit their target folate-dependent enzymes in thymidylate and purine biosynthesis to a greater extent than the monoglutamate forms (2, 3).

Changes in polyglutamylation of a specific intracellular folate cofactor may also affect the sensitivity of tumor cells to other chemotherapeutic agents, such as 5FU, not typically considered as antifolates. One cytotoxic mechanism of 5FU is the formation of a ternary complex involving a metabolite of 5FU (FdUMP), TS and 5,10-methyleneTHF, thereby inhibiting TS activity with consequent suppression of DNA synthesis (227). LV (5-formylTHF), a precursor for 5,10- methyleneTHF, potentiates the cytotoxic effect of 5FU by stabilizing this inhibitory ternary complex (227). 5,10-MethyleneTHF with longer chain length polyglutamates is better retained intracellularly and is more efficient in the formation and stabilization of this inhibitory ternary complex compared with shorter chain polyglutamates (122). Therefore, modulation of 5,10- methyleneTHF polyglutamylation may affect chemosensitivity of cancer cells to 5FU.

The effects of differences in FPGS activity and of specific FPGS modulation on drug resistance of and chemosensitivity to MTX and other antifolates (15, 19-22, 24, 26) and 5FU (18, 23, 25)

66 have been extensively studied. Generally, high FPGS activity or upregulation appears to enhance chemosensitivity of cancer cells to MTX, other antifolates and 5FU, whereas low FPGS activity or downregulation seems to be a mechanism of resistance to MTX and other antifolates as well as 5FU.

In contrast, the role of GGH modulation on chemosensitivity of cancer cells to antifolate and 5FU has not yet been clearly established (144). Increased GGH activity was identified as the mechanism of antifolate resistance in rat hepatoma cells (172, 173), human sarcoma cells (173) and human leukemia cells (174). However, ectopic overexpression of GGH after transfection of MCF7 breast cancer and HT1080 fibrosarcoma cells did not confer MTX resistance (188). Pharmacologic GGH inhibition was observed to enhance the sensitivity of human sarcoma (138) and breast cancer cells (371, 372) to MTX. GGH inhibition induced by siRNA in human DLD-1 colon cancer cells enhanced sensitivity to 5FU (5).

GGH may play an important role in modulating drug resistance and chemosensitivity of cancer cells to antifolates and 5FU by inducing changes in polyglutamylation of antifolates and a specific target intracellular folate cofactor (e.g., 5,10-methyleneTHF for 5FU), respectively. However, GGH modulation affects polyglutamylation of not only antifolates and specific target intracellular folate cofactors but also of all other intracellular folate cofactors. GGH modulation also affects total intracellular folate concentration, which is an important determinant of the cytotoxic effects of antifolates and 5FU (3, 46, 136). Therefore, the GGH modulation-induced changes in intracellular folate concentrations and folylpolyglutamylation may counterbalance the effects of the GGH modulation-induced changes in polyglutamylation of antifolates and specific 5,10-methyleneTHF. We have previously demonstrated this complex interaction in an in vitro model of FPGS overexpression and inhibition (4, 17).

In the present study, we generated an in vitro model of GGH modulation in human HCT116 colon and MDA-MB-435 breast cancer cells and determined the effects of GGH modulation on chemosensitivity to 5FU and MTX.

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4.3 Materials and Methods

4.3.1 Cell Lines and Culture

Human colon adenocarcinoma HCT116 and breast adenocarcinoma MDA-MB-435 cells were purchased from the American Type Culture Collection (Rockville, MD). Both cells were grown in RPMI-1640 medium (Invitrogen) containing 2.3 µmol/L FA supplemented with 10% fetal bovine serum (FBS, Invitrogen), 500 µg/mL Geneticin® (G418; Invitrogen), 50 units/mL penicillin with 50 µg/mL streptomycin (Invitrogen), and 0.25 µg/mL fungizone amphotericin B

(Invitrogen). Cell cultures were maintained at 37°C in 5% CO2.

Table 4.1 summarizes the phenotypic and molecular characteristics of HCT116 and MDA-MB- 435 cell lines. HCT116 cells exhibit microsatellite instability due to a mutation in the mismatch repair gene MLH1 (373), and contain a point mutation in the K-ras oncogene (374). It has been known that HCT116 has intact DNA damage-dependent and spindle-dependent checkpoints, and it is suitable for targeted homologous recombination (375-377). Furthermore, HCT116 has a single, well-defined defect in the TGF-β pathway such as a truncating mutation in TβRII (378, 379). This allows well-controlled restoration of TGF-β-mediated signaling through the introduction of a wild-type TβRII gene.

The MDA-MB-435 cell line is one of the most metastatic human breast cancer cell lines available (380). MDA-MB-435 breast cancer cells are estrogen receptor negative cells (381), which is beneficial in this proof-of-concept study. Since these cells do not require exogenous estrogen to be added in their growth media, it minimizes potential -drug interactions, facilitating a mechanistic approach to investigate the effect of GGH modulation on chemosensitivity. Furthermore, efficient transfection of this cell line has been previously established (244). Although this cell line was ultimately selected for the present study, there is debate as to the true origin of this cell line. A microarray analysis has indicated that the gene expression pattern of the human MDA-MB-435 (382) resembles that of human melanoma cell lines (383). Since then, additional evidence has shown the ability of MDA-MB-435 cells to express melanocytic markers (384-386). However, upon induction with heregulin in vitro, MDA- MB-435 cells undergo sufficient mammary epithelial differentiation to produce milk lipid

68 droplets and to express β-casein mRNA (387). Additionally, enhanced expression of NM23-H1, a metastasis suppressor gene, leads to the formation of organized mammary acinus-like spheres in 3D culture and the expression of sialomucin (epithelial membrane antigen, EMA) (388). A recent investigation confirmed the ability of MDA-MB-435 cells to co-express markers of mammary epithelium and melanocytes both in vitro and in vivo, and postulated committing lineage infidelity as an underlying mechanism for the observed dual lineage transdifferentiation (381). Furthermore, another recent study reported the robust expression of melanocyte-related genes in a variety of breast cancer cell lines including MDA-MB-435, and more importantly in freshly resected and histopathologically confirmed human breast cancer specimens (389).

Table 4. 1 Summary of phenotypic and molecular characteristics of HCT116 and MDA- MB-435 cell lines

Cell Line HCT116 MDA-MB-435 Organism Homo sapiens (Human) Homo sapiens (Human)

Gender, race, age Male, adults Female, Caucasian, 31 years

Tissue Colorectal carcinoma Previously described as ductal carcinoma Derived from metastatic site: pleural effusion

Tumorigenic Yes, in nude mice No

Morphology Epithelial Spindle shaped

Growth properties Adherent Adherent

Cellular products Carcinoembryonic antigen (CEA) 1 Tubulin, actin ng/106 cells/10 days, keratin

Molecular features Positive for keratin by Gene expression analysis generated immunoperoxidase staining questions about the origin

Positive for transforming growth factor Cross-contaminated with M14 β1 and β2 expression melanoma cell line

Gene mutations MLH1, MSH3 A8, MSH6 C8, K-ras, p14ARF, p16, BAX G8, β-catenin, DCC, Axin2, CBF2, E2F4, GRK4, Helicase q1, MBD4 10A, RAD50 9A, RIZ 9A, TGFBR-II

American Type Culture Collection: http://www.atcc.org/ATCCAdvancedCatalogSearch/tabid/112/Default.aspx

69

4.3.2 Construction and Transfection of GGH Expression Vectors

The full-length human GGH cDNA (1.2 kb; GeneBank accession No. U55206) (137) was subcloned into the Not1 site of the eukaryotic expression vector pIRESneo (Clontech, Palo Alto, CA) containing a human cytomegalovirus (CMV) promoter and a neomycin resistance gene expression cassette in the sense orientation to generate the sense GGH expression vector (Figure 4.1). The GGH-targeted siRNA was designed according to the manufacturer’s protocol (QIAGEN), and ligated into the vector between the BamH1 and HindIII restriction sites of the pSilencer neo siRNA expression vector (Ambion, Austin, TX) (Figure 4.1). pSilencer neo vectors employ RNA polymerase III promoters which generate large amounts of small RNA using simple promoter and terminator sequences. They also contain a neomycin resistance gene to enable selection. The oligonucleotides were designed encoding the desired siRNA strand: GTACTTGGAGTCTGCAGGT (forward) and ACCTGCAGACTCCAAGTAC (reverse), and ACCTGCAGACTCCAAGTAC (forward) and GTACTTGGAGTCTGCAGGT (reverse). Correct integration, orientation, and sequence of the sense GGH cDNA and GGH-targeted siRNA were confirmed by predicted fragment sizes after multiple restriction enzyme digestions and DNA sequencing.

The pIRESneo vector containing the sense GGH cDNA and the pSilencer vector containing the GGH-targeted siRNA were stably transfected into HCT116 and MDA-MB-435 cells using Lipofectin (Invitrogen) according to the manufacturer’s protocol. In separate , both cell lines were stably transfected with empty pIRESneo and pSilencer vectors as corresponding controls expressing endogenous GGH. Transfected cells were incubated with 500 µg/mL of neomycin (Invitrogen) to select for cells that expressed the various constructs. After a population of cells was selected, individual clonal cell lines were isolated and expanded. Cells were maintained in complete medium supplemented with 500 µg/mL neomycin. Several (> 10) clones expressing the sense GGH cDNA, GGH-targeted siRNA, and empty vectors were screened at random, and two independent clones of each construct were selected for further analysis. Data from three experiments using two independent clones of each construct were similar, and thus, the data from one experiment are presented. All analyses were done in triplicate, and at least three replicate experiments were done. Cells with similar passage numbers were selected for transfection with different GGH expression vectors and for subsequent functional analyses. Cells

70 expressing the sense GGH or GGH-targeted siRNA and corresponding controls with similar passage numbers were used for chemosensitivity analysis.

A B

Figure 4. 1 Maps of pIRESneo (A) and pSilencer neo (B) expression vectors

4.3.3 Western Blot Analysis

GGH and DHFR protein expression was determined by standard western analysis as previously described (4, 244), using a rabbit polyclonal antibody raised against human GGH (SDI, Newark, DE) at a dilution of 1:1,000 and against human DHFR (Sigma, Saint Louis, MO) at a dilution of 1:3,000, respectively. To confirm that the proteins were loaded equally, the membranes were stripped and reprobed with a mouse raised against human β-actin (Sigma, Oakville, ON) at a dilution of 1:3000. All western analyses were repeated using three different cell lysates. Densitometry of bands were determined using the public domain ImageJ (version 1.38) from the National Institute of Health available on the Internet at http://rsbweb.nih.gov/ij.

71

4.3.4 GGH, TS, and DHFR Enzyme Activity Assays

GGH was determined by incubating protein in cell lysate with MTX-glu4 as substrate and measuring MTX and its polyglutamates using HPLC as described (188, 390). The catalytic activity of TS was determined by the [3H] release that occurred during the conversion of [5-3H]- dUMP to dTMP as described (391). The catalytic activity of DHFR was determined using the DHFR assay kit (Sigma) as per the manufacturer’s protocol. The assay is based on the ability of DHFR to catalyze the reversible NADPH-dependent reduction of DHF to THF. Enzyme assays were performed in triplicate and repeated using three different cell lysates.

4.3.5 Intracellular Total and Long-Chain Folate Concentrations Analysis

Intracellular folate concentrations for conjugase treated and untreated samples were determined by a standard microbiological assay to determine the extent of polyglutamylation as described (4, 17). All analyses were performed in triplicate and repeated using three different cell lysates.

4.3.6 Doubling Time Calculation

Eight thousand cells per well were plated in a 96-well flat-bottom plate and grown in RPMI-1640 with 10% FBS for 72 hours. The cell population was determined using the sulforhodamine B (SRB) absorbance measurement assay. The growth rate constant k was derived kt using the equation N/N0 = e , where N0 is the A of cells at time zero, and N is the A of cells at 72 hours. The same equation was used to calculate doubling time t by setting N/N0 = 2. All analyses were performed in triplicate, and three replicate experiments were performed.

4.3.7 In Vitro Chemosensitivity Assay

In vitro chemosensitivity was determined using a modification of the SRB protein assay as described (392, 393). Briefly, 8,000 cells per 100 µL RPMI-1640 per well were seeded in

72 triplicate in 96-well flat-bottom plates. After 24 hours, an additional 100 µL of RPMI-1640 containing 5FU (InvivoGen) alone or in combination with LV (Sigma), MTX (Sigma), MTA (Eli Lily), or TMTX (USB Pharma B.V.) were added, and cells were cultured for an additional 72 hours. In both cell lines, the concentration of 5FU ranged from 1.5-25 × 10-6 mol/L, whereas the concentration of LV was held constant at 5 × 10-6 mol/L. The concentration range of HCT116 cells was 0.5-5 × 10-8 mol/L for MTX, 1-15 × 10-7 mol/L for MTA, and 2-36 × 10-9 mol/L for TMTX. The concentration range of MDA-MB-435 cells was 1.5-6 × 10-8 mol/L for MTX, 1-15 × 10-6 mol/L for MTA, and 4-42 × 10-9 mol/L for TMTX. After 72 hours, cells were fixed with trichloroacetic acid and stained with SRB protein dye. The dye was solubilized, and the A of the solution was measured at 595 nm. The results were expressed as the percentage of cell survival on the basis of the difference between A at the start and end of drug exposure according to the formula (394):

(A drug / A start drug exposure) - 1 Survival (%) = × 100 (A no drug / A start drug exposure) - 1

IC50 values (i.e., the drug concentration that corresponded to a reduction in cell survival of 50% compared with survival of untreated control cells) were calculated from plots of drug concentration vs. proportion of cells that survived. All analyses were performed in sextuplet, and at least three replicate experiments were performed for all drug treatments.

4.3.8 Statistical Analysis

For continuous variables, comparisons between cells expressing the sense GGH (Sense) and controls (Control-S) and between cells transfected with the GGH-targeted siRNA (siRNA) and controls (Control-si) were determined using the Student’s t-test. For the in vitro chemosensitivity analyses, plots of percentage of survival vs. dose demonstrated S-shaped curves, and therefore, the logit transformation [logit (p) = ln (p/[1 - p])] was used. Ordinary least squares regression was used to model the effect of log (dose) of chemotherapy and cell type on the logit- transformed proportion of cells that survived at each dose. The interaction between cell type and log (dose) was included in the model to test the hypothesis that the cell types were differentially

73 sensitive to chemotherapy. IC50 doses and their 95% confidence intervals were calculated on the loscale from the regression results as described (395), and then back-transformed to the original scale for reporting. For all analyses, results were considered statistically significant if two-tailed P-values were < 0.05. Analyses were done using SPSS Statistics 17.0 (IBM SPSS, Chicago, IL) and graphs were prepared in Microsoft Excel.

4.4 Results

4.4.1 GGH Protein Expression and GGH Enzyme Activity

In HCT116 and MDA-MB-435 cell lines, cells expressing the sense GGH (Sense) and GGH- targeted siRNA (siRNA) had significantly higher and lower steady-state levels of the GGH protein, respectively, compared with corresponding controls expressing endogenous GGH (P < 0.05; Figure 4.2). HCT116 cells expressing the sense GGH had a 4.7-fold higher (P < 0.001) and those transfected with the GGH-targeted siRNA had a 2.7-fold lower (P < 0.001) GGH activity compared with corresponding controls (Figures 4.2A and 2B). Similarly, MDA-MB-435 cells expressing the sense GGH had a 3.3-fold higher (P < 0.001) and those transfected with the GGH- targeted siRNA had a 3.4-fold lower (P = 0.001) GGH activity compared with corresponding controls (Figures 4.2C and 2D).

74

A Control-S Sense B Control-si siRNA

GGH GGH (37 kDa) (37 kDa)

β-actin β-actin (42 kDa) (42 kDa)

7 * 7

6 6 5 5

4 4

3 3

2 2

1 1 * GGH Activity (pmol/µg protein/min) GGH Activity (pmol/µg protein/min) 0 0 Control-S Sense Control-si siRNA

P < 0001 P < 0001

C Control-S Sense D Control-si siRNA

GGH GGH (37 kDa) (37 kDa)

β-actin β-actin (42 kDa) (42 kDa)

10 10 * 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 * 1 1 GGH Activity (pmol/µg protein/min) 0 GGH Activity (pmol/µg protein/min) 0 Control-S Sense Control-si siRNA P < 0.001 P = 0.001

Figure 4. 2 ɤ-Glutamyl hydrolase (GGH) protein expression and GGH enzyme activity in the GGH-modulated HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cells. *, P < 0.05 compared with corresponding control. Values are mean ± standard deviation (SD).

75

4.4.2 Concentrations of Intracellular Folate and Long-Chain Length Polyglutamates

Following conjugase treatment (which allows the measurement of total intracellular folate concentration, including short- and long-chain folylpolyglutamates), intracellular folate concentration of HCT116 cells expressing the sense GGH was 1.27 to 1.28-fold lower (P = 0.007), whereas that of cells expressing the GGH-targeted siRNA was 1.83- to 1.96-fold higher (P < 0.001), than that of those expressing endogenous GGH (Figure 4.3A). Intracellular folate concentration of HCT116 cells not treated with conjugase (which allows the determination of short-chain polyglutamates) was not significantly different between cells expressing the sense GGH and those expressing endogenous GGH (Figure 4.3A), while it was higher in HCT116 cells expressing the GGH-targeted siRNA than that of control (P = 0.003; Figure 4.3A). The concentration of long-chain polyglutamates (i.e., differences between folate concentrations of conjugase-treated and untreated samples) clearly show that HCT116 cells expressing the sense GGH had significantly lower concentrations of long-chain polyglutamates than cells expressing endogenous GGH (P = 0.027; Figure 4.3B), and HCT116 cells expressing the GGH-targeted siRNA had significantly higher concentrations of long-chain polyglutamates than the corresponding controls (P = 0.015; Figure 4.3B).

Similarly, the intracellular total folate concentration of MDA-MB-435 cells expressing the sense GGH was 2.60- to 2.69-fold lower (P < 0.001), whereas that of cells expressing the GGH- targeted siRNA was 1.39- to 1.59-fold higher (P < 0.001), than that of those expressing endogenous GGH (Figure 4.4A). Intracellular short-chain folate concentration of MDA-MB-435 cells not treated with conjugase was lower in cells expressing the sense GGH (P < 0.001) and GGH-targeted siRNA compared with corresponding controls (Sense, P < 0.001, Figure 4.4A; siRNA, P = 0.018, Figure 4.4A).

The concentration of long-chain polyglutamates demonstrate that MDA-MB-435 cells expressing the sense GGH had significantly lower concentrations of long-chain polyglutamates than cells expressing endogenous GGH (P = 0.023; Figure 4.4B), and MDA-MB-435 cells expressing the GGH-targeted siRNA had significantly higher concentrations of long-chain polyglutamates than corresponding controls (P < 0.001; Figure 4.4B). These observations confirm that GGH

76 modulation in HCT116 and MDA-MB-435 cells has appropriately affected the glutamate chain lengths of intracellular folate pool.

A 2 2 conjugase conjugase cells) cells) 6 non-conjugase 6 non-conjugase

1.5 1.5 c

a 1 1 c a a b b b 0.5 0.5

0 Folate Concentration / (ng 5 x 10 0 Folate Concentration (ng / 5 x 10 (ng 5 x / Concentration Folate Control-S Sense Control-si siRNA

P < 0.05 P < 0.05

B 1.2 1.2

1 1

0.8 0.8 cells) cells) 6 6 * 0.6 0.6

0.4 0.4 (ng /(ng 5 x 10 /(ng 5 x 10 * 0.2 0.2

0 0 Long-Chain Polyglutamates Concentration Control-S Sense Long-Chain Polyglutamates Concentration Control-si siRNA

P = 0.027 P = 0.015

Figure 4. 3 Concentrations of intracellular total folate (A) and long-chain length polyglutamates (B) in the GGH-modulated HCT116 colon cancer cells. *, P < 0.05 compared with corresponding control. Different letters within each group denote significant differences at P < 0.05. Values are mean ± SD.

77

A 2 2 conjugase conjugase

cells) non-conjugase 6

cells) non-conjugase a 6 1.5 1.5 c b 1 1 a c b d 0.5 0.5 d

Folate Concentration (ng / 5 x 10 5 x / (ng Concentration Folate Folate Concentration / (ng 5 x 10 0 0 Control-S Sense Control-si siRNA

P < 0.05 P < 0.05 B 1.2 1.2 * 1 1

0.8 0.8 cells) cells) 6

6 0.6 0.6

0.4 0.4 (ng /(ng 5 x 10 /(ng 5 x 10 * 0.2 0.2

0 0 Long-Chain Polyglutamates Concentration Long-Chain Polyglutamates Concentration Control-S Sense Control-si siRNA

P = 0.023 P < 0.001

Figure 4. 4 Concentrations of intracellular total folate (A) and long-chain length polyglutamates (B) in the GGH-modulated MDA-MB-435 breast cancer cells. *, P < 0.05 compared with corresponding control. Different letters within each group denote significant differences at P < 0.05. Values are mean ± SD.

4.4.3 TS Catalytic Enzyme Activity

TS catalytic activity in HCT116 and MDA-MB-435 cells expressing the sense GGH was significantly lower than in the corresponding cells expressing endogenous GGH (HCT116, P < 0.001, Figure 4.5A; MDA-MB-435, P = 0.005, Figure 4.5C). TS catalytic activity in both cell lines transfected with the GGH-targeted siRNA was significantly higher compared with cells transfected with the vector alone (HCT116, P = 0.031, Figure 4.5B; MDA-MB-435, P = 0.004, Figure 4.5D).

78

Figure 4. 5 Thymidylate synthase (TS) catalytic activity in the GGH-modulated HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cells. *, P < 0.05 compared with corresponding control. Values are mean ± SD.

4.4.4 DHFR Protein Expression and DHFR Enzyme Activity

HCT116 and MDA-MB-435 cells transfected with the sense GGH had lower levels of the DHFR protein (HCT116, P = 0.008, Figure 4.6A; MDA-MB-435, P = 0.044, Figure 4.7A) and lower DHFR activity (HCT116, P = 0.006, Figure 4.6A; MDA-MB-435, P = 0.018, Figure 4.7A) compared with cells expressing endogenous GGH. In both cell lines, cells transfected with the GGH-targeted siRNA had higher levels of the DHFR protein (HCT116, P = 0.013, Figure 4.6B; MDA-MB-435, P = 0.014, Figure 4.7B) and higher DHFR activity (HCT116, P < 0.001, Figure 4.6B; MDA-MB-435, P = 0.023, Figure 4.7B) than cells transfected with the vector alone.

79

Figure 4. 6 Dihydrofolate reductase (DHFR) protein expression and DHFR enzyme activity in the GGH-overexpressed (A) and GGH-inhibited (B) HCT116 colon cancer cells. *, P < 0.05 compared with corresponding control. Values are mean ± SD.

80

Figure 4. 7 Dihydrofolate reductase (DHFR) protein expression and DHFR enzyme activity in the GGH-overexpressed (A) and GGH-inhibited (B) MDA-MB-435 breast cancer cells. *, P < 0.05 compared with corresponding control. Values are mean ± SD.

81

4.4.5 Doubling Time

Growth curves of HCT116 and MDA-MB-435 cells expressing the sense GGH exhibited a significantly slower growth rate compared with corresponding controls (data not shown). Unexpectedly, HCT116 and MDA-MB-435 cells transfected with the GGH-targeted siRNA also demonstrated a significantly slower growth rate on growth curves compared with corresponding controls (data not shown). The growth curve data were confimed by doubling time.

HCT116 and MDA-MB-435 cells expressing the sense GGH exhibited a significantly increased doubling time compared with controls (HCT116, 28.8 ± 0.3 h vs. 25.5 ± 0.03 h, P < 0.001, Figure 4.8A; MDA-MB-435, 43.2 ± 0.7 h vs. 39.5 ± 0.4 h, P < 0.001, Figure 4.8C), indicating a slower growth rate. Unexpectedly, cells transfected with the GGH-targeted siRNA also had an increased doubling time compared with controls in both cell lines (HCT116, 30.0 ± 0.04 h vs. 29.3 ± 0.1 h, P < 0.001, Figure 4.8B; MDA-MB-435, 37.4 ± 0.2 h vs. 35.0 ± 0.6 h, P < 0.001, Figure 4.8D).

A 35 B 35 * 30 * 30

25 25

20 20

15 15

10 10 Doubling Time (hr) Doubling Doubling Time (hr) 5 5

0 0 Control-S Sense Control-si siRNA P < 0.001 P < 0.001 C 50 D 50 *

40 40 *

30 30

20 20 Doubling Time (hr) Doubling Doubling Time (hr) Doubling 10 10

0 0 Control-S Sense Control-si siRNA P < 0.001 P < 0.001 Figure 4. 8 Doubling time of the GGH-modulated HCT116 colon (A, B) and MDA-MB- 435 breast (C, D) cancer cells. *, P < 0.05 compared with corresponding control. Values are mean ± SD.

82

4.4.6 In Vitro Chemosensitivity

All analyses were performed in sextuplet, and at least five replicate experiments were performed for all drug treatments. Chemosensitivity data presented are representative of the overall results. Cell survival calculations are further explained in Appendix 1. As proof-of-principle, the effect of GGH overexpression and inhibition on chemosensitivity of HCT116 and MDA-MB-435 cells to MTA (positive control) and TMTX (negative control) was determined.

MTA Chemosensitivity: MTA is a novel antimetabolite that inhibits multiple enzymes involved in thymidylate and purine biosynthesis whose cytotoxic effects depend on polyglutamylation (270). Our a priori hypothesis was that GGH overexpression would decrease, whereas GGH inhibition would increase, chemosensitivity of cancer cells to MTA. In contrast to our expectation, HCT116 cells that overexpressed GGH were more sensitive, whereas HCT116 cells in which GGH was inhibited were less sensitive, to MTA compared with controls (P < 0.05; Figures 4.9A and 9F). As expected, MDA-MB-435 cells that overexpressed GGH were less sensitive to MTA compared with controls, whereas in contrast to our expectation, MDA-MB-435 cells in which GGH was inhibited were also less sensitive to MTA compared with controls (P < 0.05; Figures 4.10A and 10F).

TMTX Chemosensitivity: TMTX is a nonclassic antifolate that directly inhibits DHFR whose cytotoxic effects do not depend on polyglutamylation as it is not polyglutamylated (396). Our a priori hypothesis was that chemosensitivity of cancer cells to TMTX would largely depend on the GGH modulation-induced changes in concentrations and polyglutamylation of intracellular folates. HCT116 cells that overexpressed GGH were more sensitive to TMTX compared with controls (P < 0.05), whereas GGH inhibition had no significant effect (Figures 4.9B and 9G). In MDA-MB-435 cells, both GGH overexpression and inhibition decreased chemosensitivity to TMTX compared with corresponding controls (P < 0.05; Figures 4.10B and 10G).

5FU Chemosensitivity: For 5FU, our a priori hypothesis was that GGH overexpression would decrease the cytotoxic effect of 5FU by decreasing relative intracellular concentration of long- chain 5,10-methyleneTHF-polyglutamates, resulting in less efficient formation and stabilization of the inhibitory 5,10-methyleneTHF-TS-FdUMP ternary complex. In contrast, we hypothesized that GGH inhibition would increase the cytotoxic effect of 5FU by increasing relative

83 intracellular concentrations of long-chain 5,10-methyleneTHF-polyglutamates, resulting in more efficient formation and stabilization of the 5,10-methyleneTHF-TS-FdUMP ternary complex. Consistent with our hypothesis, chemosensitivity of both HCT116 and MDA-MB-435 cells expressing the sense GGH to 5FU+LV was significantly decreased compared with corresponding controls (P < 0.05; Figures 4.9C and 10C; Table 4.2), while that of cells transfected with the GGH-targeted siRNA to 5FU+LV was significantly increased compared with corresponding controls (P < 0.05; Figures 4.9H and 10H; Table 4.2). Interestingly, in contrast to our hypothesis and to the 5FU+LV data, GGH overexpression significantly increased, whereas GGH inhibition significantly decreased, chemosensitivity of HCT116 cells to 5FU alone compared with controls (P < 0.05; Figures 4.9D and 9I; Table 4.2). In contrast, GGH modulation had no significant effect of chemosensitivity of MDA-MB-435 cells to 5FU (Figures 4.10D and 10I; Table 4.2).

MTX Chemosensitivity: We predicted that the GGH overexpression-induced decreased MTX- polyglutamylation would decrease, whereas the GGH inhibition-induced increased MTX- polyglutamylation would enhance, the cytotoxic effect of MTX. Consistent with our hypothesis, chemosensitivity of both HCT116 and MDA-MB-435 cells expressing the sense GGH to MTX was significantly decreased compared with corresponding controls (P < 0.05; Figures 4.9E and 10E; Table 4.2). Unexpectedly, in contrast to our hypothesis, chemosensitivity of both HCT116 and MDA-MB-435 cells transfected with the GGH-targeted siRNA was also significantly decreased compared with corresponding controls (P < 0.05; Figures 4.9J and 10J; Table 4.2).

84

A B C D E

100 Control-S Control-S 100 100 100 Control-S 100 Sense Sense Control-S Control-S Sense 80 Sense Sense 80 80 80 80

60 60 60 60 60

Survival % 40 Survival %

Survival % 40 40 Survival % 40 * Survival % 40 * * * * 20 20 20 20 20

0 0 0 0 0 0 0.3 0.6 0.9 1.2 1.5 1.8 0 10 20 30 40 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 10 20 30 40 50 60 µM 5FU nM MTX µM μPemetrexedM MTA nM TMTX μM 5FU + 5 μM LV

F G H I J 100 Control-si Control-si 100 100 Control-si siRNA siRNA 100 Control-si 100 Control-si siRNA siRNA siRNA 80 80 80 80 80

60 60 * 60 60 60 Survival % 40 Survival % 40 Survival % * 40 Survival % 40 Survival % 40 * 20 20 20 20 20 *

0 0 0 0 0 0 0.3 0.6 0.9 1.2 1.5 1.8 0 10 20 30 40 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 10 20 30 40 50 60 µM Pemetrexed μM 5FU + 5 μM LV nM MTX μM MTA nM TMTX µM 5FU Figure 4. 9 In vitro chemosensitivity of HCT116 colon cancer cells transfected with either the sense GGH (Sense) or GGH- targeted siRNA (siRNA) to pemetrexed (A, F; MTA, positive control), trimetrexate (B, G; TMTX, negative control), 5-fluorouracil plus leucovorin (C, H; 5FU+LV), 5-fluorouracil alone (D, I; 5FU), or methotrexate (E, J; MTX) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH). Cells were grown in medium containing 2.3 µmol/L folic acid. Points, means; bars, SD. *, P < 0.05 statistically significant compared with corresponding control cells.

85

A B C D E

Control-S Control-S 100 100 100 Control-S 100 Sense 100 Control-S Control-S Sense Sense Sense Sense 80 * 80 80 80 80

60 60 60 60 60 *

Survival % *

40 Survival % 40 Survival % Survival % 40 40 40 Survival % *

20 20 20 20 20

0 0 0 0 0 0 3 6 9 12 15 18 0 10 20 30 40 50 0 5 10 15 20 25 30 0 5 10 15 20 25 30 10 20 30 40 50 60 70 nM MTX µM μPemetrexedM MTA nM TMTX µM 5FU + 5 µM LV µM 5FU

F G H I J 100 Control-si Control-si siRNA 100 100 Control-si 100 Control-si Control-si siRNA 100 siRNA siRNA 80 siRNA * 80 80 80 80 60 60 60 60 60 Survival % 40 Survival % 40 * 40 Survival % Survival % 40 Survival % * 40 20 * 20 20 20 20

0 0 0 0 0 0 3 6 9 12 15 18 0 10 20 30 40 50 0 5 10 15 20 25 30 0 5 10 15 20 25 30 10 20 30 40 50 60 70 µM μPemetrexedM MTA nM TMTX μM 5FU + 5 μM LV µM 5FU nM MTX

Figure 4. 10 In vitro chemosensitivity of MDA-MB-435 breast cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to pemetrexed (A, F; MTA, positive control), trimetrexate (B, G; TMTX, negative control), 5- fluorouracil plus leucovorin (C, H; 5FU+LV), 5-fluorouracil alone (D, I; 5FU), or methotrexate (E, J; MTX) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH). Cells were grown in medium containing 2.3 µmol/L folic acid. Points, means; bars, SD. *, P < 0.05 statistically significant compared with corresponding control cells.

86

Table 4. 2 IC50 values of 5FU and MTX in the GGH-modulated HCT116 colon and MDA-MB-435 breast cancer cells

HCT116 MDA-MB-435

IC50 (95% CI) IC50 (95% CI)

5FU (µM) + 5 µM LV

Sense 11.48* (11.13, 12.00) 17.15* (16.35, 18.40)

Control-S 8.42* (8.01, 8.87) 6.54* (5.96, 7.05) siRNA 8.83* (8.36, 9.49) 3.94* (3.74, 4.08)

Control-si 17.04* (16.36, 17.93) 8.91* (8.58, 9.52)

5FU (µM)

Sense 10.17* (10.04, 10.34) 6.72* (6.25, 7.13)

Control-S 21.77* (20.47, 23.71) 6.95* (6.47, 7.43) siRNA 15.34* (14.70, 16.23) 6.30* (5.85, 6.65)

Control-si 6.42* (6.10, 6.65) 6.66* (6.45, 6.87)

Methotrexate (nM)

Sense 26.37* (25.79, 27.05) 46.29* (45.22, 47.61)

Control-S 20.08* (19.38, 20.69) 30.26* (29.92, 30.66) siRNA 18.99* (18.54, 19.48) 44.10* (43.41, 44.82)

Control-si 12.73* (12.36, 13.20) 30.72* (29.84, 31.57)

*, P < 0.05 compared with corresponding controls.

87

4.5 Discussion

We developed an appropriate in vitro model of GGH overexpression and inhibition in HCT116 colon and MDA-MB-435 breast cancer cells with predictable functional consequences. Compared with controls expressing endogenous GGH, cells expressing the sense GGH had significantly higher GGH protein expression and activity, lower total intracellular folate concentrations and lower content of long-chain folylpolyglutamates. In contrast, cells transfected with the GGH-targeted siRNA had significantly lower GGH protein expression and activity, higher concentrations of total intracellular folate, and higher content of long-chain folylpolyglutamates compared with controls expressing endogenous GGH. Cells expressing the sense GGH had a significantly slower growth rate compared with controls. This is consistent with the observed decreased intracellular folate concentrations and lower content of long-chain folylpolyglutamates, which are better substrates than short-chain folylpolyglutamates for folate- dependent enzymes involved in thymidylate and purine biosynthesis. Consistent with the growth data, cells overexpressing GGH had significantly lower TS activity and DHFR protein expression and activity. Unexpectedly, however, cells transfected with the GGH-targeted siRNA also had a significantly slower growth rate compared with controls, which is not readily explained by the observed increased intracellular folate concentrations, higher content of long- chain folylpolyglutamates, increased TS activity, and increased DHFR protein expression and activity. Generally, most of the observed functional consequences of GGH overexpression and inhibition are consistent with the known biological function of GGH and provided an appropriate in vitro model to test the effect of GGH modulation on chemosensitivity of colon and breast cancer cells to 5FU and MTX.

GGH overexpression and inhibition in our model significantly modulated chemosensitivity of cancer cells to MTA whose cytotoxic effects depend on polyglutamylation (270). Our a priori hypothesis was that if the cytotoxicity of MTA depended solely on its polyglutamylation, GGH overexpression would decrease, where GGH inhibition would increase, chemosensitivity of cancer cells to MTA. However, the modulatory effect was in the expected direction only in MDA-MB-435 cells overexpressing GGH and was in the opposite direction of our a priori hypothesis in other cell constructs. These observations suggest that the GGH modulation-induced changes in MTA polyglutamylation may not be the primary determinant of chemosensitivity of HCT116 and MDA-MB-435 cells to MTA. In HCT116 cells overexpressing GGH, low total

88 folate concentrations, low content of long-chain folylpolyglutamates and decreased TS and DHFR have likely counteracted the potentially reduced cytotoxic effect of the GGH overexpression-induced decreased MTA polyglutamylation, resulting in enhanced chemosensitivity. However, in MDA-MB-435 cells overexpressing GGH, the decreased MTA polyglutamylation was sufficient to overcome the counterbalancing effect of the GGH overexpression-induced decrease in total folate and long-chain folylpolyglutamates and TS and DHFR activity, resulting in decreased chemosensitivity. In both HCT116 and MDA-MB-435 cells in which GGH is inhibited, high total folate concentrations, high content of long-chain folylpolyglutamates and increased TS and DHFR activity have likely overcome the potentially increased cytotoxic effect of the GGH inhibition-induced increased MTA polyglutamylation, resulting in decreased chemosensitivity. These data collectively suggest that the effect of GGH modulation on chemosensitivity of antifolates, whose cytotoxic effects depend on polyglutamylation, cannot be predicted solely based on the polyglutamylation of antifolates and that it may be cell-specific.

Because TMTX is not polyglutamylated and hence does not depend on polyglutamylation for its cytotoxic effects (396), chemosensitivity to TMTX should primarily depend on different concentrations and polyglutamylation of intracellular folates mediated by GGH modulation. As expected, decreased intracellular total folate concentrations, long-chain folylpolyglutamates, and TS and DHFR activity associated with GGH overexpression potentiated the cytotoxic effect of TMTX in HCT116 cells. Unexpectedly, however, GGH overexpression significantly decreased the chemosensitivity of MDA-MB-435 cells to TMTX, suggesting that factors other than changes in concentrations and polyglutamylation of intracellular folates associated with GGH overexpression are likely operative in modulating the chemosensitivity of these breast cancer cells to TMTX. As expected, increased intracellular total folate concentrations, long-chain folylpolyglutamates, and TS and DHFR activity associated with GGH inhibition decreased chemosensitivity of MDA-MB-435 cells to TMTX while the magnitude of these changes were insufficient to affect chemosensitivity of HCT116 cells to TMTX, suggesting cell-specificity.

Our a priori hypothesis concerning the effects of GGH modulation on chemosensitivity of colon and breast cancers to 5FU was based on the assumption that the GGH modulation-induced change in intracellular concentration of long-chain 5,10-methyleneTHF-polyglutamates might be the primary determinant of chemosensitivity. We hypothesized that GGH overexpression would

89 decrease the cytotoxic effect of 5FU by decreasing the relative intracellular concentration of long-chain 5,10-methyleneTHF-polyglutamates, resulting in less efficient formation and stabilization of the inhibitory 5,10-methyleneTHF-TS-FdUMP ternary complex. When 5FU was given alone, in contrast to our expectation, GGH overexpression significantly enhanced the chemosensitivity of HCT116 cells to 5FU. This suggests that decreased long-chain 5,10- methyleneTHF-polyglutamates and hence less TS inhibiting effect was not sufficient to override the potentiating effect on chemosensitivity to 5FU associated with decreased total intracellular folate concentrations and contents of long-chain polyglutamates of other intracellular folate derivatives and decreased TS and DHFR. Alternatively, decreased total intracellular folate concentrations in response to GGH overexpression might be associated with increased chemosensitivity of HCT116 cells to 5FU since 5FU functions via one or more mechanisms other than TS inhibition. Two metabolites of 5FU, 5FdUTP and 5FUTP, can be incorporated into DNA and RNA, respectively, resulting in DNA instability and interfering with RNA processing and function (227). Folate deficiency can lead to imbalances in nucleotide pools, causing DNA damage and apoptosis (397, 398). Indeed, folate deficiency has been shown to increase chemosensitivity to 5FU alone or in combination with LV in human cancer cell lines and in mouse tumor models presumably associated with the decrease in total intracellular folate concentrations (46, 399). In MDA-MB-435 cells, the competing effects of decreased intracellular concentrations of total folate and long-chain folylpolyglutamates and of less efficient and stable TS inhibition associated with decreased long-chain 5,10-methyleneTHF-polyglutamates appear to have cancelled each other out. However, when a precursor for 5,10-methyleneTHF (i.e., LV) was supplied exogenously, thereby significantly increasing the intracellular pool of 5,10- methyleneTHF, less efficient and stable TS inhibition associated with the GGH overexpression- induced decreased long-chain 5,10-methyleneTHF-polyglutamates overcame the counterbalancing effect of decreased intracellular concentrations of total folate and long-chain polyglutamates of other intracellular folate derivatives, leading to decreased chemosensitivity of both colon and breast cancer cells to 5FU.

A similar pattern of 5FU chemosensitivity was also observed for GGH inhibition. We hypothesized that GGH inhibition would increase the cytotoxic effect of 5FU by increasing relative intracellular concentrations of long-chain 5,10-methyleneTHF-polyglutamates, resulting in more efficient formation and stabilization of the 5,10-methyleneTHF-TS-FdUMP ternary complex. When 5FU was given alone, in contrast to our expectation, GGH inhibition

90 significantly decreased chemosensitivity of HCT116 cells to 5FU. This suggests that increased long-chain 5,10-methyleneTHF-polyglutamates and hence increased TS inhibiting effect was not sufficient to overcome the decreased effect on chemosensitivity to 5FU associated with increased total intracellular folate concentrations and contents of long-chain polyglutamates of other intracellular folate derivatives and increased TS and DHFR, which result in increased thymidylate and purine biosynthesis. In MDA-MB-435 cells, the competing effects of increased intracellular concentrations of total folate and long-chain folylpolyglutamates and of more efficient and stable TS inhibition associated with increased long-chain 5,10-methyleneTHF- polyglutamates appear to have cancelled each other out. However, when a precursor for 5,10- methyleneTHF (i.e., LV) was supplied exogenously, thereby significantly increasing the intracellular pool of 5,10-methyleneTHF, more efficient and stable TS inhibition associated with the GGH inhibition-induced increased long-chain 5,10-methyleneTHF-polyglutamates overcame the counterbalancing effect of increased intracellular concentrations of total folate and long-chain polyglutamates of other intracellular folate derivatives, leading to enhanced chemosensitivity of both colon and breast cancer cells to 5FU.

It appears that the GGH overexpression-induced decreased MTX-polyglutamylation was the primary determinant of the observed decreased chemosensitivity of HCT116 and MDA-MB-435 cells to MTX. The GGH overexpression-induced decreased MTX-polyglutamylation was sufficient to overcome the counterbalancing effect of decreased intracellular concentrations of total folate and long-chain folylpolyglutamates and decreased TS and DHFR activity associated with GGH overexpression. In contrast, in the GGH inhibition system, increased intracellular concentrations of total folate and long-chain folylpolyglutamates and increased TS and DHFR activity were the primary determinants of the observed decreased chemosensitivity of colon and breast cancer cells to MTX rather than the GGH inhibition-induced increased MTX- polyglutamylation, which should have enhanced MTX chemosensitivity.

Clinical studies investigating the role of GGH in modulating chemosensitivity to antifolates and 5FU have begun to emerge. Low GGH expression was reported to correlate with a good response to 5FU-based chemotherapy in patients with metastatic CRC (183). In patients with advanced pancreatic cancer treated with an oral fluoropyrimidine derivative, S-1, low GGH expression was associated with improved overall survival (400). In acute myelogenous leukemia, it was found that high GGH activity may paly a role in inherent drug resistance to MTX (171). High GGH

91 expression was shown to be associated with a higher risk of developing advanced toxicity to MTA in patients with advanced breast cancer (271). A recent study has reported that high GGH protein level is associated with poor prognosis and unfavourable clinical outcomes in patients with invasive breast cancer (401). Furthermore, several recently identified and characterized functionally significant genetic and epigenetic polymorphisms of GGH have been reported to predict response to and toxicity of antifolate-based treatment in patients with several cancers (161, 164, 402-404) and inflammatory arthritis (160, 176, 177, 405-407).

Intracellular folate and antifolate accumulation and metabolism, including polyglutamylation, are affected by multiple enzymes in the highly complex folate metabolic pathway (144). Furthermore, changes in a single enzyme such as GGH induce a cascade of adaptive and compensatory changes in other enzymes in order to maintain folate and antifolate homeostasis (1). Thus, the attribution of changes in chemosensitivity of cancer cells to antifolates and 5FU to a single enzyme such as GGH is difficult. Most likely, GGH modulation is but one of several factors that affect cancer cells’ sensitivity to antifolates and 5FU. In addition to changes in polyglutamylation of 5,10-methyleneTHF and antifolates, GGH modulation might have also induced changes in drug uptake into and accumulation in the cells; the forward polyglutamylation reaction by FPGS; intracellular concentration and polyglutamylation of other folate cofactors; intracellular distribution of the various folate species between cytoplasm, lysosome, and mitochondria; and/or transport mechanisms of folates and antifolates into the lysosomes where GGH resides and out of the cells by various ABC transporters (144). These adaptive and compensatory changes have likely played a role in modifying the effects of GGH modulation on chemosensitivity of cancer cells to antifolates and 5FU. Some of the differences between the two cell lines are likely caused by differences in this complexity.

In conclusion, as proof-of-principle, we provide functional evidence that GGH modulation affects chemosensitivity of colon and breast cancer cells to 5FU and MTX. Our data also suggest that the effects of GGH modulation on the chemosensitivity of colon and breast cancer cells to 5FU and MTX are highly complex and cannot be predicted solely based on the GGH modulation-induced changes in polyglutamylation of 5,10-methyleneTHF and MTX, respectively. GGH modulation was associated with changes in chemosensitivity of colon and breast cancer cells to 5FU in the expected direction based on the GGH modulation-induced changes in polyglutamylation of 5,10-methyleneTHF only when a large amount of a precursor

92 for 5,10-methyleneTHF (i.e., LV) was supplied exogenously. The GGH modulation-induced changes in MTX-polyglutamylation appeared to be the primary determinant of chemosensitivity of colon and breast cells to MTX in the GGH overexpression system but not in the GGH inhibition system. Notwithstanding the complexity of the GGH modulation-induced changes in folate and antifolate accumulation and metabolism, the potential role of GGH modulation in 5FU- and antifolate-based cancer chemotherapy is worthy of further exploration.

CHAPTER 5: STUDY 2 –

THE EFFECTS OF GGH MODULATION AND FOLATE ON CHEMOSENSITIVITY OF HUMAN COLON AND BREAST CANCER CELLS TO CHEMOTHERAPEUTICS IN IN VITRO AND IN VIVO MODELS

Modified from: Sung-Eun Kim, Peter D. Cole, Robert C. Cho, Anna Ly, Lisa Ishiguro, Kyoung-Jin Sohn, Ruth Croxford, Barton A. Kamen, and Young-In Kim. Effects of folate and γ-glutamyl hydrolase modulation on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and antifolates in vitro and in vivo. Submitted to British Journal of Cancer (under review)

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5.1 Abstract

Background: GGH removes terminal glutamates of polyglutamylated folates and antifolates, thereby facilitating the hydrolysis and efflux of folates and antifolates from the cell. Therefore, GGH plays an important role in regulating intracellular folates and antifolates for optimal folate- dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects. We have previously reported that GGH modulation significantly affects chemosensitivity of colon and breast cancer cells to antifolates and 5FU by changing intracellular retention of antifolates and by changing intracellular retention of a specific folate cofactor for 5FU cytotoxicity, respectively. We investigated whether GGH modulation affects chemosensitivity of cancer cells grown in physiological concentrations and nutritionally relevant forms of folate to antifolates and 5FU and whether the in vitro observations can be extended to in vivo.

Methods: Human HCT116 colon and MDA-MB-435 breast cancer cells were stably transfected with the sense GGH cDNA or GGH-targeted siRNA, respectively, to generate an in vitro model of GGH overexpression and inhibition. In vitro chemosensitivity to antifolates and 5FU was determined by the sulforhodamine B assay at 50 nM and 100 nM 5-MTHF. In vivo chemosensitivity of HCT116 cells to 5FU at different dietary supplemental levels of FA was determined in nude mice to confirm in vitro chemosensitivity data.

Results: GGH overexpression decreased chemosensitivity of MDA-MB-435 breast cancer cells to 5FU+LV and MTX at 50 nM and 100 nM 5-MTHF as expected. The GGH overexpression- induced changes in chemosensitivity of colon and breast cancer cells to 5FU+LV and MTX were in the same direction at different folate concentrations. Generally, GGH inhibition demonstrated the increased chemosensitivity of colon and breast cancer cells to 5FU+LV at both concentrations. IC50 data demonstrated that an increased concentration of 5FU and MTX was required to achieve 50% inhibition among cells grown in higher folate concentrations. Compared with xenografts expressing endogenous GGH, xenografts expressing the sense GGH were more sensitive to 5FU+LV at the 2 mg FA/kg control diet but less sensitive to 5FU+LV at the 8 mg FA/kg supplemented diet, which are consistent with the in vitro chemosensitivity of the GGH- overexpressed HCT116 cells to 5FU+LV at 50 nM 5-MTHF and 2.3 μM FA (Chapter 4), respectively.

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Conclusions: GGH modulatoin can affect chemosensitivity of cancer cells to 5FU and antifolates in physiologically relevant concentrations and form of folate, and in vitro observations were confirmed in vivo. Our data suggest that the GGH modulation-induced changes in chemosensitivity of colon and breast cancer cells to antifolates and 5FU are highly complex and the effects appeared to depend not only on the GGH modulation-induced changes in polyglutamylation of a specific folate cofactor or antifolates but also on total intracellular folate pools and polyglutamylation of other intracellular folate cofactors and on adaptive and compensatory changes in other enzymes involved in intracellular folate and antifolate accumulation and metabolism in response to GGH modulation.

5.2 Introduction

Folate plays an essential role in DNA synthesis and methylation as an important mediator of one- carbon transfer reactions (1). Folate and antifolates are retained intracellularly by the FPGS- induced polyglutamylation, and exported out of the cell after the γ-polyglutamate tails attached to folate and antifolates are hydrolyzed by GGH to produce the monoglutamate form (1, 137). GGH (EC 3.4.19.9), an acidic lysosomal enzyme, is present in serum, bile, and pancreatic secretions and also secreted by normal and tumor cells in vitro (139, 140, 147). Increased GGH expression occurs in cancers of the breast, colorectal, bladder, and urothelial, suggesting that GGH expression levels could be a useful biomarker of various cancers for diagnostic purposes (129, 148, 154, 155, 157, 158). Several SNPs identified in the GGH gene appear to have functional and pharmacological consequences (159-164). Furthermore, it has been reported that epigenetic regulation alters GGH activity and MTX polyglutamate accumulation in human leukemia cells (165).

Polyglutamylated folates are retained in cells longer and are better substrates than their monoglutamate counterparts for folate dependent enzymes involved in thymidylate and purine biosynthesis (2). Likewise, 5,10-methyleneTHF, a folate cofactor necessary for the cytotoxic effects of 5FU, with longer chain length polyglutamates is better retained intracellularly and is more efficient in the formation and stabilization of the inhibitory ternary complex (5FdUMP-TS- 5,10-methyleneTHF) compared with shorter chain polyglutamates (122). Also, polyglutamylated

96 antifolates generally have an affinity for, and thus inhibit their target folate-dependent enzymes, which are involved in the de novo nucleotide synthesis, more effectively than the monoglutamate form (2, 3). Therefore, GGH as well as FPGS may play an important role in modulating the chemosensitivity of cancer cells to 5FU and antifolates by inducing changes in polyglutamylation of a specific target intracellular folate cofactor and antifolates, respectively (144, 172-174, 389). In addition to the effect on the polyglutamylation of folate and antifolates, GGH and FPGS induce changes in intracellular folate concentrations (4, 5, 17, 295). Intracellular folate status is a critical determinant of the chemosensitivity of cancer cells to chemotherapeutic agents, which are designed to interrupt intracellular folate metabolism and DNA synthesis (3, 5, 46, 136). Collectively, GGH and FPGS are important enzymes for the maintenance of intracellular homeostasis of folates and antifolates for optimal folate-dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects, respectively.

There is accumulating evidence which demonstrates that FPGS modulation affects the sensitivity of tumor cells to chemotherapeutic agents. Generally, high FPGS expression/activity is shown to increase polyglutamylation and chemosensitivity of antifolates and 5FU (4, 15-17), whereas low FPGS expression/activity is associated with drug resistance (4, 17-26). However, it is largely unknown whether GGH modulation can affect chemosensitivity of cancer cells to chemotherapeutic agents. Increased GGH activity was identified as the mechanism of antifolate resistance in rat hepatoma cells (147, 172, 182, 408) and human sarcoma and leukemia cells (173, 174). However, GGH overexpression alone was not sufficient to confer resistance to MTX in human breast and sarcoma cells (188). Pharmacologic GGH inhibition has been shown to increase the sensitivity to MTX in human sarcoma (138) and breast cancer cells (371, 372). The siRNA-induced decrease in GGH expression exhibits enhanced 5FU efficacy in human colon cancer cells (5).

A growing body of evidence from clinical studies also suggests the role of GGH in modulating chemosensitivity to antifolates and 5FU. High GGH activity may be in part responsible for the inherent drug resistance to MTX exhibited in acute myelogenous leukemia (171). High GGH expression was associated with a higher risk of developing advanced toxicity to MTA in patients with advanced breast cancer (271). Recently, a high GGH protein level was shown to be associated with a poor prognosis and unfavourable clinical outcomes in patients with invasive breast cancer (401). In contrast, low GGH expression was correlated with a positive response to

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5FU-based chemotherapy in patients with metastatic CRC (183). Low GGH expression and high 5,10-methyleneTHF levels were found in CIMP+ CRC, which was associated with an improved response to 5FU compared with CIMP- CRC (186, 187). Low GGH expression was also associated with improved overall survival in patients with advanced pancreatic cancer treated with an oral fluoropyrimidine derivative, S-1 (400). Furthermore, there have been several investigations of the effects of several polymorphisms of GGH on the efficacy and toxicity of chemotherapy. Polymorphisms in the GGH gene were reported to be associated with the efficacy and toxicity of MTX treatment in ALL patients (164, 402), the overall survival of MTA in patients with lung cancer (404), and the response to platinum-based chemotherapy in patients with cervical cancer (161, 403). Additionally, genetic variants of GGH have been shown to predict response to and toxicity of MTX-based treatment in patients with inflammatory arthritis (160, 176, 177, 405-407).

We have previously reported that the FPGS modulation-induced changes in the chemosensitivity of cancer cells to 5FU amd MTX are further influenced by exogenous folate concentrations (4). In addition, as presented in Chapter 4, we have developed an in vitro model of GGH overexpression and inhibition in human HCT116 colon and MDA-MB-435 breast cancer cells with predictable functional consequences, and have also demonstrated that GGH modulation significantly affects chemosensitivity to antifolates and 5FU in cancer cells cultured in standard FA media. Therefore, in the present study, we investigated whether GGH modulation would affect the chemosensitivity of colon and breast cancer cells to antifolates and 5FU at nutritionally relevant and physiological levels of 5-MTHF, which is the physiological form of folate in the circulation. The levels of 5-MTHF chosen for this study reflect human serum folate levels in the postfortification era, both with and without FA supplementation (6-8) considering that ~50% of cancer patients and long-term cancer survivors use FA-containing supplements (9, 10). To confirm the results of the in vitro chemosensitivity test, we also examined whether GGH modulation and different dietary supplemental levels of FA would affect the in vivo chemosensitivity of HCT116 cells to 5FU in nude mice.

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5.3 Materials and Methods

5.3.1 Cell Lines and Culture

HCT116 and MDA-MB-435 cells were grown in complete RPMI-1640 medium without FA (Invitrogen) supplemented with 10% dialyzed FBS (Invitrogen), 500 µg/mL Geneticin® (G418; Invitrogen), 50 units/mL penicillin with 50 µg/mL streptomycin (Invitrogen), 0.25 µg/mL fungizone amphotericin B (Invitrogen), and either 50 nM or 100 nM 5-MTHF (Schircks Laboratories, Jona, Switzerland) to represent nutritionally relevant and physiological folate levels. Dialyzed FBS was utilized to minimize the contribution of FA contained in the FBS since dialyzed serum contained only 0.6 nM FA which is physiologically sufficient to sustain growth of mammalian cell lines in culture, including HCT116 and MDA-MB-435 cells (409). Cultures were maintained at 37°C in 5% CO2.

Medium supplemented with 50 nM 5-MTHF represented normal folate concentrations in the circulation, whereas those supplemented with 100 nM 5-MTHF represented high folate concentration after supplementary folate intake (8). Serum folate concentrations of young Canadian women after FA fortification were 33.0 ± 7.9 nM (410). In an analysis from NHANES 1999-2010 data, postfortification serum folate concentrations of the United States population were 41.0 ± 0.3 nM and were ~30 nM higher in supplement users than in nonusers (8). Likewise, the nutritionally relevant and physiological folate levels chosen for this study reflect human serum folate levels in the postfortification era, both with and without FA supplementation (6-8) considering that ~50% of cancer patients and long-term cancer survivors use FA-containing supplements (9, 10).

5.3.2 Construction and Transfection of GGH Expression Vectors

Construction and transfection of GGH expression vectors was performed as described in Section 4.3.2. Cells with similar passage numbers were selected for transfection with different GGH expression vectors and for subsequent functional analyses. Cells expressing the sense GGH or GGH-targeted siRNA and corresponding controls with similar passage numbers were used for chemosensitivity analysis.

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5.3.3 Intracellular Total and Long-Chain Folate Concentrations Analysis

Intracellular folate levels of HCT116 and MDA-MB-435 cell lines under 50 nM and 100 nM of 5-MTHF were determined as described in Section 4.3.5.

5.3.4 Doubling Time Calculation

Doubling time of HCT116 and MDA-MB-435 cell lines under 50 nM and 100 nM of 5-MTHF were determined as described in Section 4.3.6.

5.3.5 In Vitro Chemosensitivity Assay

The same set of experiments described in Section 4.3.7 were performed at more nutritionally relevant and physiological levels of folate using 5-MTHF, which is the physiological form of folate in the circulation.

5.3.6 In Vivo Chemosensitivity Study

6-8 weeks, BALB/c nu/nu mice (n=20) 2 mg/kg FA diet

1 week

Cell injection (S.C): HCT116 Control-S & Sense

2-3 weeks

Drug treatment

Control: Supplemented: 2 mg/kg FA (n=10) 8 mg/kg FA (n=10)

 Tumor size  Body weight 0.9 % NaCl 5FU+LV 0.9% NaCl 5FU+LV  Food intake (n=5) (n=5) (n=5) (n=5)

3-4 weeks

Necropsy  Plasma: folate/homocysteine concentrations  Tumor: foate concentration, TS enzyme activity, immunohistochemistry (Ki-67, TUNEL) Figure 5. 1 Experimental design for in vivo chemosensitivity study

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Twenty 6-week-old male BALB/c nu/nu mice (Charles River, Wilmington, MA) received subcutaneous injections in each flanks of HCT116 colon cancer cells expressing endogenous GGH and the sense GGH (1 × 106 cells/site/mouse) in 100 μL of serum-free RPMI-1640 medium after acclimatization to 2 mg FA/kg diet for one week. When xenografts reached a volume of 80- 200 mm3, mice were randomly assigned to one of four groups (n = 5) and treatment was initiated: (i) treated with 0.9% NaCl fed 2 mg FA/kg diet, (ii) treated with 5FU+LV fed 2 mg FA/kg diet, (iii) treated with 0.9% NaCl fed 8 mg FA/kg diet, and (iv) treated with 5FU+LV fed 8 mg FA/kg diet. 5FU (Sigma, 20 mg/kg/day) dissolved in 100 μL of 0.9% NaCl was administered via intraperitoneal injection for five consecutive days for one week. Leucovorin (Sigma, 1 mg/kg/day) dissolved in 100 μL of 0.9% NaCl was administered 1 hour before 5FU administration via intraperitoneal injection. Control mice for each group received intraperitoneal injections of 0.9% NaCl (Figure 5.1).

The concentrations of 5FU and LV were determined based on the Mayo Clinic regimen (20 mg/m2 LV [0.7 mg/kg] + 425 mg/m2 5FU [14 mg/kg]) since this is a well-recognized and commonly used schedule with proven efficacy (411, 412). In addition, these concentrations of 5FU and LV are used for the comparator arm required by the U.S. Food and Drug Administration in registration trials for new CRC therapies (412). Amino acid-defined diets containing different levels of FA constitute a standard method of providing supplemental dietary folate in rodents (413). The control diet, containing 2 mg FA/kg, is generally accepted as the basal dietary requirement (BDR) for rodents (414). This diet contains approximately 4000 kcal/kg diet, which translates to 0.5-1 mg of FA in 2000 kcal. This level of FA expressed relative to caloric content very closely approximates the RDA of 0.4 mg DFE in humans consuming a daily average of 2000 kcal (415) and thus, was selected to parallel the RDA for folate in humans. The supplemented diet was prepared to contain 8 mg FA/kg diet (4× BDR). This level represents a daily intake of 1.6 mg/day in humans, and approximates a total folate intake from fortified foods and regular supplement use of 1.0 mg FA (9, 416). The detailed composition of the diets has been published previously (417) and presented in Appendix 2. Sterilized diets and water were provided ad libitum. Food intake and body weights were recorded twice a week.

Tumor size was measured with a digital caliper twice a week. The estimated tumor volume (V) was calculated based on the formula W2 × L × 0.5, where W represents the largest tumor diameter in centimeters and L represents the next largest tumor diameter (418). The individual

101 relative tumor volume (RTV) was calculated as Vx/V1, where Vx is the volume in cubic millimeters at a given time and V1 is the volume at the start of treatment. Results are expressed as the mean daily percent change in tumor volume for each group of mice. At necropsy, blood was drawn for plasma folate and Hcy concentration analyses. The protocol was approved by the Animal Care Ethics Committee of the University of Toronto.

5.3.7 Plasma Folate and Homocysteine Concentration Analyses

Plasma folate concentrations were determined by a standard microbiological microtitre plate assay (419, 420). Total plasma Hcy concentrations were determined using the Axis Homocysteine Enzyme Immunoassay kit (Abbott Laboratories) (421).

5.3.8 Xenograft Epithelial Proliferation and Apoptosis

Xenograft epithelial proliferation was determined by staining histological sections with monoclonal antibodies against Ki-67, a nuclear protein expressed in proliferating cells (419, 421). The Ki-67 index was expressed as a percent of positively stained nuclei in relation to the total number of cells considered. Xenograft epithelial apoptosis was determined by terminal uridine deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay using the ApopTag Peroxidase In Situ Apoptosis Detection Kit S7100 (Millipore, Billerica, MA) (419, 421). The apoptopic index was based on the percentage of positively stained cells out of the total number of cells in each slide.

5.3.9 Statistical Analysis

As a preliminary test, differences between intracellular folate concentrations under 50 nM and 100 nM 5-MTHF were analyzed by one-way ANOVA with Tukey’s test for pairwise comparisons. Pearson’s correlation analysis was performed to measure a correlation between continuous variables of intracellular folate concentrations. For continuous variables, comparisons between cells expressing the sense GGH (Sense) and controls (Control-S) and between cells transfected with the GGH-targeted siRNA (siRNA) and controls (Control-si) were determined

102 using the Student’s t-test. For the in vitro chemosensitivity analyses, plots of percentage of survival vs. dose demonstrated S-shaped curves, and therefore the logit transformation [logit (p) = ln (p/[1-p])] was used. Ordinary least squares regression was used to model the effect of log (dose) of chemotherapy and cell type on the logit-transformed proportion of cells that survived at each dose. The interaction between cell type and log (dose) was included in the model to test the hypothesis that the cell types were differentially sensitive to chemotherapy. IC50 doses and their 95% confidence intervals were calculated on the log scale from the regression results as described (395), and then back-transformed to the original scale for reporting.

For the in vivo chemosensitivity analyses, the dependent variable was tumor volume, which was log-transformed due to its skewed distribution. A generalized linear regression was used to account for the fact that each mouse had been injected with two different types of cells, and in addition, each mouse was measured repeatedly over time. The resulting slopes estimate the change in log (volume) per day and, when back-transformed, estimate the growth rate per day. The effect of diet, treatment, and/or cell types on rate of growth was included due to a significant interaction between the effect of time and the effect of one or more of the other three factors. Differences among groups in food intake and body weight were analyzed using generalized linear models to take into account the repeated measures aspects of the data with the autoregressive correlation structure. Plasma folate and Hcy concentrations were analyzed by two-way ANOVA with diet and treatment as the independent variables. Xenograft epithelial proliferation and apoptosis were analyzed by the repeated measures analysis with the inclusion of an interaction term and diet, treatment, and xenograft type as the independent variables. For all analyses, results were considered statistically significant if two-tailed P-values were < 0.05. Analyses were performed using SPSS Statistics 17.0 (IBM SPSS, Chicago, IL) and SAS version 9.1 (SAS Institute, Cary, NC) and graphs were prepared in Microsoft Excel.

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5.4 Results

5.4.1 Concentrations of Intracellular Folate and Long-Chain Length Polyglutamates

We measured the concentrations of intracellular total folate and long-chain polyglutamate as preliminary data to confirm that intracellular folate concentrations would represent different concentrations of media over time for the in vitro chemosensitivity test. Overall, intracellular total and long-chain polyglutamate concentrations in Day 6 were lower compared with Day 3, and those at 50 nM 5-MTHF were lower compared with 100 nM 5-MTHF in both cell lines; GGH overexpression demonstrated lower, where GGH inhibition was associated with higher, concentrations of total and long-chain polyglutamates compared with corresponding controls (Tables 5.1 and 5.2). Collectively, these obervations confirm that intracellular folate concentrations reflected nutritionally relevant and physiological folate levels (50 nM and 100 nM 5-MTHF) over time and provided an appropriate condition for the in vitro chemosensitivity test.

5.4.2 Doubling Time

HCT116 and MDA-MB-435 cells expressing the sense GGH exhibited a significantly increased doubling time compared with corresponding controls at 50 nM and 100 nM 5-MTHF (HCT116, 39.2 ± 0.2 h [Sense] vs. 37.1 ± 0.3 h [Control-S] at 50 nM, 39.6 ± 0.2 h [Sense] vs. 33.8 ± 0.1 h [Control-S] at 100 nM, P < 0.001; MDA-MB-435, 48.8 ± 0.3 h [Sense] vs. 43.9 ± 0.4 h [Control- S] at 50 nM, 47.0 ± 0.3 h [Sense] vs. 41.7 ± 0.3 h [Control-S] at 100 nM, P < 0.001), indicating a slower growth rate. As expected, HCT116 cells transfected with the GGH-targeted siRNA had a decreased doubling time compared with corresponding controls at both concentrations of media (37.6 ± 0.2 h [siRNA] vs. 40.9 ± 0.2 h [Control-si] at 50 nM, 37.5 ± 0.1 h [siRNA] vs. 39.3 ± 0.2 h [Control-si] at 100 nM, P < 0.001). Interestingly, MDA-MB-435 cells in which GGH was inhibited showed an increased doubling time at 100 nM (43.1 ± 0.3 h [siRNA] vs. 42.3 ± 0.5 h [Control-si], P < 0.001) and decreased doubling time at 50 nM (39.7 ± 0.4 h [siRNA] vs. 43.9 ± 0.5 h [Control-si], P < 0.001) compared with corresponding controls.

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Table 5. 1 Intracellular folate concentrations of the GGH-overexpressed HCT116 and MDA-MB-435 cells at different concentrations of media

Control-S Sense

Day 3 Day 6 Day 3 Day 6

50 nM 100 nM 50 nM 100 nM 50 nM 100 nM 50 nM 100 nM

5-MTHF 5-MTHF 5-MTHF 5-MTHF 5-MTHF 5-MTHF 5-MTHF 5-MTHF

HCT116 Total folate 0.839 ± 0.936 ± 0.325 ± 0.373 ± 0.905 ± 0.945 ± 0.320 ± 0.422 ± (ng/5 × 106 cells) 0.052a 0.053ab 0.037c 0.008c 0.017a 0.028ab 0.021c 0.013c Long-chain polyglutamates 0.191 ± 0.406 ± 0.105 ± 0.246 ± 0.070 ± 0.179 ± 0.035 ± 0.099 ± (ng/5 × 106 cells) 0.052ab 0.025c 0.035bd 0.001ae 0.010bd 0.033ab 0.018d 0.021bd

MDA-MB-435 Total folate 0.576 ± 0.641 ± 0.335 ± 0.479 ± 0.243 ± 0.253 ± 0.225 ± 0.396 ± (ng/5 × 106 cells) 0.008a 0.072a 0.042bcd 0.069ab 0.017cd 0.007cd 0.012d 0.051bc Long-chain polyglutamates 0.211 ± 0.337 ± 0.142 ± 0.226 ± 0.099 ± 0.148 ± 0.077 ± 0.130 ± (ng/5 × 106 cells) 0.043ab 0.047ac 0.012b 0.008ab 0.010b 0.004b 0.011b 0.015b

Results are expressed as mean ± SD. Means in row with different letters significantly differ at P < 0.05.

HCT116: Total folate*Day: Conotrol-S, r = -0.98, P < 0.01; Sense, r = -0.93, P < 0.01 Polyglutamates*Day: Control-S, r= -0.84, P = 0.002; Sense, r = -0.85, P = 0.002 Polyglutamates*Media: Control-S, r= -0.93, P < 0.01; Sense, r = -0.91, P < 0.01

MDA-MB-435: Total folate*Day: Conotrol-S, r = -0.90, P < 0.01; Sense, r = -0.76, P = 0.012 Total folate*Media: Conotrol-S, r = -0.84, P = 0.002; Sense, r = -0.85, P = 0.002 Polyglutamates*Day: Control-S, r= -0.80, P = 0.005; Sense, r = -0.79, P = 0.007 Polyglutamates*Media: Control-S, r= -0.84, P = 0.003; Sense, r = -0.96, P < 0.01

105 Table 5. 2 Intracellular folate concentrations of the GGH-inhibited HCT116 and MDA-MB-435 cells at different concentrations of media

Control-si siRNA

Day 3 Day 6 Day 3 Day 6

50 nM 100 nM 50 nM 100 nM 50 nM 100 nM 50 nM 100 nM

5-MTHF 5-MTHF 5-MTHF 5-MTHF 5-MTHF 5-MTHF 5-MTHF 5-MTHF

HCT116 Total folate 0.339 ± 0.563 ± 0.243 ± 0.325 ± 0.693 ± 0.725 ± 0.491 ± 0.553 ± (ng/5 × 106 cells) 0.004a 0.013b 0.020a 0.013a 0.031c 0.024c 0.030b 0.018b Long-chain polyglutamates 0.168 ± 0.226 ± 0.092 ± 0.153 ± 0.231 ± 0.448 ± 0.166 ± 0.318 ± (ng/5 × 106 cells) 0.010ab 0.008ac 0.002d 0.001b 0.003c 0.008e 0.010b 0.010f

MDA-MB-435 Total folate 0.487 ± 0.499 ± 0.363 ± 0.319 ± 1.104 ± 1.218 ± 0.585 ± 0.635 ± (ng/5 × 106 cells) 0.013abc 0.025ab 0.025bc 0.004c 0.088d 0.081d 0.046a 0.016a Long-chain polyglutamates 0.214 ± 0.340 ± 0.154 ± 0.255 ± 0.565 ± 1.058 ± 0.337 ± 0.519 ± (ng/5 × 106 cells) 0.022a 0.008abc 0.016a 0.003ac 0.107b 0.071d 0.012abc 0.020bc

Results are expressed as mean ± SD. Means in row with different letters significantly differ at P < 0.05.

HCT116: Total folate*Day: Conotrol-si, r = -0.89, P = 0.001; siRNA, r = -0.93, P < 0.01 Total folate*Media: Conotrol-si, r = -0.87, P = 0.001; siRNA, r = -0.81, P = 0.005 Polyglutamates*Day: Control-si, r= -0.92, P < 0.01; siRNA, r = -0.81, P = 0.005 Polyglutamates*Media: Control-si, r= -0.86, P = 0.001; siRNA, r = -0.95, P < 0.01

MDA-MB-435: Total folate*Day: Conotrol-si, r = -0.91, P < 0.01; siRNA, r = -0.95, P < 0.01 Total folate*Media: Conotrol-si, r = -0.77, P = 0.010; siRNA, r = -0.78, P = 0.007 Polyglutamates*Day: Control-si, r= -0.86, P = 0.002; siRNA, r = -0.89, P = 0.001 Polyglutamates*Media: Control-si, r= -0.93, P < 0.01; siRNA, r = -0.86, P = 0.001

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5.4.3 In Vitro Chemosensitivity

As proof-of-principle, the effect of GGH overexpression and inhibition on chemosensitivity of HCT116 and MDA-MB-435 cells to MTA (positive control) and TMTX (negative control) was determined at different concentrations of media.

MTA Chemosensitivity: MTA is a novel antimetabolite that inhibits multiple enzymes involved in thymidylate and purine biosynthesis whose cytotoxic effects depend on polyglutamylation (270). Our a priori hypothesis was that GGH overexpression would decrease, where GGH inhibition would increase, chemosensitivity of cancer cells to MTA due to the effect of polyglutamylation on its cytotoxicity. In contrast to our expectation, HCT116 cells that overexpressed GGH were more sensitive to MTA compared with controls at 50 nM and 100 nM 5-MTHF (P < 0.05; Figures 5.2A and 2B). As expected, chemosensitivity of HCT116 cells transfected with the GGH-targeted siRNA to MTA was significantly increased compared with controls at 50 nM 5-MTHF (P < 0.05; Figure 5.2C), whereas in contrast to our expectation, HCT116 cells in which GGH was inhibited were less sensitive to MTA compared with controls at 100 nM 5-MTHF (P < 0.05; Figure 5.2D). At both concentrations, MDA-MB-435 cells that overexpressed GGH were less sensitive to MTA compared with controls as expected (P < 0.05; Figures 5.2E and 2F), whereas MDA-MB-435 cells in which GGH was inhibited were also less sensitive to MTA compared with controls (P < 0.05; Figures 5.2G and 2H).

TMTX Chemosensitivity: TMTX is a nonclassic antifolate that directly inhibits DHFR whose cytotoxic effects do not depend on polyglutamylation as it is not polyglutamylated (396). We hypothesized that chemosensitivity of cancer cells to TMTX would largely depend on the GGH modulation-induced changes in concentrations and polyglutamylation of intracellular folates. In both cell lines, GGH overexpression showed decreased chemosensitivity to TMTX compared with controls at 50 nM and 100 nM 5-MTHF (P < 0.05; Figures 5.3A, 3B, 3E, and 3F). Both HCT116 and MDA-MB-435 cells transfected with the GGH-targeted siRNA were also less sensitive to TMTX compared with controls at 50 nM and 100 nM 5-MTHF as expected (P < 0.05; Figures 5.3C, 3D, 3G, and 3H).

MTX Chemosensitivity: We predicted that the GGH overexpression-induced decreased MTX- polyglutamylation would decrease, whereas the GGH inhibition-induced increased MTX-

107 polyglutamylation would enhance, the cytotoxic effect of MTX. Interestingly, GGH overexpression increased chemosensitivity of HCT116 cells to MTX compared with controls at 50 nM 5-MTHF (P < 0.05; Figure 5.4A; Table 5.3), while this effect was not observed at 100 nM 5-MTHF (Figure 5.4B; Table 5.3). Chemosensitivity of HCT116 cells transfected with the GGH-targeted siRNA to MTX was significantly decreased compared with controls at both concentrations (P < 0.05; Figures 5.4C and 4D; Table 5.3). Consistent with our hypothesis, chemosensitivity of MDA-MB-435 cells expressing the sense GGH to MTX was significantly decreased compared with controls at 50 nM and 100 nM 5-MTHF (P < 0.05; Figures 5.4E and 4F; Table 5.4). Unexpectedly, in contrast to our hypothesis, chemosensitivity of MDA-MB-435 cells transfected with the GGH-targeted siRNA to MTX was also significantly decreased compared with corresponding controls at both concentrations (P < 0.05; Figures 5.4G and 4H; Table 5.4).

5FU Chemosensitivity: Our a priori hypothesis was that GGH overexpression would decrease the cytotoxic effect of 5FU by decreasing relative intracellular concentration of long-chain 5,10- methyleneTHF-polyglutamates, resulting in the less efficient formation and stabilization of the inhibitory 5,10-methyleneTHF-TS-FdUMP ternary complex. In contrast, we hypothesized that GGH inhibition would increase the cytotoxic effect of 5FU by increasing relative intracellular concentrations of long-chain 5,10-methyleneTHF-polyglutamates, resulting in the more efficient formation and stabilization of the 5,10-methyleneTHF-TS-FdUMP ternary complex. Similar to the results of chemosensitivity of HCT116 cells expressing the sense GGH to MTX, GGH overexpression significantly increased chemosensitivity of HCT116 cells to 5FU+LV compared with controls at 50 nM 5-MTHF (P < 0.05; Figure 5.5A; Table 5.3), while it had no significant effect of chemosensitivity of HCT116 cells to 5FU+LV at 100 nM 5-MTHF (Figure 5.5B; Table 5.3). Consistent with our hypothesis, chemosensitivity of HCT116 cells transfected with the GGH-targeted siRNA to 5FU+LV was significantly increased compared with controls at 50 nM and 100 nM 5-MTHF (P < 0.05; Figures 5.5C and 5D; Table 5.3). Consistent with our hypothesis and with MTX data, MDA-MB-435 cells expressing the sense GGH were less sensitive to 5FU+LV compared with controls at both concentrations (P < 0.05; Figures 5.5E and 5F; Table 5.4). GGH inhibition had no significant effect of MDA-MB-435 cells to 5FU+LV at 50 nM 5-MTHF (Figure 5.5G), while chemosensitivity of MDA-MB-435 cells in which GGH

108 was inhibited to 5FU+LV was increased compared with controls at 100 nM 5-MTHF as expected (P < 0.05; Figure 5.5H; Table 5.4).

Interestingly, in contrast to our hypothesis, GGH overexpression increased chemosensitivity of HCT116 cells to 5FU alone compared with controls at 50 nM and 100 nM 5-MTHF (P < 0.05; Figures 5.6A and 6B; Table 5.3), whereas GGH inhibition had no significant effect of chemosensitivity of HCT116 cells to 5FU alone at 50 nM 5-MTHF (Figure 5.6C; Table 5.3). In contrast to our hypothesis and to 5FU+LV data, GGH inhibition significantly decreased chemosensitivity of HCT116 cells to 5FU alone compared with controls at 100 nM 5-MTHF (P < 0.05; Figure 5.6D; Table 5.3). In MDA-MB-435 cells, GGH overexpression decreased chemosensitivity to 5FU alone compared with controls at 50 nM 5-MTHF as expected (P < 0.05; Figure 5.6E; Table 5.4), whereas it had no significant effect of chemosensitivity to 5FU alone at 100 nM 5-MTHF (Figure 5.6F; Table 5.4). Chemosensitivity of MDA-MB-435 cells transfected with the GGH-targeted siRNA to 5FU alone was increased compared with controls at 100 nM 5- MTHF consistent with our expectation (P < 0.05; Figure 5.6H; Table 5.4), while this effect was not observed at 50 nM 5-MTHF (Figure 5.6G; Table 5.4).

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Figure 5. 2 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to pemetrexed (MTA; positive control) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH). Cells were grown in medium containing 50 nM (A, C, E, G) or 100 nM 5-MTHF (B, D, F, H). Points, means; bars, SD. *, P < 0.05 statistically significant compared with corresponding control cells.

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Figure 5. 3 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to trimetrexate (TMTX; negative control) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH). Cells were grown in medium containing 50 nM (A, C, E, G) or 100 nM 5-MTHF (B, D, F, H). Points, means; bars, SD. *, P < 0.05 statistically significant compared with corresponding control cells.

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Figure 5. 4 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to methotrexate (MTX) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH). Cells were grown in medium containing 50 nM (A, C, E, G) or 100 nM 5-MTHF (B, D, F, H). Points, means; bars, SD. *, P < 0.05 statistically significant compared with corresponding control cells.

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Figure 5. 5 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to 5-fluorouracil (5FU) plus leucovorin (LV) in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH). Cells were grown in medium containing 50 nM (A, C, E, G) or 100 nM 5-MTHF (B, D, F, H). Points, means; bars, SD. *, P < 0.05 statistically significant compared with corresponding control cells.

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Figure 5. 6 In vitro chemosensitivity of HCT116 colon (A-D) and MDA-MB-435 breast (E-H) cancer cells transfected with either the sense GGH (Sense) or GGH-targeted siRNA (siRNA) to 5-fluorouracil (5FU) alone in comparison to cells transfected with the vector alone (Control-S or Control-si; endogenous GGH). Cells were grown in medium containing 50 nM (A, C, E, G) or 100 nM 5-MTHF (B, D, F, H). Points, means; bars, SD. *, P < 0.05 statistically significant compared with corresponding control cells.

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Table 5. 3 IC50 values of 5FU and MTX in HCT116 colon cancer cells transfected with the sense GGH and GGH-targeted siRNA in comparison with corresponding control cells expressing endogenous GGH at different concentrations of media

50 nM 5-MTHF 100 nM 5-MTHF

IC50 (95% CI) IC50 (95% CI)

5FU (µM) + 5 µM LV

Sense 4.60* (4.37, 4.90) 7.60* (7.17, 8.07)

Control-S 7.78* (7.04, 8.91) 7.82* (7.21, 9.40)

siRNA 5.22* (5.02, 5.46) 5.20* (4.85, 5.46)

Control-si 9.09* (8.57, 9.51) 10.63* (10.19, 11.15)

5FU (µM)

Sense 5.81* (5.20, 6.26) 4.53* (3.81, 5.03)

Control-S 7.43* (7.08, 7.79) 7.78* (7.57, 7.93)

siRNA 7.91* (7.35, 8.58) 9.62* (8.91, 10.23)

Control-si 6.84* (6.16, 7.51) 7.08* (6.87, 7.25)

Methotrexate (nM)

Sense 11.75* (11.44, 12.15) 12.78* (12.21, 13.59)

Control-S 12.39* (12.04, 12.86) 13.03* (12.58, 13.62)

siRNA 12.80* (12.18, 13.35) 13.64* (12.91, 14.63)

Control-si 12.40* (11.92, 13.07) 13.14* (12.63, 13.80)

*, P < 0.05 compared with corresponding controls.

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Table 5. 4 IC50 values of 5FU and MTX in MDA-MB-435 breast cancer cells transfected with the sense GGH and GGH-targeted siRNA in comparison with corresponding control cells expressing endogenous GGH at different concentrations of media

50 nM 5-MTHF 100 nM 5-MTHF

IC50 (95% CI) IC50 (95% CI)

5FU (µM) + 5 µM LV

Sense 8.84* (7.90, 11.21) 11.00* (10.85, 11.23)

Control-S 6.47* (5.95, 6.70) 6.11* (5.38, 6.45)

siRNA 8.26* (7.77, 8.91) 6.43* (6.29, 6.58)

Control-si 8.87* (8.17, 10.02) 9.64* (9.40, 9.93)

5FU (µM)

Sense 3.56* (3.31, 3.76) 2.33* (1.85, 2.67)

Control-S 2.39* (2.34, 2.43) 2.65* (2.33, 2.91)

siRNA 5.72* (5.53, 6.08) 3.57* (3.34, 3.53)

Control-si 5.18* (4.93, 5.50) 6.08* (5.81, 6.57)

Methotrexate (nM)

Sense 40.34* (39.80, 40.92) 44.75* (44.06, 45.50)

Control-S 38.45* (37.75, 39.13) 39.65* (38.91, 40.30)

siRNA 36.53* (35.64, 37.69) 44.96* (43.56, 46.79)

Control-si 27.02* (26.21, 27.74) 34.46* (33.78, 35.22)

*, P < 0.05 compared with corresponding controls.

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5.4.4 In Vivo Chemosensitivity

We next investigated whether GGH modulation and different dietary supplemental levels of FA would affect the in vivo chemosensitivity of HCT116 cells to 5FU+LV in nude mice in order to confirm our in vitro chemosensitivity data. LV was added to simulate the standard 5FU-based chemotherapy used in the treatment of CRC and thus, in vivo chemosensitivity to 5FU alone was not tested. We chose HCT116 GGH overexpression system for in vivo study since the magnitude of the cell survival difference was more pronounced in this system compared with other systems.

Food Intake and Body Weight

The mean food intake per day between two different FA diet groups did not differ significantly, and 5FU+LV treatment did not affect food intake (Figure 5.7A). The effect of both diet and 5FU+LV treatment on body weight remained significant and varied over time (P-interaction for diet × day = 0.03; P-interaction for treatment × day = 0.01). Overall, the mice treated with saline lost weight over time whereas the mice treated with 5FU+LV lost weight initially and regained it back (Figure 5.7B).

A B

Diet*Treatment: NS (P = 0.67) Treatment: NS (P = 0.39) Diet*Treatment: NS (P = 0.17) Day*Diet: NS (P = 0.54) Diet: NS (P = 0.37) Day*Diet: P = 0.03 Day*Treatment: NS (P = 0.07) Day: NS (P = 0.32) Day*Treatment: P = 0.01

Figure 5. 7 Food intake (A) and body weight (B) of mice from four different diet and treatment groups. Values are mean ± standard error of the mean (SEM).

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Plasma Folate and Homocysteine Concentrations

Plasma folate concentrations of the FA supplemented mice were significantly higher than that of the mice on the control diet (P < 0.001; Figure 5.8A) while plasma Hcy concentrations did not differ between two diets groups (Figure 5.8B). This observation suggests that plasma folate status of mice reflected their exposure to dietary FA during the study.

A B

*

Treatment: NS (P = 0.50) Treatment: NS (P = 0.99) Diet: NS (P = 0.92)

Figure 5. 8 Plasma folate (A) and homocysteine (B) concentrations of mice at different folic acid diets. *, P < 0.001 compared with FA supplemented diet. Values are mean ± SEM.

Growth Rate of Xenograft

The xenografts on the 2 mg FA control diet grew faster than those on the 8 mg FA supplemented diet (average growth rate = 10.3%/day [95% CI = 9.1 to 11.5%/day] vs. 7.8%/day [95% CI = 7.0 to 8.5%/day]; P < 0.001; P-interaction for diet × day = 0.01; Figure 5.9). For each diet, the difference in the growth rates of xenografts between cell types (Sense and Control-S) depended on treatment, and the effect of treatment depended on the cell types (P-interaction for treatment × cell types = 0.04 at 2 mg FA diet and < 0.0001 at 8 mg FA diet).

When the growth rates of Sense and Control-S xenografts in mice injected with 0.9% NaCl were compared, Sense xenografts grew slower than Control-S xenografts at both FA diet groups (2 mg FA diet, RTV = 0.78 [95% CI = 0.65 to 0.94], P = 0.011; 8 mg FA diet, RTV = 0.55 [95% CI =

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0.52 to 0.58], P < 0.0001); Figures 5.9A and 9C). These data were consistent with the in vitro observations demonstrating HCT116 cells expressing the sense GGH showed decreased intracellular total and long-chain polyglutamylated folate concentrations, lower TS and DHFR activity, and increased doubling time compared with corresponding controls (Figures 4.3A, 5A, 6A, and 8A). The growth rate of HCT116 xenografts expressing the sense GGH was inhibited more effectively by 5FU+LV at the 2 mg FA control diet compared with those expressing endogenous GGH (38% inhibition [95% CI = 32% to 43% inhibition], P < 0.0001; Figure 5.9B), whereas, at the 8 mg FA supplemented diet, the growth rate of xenografts expressing endogenous GGH was inhibited more effectively by 5FU+ LV compared with those expressing the sense GGH (36% inhibition [95% CI = 21% to 48% inhibition], P < 0.0001; Figure 5.9D). The in vivo chemosensitivity of these results support the in vitro observations that HCT116 cells that overexpressed GGH have slower growth rates compared with cells expressing endogenous GGH at 50 nM and 100 nM 5-MTHF. The increased in vivo chemosensitivity at the 2 mg FA control diet was consistent with the in vitro chemosensitivity at 50 nM 5-MTHF as expected (Figure 5.5A; Table 5.3). Interestingly, the observed decreased in vivo chemosensitivity at the 8 mg FA supplemented diet was in agreement with the in vitro chemosensitivity at 2.3 μM FA (Figure 4.9C; Table 4.2), not at 100 nM 5-MTHF. We observed that GGH overexpression had no significant effect of chemosensitivity of HCT116 cells to 5FU+LV at 100 nM 5-MTHF (Figure 5.5B; Table 5.3).

A B

Figure 5. 9 Relative tumor volume of HCT116 colon cancer xenografts expressing either GGH overexpression (Sense) or endogenous GGH (Control-S) treated with saline or 5FU+LV at 2 mg FA/kg control (A) and 8 mg FA/kg supplemented (B) diets. Values are mean ± SEM.

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Xenograft Epithelial Proliferation and Apoptosis

We expected that xenografts demonstrating a faster growth rate would be associated with higher proliferation and lower apoptosis, while those showing a slower growth rate would exhibit lower proliferation and higher apoptosis. Interestingly, we generally observed that xenografts expressing endogenous GGH associated with a faster growth rate (Figure 4.8A, Chapter 4) showed lower proliferation and higher apoptosis compared with those expressing the sense GGH (P < 0.05; Figures 5.10 and 5.11). Similarly, in the 2 mg FA control diet, xenografts expressing the sense GGH treated with 5FU+LV demonstrating the slowest growth rate (Figure 5.9A) were associated with the increased proliferation and decreased apoptosis compared with those expressing endogenous GGH regardless of treatments (5FU+LV vs. 0.9% NaCl) (P < 0.05; Figures 5.10 and 5.11). The discrepancy between the expectation and the outcome may be speculated based on a Gompertzian model of cancer cell growth. It has been known that during the early stages of tumor expansion, growth is exponential, but with enlargement, tumor growth slows at the late stages (422, 423). HCT116 cells expressing endogenous GGH grew faster than those transfected with the sense GGH (Figure 4.8A, Chapter 4), and thus it is likely that xenografts expressing endogenous GGH was associated with the late stages of tumor on Day 21. In contrast, xenografts expressing the sense GGH appeared to be relevant to the earlier stages of tumor on Day 21 due to their slower growth rate compared with those expressiong endogenous GGH. Accordingly, it is presumed that xenografts expressing endogenous GGH associated with the late stages of tumor might be less proliferative and more likely to undergo apoptosis, while those expressing the sense GGH related with the early stages of tumor represented an increase in proliferation and less of a chance to undergo apoptosis. It may be advisable to perform the immunohistochemistry analysis using xenografts sections at 48-72 hours after 5FU treatment (Day 3) or when the size of xenografts is similar in order to investigate the effects of GGH overexpression and treatment on proliferation and apoptosis in vivo study.

Interestingly, no significant differences were observed in tumor proliferation between treatments within each xenograft on the 8 mg FA supplemented diet (Figure 5.10). There was a significant interaction between xenograft type (Sense vs. Control-S) and treatment (5FU+LV vs. 0.9% NaCl) (epithelial proliferation, P = 0.03; apoptosis, P = 0.04), suggesting that the effect of xenograft type depended on treatment and similarly the effect of treatment depended on

120 xenograft type. For the degree of xenograft epithelial proliferation, a significant effect of diet was observed (P = 0.01), indicating diet also affected xenograft proliferation (Figure 5.10).

For the level of xenograft apoptosis, the effect of diet was not significant (P = 0.46). As shown in Figure 5.11C, no significant differences were observed in the degree of apoptosis between treatments (5FU+LV vs. 0.9% NaCl) within each xenograft. Xenografts expressing endogenous GGH treated with 5FU+LV had a significantly higher level of apoptosis than those expressing the sense GGH regardless of treatments (P < 0.05; Figure 5.11C). Xenografts expressing the sense GGH treated with 5FU+LV showed a significantly lower level of apoptosis than those expressing endogenous GGH regardless of treatments (P < 0.05; Figure 5.11C).

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A 2 mg FA Control diet 8 mg FA supplemented diet

0.9% NaCl 5FU+LV 0.9% NaCl 5FU+LV

Control-S

Sense

B

c bc bc

ab ab ab

a

a

P-interaction for xenograft type x treatment = 0.03 Diet: P = 0.01

Figure 5. 10 Representative images of Ki-67-stained xenografts (A) and percentage of Ki- 67-positive cells (B) of HCT116 colon cancer xenografts expressing either GGH overexpression (Sense) or endogenous GGH (Control-S). Different letters within each group denote significant difference at P < 0.05. Values are mean ± SEM.

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A 2 mg FA Control diet 8 mg FA supplemented diet

0.9% NaCl 5FU+LV 0.9% NaCl 5FU+LV

Control-S

Sense

B C

a a ab ab

ab bc abc abc abc bc c

c

P-interaction for xenograft type x treatment = 0.04 Diet: NS (P = 0.46)

Figure 5. 11 Representative images of xenografts apoptosis as determined by TUNEL assay (A) and percentage of TUNEL-positive cells (B, C) of HCT116 colon cancer xenografts expressing either GGH overexpression (Sense) or endogenous GGH (Control-S). Since the effect of diet was not significant (B; P = 0.46), the degree of apoptosis between xenograft type and treatment was analysed (C). Different letters within each group denote significant difference at P < 0.05. Values are mean ± SEM.

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5.5 Discussion

We have previously developed an appropriate in vitro model of GGH overexpression and inhibition in HCT116 colon and MDA-MB-435 breast cancer cells with predictable functional consequences, and found that GGH modulation significantly affects the chemosensitivity of colon and breast cancer cells to antifolates and 5FU by altering the intracellular retention of antifolates and a folate cofactor (e.g., 5,10-methyleneTHF for 5FU cytotoxicity), respectively (Chapter 4). The objectives of the present study were to confirm our previous findings at more physiological and nutritionally relevant levels and form of folate and also to confirm in vitro findings in vivo. We have previously observed that both HCT116 and MDA-MB-435 cells expressing the sense GGH had a significantly slower growth rate, lower concentrations of total intracellular folate, lower content of long-chain folylpolyglutamates, lower TS activity, and lower DHFR protein expression and activity compared with corresponding controls at 2.3 μM FA (Chapter 4). In the present study, both cells expressing the sense GGH also had a significantly slower growth rate at 50 nM and 100 nM 5-MTHF consistent with the previously observed functional consequences of GGH overexpression. Unexpectedly, at 2.3 μM FA, both HCT116 and MDA-MB-435 cells transfected with the GGH-targeted siRNA also demonstrated a slower growth rate compared with controls, although GGH inhibition was associated with significantly higher concentrations of total intracellular folate, higher content of long-chain folylpolyglutamates, higher TS activity, and higher DHFR protein expression and activity compared with controls expressing endogenous GGH (Chapter 4). In contrast, HCT116 cells in which GGH was inhibited showed a significantly faster growth rate at 50 nM and 100 nM 5- MTHF as expected. This is consistent with the observed functional consequenses associated with GGH inhibition including increased TS and DHFR activity as well as increased intracellular folate concentrations and higher content of long-chain folylpolyglutamates, which are better substrates than short-chain folylpolyglutamates for folate-dependent enzymes involved in thymidylate and purine biosynthesis (Chapter 4). Similarly, the GGH-inhibited MDA-MB-435 cells showed a faster growth rate at 50 nM 5-MTHF. Generally, most of the observed growth rates of GGH overexpression and inhibition are consistent with the known biological function of GGH at nutritionally and physiological relevant levels and form of folate (i.e., 5-MTHF).

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GGH modulation in our model significantly affected chemosensitivity of cancer cells to MTA whose cytotoxic effects depend on polyglutamylation (270). Our a priori hypothesis was that if the cytotoxicity of MTA depended solely on its polyglutamylation, GGH overexpression would decrease, where GGH inhibition would increase, chemosensitivity of cancer cells to MTA. However, the modulatory effect was in the expected direction only in MDA-MB-435 cells overexpressing GGH at both concentrations and in HCT116 cells in which GGH was inhibited at 50 nM 5-MTHF, and was in the opposite direction of our a priori hypothesis in other cell constructs. These observations suggest that the GGH modulation-induced changes in MTA polyglutamylation may not be the primary determinant of chemosensitivity of HCT116 and MDA-MB-435 cells to MTA. In HCT116 cells overexpressing GGH, low total folate concentrations, low content of long-chain folylpolyglutamates and decreased TS and DHFR have likely counteracted the potentially reduced cytotoxic effect of the GGH overexpression-induced decreased MTA polyglutamylation, resulting in enhanced chemosensitivity. However, in MDA- MB-435 cells overexpressing GGH, the decreased MTA polyglutamylation was sufficient to overcome the counterbalancing effect of the GGH overexpression-induced decrease in total folate and long-chain folylpolyglutamates and TS and DHFR activity, resulting in decreased chemosensitivity. In HCT116 (at 100 nM 5-MTHF) and MDA-MB-435 cells (at both concentrations) in which GGH was inhibited, high total folate concentrations, high content of long-chain folylpolyglutamates and increased TS and DHFR activity have likely overcome the potentially increased cytotoxic effect of the GGH inhibition-induced increased MTA polyglutamylation, resulting in decreased chemosensitivity. However, in HCT116 cells at 50 nM 5-MTHF in which GGH was inhibited, the increased MTA polyglutamylation was sufficient to overcome the counterbalancing effect of the GGH inhibition-induced increase in total folate and long-chain folylpolyglutamates and TS and DHFR activity, resulting in enhanced chemosensitivity. These data collectively suggest that the effect of GGH modulation on chemosensitivity of antifolates, whose cytotoxic effects depend on polyglutamylation, cannot be predicted solely based on the polyglutamylation of antifolates and that it may be cell-specific.

We hypothesized that chemosensitivity to TMTX should primarily depend on different concentrations and polyglutamylation of intracellular folates mediated by GGH modulation, because TMTX is not polyglutamylated and hence does not depend on polyglutamylation for its cytotoxic effects (396). Unexpectedly, GGH overexpression significantly decreased

125 chemosensitivity of HCT116 and MDA-MB-435 to TMTX at both concentrations, suggesting that factors other than changes in concentrations and polyglutamylation of intracellular folates associated with GGH overexpression are likely operative in modulating chemosensitivity of these cancer cells to TMTX. Consistent with our hypothesis, increased intracellular total folate concentrations, long-chain folylpolyglutamates, and TS and DHFR activity associated with GGH inhibition decreased chemosensitivity to TMTX in HCT116 and MDA-MB-435 cells at both concentrations. At 2.3 μM FA, the magnitude of these changes were insufficient to affect chemosensitivity of HCT116 cells to TMTX, while GGH inhibition decreased TMTX chemosensitivity in MDA-MB-435 cells (Chapter 4), suggesting that exogenous folate concentrations further influence the GGH modulation-induced chemosensitivity and it may be cell-specific.

In the previous study, it appears that the GGH overexpression-induced decreased MTX- polyglutamylation was the primary determinant of the observed decreased chemosensitivity of HCT116 and MDA-MB-435 cells to MTX at 2.3 μM FA (Chapter 4). The GGH overexpression-induced decreased MTX-polyglutamylation was also sufficient to overcome the counterbalancing effect of decreased intracellular concentrations of total folate and long-chain folylpolyglutamates and decreased TS and DHFR activity associated with GGH overexpression in MDA-MB-435 cells at 50 nM and 100 nM 5-MTHF. However, in HCT116 cells, this effect was not observed at both concentrations, suggesting that the magnitude of intracellular folate depletion associated with GGH overexpression at these folate concentrations was not sufficient to modulate chemosensitivity to MTX. In contrast, in both the GGH-inhibited HCT116 and MDA-MB-435 cells, increased intracellular concentrations of total folate and long-chain folylpolyglutamates and increased TS and DHFR activity were the primary determinants of the observed decreased chemosensitivity of cancer cells to MTX rather than the GGH inhibition- induced increased MTX-polyglutamylation, which should have enhanced MTX chemosensitivity.

IC50 values indicate that an increased concentration of MTX was required to achieve 50% inhibition among cells grown in higher concentrations of folate. This suggests that increased total intracellular folate concentrations and higher contents of folylpolyglutamates associated with GGH inhibition, which would provide an increased amount of substrates for nucleotide biosynthesis, had a competitive effect on increased MTX polyglutamylation. Furthermore, folate and MTX share cell entry mechanisms, and folate has a higher affinity for DHFR than MTX (2,

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3). Therefore, at high medium folate concentrations, more folate will enter cells and bind preferentially to DHFR, resulting in higher concentrations of MTX being required for its cytotoxic effects in high folate environments.

Our a priori hypothesis concerning the effects of GGH modulation on chemosensitivity of colon and breast cancers to 5FU was based on the assumption that the GGH modulation-induced changes in intracellular concentrations of long-chain 5,10-methyleneTHF-polyglutamates might be the primary determinant of chemosensitivity. We hypothesized that GGH overexpression would decrease the cytotoxic effect of 5FU by decreasing relative intracellular concentrations of long-chain 5,10-methyleneTHF-polyglutamates, resulting in less efficient formation and stabilization of the inhibitory 5,10-methyleneTHF-TS-FdUMP ternary complex. When 5FU was administered alone, in contrast to our expectation, GGH overexpression enhanced chemosensitivity of HCT116 cells to 5FU at both concentrations. This suggests that decreased long-chain 5,10-methyleneTHF-polyglutamates and hence a decreased TS inhibitory effect was not sufficient to override the potentiating effect on chemosensitivity to 5FU associated with decreased total intracellular folate concentrations and contents of long-chain polyglutamates of other intracellular folate derivatives and decreased TS and DHFR. In MDA-MB-435 cells, the competing effects of decreased intracellular concentrations of total folate and long-chain folylpolyglutamates and of less efficient and stable TS inhibition associated with decreased long- chain 5,10-methyleneTHF-polyglutamates appear to have cancelled each other out at 100 nM 5- MTHF. Decreased chemosensitivity to 5FU associated with GGH overexpression seems to overwhelm the counteracting effect of decreased intracellular concentrations of total folate and long-chain polyglutamates of other intracellular folate derivatives at 50 nM 5-MTHF. However, when a precursor for 5,10-methyleneTHF (i.e., LV) was supplied exogenously, thereby significantly increasing the intracellular pool of 5,10-methyleneTHF, less efficient and stable TS inhibition associated with the GGH overexpression-induced decreased long-chain 5,10- methyleneTHF-polyglutamates overcame the counterbalancing effect of decreased intracellular concentrations of total folate and long-chain polyglutamates of other intracellular folate derivatives, leading to decreased 5FU chemosensitivity of MDA-MB-435 cells at both concentrations. Interestingly, in HCT116 cells, this decreased cytotoxic effect of 5FU+LV was not observed at 100 nM 5-MTHF, whereas GGH overexpression was associated with increased chemosensitivity to 5FU+LV at 50 nM 5-MTHF. This suggests that decreased long-chain 5,10-

127 methyleneTHF-polyglutamates and hence less TS inhibiting effect associated with GGH overexpression was not sufficient to overcome the potentiating effect on chemosensitivity to 5FU associated with decreased total intracellular folate concentrations and contents of long-chain polyglutamates of other intracellular folate derivatives at 50 nM 5-MTHF. Furthermore, the observed in vitro chemosensitivity of HCT116 cells overexpressing GGH to 5FU+LV at different folate concentrations was confirmed in an in vivo model. Compared with xenografts expressing endogenous GGH, xenografts expressing the sense GGH were more sensitive to 5FU+LV at the 2 mg FA/kg control diet but less sensitive to 5FU+LV at the 8 mg FA/kg supplemented diet, which are consistent with the in vitro chemosensitivity of the GGH- overexpressed HCT116 cells to 5FU+LV at 50 nM 5-MTHF and 2.3 μM FA (Chapter 4), respectively.

A similar pattern of 5FU chemosensitivity was also observed for GGH inhibition. We hypothesized that GGH inhibition would increase the cytotoxic effect of 5FU by increasing relative intracellular concentrations of long-chain 5,10-methyleneTHF-polyglutamates, resulting in a more efficient formation and stabilization of the 5,10-methyleneTHF-TS-FdUMP ternary complex. When 5FU was given alone, in contrast to our expectation, GGH inhibition significantly decreased chemosensitivity of HCT116 cells to 5FU at 100 nM 5-MTHF. This suggests that increased long-chain 5,10-methyleneTHF-polyglutamates and hence increased TS inhibiting effect were not sufficient to overcome the decreased effect on chemosensitivity to 5FU associated with increased total intracellular folate concentrations and contents of long-chain polyglutamates of other intracellular folate derivatives and increased TS and DHFR, which result in increased thymidylate and purine biosynthesis. In both cell lines, the competing effects of increased intracellular concentrations of total folate and long-chain folylpolyglutamates and of more efficient and stable TS inhibition associated with increased long-chain 5,10- methyleneTHF-polyglutamates appear to have cancelled each other out at 50 nM 5-MTHF. In MDA-MB-435 cells, GGH inhibition significantly increased chemosensitivity to 5FU at 100 nM 5-MTHF, suggesting that increased long-chain 5,10-methyleneTHF-polyglutamates and hence increased TS inhibiting effect were sufficient to override the decreased effect on chemosensitivity to 5FU associated with increased total intracellular folate concentrations and contents of long-chain polyglutamates of other intracellular folate derivatives. However, when a precursor for 5,10-methyleneTHF (i.e., LV) was supplied exogenously, thereby significantly

128 increasing the intracellular pool of 5,10-methyleneTHF, more efficient and stable TS inhibition associated with the GGH inhibition-induced increased long-chain 5,10-methyleneTHF- polyglutamates overcame the counterbalancing effect of increased intracellular concentrations of total folate and long-chain polyglutamates of other intracellular folate derivatives, leading to enhanced chemosensitivity of both colon and breast cancer cells to 5FU at both concentrations except for MDA-MB-435 cells at 50 nM 5-MTHF. Generally, IC50 values indicate that an increased concentration of 5FU was required to achieve 50% inhibition among cells expressing the sense GGH grown in higher concentrations of folate. This suggests that FA supplementation may interfere with the sensitivity of 5FU and may cause drug resistance in the GGH overexpression system (12, 13).

Accumulating evidence from recent clinical studies supports the role of GGH in modulating chemosensitivity to antifolates and 5FU. Low GGH expression was associated with a good response to 5FU-based chemotherapy in patients with metastatic CRC (183). CIMP+ CRC associated with low GGH expression and high 5,10-methyleneTHF levels has a higher response to 5FU than CIMP- CRC (186, 187). FPGS levels were not significantly different, however, between CIMP+ and CIMP- CRC (187), suggesting that FPGS has less of an influence than GGH in modulating the chemosensitivity to 5FU. In patients with advanced pancreatic cancer treated with an oral fluoropyrimidine derivative, S-1, low GGH expression was associated with improved overall survival (400). In contrast, high GGH expression was shown to be associated with a higher risk of developing advanced toxicity to MTA in patients with advanced breast cancer (271). A recent study has reported that a high GGH protein level is associated with poor prognosis and unfavourable clinical outcomes in patients with invasive breast cancer (401). Furthermore, several recently identified and characterized genetic and epigenetic polymorphisms of GGH have been reported to predict a response to MTX-based treatment in patients with cancer and inflammatory arthritis (133, 161, 171, 177). It has been shown that decreased GGH activity was also associated with high MTX accumulation in the ALL patients with +452C>T SNP (164).

In conclusion, our data suggests that the GGH-modulated chemosensitivity of cancer cells to antifolates and 5FU is highly complex and appeared to depend not only on the GGH modulation- induced changes in polyglutamylation of antifolates and a specific target intracellular folate cofactor such as 5,10-methyleneTHF, respectively, but also on total intracellular folate pools and polyglutamylation of other intracellular folate cofactors as well as adaptive and compensatory

129 changes in other enzymes involved in intracellular folate and antifolate accumulation and metabolism in response to GGH modulation. The GGH overexpression-induced changes in chemosensitivity of colon and breast cancer cells to 5FU+LV and MTX were in the same direction at different folate concentrations. Our IC50 data also suggest that an increased concentration of 5FU and MTX was required to achieve 50% inhibition among cells grown in higher folate concentrations, supporting the hypothesis that FA supplementation might be associated with drug resistance. We provide evidence that both GGH modulation and folate status may be important clinical determinants for providing appropriate and effective tailored cancer therapy, which requires further exploration.

Table 5. 5 Summary of the in vitro chemosensitivity of the GGH-modulated HCT116 and MDA-MB-435 cells at different folate levels (Chapters 4 and 5)

The thickness of arrows indicates the magnitude of cell survival difference between Sense and Control-S and between siRNA and Control-si. The result highlighted with blue line denotes our expected outcome.

CHAPTER 6: STUDY 3 – THE EFFECT OF GGH MODULATION ON GLOBAL AND GENE- SPECIFIC DNA METHYLATION AND GENE EXPRESSION IN HUMAN COLON AND BREAST CANCER CELLS

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6.1 Abstract

Background: Folate mediates the transfer of one-carbon units for the generation of SAM, the primary methyl group donor for most biological methylation reactions including DNA methylation, which is catalyzed by DNMT. Both genomic DNA hypomethylation and gene- specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis. DNA methylation and DNMT are potential therapeutic targets and may modify the effect of specific chemotherapeutic agents. GGH plays an important role in folate homeostasis by catalyzing the hydrolysis of polyglutamylated folate into monoglutamates, thereby facilitating the export of folate from cells. We investigated whether GGH modulation would affect global and gene-specific methylation and gene expression profiles in human HCT116 colon and MDA-MB-435 breast cancer cells.

Methods: We generated an in vitro model of GGH overexpression and inhibition in human HCT116 colon and MDA-MB-435 breast cancer cells by stably transfecting the cells with the sense GGH cDNA or GGH-targeted siRNA, respectively. Global DNA methylation and DNMT activity were determined. Illumina HT-12 and Infinium Methylation assays were performed, and Ingenuity Pathway Analysis was used for functional analysis. mRNA expression of selected genes, expression of which was regulated by DNA methylation, was confirmed by qRT-PCR.

Results: In both cell lines, GGH overexpression was associated with significantly lower global DNA methylation and DNMT activity compared with controls, while GGH inhibition was associated with significantly higher global DNA methylation and DNMT activity compared with controls. In HCT116 cells, we identified 152 differentially expressed genes (most commonly involved pathways: cellular movement and cell death) associated with GGH overexpression, and 321 differentially expressed genes (most commonly involved pathways: cell death and cell cycle) associated with GGH inhibition; 905 and 1869 genes showed altered CpG promoter methylation associated with GGH overexpression and inhibition, respectively; and an integrated analysis revealed 5 and 16 genes, expression of which was regulated by CpG promoter methylation changes associated with GGH overexpression and inhibition, respectively. In MDA-MB-435 cells, we identified 1383 differentially expressed genes (most commonly involved pathways: cell-to-cell signaling and interaction and cellular movement) associated with GGH

132 overexpression, and 859 differentially expressed genes (most commonly involved pathways: cell death and cellular development) associated with GGH inhibition; 2394 and 2666 genes showed altered CpG promoter methylation associated with GGH overexpression and inhibition, respectively; and an integrated analysis revealed 101 and 47 genes, expression of which was regulated by CpG promoter methylation changes associated with GGH overexpression and inhibition, respectively.

Conclusions: Our data indicate that GGH modulation can affect global DNA methylation and DNMT activity as well as differential gene expression and CpG promoter DNA methylation involved in important biological pathways, and some of the observed altered gene expression appear to be regulated by DNA methylation.

6.2 Introduction

Folate mediates the transfer of one-carbon units involved in thymidylate and purine biosynthesis, the methionine cycle, and biological methylation reactions (1, 29). As an essential cofactor for de novo nucleotide biosynthesis, folate plays an important role in DNA synthesis, stability and integrity, and repair (1, 36). Furthermore, folate provides SAM, the primary methyl group donor for most biological methylations including DNA methylation, which is catalyzed by DNMT (1, 27). Both global DNA hypomethylation and gene-specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis (29). DNA methylation and DNMT are also potential therapeutic targets due to the reversible nature of epigenetic alterations, and modify the effect of specific chemotherapeutic agents (30). DNA methylation inhibitors, such as 5-aza-CR and 5-aza-CdR, are incorporated into the DNA of tumor cells during replication and thereby, they inhibit DNA methylation by trapping DNMT onto DNA, leading to inhibition of tumor growth by reactivation of tumor suppressor genes that are aberrantly silenced in cancer (297, 339). A recent study reported that treatment of cisplatin- resistant cells with 5-aza-CdR induced resensitization to cisplatin and re-expression of downregulated genes such as MLH1, a mismatch repair gene (424). In addition, growing evidence suggests that DNA methylation could provide a good molecular marker to predict sensitivity to chemotherapy since aberrant DNA methylation might affect chemosensitivity of

133 cancers by altering expression of genes critical to the drug response. Methylation of the MGMT promoter is predictive for favorable outcomes in patients with malignant glioma treated with alkylating agents (344). In a clinical trial, inactivation of MGMT by promoter hypermethylation was associated with longer survival in glioblastoma patients treated with alkylating agents (346). Furthermore, CRC cell lines displaying microsatellite instability are resistant to 5FU due to methylation of hMLH1, but they become susceptible to treatment upon exposure to 5-aza-CdR (349). Therefore, methylation of hMLH1 appears to be a predictive molecular marker of the sensitivity of CRC to 5FU. Simiarly, inactivation of hMLH1 by methylation in some ovarian tumors results in the loss of cisplatin sensitivity (350, 351), whereas hMLH1 expression and sensitivity to cisplatin were restored after treatment with 5-aza-CdR (352). Hypermethylation of WRN, a gene related with RecQ-like helicase involved in the maintenance of genetic stability, in CRC is known as a useful predictor of response to topoisomerase inhibitors (34, 425). Methylation of CHFR, a mitotic checkpoint gene, could also serve as a useful predictive marker of the sensitivity of tumors to microtubule inhibitors (426-428). Hypermethylation of p73, a homolog of the p53 tumor suppressor gene, was also associated with increased sensitivity to cisplatin in NCI-60 cells, but the mechanism responsible for these effects remains unknown (353). A loss of IGFBP3 expression mediated by promoter hypermethylation showed reduced sensitivity to cisplatin in NSCLC cells and in patients (429).

Intracellular folate homeostasis is maintained by FPGS that facilitates intracellular retention of folate by polyglutamylation and by GGH that catalyzes the hydrolysis of polyglutamylated folate into monoglutamates, thereby facilitating the export of folate out of the cell (1). Polyglutamylation is also important in DNA methylation as polyglutamylated folates are better substrates for folate-dependent enzymes such as MTHFR and MS that are involved in the generation of SAM, which is a substrate for DNA methylation mediated by DNMT (2, 28). And hence, GGH modulation may affect DNA methylation at global and gene-specific levels with consequent functional ramifications. As mentioned earlier, DNA methylation and DNMT are also potential therapeutic targets that may modify the effect of specific chemotherapeutic agents (30), suggesting that the GGH-modulated DNA methylation changes might influence chemosensitivity to chemotherapeutic agents. Therefore, the primary objective of this study was to investigate whether GGH modulation would affect global and gene-specific DNA methylation

134 and DNMT activity, and the secondary objective was to interrogate the effect of GGH modulation on gene expression in HCT116 and MDA-MB-435 cell lines.

We hypothesized that (1) GGH overexpression would decrease global DNA methylation and DNMT activity, whereas GGH inhibition would increase global DNA methylation and DNMTactivity; and (2) GGH overexpression and inhibition would further affect the degree of promoter methylation and the differential expression of genes.

6.3 Materials and Methods

6.3.1 Cell Lines and Culture

Cells were maintained in RPMI-1640 medium as described in Section 4.3.1.

6.3.2 Construction and Transfection of GGH Expression Vector

Construction and transfection of GGH expression vectors were performed as described in Section 4.3.2. In order to obviate the potential confounding effect of different cell passage numbers on DNA methylation, cells expressing the sense GGH or GGH-targeted siRNA and corresponding controls with similar passage numbers were used for DNA methylation analysis.

6.3.3 Global DNA Methylation Analysis

Genomic DNA Isolation:

Total genomic DNA was extracted by a standard technique as previously described (430). The final preparation had a ratio of A260:A280 between 1.8 and 2.0, indicating high quality and free of RNA and protein contamination of DNA sample. The concentration of each DNA sample was determined as the mean of three independent spectrophotometric readings. The DNA samples were stored at -4ºC until further analysis.

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Global DNA Methylation:

The methylation status of CpG sites in genomic DNA was determined by the in vitro methyl acceptance assay using [3H-methyl] SAM (New England Nuclear, Boston, MA) as a methyl donor and a prokaryotic CpG DNMT, Sss1 (New England Biolabs, Beverly, MA), as previously described (431, 432). The manner in which this assay is performed produces an inverse relationship between the endogenous DNA methylation status and exogenous [3H-methyl] incorporation. All analyses were performed in quadruplicate and repeated using two independent cell lysates.

6.3.4 DNA Methyltransferase Enzyme Activity Assay

Total cellular CpG DNMT activity was measured by incubating cell lysate containing 10 μg of protein with 0.5 μg of poly[d(I-C)·d(I-C)] template (Sigma-Aldrich, St.Louis, MO), 3 μCi [3H]- SAM (New England Nuclear, Boston, MA), and lysis buffer in a total volume of 20 μL for 2 hours at 37°C as described previously (431, 432). Each reaction was performed in triplicate and the assay was repeated three times.

6.3.5 Gene-Specific Promoter CpG Island Methylation Analysis

The Illumina Infinium HumanMethylation27 BeadChip was used to interrogate the DNA methylation status of 27,578 individual CpG sites located at promoter regions of 14,495 genes (360). Briefly, 1 µg of genomic DNA was bisulfite-converted using the EZ-96 DNA Methylation Kit (Zymo Research) according to the manufacturer’s instructions. Unmethylated cytosines are deaminated to uracil in the presence of bisulfite, while methylated cytosines are refractory to the effects of bisulfite and remain as cytosine. The bisulfite conversion included a thermocycling program with a short denaturation step (16 cycles of 95ºC for 30 seconds followed by 50ºC for 1 hour). The amount of bisulfite-converted DNA and completeness of bisulfite conversion was assessed using a panel of MethyLight-based quality control (QC) reactions as previously described (433). All of the samples passed our QC tests and were used for the Infinium DNA methylation assay. A measure of the level of DNA methylation at each CpG site was scored as beta (β) values using BeadStudio software. DNA methylation β-values represent the ratio of the

136 intensity of the methylated bead type to the combined locus intensity ranging from 0 to 1. Values close to 0 indicate low levels of DNA methylation, while values close to 1 indicate high levels of DNA methylation (360). The detection P-values measure the difference of the signal intensities at the interrogated CpG site compared with those from a set of 16 negative control probes embedded in the assay. We identified all data points with a detection P-value > 0.05 as not statistically significantly different from background measurements, and therefore were not considered trustworthy measures of DNA methylation. These data points were replaced by “NA” values as previously described (434). Detailed information on this assay and data filtering and normalization is described in Appendix 3. Statistical analysis and data visualization were carried out using the R/Biocoductor software packages (http://www.bioconductor.org). The Illumina Infinium DNA methylation β-values were represented graphically using a heatmap, generated by the R/Bioconductor packages called heatmap.plus and matlab.

6.3.6 Gene Expression Analysis

RNA Isolation:

RNAs from the GGH-modulated HCT116 and MDA-MB-435 cells were isolated using the RNeasy Microarray Tissue Mini Kit (Qiagen, Cat#: 73304) according to the manufacturer’s protocol. The RNA concentration was measured using a spectrophotometer. All samples had a ratio of A260:A280 nm between 1.9 and 2.1, indicating pure RNA. The purified RNA was stored at -20ºC for further analysis.

RNA Concentration and Integrity:

Total RNA was assessed for the RNA quality verification and microarray hybridization. The Agilent 2100 Bioanalyzer (Agilent Technologies), a microfluidics-based platform, was used for sizing, quantification and quality of RNA. The RNA Integrity Number (RIN) score was generated on the Agilent software. For the microarray analysis, the RNA quality for all of the samples had a RIN score ≥ 7.

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RNA Amplification:

The Illumina® TotalPrepTM-96 RNA Amplification Kit (Ambion, Lot#: 1008019) was used for generating biotinylated, amplified cRNA for hybridization with Illumina HumanHT-12 v4.0 Expression BeadChip according to the manufacturer’s protocol. The cRNA yield was quantified by NanoDrop (NanoDrop Technologies).

Hybridization:

A total of 750 ng of purified biotinylated cRNA generated from the samples were randomized in triplicate and used to hybridize onto the Illumina HumanHT-12 v4.0 BeadChip. Each array on this BeadChip targets 31,335 annotated genes and includes 47,231 probes designed to cover content from NCBI RefSeq Release 38 (November 7, 2009), as well as legacy UniGene content. Twelve samples are hybridized to one slide for higher throughput and reduced sample-to-sample variability since gaskets separate each array. The BeadChip was incubated at 58°C, with rotation speed 5 for 18 hours for hybridization. After washing and staining, the BeadChip was scanned on the iScan (Illumina), a laser based imaging system with 2 lasers (red: Cy5 and green: Cy3) for detecting fluorescence information on BeadChips. Although the iScan can scan both red and green intensities, the Illumina gene expression array is a single colour array; therefore, it scans with a green laser. The intensities files were quantified in GenomeStudio® (Illumina, version 2010.2) to generate raw intensities files without normalization algorithims. All samples passed Illumina's sample dependent and independent QC metrics. Raw intensities data files need to be normalized to adjust samples signals in order to minimize the effects of variation arising from non-biological factors. For our study, the data normalization was performed using GeneSpring (Agilent Technologies) prior to data analysis.

Gene Expression Profiles Analysis:

For the microarray analysis, data were checked for overall quality using R version 2.13.2 with the Bioconductor framework and the LUMI package (http://www.bioconductor.org). Data were imported in GeneSpring GX 11.5 (Agilent Technologies) for analysis, and data were normalized using a standard (for Illumina arrays) quantile based normalization followed by a ‘per probe’ median centered normalization. All data were log2-transformed for analysis and visualization. Data were initially filtered in order to remove any confounding effects that probes which show

138 no signal may have on subsequent analysis. Only probes in the upper 80th percentile of the distribution of intensities in 100% of sample from any of the groups were retained following filtering. Normalization and filtering of data was performed separately for each subset of samples being used in a particular analysis. For comparisons between cells expressing the sense GGH (Sense) and control (Control-S) and between cells transfected with the GGH-targeted siRNA (siRNA) and control (Control-si), an unpaired t-test using a false discovery rate (FDR) Benjamini and Hochberg multiple testing correction with a P-value cut-off of 0.05 was performed. Multiple testing corrections adjust P-values derived from multiple statistical tests to correct for occurrence of false positives. In the microarray data analysis, “false positives” are genes that are found to be statistically different between conditions, but are not in reality. The incidence of false positives is proportional to the number of tests performed and the critical significance level (P-value cut-off). Likewise, multiple testing correction adjusts the individual P-value for each gene to keep overall error rate (or false positive rate) to less than or equal to the user-specified P-value cut-off (435, 436). The Benjamini and Hochberg FDR is the least stringent of all corrections and provides a good balance between discovery of statistically significant genes and limitation of false positive occurrences. The more stringent a multiple testing correction, the less false positive genes are allowed (435, 436). A one-way ANOVA with a FDR corrected P-value ≤ 0.05 was performed to compare differences in gene expression among the GGH-overexpressed and inhibited cells, and their corresponding controls within each cell line. Two-way unsupervised hierarchical clustering of GGH overexpression and inhibition in HCT116 and MDA-MB-435 cells was performed using a Pearson centered distance metric under average linkage tree building rules. Results from data analyses were visualized to allow ease of data mining with the above mentioned dendrogram heat plots; red represents elevated gene expression and green represents decreased gene expression. All cluster diagrams are presented in Microsoft EXCEL spreadsheet format. More detailed information about the process for this assay is described in Appendix 4.

6.3.7 Integrated Analysis of DNA Methylation and Gene Expression Data

We merged the DNA methylation and gene expression data set using Gene IDs. We used a β-value difference (|Δβ|) of 0.20 as a threshold for differential DNA methylation. This threshold of |Δβ| = 0.20 was determined previously as a stringent estimate of Δβ detection

139 sensitivity across the range of β-values (360). Gene expression data with a fold change > 1.3 or < -1.3 and one-way ANOVA with a Benjamini and Hochberg corrected P-value ≤ 0.05 were used for integrated analysis. We set 1.3 as a fold change not to overlook small changes in response to GGH modulation as we identified a small number of genes differentially expressed especially in the GGH-modulated HCT116 cells.

6.3.8 Ingenuity Pathway Analysis

The functional analysis was performed using Ingenuity Pathway Analysis (IPA, Ingenuit y® Systems, Redwood City, CA; http://www.ingenuity.com) to identify biological functions and/or disease that were most significant to genes differentially methylated and/or regulated in each system. Genes with a threshold of |Δβ| = 0.20 and/or fold change cut-off of 1.3 and were associated with biological functions and/or diseases in the Ingenuity Knowledge Base were considered for the analysis. The Ingenuity Knowledge Base contains findings and annotations from the literature as well as other sources including EntrezGene, RefSeq, OMIM, GWAS database, , Tissue Expression Body Atlas, NCI-60 Cell Line Expression Atlas, KEGG Metabolic pathway information and so on. The right-tailed Fisher’s exact test was used to calculate a P‐value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone.

The functional analysis of a network was also performed using IPA in order to further examine regulatory relationships between differentially methylated and/or regulated genes and biological processes. The network genes associated with biological functions and/or diseases in the Ingenuity Knowledge Base were considered for the analysis. The right‐tailed Fisher’s exact test was used to calculate a P‐value determining the probability that each biological function and/or disease assigned to that network is due to chance alone. IPA computes a score for each network according to the fit of that network to the user-defined set of Focus Genes. The score is derived from a P-value and displayed as the negative log of that value. This score indicates the likelihood of the Focus Genes in a network being found together due to random chance. A score of 2 indicates that there is a 1 in 100 chance that the Focus Genes are together in a network due to random chance. Therefore, scores of 2 or higher have at least a 99% confidence of not being

140 generated by random chance alone.

6.3.9 Quantitative Reverse Transcriptase-Polymerase Chain Reaction

Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) was performed to confirm the data obtained from gene expression analysis using the Illumina HumanHT-12 v4.0 BeadChip described in Section 6.3.6. Total RNA was isolated as described in Section 6.3.6, and template RNA was reversely transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). Synthesized cDNAs, the reverse-transcription reactions, were then stored at -20°C until use for qRT-PCR. Selected primer sequences were synthesized by the Integrated DNA Technologies (Coralville, IA), and are presented in Table 6. 1. The reactions were run in triplicate on MicroAmp Optical 384-well plates (Applied Biosystems, Life Technologies) and their amplifications were tracked by SYBR Green fluorescent dye (Applied Biosystems, Life Technologies). Completed plates were spun at 1200 rpm for 2 minutes at 4°C and then placed in the ViiA-7TM Real-Time PCR System (Applied Biosystems, Life Technologies). The reaction conditions for stage one were as follows: 2 minutes at 50°C, followed by 10 minutes at 95°C to activate the polymerase. This stage was followed by 40 cycles beginning with 15 seconds at 95°C to denature the target strand followed by 1 minute at 60°C to allow for the polymerase to anneal to and extend the target strand. Finally a melt curve followed consisting of 15 seconds at 95°C then 1 minute at 60°C. Relative gene expression data was analyzed using the comparative threshold (Ct) method as described previously (437). The delta Ct (ΔCt) method requires an internal control (housekeeping genes, GAPDH or β-actin) to normalize the number of reactions for the amount of cDNA added to the reaction. In addition, the ΔΔCt method requires a calibrator, an untreatead control (Control-S or Control-si), thereby providing the fold change in gene expression normalized to the endogenous reference gene and relative to the untreated control (437). In brief, the average of the three Ct values for each gene was calculated yielding the Ct mean value since the experiments were performed in triplicate. The Ct mean value of GAPDH or β-actin was subtracted from the Ct mean value of each gene of interest and the difference yielded ΔCt. In order to determine relative quantification, the ΔΔCt value was calculated by subtracting the ΔCt mean value of Control-S or Control-si from the ΔCt mean value of each gene of interest. Then, the ΔΔCt value was inserted into the equation 2-ΔΔCt in order to convey this gene expression numerically.

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Table 6. 1 Primer sequences for genes selected for qRT-PCR

Gene Forward Primer Reverse Primer Symbol

AKR1C3 5'-GGGATCTCAACGAGACAAACG-3' 5'-AAAGGACTGGGTCCTCCAAGA-3'

ALDH1A3 5'-TCTCGACAAAGCCCTGAAGT-3' 5'-GTCCGATGTTTGAGGAAGGA-3'

APOD 5'-CCACCCCAGTTAACCTCACA-3' 5'-GTGCCGATGGCATAAACC-3'

5'-TGACGGGGTCACCCACACTGTGCCC 5'-CTAGAAGCATTTGCGGTGGACGAT β-actin ATCTA-3' GGAGGG-3'

BCHE 5'-CGGGTTGAAAGAGTTATTGTAG-3' 5'-GAACCCACTGAAGAGCCAAC-3'

CCND1 5'-GGACAACGGGCGGATAGAG-3' 5'-CACAGTCATCCCAGGGTTTAACA-3'

CDKN1A 5'-ATGTCAGAACCGGCTGGGGAT-3' 5'-TAGGGCTTCCTCTTGGAGAAG-3'

CRABP2 5'-GACCTCGTGGACCAGAGAACTG-3' 5'-CCTGGTGCACACAACGTCAT-3'

FGFBP1 5'-AGAACAAGGTGAACGCCCAGC-3' 5'-CCCGGGCCTGCTTTTCTGCTT-3'

GAPDH 5'-ACCACAGTCCATGCCATCAC-3' 5'-TCCACCACCCTGTTGCTGTA-3'

HLA-DRA 5'-TGGACAAAGCCAACCTGGAAA-3' 5'-AGGACGTTGGGCTCTCTCAG-3'

IGFBP7 5'-CACTGGTGCCCAGGTGTACT-3' 5'-TTGGATGCATGGCACTCACA-3'

IL11RA 5'-CCTGCGAGCCAGCTGGACA-3' 5'-ACTAGTGTCCCTGAGACACCT-3'

MMP7 5'-AACTCCCGCGTCATAGAAAT-3' 5'-GATACGATCCTGTAGGTGAC-3'

MSRB2 5'-CAAGGAAGCAGGAATGTATCA-3' 5'-ATGGTCAGTGTTTCCTTGGTTT-3'

NNMT 5'-GTTTGGTTCTAGGCACTCTG-3' 5'-GCAGGTTCTGGTCTGAGTAG-3'

PCOLCE 5'-GACTGCGACAGTGAAGTCCA-3' 5'-GCTTGCAAGGCACGTAAAAC-3'

S100A4 5'-CCACAAGTACTCGGGCAAAG-3' 5'-GTCCCTGTTGCTGTCCAAGT-3'

TYR 5'-CTCAAAGCAGCATGCACAAT-3' 5'-GCCCAGATCTTTGGATGAAA-3'

ZNF91 5'-GCCCTGGAATATGAAGCAACA-3' 5'-TCTGGCCAAAAGTCTTGAGGA-3'

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6.3.10 Statistical Analysis

For global DNA methylation, DNMT enzyme activity, and qRT-PCR analysis, comparisons between cells expressing the sense GGH (Sense) and controls (Control-S) and between cells transfected with the GGH-targeted siRNA (siRNA) and controls (Control-si) were determined using the Student’s t-test. The results were considered statistically significant if two-tailed P- values were < 0.05. Analyses were done using SPSS Statistics 17.0 (IBM SPSS, Chicago, IL) and graphs were prepared in Microsoft Excel.

With respect to gene-specific CpG promoter methylation, detailed information on data filtering and normalization and statistical analysis is presented in Section 6.3.5 and Appendix 3. The analysis of gene expression profiles is described in Section 6.3.6 and Appendix 4.

6.4 Results

6.4.1 Global DNA Methylation

In both cell lines, GGH overexpression was associated with significantly lower global DNA methylation than controls (HCT116, 16% lower, P = 0.024, Figure 6.1A; MDA-MB-435, 22% lower, P < 0.001, Figure 6.1C), while GGH inhibition was associated with significantly higher global DNA methylation than controls (HCT116, 15% higher, P = 0.013, Figure 6.1B; MDA- MB-435, 7% higher, P = 0.013, Figure 6.1D).

6.4.2 DNA Methyltransferase Enzyme Activity

Similar to global DNA methylation results, GGH overexpression showed significantly lower DNMT activity than controls (HCT116, 66% lower, P < 0.001, Figure 6.2A; MDA-MB-435, 26% lower, P = 0.003, Figure 6.2C), while GGH inhibition demonstrated significantly higher DNMT activity than controls (HCT116, 47% higher, P < 0.001, Figure 6.2B; MDA-MB-435, 27% higher, P = 0.002, Figure 6.2D).

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Figure 6. 1 Global DNA methylation in the GGH-modulated HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cells. The in vitro methyl acceptance assay produces an inverse relationship between the endogenous DNA methylation status and exogenous [3H- methyl] incorporation into DNA. Control-S, cells expressing endogenous GGH; Sense, cells transfected with the sense GGH cDNA; Control-si, cells expressing endogenous GGH; siRNA, cells transfected with the GGH-targeted siRNA. *, P < 0.05 compared with corresponding control. Values are mean ± SD.

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Figure 6. 2 DNA methyltransferase enzyme activity in the GGH-modulated HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cells. The assay produces a positive relationship between the endogenous enzyme activity and exogenous [3H-methyl] incorporation into DNA. Control-S, cells expressing endogenous GGH; Sense, cells transfected with the sense GGH cDNA; Control-si, cells expressing endogenous GGH; siRNA, cells transfected with the GGH-targeted siRNA. *, P < 0.05 compared with corresponding control. Values are mean ± SD.

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6.4.3 Gene-Specific Promoter CpG Island Methylation

Scatter plots of DNA methylation β-values showed differentially methylated loci between Sense and Control-S and between siRNA and Control-si as measured by Illumina Infinium HumanMethylation 27 BeadChip. The presence of more spread in each plot represents higher degree of hyper- or hypomethylation changes. MDA-MB-435 cells showed more CpG methylation alterations in response to GGH modulation than HCT116 cells (Figure 6.3).

A B

R2 = 0.956 R2 = 0.925

C D

R2 = 0.91 R2 = 0.888

Figure 6. 3 Scatter plots of DNA methylation β-value of GGH overexpression and inhibition in HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cell lines. Each dot indicates β-value at the particular gene locus. β-Value represents the ratio of the intensity of the methylated bead type to the combined locus intensity (methylated+unmethylated) ranging from 0 to 1. Values close to 0 indicate low levels of DNA methylation, while values close to 1 indicate high levels of DNA methylation. The presence of more spread in each graph represents higher degree of hyper- or hypomethylation changes.

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To illustrate the representative distribution of methylation levels across more than 27,000 CpG sites, Figures 6.4 and 6.5 displayed histograms of β-values in the GGH-modulated HCT116 and MDA-MB-435 cells. The number of CpG sites methylated at different β-values in the GGH- modulated HCT116 and MDA-MB-435 cell lines showed a bimodal distribution (Figures 6.4 and 6.5). This histogram indicated that MDA-MB-435 cells were associated with more CpG methylation alterations than HCT116 cells in response to GGH modulation (Figures 6.4 and 6.5). These obervations were consistent with the results previously mentioned (Figure 6.3). Overall, in both cell lines, GGH overexpression showed less methylation alterations than control, while GGH inhibition was associated with more methylation changes compared with control (Figures 6.4 and 6.5).

Figure 6. 4 Number of CpG sites methylated at different β-values in the GGH-modulated HCT116 colon cancer cells. The histogram indicates the number of CpG sites methylated at different β-values, and represents the distribution of β-values for 27,578 CpG sites in each system.

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Figure 6. 5 Number of CpG sites methylated at different β-values in the GGH-modulated MDA-MB-435 breast cancer cells. The histogram indicates the number of CpG sites methylated at different β-values, and represents the distribution of β-values for 27,578 CpG sites in each system.

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A two-dimensional hierarchical clustering represented the patterns of Infinium DNA methylation β-value in the GGH-modulated HCT116 (Figure 6.6) and MDA-MB-435 cell lines (Figure 6.7). It revealed distinctively different DNA methylation alteration profiles between GGH overexpression and inhibition in both cell lines (Figures 6.6 and 6.7).

Figure 6. 6 Two-dimensional hierarchical clustering of DNA methylation β-value in the GGH-modulated HCT116 cell line. A color gradient from dark blue to red indicated the low and high methylation, respectively.

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Figure 6. 7 Two-dimensional hierarchical clustering of DNA methylation β-value in the GGH-modulated MDA-MB-435 cell line. A color gradient from dark blue to red indicated the low and high methylation, respectively.

Table 6.2 represents the number of genes differentially methylated in each of the cell lines. Hyper- or hypomethylation was calculated by subtracting the β-value of corresponding control from the β-value of Sense or siRNA. We determined 0.2 as the β-value difference at 99% confidence based on intra- and inter- assay variations. In the HCT116 cell line, we identified 905 genes that were differentially methylated (546 hypermethylated and 359 hypomethylated) in response to GGH overexpression, while 1869 genes were differentially methylated (998 hypermethylated and 871 hypomethylated) in response to GGH inhibition (Table 6.2). As for the

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MDA-MB-435 cell line, we identified 2394 genes that were differentially methylated (1058 hypermethylated and 1336 hypomethylated) in response to GGH overexpression, while 2666 genes were differentially methylated (1122 hypermethylated and 1544 hypomethylated) in response to GGH inhibition (Table 6.2). The number of genes differentially methylated when we applied 0.5 as a more stringent criterion for β-value difference is also shown in Table 6.2.

Table 6. 2 Summary of number of genes differentially methylated in the GGH- modulated HCT116 and MDA-MB-435 cell lines

GGH Overexpression GGH Inhibition

Hyper- Hypo- Hyper- Hypo- Total Total methylation methylation methylation methylation

HCT116 β-value difference |0.2| 546 359 905 998 871 1869 (3.8%) (2.5%) (6.3%) (6.9%) (6.0%) (12.9%) β-value difference |0.5| 15 4 19 95 73 168 (0.10%) (0.03%) (0.13%) (0.7%) (0.5%) (1.2%) MDA-MB-435

β-value difference |0.2| 1058 1336 2394 1122 1544 2666 (7.3%) (9.2%) (16.5%) (7.7%) (10.7%) (18.4%) β-value difference |0.5| 80 100 180 129 240 369 (0.6%) (0.7%) (1.3%) (0.9%) (1.7%) (2.6%)

The numbers in brackets indicate the percentage of genes differentially methylated relative to total genes targeted in the Infinium assay.

6.4.3.1 Genes Differentially Methylated in the GGH-Modulated HCT116 Cells

We performed a functional analysis using IPA to identify biological and disease processes most relevant to differentially methylated genes in each system. Some genes were assigned to more than one category. The top five molecular and cellular functions are presented based on significance. In the GGH-overexpressed HCT116 cells, the hypermethylated genes were associated with cell morphology, cellular development, antigen presentation, cell-to-cell signaling and interaction, and cell death, while the hypomethylated genes were involved in molecular transport, cellular assembly and organization, cell-to-cell signaling and interaction, cellular growth and proliferation, and antigen presentation (Table 6.3). As for the GGH-inhibited HCT116 cells, major function categories of the hypermethylated genes included cell-to-cell

151 signaling and interaction, amino acid metabolism, drug metabolism, molecular transport, and small molecule biochemistry, whereas those of the hypomethylated genes consisted of lipid metabolism, molecular transport, small molecule biochemistry, cellular movement, and carbohydrate metabolism (Table 6.3).

Table 6. 3 The top molecular and cellular functions associated with differentially methylated genes in the GGH-modulated HCT116 colon cancer cells

Hypermethylation Hypomethylation No. of No. of Category P-value Category P-value Genes Genes GGH Overexpression Cell Morphology 5.76E-04 - 21 Molecular Transport 7.95E-05 - 23 3.16E-02 4.97E-02 Cellular Development 8.57E-04 - 36 Cellular Assembly and 3.60E-04 - 10 4.76E-02 Organization 3.76E-02 Antigen Presentation 4.13E-03 - 5 Cell-To-Cell Signaling 3.46E-03 - 20 2.93E-02 and Interaction 4.65E-02 Cell-To-Cell Signaling and 4.13E-03 - 31 Cellular Growth and 3.53E-03 - 10 Interaction 4.03E-02 Proliferation 3.76E-02 Cell Death 4.94E-03 - 22 Antigen Presentation 4.46E-03 - 6 3.95E-02 2.44E-02

GGH Inhibition Cell-To-Cell Signaling and 8.88E-05 - 73 Lipid Metabolism 9.96E-05 - 26 Interaction 4.94E-02 4.85E-02 Amino Acid Metabolism 1.08E-04 - 17 Molecular Transport 9.96E-05 - 52 7.78E-03 4.85E-02 Drug Metabolism 1.08E-04 - 13 Small Molecule 9.96E-05 - 39 4.94E-02 Biochemistry 4.85E-02 Molecular Transport 1.08E-04 - 27 Cellular Movement 1.63E-04 - 84 4.94E-02 4.85E-02 Small Molecule 1.08E-04 - 82 Carbohydrate Metabolism 5.69E-04 - 19 Biochemistry 4.94E-02 4.85E-02

To examine further regulatory relationships between differentially methylated genes and biological processes, the functional analysis of a network was performed using IPA. The network analysis identified the biological functions and/or diseases that were most significant to the genes in the network. The list of top networks in the GGH-modulated HCT116 cells is presented in Table 6.4.

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Table 6. 4 The top networks matched by the genes differentially methylated in the GGH- modulated HCT116 colon cancer cells

Hypermethylation Hypomethylation Focus Focus No Top Functions Score Top Functions Score Genes Genes GGH Overexpression 1 Immunological Disease, 37 28 Lipid Metabolism, Small 43 28 Hematological System Molecule Biochemistry, Development and Function, Molecular Transport Organismal Functions 2 Cell Death, Skeletal and 37 28 Cellular Movement, Cell 32 23 Muscular System Development Signaling, Cell Death and Function, Cellular Compromise 3 Reproductive System 31 25 Carbohydrate Metabolism, 17 15 Development and Function, Cell Molecular Transport, Small Morphology, Hematopoiesis Molecule Biochemistry 4 Hereditary Disorder, 16 16 Cell-To-Cell Signaling and 15 14 Immunological Disease, Interaction, Cell-mediated Cardiovascular System Immune Response, Cellular Development and Function Movement 5 Tissue Development, 16 16 Developmental Disorder, 15 14 Cardiovascular System Hereditary Disorder, Metabolic Development and Function, Disease Organismal Development GGH Inhibition 1 Cell Cycle, DNA Replication, 32 29 Cancer, Tissue Morphology, 37 31 Recombination, and Repair, Cell Death Cellular Compromise 2 Antigen Presentation, Cellular 30 28 Cell-To-Cell Signaling and 32 29 Movement, Hematological Interaction, Hematological System Development and System Development and Function Function, Inflammatory Response 3 Amino Acid Metabolism, Post- 26 26 Connective Tissue Disorders, 27 26 Translational Modification, Dermatological Diseases and Small Molecule Biochemistry Conditions, Developmental Disorder 4 Cellular Function and 23 24 Inflammatory Response, 27 26 Maintenance, Tissue Lymphoid Tissue Structure and Development, Cellular Development, Organ Development Morphology 5 Connective Tissue Development 21 23 Cellular Movement, Nervous 25 25 and Function, Reproductive System Development and System Development and Function, Cell Morphology Function, Tissue Morphology The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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6.4.3.2 Genes Differentially Methylated in the GGH-Modulated MDA-MB-435 Cells

Genes with functions related to cell-to-cell signaling and interaction, cellular movement, and molecular transport were found to be differentially methylated in the GGH-overexpressed MDA- MB-435 cells (Table 6.5). In the GGH-inhibited MDA-MB-435 cells, the hypermethylated genes were associated with cellular movement, cell-to-cell signaling and interaction, cell morphology, cell death, and lipid metabolism, while the hypomethylated genes were involved in cell signaling, molecular transport, vitamin amd mineral metabolism, , and small molecule biochemistry (Table 6.5). The list of top networks generated by mapping the focus genes that were differentially methylated in the GGH-modulated MDA-MB-435 cells is shown in Table 6.6.

Table 6. 5 The top molecular and cellular functions associated with differentially methylated genes in the GGH-modulated MDA-MB-435 breast cancer cells

Hypermethylation Hypomethylation No. of No. of Category P-value Category P-value Genes Genes GGH Overexpression Cell-To-Cell Signaling and 6.59E-09 - 108 Cell-To-Cell Signaling 3.01E-08 - 120 Interaction 1.34E-02 and Interaction 1.72E-02 Cellular Movement 2.89E-08 - 112 Cellular Growth and 3.21E-07 - 250 1.28E-02 Proliferation 1.72E-02 Lipid Metabolism 1.99E-06 - 69 Cellular Movement 4.93E-07 - 129 1.17E-02 1.72E-02 Molecular Transport 1.99E-06 - 132 Cell Signaling 1.05E-06 - 112 1.17E-02 1.57E-02 Small Molecule 1.99E-06 - 110 Molecular Transport 1.05E-06 - 100 Biochemistry 1.17E-02 1.15E-02 GGH Inhibition Cellular Movement 7.84E-07 - 119 Cell Signaling 1.71E-09 - 146 3.59E-02 7.31E-03 Cell-To-Cell Signaling and 2.93E-06 - 103 Molecular Transport 1.71E-09 - 194 Interaction 2.55E-02 1.06E-02 Cell Morphology 1.85E-05 - 72 Vitamin and Mineral 1.71E-09 - 108 3.37E-02 Metabolism 1.57E-02 Cell Death 9.73E-05 - 182 Nucleic Acid Metabolism 1.20E-08 - 62 3.40E-02 1.23E-02 Lipid Metabolism 1.87E-04 - 77 Small Molecule 1.20E-08 - 149 3.51E-02 Biochemistry 1.57E-02

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Table 6. 6 The top networks matched by the genes differentially methylated in the GGH- modulated MDA-MB-435 breast cancer cells

Hypermethylation Hypomethylation Focus Focus No Top Functions Score Top Functions Score Genes Genes GGH Overexpression 1 Development 31 29 Cell-To-Cell Signaling and 34 32 and Function, Lipid Interaction, Cellular Growth and Metabolism, Small Molecule Proliferation, Hematological Biochemistry System Development and Function 2 Inflammatory Response, 31 29 Cellular Movement, Cell 30 30 Cellular Movement, Immune Signaling, Molecular Transport Cell Trafficking 3 Molecular Transport, Cell 27 27 Cell Death, Cellular 30 30 Signaling, Vitamin and Mineral Development, Cellular Growth Metabolism and Proliferation 4 Molecular Transport, Nucleic 27 27 Cell Morphology, Humoral 28 29 Acid Metabolism, Small Immune Response, Molecule Biochemistry Hematological System Development and Function 5 Cell Death, Cell-To-Cell 16 20 Cell Cycle, DNA Replication, 26 28 Signaling and Interaction, Recombination, and Repair, Inflammatory Response Cancer

GGH Inhibition 1 Cell Death, Cancer, Tumor 34 31 Cellular Movement, 32 32 Morphology Hematological System Development and Function, Antigen Presentation 2 Cellular Movement, Lipid 32 30 Cell Signaling, Molecular 28 30 Metabolism, Molecular Transport, Vitamin and Mineral Transport Metabolism 3 Dermatological Diseases and 30 29 Developmental Disorder, 28 30 Conditions, Hereditary Embryonic Development, Organ Disorder, Inflammatory Disease Development 4 Cell Signaling, Molecular 28 28 Molecular Transport, Organ 26 29 Transport, Vitamin and Mineral Morphology, Reproductive Metabolism System Development and Function 5 Cancer, Reproductive System 25 26 Cell-To-Cell Signaling and 25 28 Disease, Endocrine System Interaction, Hematological Disorders System Development and Function, Cellular Development

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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6.4.3.3 Common Genes Differentially Methylated in the GGH-Modulated HCT116 and MDA-MB-435 Cells

A venn diagram was used to depict the number of genes commonly differentially methylated in the GGH-modulated HCT116 and MDA-MB-435 cell lines (Figures 6.8 and 6.9). Sixty-one hyper- and 54 hypomethylated genes were common between the HCT116 and MDA-MB-435 cell lines in response to GGH overexpression (Figure 6.8), whereas 117 hyper- and 129 hypomethylated genes were common between these cell lines in response to GGH inhibition (Figure 6.9). The proportion of differentially methylated CpG loci located within CpG island and non-CpG island regions is also presented in Figures 6.8 and 6.9. Generally, the proportion of CpG loci in non-CpG island regions was greater than those within CpG island regions.

Figure 6. 8 Number of genes commonly differentially methylated in HCT116 and MDA- MB-435 cells and distribution of differentially methylated CpG loci in response to GGH overexpression

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Figure 6. 9 Number of genes commonly differentially methylated in HCT116 and MDA- MB-435 cells and distribution of differentially methylated CpG loci in response to GGH inhibition

In the GGH-overexpressed HCT116 and MDA-MB-435 cell lines, the commonly hypermethylated genes were associated with cell death, cell cycle, cell morphology, cellular assembly and organization, and cellular compromise, while the commonly hypomethylated genes were related to cellular assembly and organization, cellular development, cellular growth and proliferation, amino acid metabolism, and cell cycle (Table 6.7). As for the GGH-inhibited HCT116 and MDA-MB-435 cell lines, major function categories of the commonly hypermethylated genes included cell signaling, small molecule biochemistry, carbohydrate metabolism, cell-to-cell signaling and interaction, and lipid metabolism, whereas genes hypomethylated in common consisted of cellular movement, antigen presentation, lipid metabolism, molecular transport, and small molecule biochemistry (Table 6.7). The list of top networks generated by mapping the focus genes that were commonly differentially methylated in both the GGH-modulated HCT116 and MDA-MB-435 cells is presented in Tables 6.8 and 6.9.

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Table 6. 7 The top molecular and cellular functions associated with genes with commonly differentially methylated in response to GGH modulation in both the HCT116 and MDA- MB-435 cells

GGH Overexpression GGH Inhibition No. of No. of Category P-value Category P-value Genes Genes Hypermethylated Cell Death 1.29E-03 - 7 Cell Signaling 1.48E-04 - 13 4.61E-02 4.56E-02 Cell Cycle 3.36E-03 - 3 Small Molecule 1.48E-04 - 22 2.99E-02 Biochemistry 4.56E-02 Cell Morphology 3.36E-03 - 4 Carbohydrate Metabolism 4.00E-04 - 10 4.61E-02 4.56E-02 Cellular Assembly and 3.36E-03 - 2 Cell-To-Cell Signaling 1.29E-03 - 18 Organization 3.64E-02 and Interaction 3.45E-02 Cellular Compromise 3.36E-03 - 1 Lipid Metabolism 1.34E-03 - 10 3.36E-03 4.56E-02

Hypomethylated Cellular Assembly and 2.75E-05 - 3 Cellular Movement 2.68E-04 - 19 Organization 3.62E-02 4.69E-02

Cellular Development 1.94E-03 - 6 Antigen Presentation 6.62E-04 - 8 3.33E-02 4.69E-02 Cellular Growth and 1.94E-03 - 5 Lipid Metabolism 1.29E-03 - 12 Proliferation 3.33E-02 4.90E-02 Amino Acid Metabolism 3.07E-03 - 2 Molecular Transport 1.29E-03 - 20 9.18E-03 4.69E-02 Cell Cycle 3.07E-03 - 4 Small Molecule 1.29E-03 - 25 3.33E-02 Biochemistry 4.90E-02

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Table 6. 8 The top networks matched by the genes differentially methylated in both the GGH-overexpressed HCT116 and MDA-MB-435 cells

Hypermethylation Hypomethylation Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cell Cycle, Cellular Movement, 23 12 Cell Death, Inflammatory 21 11 Hematological System Response, Respiratory Disease Development and Function 2 Cellular Movement, Immune 7 5 Lipid Metabolism, Small 21 11 Cell Trafficking, Inflammatory Molecule Biochemistry, Response Reproductive System Disease 3 Cancer, Hematological Disease, 2 1 Cellular Movement, Infectious 2 1 Endocrine System Disorders Disease, Cellular Development 4 Carbohydrate Metabolism, Cell 2 1 Cell-To-Cell Signaling and 2 1 Death, Cellular Assembly and Interaction, Cellular Assembly Organization and Organization, Cellular Function and Maintenance 5 Cell Death, Cellular 2 1 Development, Connective Tissue Disorders The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

Table 6. 9 The top networks matched by the genes differentially methylated in both the GGH-inhibited HCT116 and MDA-MB-435 cells

Hypermethylation Hypomethylation Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cell-To-Cell Signaling and 46 24 Cellular Movement, 39 22 Interaction, Hematopoiesis, Hematological System Cellular Function and Development and Function, Maintenance Immune Cell Trafficking 2 Dermatological Diseases and 14 10 Cell Morphology, Cancer, 15 11 Conditions, Connective Tissue Hematological Disease Disorders, Infectious Disease 3 Cell-mediated Immune 12 9 Metabolic Disease, 15 11 Response, Cellular Movement, Cardiovascular Disease, Tissue Hematological System Morphology Development and Function 4 Connective Tissue Disorders, 2 1 Developmental Disorder, Cell 13 10 Developmental Disorder, Death, Cell Morphology Hereditary Disorder 5 Cancer, Hematological Disease, 2 1 Cell Cycle, Cancer, 2 1 Endocrine System Disorders Hematological Disease The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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6.4.4 Gene Expression

A two-dimensional hierarchical clustering showed the patterns of gene expression fold change for 47,231 probes in the GGH-modulated HCT116 (Figure 6.10) and MDA-MB-435 cell lines (Figure 6.11). It demonstrated distinctively different patterns of gene expression in the GGH- modulated HCT116 and MDA-MB-435 cell lines (Figures 6.10 and 6.11).

The number of the genes differentially expressed in the GGH-modulated HCT116 and MDA- MB-435 cells is presented in Table 6.10. We determined the number of genes with a fold change > 1.3 or < -1.3 using an one-way ANOVA with the FDR corrected P-value ≤ 0.05. In the HCT116 cell line, we identified 152 genes that were differentially expressed (91 downregulated and 61 upregulated) in response to GGH overexpression, while 321 genes were differentially expressed (139 downregulated and 182 upregulated) in response to GGH inhibition (Table 6.10). As for the MDA-MB-435 cell line, we identified 1383 genes that were differentially expressed (628 downregulated and 755 upregulated) in response to GGH overexpression, while 859 genes were differentially expressed (402 downregulated and 457 upregulated) in response to GGH inhibition (Table 6.10). The top fifty genes most differentially expressed in the GGH-modulated HCT116 and MDA-MB-435 cell lines are shown in Appendix 5.

Table 6. 10 Summary of number of genes differentially expressed in the GGH-modulated HCT116 and MDA-MB-435 cell lines

GGH Overexpression GGH Inhibition

Down- Up- Down- Up- Total Total regulation regulation regulation regulation

HCT116 FC |1.3| 91 61 152 139 182 321 (0.3%) (0.2%) (0.5%) (0.4%) (0.6%) (1.0%)

FC |1.5| 36 21 57 58 80 138

(0.11%) (0.07%) (0.18%) (0.18%) (0.26%) (0.44%)

MDA-MB-435 FC |1.3| 628 755 1383 402 457 859 (2.0%) (2.4%) (4.4%) (1.3%) (1.4%) (2.7%) FC |1.5| 362 426 788 154 210 364 (1.2%) (1.3%) (2.5%) (0.5%) (0.7%) (1.2%)

FC, fold change; The numbers in brackets indicate the percentage of genes differentially expressed relative to total genes targeted in the Illumina HumanHT-12 v4.0 BeadChip.

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Figure 6. 10 Two-dimensional hierarchical clustering of gene expression fold change in the GGH-modulated HCT116 cell line. A color gradient from green to red indicated the low and high fold change, respectively.

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Figure 6. 11 Two-dimensional hierarchical clustering of gene expression fold change in the GGH-modulated MDA-MB-435 cell line. A color gradient from green to red indicated the low and high fold change, respectively.

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6.4.4.1 Genes Differentially Expressed in the GGH-Modulated HCT116 Cells

As a result of the classification according to function using IPA, genes involved in cellular movement, cell death, carbohydrate metabolism, cellular function and maintenance, and small molecular biochemistry were identified in the GGH-overexpressed HCT116 cells, while genes associated with cell death, cell cycle, cellular movement, cellular development, and cellular growth and proliferation were identified in the GGH-inhibited HCT116 cells (Table 6.11). The list of genes for each top function is shown in Appendix 6. The top ten genes most differentially expressed in the GGH-modulated HCT116 cells are shown in Tables 6.12 and 6.13.

Assignment of biological processes and subsequent construction of networks was performed using IPA. A table with functions and significance scores of the top five functions is presented in Table 6.14. The focus genes indicate the number of genes from our data set involved in each of the networks. The functions of network showing the highest score included cellular movement, dermatological disease and conditions, and carbohydrate metabolism in the HCT116 cells that overexpressed GGH, whereas those of the highest-scoring network included hereditary disorder, neurological disease, and cardiovascular system development and function in the HCT116 cells in which GGH was inhibited (Table 6.14). The list of genes for each top network in the GGH- modulated HCT116 is shown in Appendix 7.

Table 6. 11 The top molecular and cellular functions associated with differentially expressed genes in the GGH-modulated HCT116 colon cancer cells

GGH Overexpression GGH Inhibition No. of No. of Category P-value Category P-value Genes Genes Cellular Movement 2.74E-04 - 22 Cell Death 2.11E-06 - 77 4.72E-02 3.93E-02 Cell Death 9.04E-04 - 37 Cell Cycle 2.91E-05 - 39 4.72E-02 3.93E-02 Carbohydrate Metabolism 1.02E-03 - 6 Cellular Movement 3.65E-05 - 41 2.86E-02 3.93E-02 Cellular Function and 1.02E-03 - 11 Cellular Development 6.74E-05 - 55 Maintenance 4.72E-02 3.93E-02 Small Molecule Biochemistry 1.02E-03 - 15 Cellular Growth and 6.74E-05 - 70 4.72E-02 Proliferation 3.93E-02

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Table 6. 12 List of the top differentially expressed genes in the GGH-overexpressed HCT116 colon cancer cells

Gene Fold Description Accession Symbol Change Downregulated POLE4 -6.53 polymerase (DNA-directed), epsilon 4 (p12 subunit) NM_019896.2 PVRL3* -3.86 poliovirus receptor-related 3 NM_015480.1 NAP1L1* -3.70 nucleosome assembly protein 1-like 1 NM_139207.1 PHLDB2* -2.74 pleckstrin homology-like domain, family B, member 2 NM_145753.1 TNFRSF6B* -2.61 tumor necrosis factor receptor superfamily, member 6b, NM_032945.2 decoy AKAP12* -2.44 A kinase (PRKA) anchor protein (gravin) 12 NM_144497.1 SUSD2 -2.27 sushi domain containing 2 NM_019601.3 BMP4* -2.19 bone morphogenetic protein 4 NM_130851.1 CDK6 -2.05 cyclin-dependent kinase 6 NM_001259.5 RND3 -1.99 Rho family GTPase 3 NM_005168.3 Upregulated FGFBP1 4.08 fibroblast growth factor binding protein 1 NM_005130.3 RERG 2.97 RAS-like, estrogen-regulated, growth inhibitor NM_032918.1 GDF15 2.44 growth differentiation factor 15 NM_004864.1 SCG2 2.21 secretogranin II (chromogranin C) NM_003469.3 LCN2 1.93 lipocalin 2 NM_005564.3 UPP1 1.90 uridine phosphorylase 1 NM_003364.2 PLAU 1.89 plasminogen activator, urokinase NM_002658.2 GAS6 1.86 growth arrest-specific 6 NM_000820.1 HIST1H2BK 1.81 histone cluster 1, H2bk NM_080593.1 PBX1 1.68 pre-B-cell leukemia homeobox 1 NM_002585.1

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

164

Table 6. 13 List of the top differentially expressed genes in the GGH-inhibited HCT116 colon cancer cells

Gene Fold Description Accession Symbol Change Downregulated ANXA10 -6.85 annexin A10 NM_007193.3 ZBED2 -3.48 zinc finger, BED-type containing 2 NM_024508.3 DHRS2* -3.33 dehydrogenase/reductase (SDR family) member 2 NM_182908.3 KRT80 -2.76 keratin 80 NM_182507.2 TACSTD2 -2.38 tumor-associated calcium signal transducer 2 NM_002353.1 PHLDB2* -2.23 pleckstrin homology-like domain, family B, member 2 NM_145753.1 NR4A2 -2.22 nuclear receptor subfamily 4, group A, member 2 NM_006186.2 NCAPD3 -2.07 non-SMC condensin II complex, subunit D3 NM_015261.2 HYOU1* -2.01 hypoxia up-regulated 1 NM_006389.2 APPL1 -1.99 adaptor protein, phosphotyrosine interaction, PH domain NM_012096.2 and leucine zipper containing 1 Upregulated IFI27 3.91 interferon, alpha-inducible protein 27 NM_005532.3 PDE4B* 3.24 phosphodiesterase 4B, cAMP-specific (phosphodiesterase NM_002600.3 E4 dunce homolog, Drosophila) CA2* 3.08 carbonic anhydrase II NM_000067.1 SNTB1 2.75 syntrophin, beta 1 (dystrophin-associated protein A1, NM_021021.2 59kDa, basic component 1) TNFRSF6B* 2.49 tumor necrosis factor receptor superfamily, member 6b, NM_032945.2 decoy CALB2 2.47 calbindin 2 NM_007088.2 ABLIM1* 2.37 actin binding LIM protein 1 NM_006720.3 SUMO3 2.36 SMT3 suppressor of mif two 3 homolog 3 NM_006936.2 KCNS3* 2.13 potassium voltage-gated channel, delayed-rectifier, NM_002252.3 subfamily S, member 3 FAT1* 2.04 FAT tumor suppressor homolog 1 (Drosophila) NM_005245.3

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

165

Table 6. 14 The top networks matched by the genes differentially expressed in the GGH- modulated HCT116 colon cancer cells

GGH Overexpression GGH Inhibition

Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cellular Movement, 40 24 Hereditary Disorder, 37 27 Dermatological Diseases and Neurological Disease, Conditions, Carbohydrate Cardiovascular System Metabolism Development and Function 2 Cell Cycle, Cancer, 20 15 Cell Death, Cellular Movement, 35 26 Hematological Disease Hematological System Development and Function 3 DNA Replication, 17 13 Cancer, Cell Cycle, Connective 29 23 Recombination, and Repair, Tissue Development and Cancer, Renal and Urological Function Disease 4 Cellular Growth and 15 12 Cell Cycle, Infectious Disease, 25 21 Proliferation, Cancer, Cell Cellular Response to Death Therapeutics 5 Cell-To-Cell Signaling and 15 12 Cellular Assembly and 22 19 Interaction, Hematological Organization, Carbohydrate System Development and Metabolism, Drug Metabolism Function, Immune Cell Trafficking

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

166

6.4.4.2 Genes Differentially Expressed in the GGH-Modulated MDA-MB-435 Cells

A functional characterization revealed genes involved in cell-to-cell signaling and interaction, cellular movement, cell death, cellular development, and cellular growth and proliferation were identified in the GGH-overexpressed MDA-MB-435 cells, while genes associated with cell death, cellular development, cellular growth and proliferation, cellular movement, and cell morphology were identified in the GGH-inhibited MDA-MB-435 cells (Table 6.15). The list of genes for each top function is shown in Appendix 6. The top ten genes most differentially expressed in the GGH-modulated MDA-MB-435 cells are shown in Tables 6.16 and 6.17.

As a result of network construction for biological processes using IPA, the functions of network showing the highest score included infectious disease, dermatological disease and conditions, and tissue morphology in the GGH-overexpressed MDA-MB-435 cells, whereas those of the highest-scoring network included developmental disorder, renal and urological disease, and protein synthesis in MDA-MB-435 cells in which GGH was inhibited (Table 6.18). A table with functions, involved genes, and significance scores of top five functions is presented in Table 6.18. The list of genes for each top network in the GGH-modulated MDA-MB-435 cells is shown in Appendix 7.

Table 6. 15 The top molecular and cellular functions associated with differentially expressed genes in the GGH-modulated MDA-MB-435 breast cancer cells

GGH Overexpression GGH Inhibition No. of No. of Category P-value Category P-value Genes Genes Cell-To-Cell Signaling and 6.49E-08 - 73 Cell Death 1.62E-08 - 173 Interaction 4.86E-02 3.18E-02 Cellular Movement 7.16E-07 - 157 Cellular Development 2.76E-08 - 138 4.89E-02 3.18E-02 Cell Death 2.20E-06 - 241 Cellular Growth and 2.76E-08 - 173 4.89E-02 Proliferation 3.18E-02 Cellular Development 3.36E-04 - 175 Cellular Movement 5.57E-07 - 101 4.65E-02 2.21E-02 Cellular Growth and 3.36E-04 - 241 Cell Morphology 1.40E-05 - 57 Proliferation 4.86E-02 2.88E-02

167

Table 6. 16 List of the top differentially expressed genes in the GGH-overexpressed MDA-MB-435 breast cancer cells

Gene Fold Description Accession Symbol Change Downregulated TYR -31.95 tyrosinase (oculocutaneous albinism IA) NM_000372.4 TSPAN7* -21.45 tetraspanin 7 NM_004615.2 SNCA* -11.04 synuclein, alpha (non A4 component of amyloid NM_007308.1 precursor) IGSF11 -9.74 immunoglobulin superfamily, member 11 NM_001015887.1 ALDH1A1* -9.45 aldehyde dehydrogenase 1 family, member A1 NM_000689.3 DCT -9.42 dopachrome tautomerase (dopachrome delta-, NM_001922.2 -related protein 2) CHCHD6 -8.00 coiled-coil-helix-coiled-coil-helix domain containing 6 NM_032343.1 TRIM48 -7.89 tripartite motif-containing 48 NM_024114.2 DYNC1I1 -5.83 dynein, cytoplasmic 1, intermediate chain 1 NM_004411.3 GPM6B* -5.59 M6B NM_001001995.1 Upregulated S100A4* 13.13 S100 calcium binding protein A4 NM_019554.2 FST* 10.32 follistatin NM_013409.1 FGFRL1* 6.83 fibroblast growth factor receptor-like 1 NM_021923.3 PLOD2* 6.56 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 NM_000935.2 HLA-DQA1 6.26 PREDICTED: major histocompatibility complex, class II, XM_936128.2 DQ alpha 1, transcript variant 10 CNN3 5.93 calponin 3, acidic NM_001839.2 COL13A1* 5.76 collagen, type XIII, alpha 1 NM_080805.2 SERPINA3 5.74 serpin peptidase inhibitor, clade A (alpha-1 NM_001085.4 antiproteinase, antitrypsin), member 3 AKR1C3 5.49 aldo-keto reductase family 1, member C3 (3-alpha NM_003739.4 hydroxysteroid dehydrogenase, type II) AHNAK* 5.17 AHNAK nucleoprotein NM_024060.2

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

168

Table 6. 17 List of the top differentially expressed genes in the GGH-inhibited MDA- MB-435 breast cancer cells

Gene Fold Description Accession Symbol Change Downregulated CTHRC1* -4.44 collagen triple helix repeat containing 1 NM_138455.2 NNMT -3.24 nicotinamide N-methyltransferase NM_006169.2 HLA-DOA -3.09 major histocompatibility complex, class II, DO alpha NM_002119.3 FSCN1 -3.03 fascin homolog 1, actin-bundling protein NM_003088.2 (Strongylocentrotus purpuratus) CAP2 -2.75 CAP, adenylate cyclase-associated protein, 2 (yeast) NM_006366.2 ARHGEF3 -2.68 Rho guanine nucleotide exchange factor (GEF) 3 NM_019555.1 CDC42EP5 -2.53 CDC42 effector protein (Rho GTPase binding) 5 NM_145057.2 LMCD1 -2.40 LIM and cysteine-rich domains 1 NM_014583.2 CHN1 -2.30 chimerin (chimaerin) 1 NM_001025201.1 SLC2A3 -2.24 solute carrier family 2 (facilitated glucose transporter), NM_006931.1 member 3 Upregulated CXorf26 10.42 chromosome X open reading frame 26 NM_016500.3 SPP1* 5.88 secreted phosphoprotein 1 NM_001040058.1 SLC30A1* 5.55 solute carrier family 30 (zinc transporter), member 1 NM_021194.2 CCL20 4.88 chemokine (C-C motif) ligand 20 NM_004591.1 PRSS7* 4.66 , serine, 7 (enterokinase) NM_002772.1 IL8* 4.15 interleukin 8 NM_000584.2 TYR 4.07 tyrosinase (oculocutaneous albinism IA) NM_000372.4 SLITRK4 3.74 SLIT and NTRK-like family, member 4 NM_173078.2 LAIR2* 3.57 leukocyte-associated immunoglobulin-like receptor 2 NM_021270.2 FAM133A 3.45 family with sequence similarity 133, member A NM_173698.1

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

169

Table 6. 18 The top networks matched by the genes differentially expressed in the GGH- modulated MDA-MB-435 breast cancer cells

GGH Overexpression GGH Inhibition

Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Infectious Disease, 37 35 Developmental Disorder, Renal 34 31 Dermatological Diseases and and Urological Disease, Protein Conditions, Tissue Morphology Synthesis 2 Cellular Development, 30 32 Cellular Movement, Cellular 32 30 Developmental Disorder, Growth and Proliferation, Cell Hereditary Disorder Death 3 Molecular Transport, Cell 30 32 Tumor Morphology, Cell Cycle, 32 30 Death, Tumor Morphology Connective Tissue Development and Function 4 Cellular Movement, Cell-To- 26 30 Cellular Movement, 30 29 Cell Signaling and Interaction, Hematological System Tissue Development Development and Function, Immune Cell Trafficking 5 Cell Death, Dermatological 26 30 Cell Death, Cellular Assembly 25 26 Diseases and Conditions, and Organization, Cellular Immunological Disease Compromise

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

6.4.4.3 Genes Commonly Differentially Expressed in the GGH-Modulated HCT116 and MDA-MB-435 Cells

A venn diagram shows the number of genes commonly differentially expressed in both the GGH-modulated HCT116 and MDA-MB-435 cell lines (Figure 6.12). Nine down- and 10 upregulated genes were common between HCT116 and MDA-MB-435 cell lines in response to GGH overexpression (Figure 6.12A), while 11 down- and 12 upregulated genes were common between these cell lines in response to GGH inhibition (Figure 6.12B).

170

A

B

Figure 6. 12 Number of genes commonly differentially expressed in both the HCT116 and MDA-MB-435 cells in response to GGH overexpression (A) and GGH inhibition (B)

In the GGH-overexpressed HCT116 and MDA-MB-435 cell lines, the commonly downregulated genes were associated with cellular growth and proliferation, cell cycle, gene expression, carbohydrate metabolism, and cellular function and maintenance, whereas the commonly upregulated genes were related to cell cycle, cellular development, molecular transport, small molecule biochemistry, and cellular growth and proliferation (Table 6.19). As for the GGH- inhibited HCT116 and MDA-MB-435 cell lines, the major function categories of the commonly downregulated genes included energy production, lipid metabolism, small molecule biochemistry, cell morphology, and cellular development, while those of genes upregulated in common consisted of cell cycle, cell death, cellular development, cellular growth and proliferation, and cell morphology (Table 6.19). The list of top networks generated by mapping the focus genes

171 that were commonly differentially expressed in both the GGH-modulated HCT116 and MDA- MB-435 cells is presented in Tables 6.20 and 6.21.

Table 6. 19 The top molecular and cellular functions associated with genes that are commonly differentially expressed in the GGH-modulated HCT116 and MDA-MB-435 cells

GGH Overexpression GGH Inhibition No. of No. of Category P-value Category P-value Genes Genes Downregulated Cellular Growth and 1.08E-03 – 1 Energy Production 4.63E-04 – 2 Proliferation 1.08E-03 1.50E-02 Cell Cycle 2.70E-03 – 1 Lipid Metabolism 4.63E-04 – 2 6.46E-03 1.50E-02 Gene Expression 4.85E-03 – 1 Small Molecule 4.63E-04 – 2 4.85E-03 Biochemistry 4.89E-02 Carbohydrate Metabolism 6.46E-03 – 1 Cell Morphology 6.58E-04 – 2 1.18E-02 1.89E-02 Cellular Function and 1.18E-02 – 1 Cellular Development 6.58E-04 – 4 Maintenance 1.18E-02 2.07E-02 Upregulated Cell Cycle 6.58E-04 – 1 Cell Cycle 2.11E-05 – 2 1.96E-02 2.46E-02 Cellular Development 6.58E-04 – 1 Cell Death 5.46E-04 - 4 1.96E-02 4.33E-02 Molecular Transport 6.58E-04 – 3 Cellular Development 6.49E-04 - 4 2.28E-02 4.18E-02 Small Molecule Biochemistry 6.58E-04 – 1 Cellular Growth and 6.49E-04 - 4 6.58E-04 Proliferation 4.18E-02 Cellular Growth and 1.97E-03 – 1 Cell Morphology 8.04E-04 – 2 Proliferation 1.97E-03 1.12E-02

172

Table 6. 20 The top networks matched by the genes differentially expressed in both the GGH-overexpressed HCT116 and MDA-MB-435 cells

Downregulation Upregulation Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Ophthalmic Disease, 3 1 Cell Morphology, 3 1 Gastrointestinal Disease, Developmental Disorder, Connective Tissue Disorders Digestive System Development and Function 2 Cellular Assembly and 3 1 Cell Cycle, Cancer, Organismal 2 1 Organization, Hair and Skin Injury and Abnormalities Development and Function, Developmental Disorder 3 Cell Cycle, Embryonic 3 1 Cell Cycle, DNA Replication, 2 1 Development, Tissue Recombination, and Repair, Morphology Cancer 4 Cell Cycle, Cell-To-Cell 3 1 Cell-mediated Immune 2 1 Signaling and Interaction, Response, Cellular Connective Tissue Development Development, Cellular Function and Function and Maintenance 5 Cell Death, Cellular Function 2 1 and Maintenance, Cellular Movement The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

Table 6. 21 The top networks matched by the genes differentially expressed in both the GGH-inhibited HCT116 and MDA-MB-435 cells

Downregulation Upregulation Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cell Death, Cellular Movement, 20 8 Cancer, Gastrointestinal 13 6 Cellular Growth and Disease, Cell Cycle Proliferation 2 Cell Death, Hair and Skin 3 1 Development and Function, Cancer 3 Organ Morphology, Skeletal 3 1 and Muscular System Development and Function, Tissue Development The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

173

6.4.5 Integrated Analysis of Gene Expression and Promoter DNA Methylation Changes

We performed the integrated analysis of differentially expressed genes and differentially methylated genes in the GGH-modulated HCT116 and MDA-MB-435 cells to identify genes, the differential expression of which was regulated by DNA methylation in response to GGH modulation. As shown in Figure 6.13, β-value difference and log2-transformed gene expression value difference between Sense and Control-S and between siRNA and Control-si are plotted on x-axis and y-axis, respectively. Red data points highlight those genes that are hypermethylated with a β-value difference > 0.2 and show < -1.3 fold change in their expression levels, while green data points indicate those genes that are hypomethylated with a β-value difference < -0.2 and show > 1.3 fold change in their expression levels. The number of genes with altered expression and CpG promoter DNA methylation in the GGH-modulated HCT116 and MDA- MB-435 cells are summarized in Table 6.22. We detected 21 and 148 genes whose expression was inversely regulated by promoter DNA methylation changes in the GGH-modulated HCT116 and MDA-MB-435 cells, respectively (Figure 6.13; Table 6.22). The list of genes with altered promoter DNA methylation and expression in the GGH-modulated HCT116 and MDA-MB-435 cell lines is shown in Appendix 8.

174

A B

C D

Figure 6. 13 Integrated analysis of gene expression and promoter DNA methylation changes between Sense and Control-S (A, C) and between siRNA and Control-si (B, D) in the GGH-modulated HCT116 (A, B) and MDA-MB-435 cells (C, D). Red data points highlight those genes that are hypermethylated with β-value difference > 0.2 and show < -1.3 fold change in their expression levels, while green data points indicate those genes that are hypomethylated with β-value difference < -0.2 and show > 1.3 fold change in their expression levels.

175

Table 6. 22 Summary of number of genes with altered expression and promoter DNA methylation in the GGH-modulated HCT116 and MDA-MB-435 cell lines

GGH Overexpression GGH Inhibition

Hypermethylated & Hypomethylated & Hypermethylated & Hypomethylated & Downregulated/ Upregulated/ Downregulated/ Upregulated/ Downregulated (%) Upregulated (%) Downregulated (%) Upregulated (%) HCT116 4/91 (4.4%) 1/61 (1.6%) 3/139 (2.2%) 13/182 (7.1%)

MDA-MB-435 26/628 (4.1%) 75/755 (9.9%) 17/402 (4.2%) 30/457 (6.6%)

The numbers in brackets indicate the percentage of genes whose expression was inversely regulated by promoter DNA methylation relative to genes differentially expressed.

6.4.5.1 Genes Regulated by DNA Methylation in the GGH-Modulated HCT116 Cells

To evaluate which biological and disease processes are most relevant to these genes, we performed the functional analysis using IPA. In the GGH-overexpressed HCT116 cells, FGFBP1 (fibroblast growth facto binding protein 1, hypomethylated and upregulated) was associated with cellular growth and proliferation (Table 6.23). As for the GGH-inhibited HCT116 cells, hypermethylated and downregulated genes were involved in cell-to-cell signaling and interaction, cellular development, cellular growth and proliferation, gene expression, and lipid metabolism, while hypomethylated and upregulated genes were associated with lipid metabolism, small molecule biochemistry, carbohydrate metabolism, cellular movement, and drug metabolism (Table 6.23). The list of top genes with altered promoter DNA methylation and expression in the GGH-modulated HCT116 cells is shown in Tables 6.24 and 6.25.

176

Table 6. 23 The top molecular and cellular functions associated with genes with altered expression and promoter methylation in the GGH-modulated HCT116 colon cancer cells GGH Overexpression GGH Inhibition No. of No. of Category P-value Category P-value Genes Genes Hypermethylated and Downregulated Cell-To-Cell Signaling 1.52E-04 - 1 and Interaction 5.78E-03 Cellular Development 1.52E-04 - 1 2.78E-02 Cellular Growth and 1.52E-04 - 1 Proliferation 3.13E-02 Gene Expression 1.52E-04 - 1 1.52E-04 Lipid Metabolism 1.52E-04 - 1 6.09E-04 Hypomethylated and Upregulated Cellular Growth and 8.22E-03 - 1 Lipid Metabolism 9.90E-04 - 3 Proliferation 8.22E-03 4.17E-02 Small Molecule 9.90E-04 - 5 Biochemistry 4.17E-02 Carbohydrate Metabolism 1.98E-03 - 1 7.89E-03 Cellular Movement 1.98E-03 - 1 1.98E-03 Drug Metabolism 1.98E-03 - 2 7.89E-03

Table 6. 24 List of the top genes with altered promoter methylation and expression in the GGH-overexpressed HCT116 colon cancer cells

DNA Methylation Gene Expression Gene Entrez Description Symbol β-Value Difference Fold Change Gene ID (vs. Control) (vs. Control) Hypermethylated and Downregulated SUSD2 sushi domain containing 2 0.26 -2.27 56241 MNS1 meiosis-specific nuclear structural 1 0.45 -1.70 55329 ZNF91 zinc finger protein 91 0.28 -1.70 7644 HLA-DRA major histocompatibility complex, 0.33 -1.37 3122 class II, DR alpha Hypomethylated and Upregulated FGFBP1 fibroblast growth factor binding -0.28 4.08 9982 protein 1

177

Table 6. 25 List of the top genes with altered promoter methylation and expression in the GGH-inhibited HCT116 colon cancer cells

DNA Methylation Gene Expression Gene Entrez Description Symbol β-Value Difference Fold Change Gene ID (vs. Control) (vs. Control) Hypermethylated and Downregulated IGFBP3* insulin-like growth factor binding 0.29 -1.84 3486 protein 3 SUSD2 sushi domain containing 2 0.23 -1.49 56241 ALDH1A3 aldehyde dehydrogenase 1 family, 0.47 -1.34 220 member A3 Hypomethylated and Upregulated SNTB1 syntrophin, beta 1 (dystrophin- -0.34 2.75 6641 associated protein A1, 59kDa, basic component 1) FAT1* FAT tumor suppressor homolog 1 -0.23 2.04 2195 (Drosophila) HIST1H2BK histone cluster 1, H2bk -0.34 1.87 85236 CRABP2 cellular retinoic acid binding -0.33 1.72 1382 protein 2 OSR1* odd-skipped related 1 (Drosophila) -0.54 1.69 130497 PRSS3 protease, serine, 3 (mesotrypsin) -0.25 1.60 5646 (PRSS3) SERPINB1 serpin peptidase inhibitor, clade B -0.37 1.55 1992 (ovalbumin), member 1 GTF3C1 general transcription factor IIIC, -0.21 1.52 2975 polypeptide 1, alpha 220kDa AKR1C3 aldo-keto reductase family 1, -0.30 1.47 8644 member C3 (3-alpha hydroxysteroid dehydrogenase, type II) CYB5A cytochrome b5 type A -0.25 1.47 1528 (microsomal)

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

The result of network analysis for biological processes by mapping focus genes whose expression was inversely regulated by CpG promoter methylation in the GGH-modulated HCT116 cells is presented in Table 6.26. In the GGH-overexpressed HCT116 cells, the functions of network shown the highest score included cell morphology, connective tissue

178 development and function, and digestive system development and function in hypermethylated and downregulated genes, whereas those of the highest-scoring network in hypomethylated and upregulated genes included cell cycle, DNA replication, recombination, and repair, and cellular compromise (Table 6.26). As for the GGH-inhibited HCT116 cells, the functions of network with the highest score included lipid metabolism, molecular transport, and small molecule biochemistry in hypermethylated and downregulated genes, while those of the highest-scoring network in hypomethylated and upregulated genes included cancer, reproductive system disease, and cellular development (Table 6.26). The list of genes for each top network in the GGH- modulated HCT116 cells is presented in Appendix 9.

Table 6. 26 The top networks matched by the genes with altered expression and promoter methylation in the GGH-modulated HCT116 colon cancer cells GGH Overexpression GGH Inhibition Focus Focus No Top Functions Score Top Functions Score Genes Genes Hypermethylated and Downregulated 1 Cell Morphology, Connective 3 1 Lipid Metabolism, Molecular 3 1 Tissue Development and Transport, Small Molecule Function, Digestive System Biochemistry Development and Function 2 Cell Cycle, Cell-To-Cell 3 1 Gene Expression, Organismal 2 1 Signaling and Interaction, Development, Cellular Growth Connective Tissue Development and Proliferation and Function 3 Genetic Disorder, 2 1 Immunological Disease, Gene Expression Hypomethylated and Upregulated 1 Cell Cycle, DNA Replication, 3 1 Cancer, Reproductive System 9 5 Recombination, and Repair, Disease, Cellular Development Cellular Compromise 2 Embryonic Development, 3 1 Organismal Development, Tissue Development 3 Developmental Disorder, 2 1 Endocrine System Disorders, Gastrointestinal Disease 4 Cancer, Embryonic 2 1 Development, Tissue Development 5 Reproductive System 2 1 Development and Function, Drug Metabolism, Lipid Metabolism The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

179

6.4.5.2 Genes Regulated by DNA Methylation in the GGH-Modulated MDA- MB-435 Cells The same set of analyses were performed for genes with altered promoter methylation and expression in MDA-MB-435 cells. In the MDA-MB-435 cells that overexpressed GGH, genes hypermethylated and downregulated were associated with cell signaling, cellular assembly and organization, cellular movement, drug metabolism, and lipid metabolism, while hypomethylated and upregulated genes were involved in cellular assembly and organization, cell-to-cell signaling and interaction, cellular growth and proliferation, cellular development, and cell death (Table 6.27). As for the MDA-MB-435 cells in which GGH is inhibited, hypermethylated and downregulated genes were involved in cell morphology, cellular development, gene expression, cellular assembly and organization, and cell-to-cell signaling and interaction, while hypomethylated and upregulated genes were associated with cell morphology, cell cycle, cellular growth and proliferation, cellular development, and cellular movement (Table 6.27). The list of top genes with altered promoter DNA methylation and expression in the GGH-modulated MDA- MB-435 cells is shown in Tables 6.28 and 6.29.

Table 6. 27 The top molecular and cellular functions associated with genes with altered expression and promoter methylation in the GGH-modulated MDA-MB-435 breast cancer cells GGH Overexpression GGH Inhibition No. of No. of Category P-value Category P-value Genes Genes Hypermethylated and Downregulated Cell Signaling 1.90E-03 - 4 Cell Morphology 6.45E-03 - 1 4.29E-02 2.18E-02 Cellular Assembly and 1.90E-03 - 2 Cellular Development 6.45E-03 - 2 Organization 5.70E-03 2.18E-02 Cellular Movement 1.90E-03 - 2 Gene Expression 6.45E-03 - 1 4.11E-02 6.45E-03 Drug Metabolism 1.90E-03 - 1 Cellular Assembly and 1.42E-02 - 2 1.32E-02 Organization 3.31E-02 Lipid Metabolism 1.90E-03 - 2 Cell-To-Cell Signaling 1.54E-02 - 1 1.32E-02 and Interaction 1.54E-02 Hypomethylated and Upregulated Cellular Assembly and 1.59E-03 - 7 Cell Morphology 3.49E-06 - 7 Organization 4.86E-02 3.31E-02 Cell-To-Cell Signaling and 2.25E-03 - 7 Cell Cycle 3.77E-06 - 6 Interaction 4.43E-02 4.27E-02 Cellular Growth and 2.41E-03 - 17 Cellular Growth and 4.71E-05 - 14 Proliferation 4.46E-02 Proliferation 4.65E-02 Cellular Development 2.50E-03 - 8 Cellular Development 7.86E-05 - 8 3.88E-02 4.75E-02 Cell Death 4.24E-03 - 13 Cellular Movement 2.05E-04 - 7 4.65E-02 4.27E-02

180

Table 6. 28 List of the top genes with altered promoter methylation and expression in the GGH-overexpressed MDA-MB-435 breast cancer cells

DNA Methylation Gene Expression Gene Entrez Description Symbol β-Value Difference Fold Change Gene ID (vs. Control) (vs. Control) Hypermethylated and Downregulated TYR tyrosinase (oculocutaneous albinism IA) 0.26 -31.95 7299 TSPAN7* tetraspanin 7 0.34 -21.45 7102 APOD apolipoprotein D 0.23 -5.41 347 CTSL2 cathepsin L2 0.23 -3.78 1515 PLSCR1 phospholipid scramblase 1 0.28 -3.63 5359 GPR143 G protein-coupled receptor 143 0.55 -3.44 4935 MSRB2 methionine sulfoxide reductase B2 0.46 -3.20 22921 KCNAB1 potassium voltage-gated channel, 0.36 -2.95 7881 shaker-related subfamily, beta member 1 EDNRB endothelin receptor type B 0.25 -2.61 1910 CMTM8 CKLF-like MARVEL transmembrane 0.25 -2.35 152189 domain containing 8 Hypomethylated and Upregulated S100A4* S100 calcium binding protein A4 -0.24 13.13 6275 FGFRL1* fibroblast growth factor receptor-like 1 -0.21 6.83 53834 PARVA* parvin, alpha -0.29 5.04 55742 NNMT nicotinamide N-methyltransferase -0.37 4.80 4837 HTATIP2 HIV-1 Tat interactive protein 2, 30kDa -0.35 4.75 10553 CRYAB crystallin, alpha B -0.46 4.48 1410 CPVL* carboxypeptidase, vitellogenic-like -0.46 4.11 54504 KLRC2 killer cell lectin-like receptor subfamily -0.34 4.09 3822 C, member 2 COL8A1* collagen, type VIII, alpha 1 -0.33 3.93 1295 RAC2 ras-related C3 botulinum toxin substrate -0.28 3.93 5880 2 (rho family, small GTP binding protein Rac2)

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

181

Table 6. 29 List of the top genes with altered promoter methylation and expression in the GGH-inhibited MDA-MB-435 breast cancer cell

DNA Methylation Gene Expression Gene Entrez Description Symbol β-Value Difference Fold Change Gene ID (vs. Control) (vs. Control) Hypermethylated and Downregulated CTHRC1* collagen triple helix repeat 0.55 -4.44 115908 containing 1 CDC42EP5 CDC42 effector protein (Rho 0.24 -2.53 148170 GTPase binding) 5 LMCD1 LIM and cysteine-rich domains 1 0.40 -2.40 29995 AMPH amphiphysin 0.35 -2.17 273 HLA-DMA major histocompatibility complex, 0.21 -1.98 3108 class II, DM alpha PCOLCE* procollagen C-endopeptidase 0.27 -1.69 5118 enhancer IL11RA interleukin 11 receptor, alpha 0.21 -1.63 3590 SORBS2* sorbin and SH3 domain containing 2 0.28 -1.60 8470 GYG2 glycogenin 2 0.24 -1.50 8908 AUTS2 autism susceptibility candidate 2 0.31 -1.50 26053 Hypomethylated and Upregulated CXorf26* chromosome X open reading frame 26 -0.40 10.42 51260 BCHE* butyrylcholinesterase -0.22 2.77 590 CDKN1A cyclin-dependent kinase inhibitor -0.29 2.57 1026 1A (p21, Cip1) DYNLT3* dynein, light chain, Tctex-type 3 -0.49 2.34 6990 APCDD1L adenomatosis polyposis coli down- -0.22 1.93 164284 regulated 1-like EDNRB endothelin receptor type B -0.85 1.89 1910 SLC22A18AS solute carrier family 22 (organic -0.25 1.86 5003 cation transporter), member 18 antisense MRGPRX4 MAS-related GPR, member X4 -0.35 1.83 117196 LEPREL1 leprecan-like 1 -0.21 1.81 55214 RENBP renin binding protein -0.52 1.73 5973

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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The top networks matched by genes with altered promoter methylation and expression in the GGH-modulated MDA-MB-435 cells are presented in Table 6.30. In the GGH-overexpressed MDA-MB-435 cells, the functions of network shown the highest score included cell morphology, reproductive system disease, and cardiovascular system development and function in hypermethylated and downregulated genes, while those of the highest-scoring network in hypomethylated and upregulated genes included cellular movement, cardiovascular system development and function, and cancer (Table 6.30). As for the GGH-inhibited MDA-MB-435 cells, the functions of network with the highest score included cellular development, cell-to-cell signaling and interaction, and hematological system development and function in hypermethylated and downregulated genes, whereas those of the highest-scoring network in hypomethylated and upregulated genes included cellular growth and proliferation, tumor morphology, and cell death (Table 6.30). The list of genes for each top network in the GGH- modulated MDA-MB-435 cells is presented in Appendix 9.

Table 6. 30 The top networks matched by the genes with altered expression and promoter methylation in the GGH-modulated MDA-MB-435 breast cancer cells

GGH Overexpression GGH Inhibition Focus Focus No Top Functions Score Top Functions Score Genes Genes Hypermethylated and Downregulated 1 Cell Morphology, Reproductive 28 13 Cellular Development, Cell-To- 17 8 System Disease, Cardiovascular Cell Signaling and Interaction, System Development and Hematological System Function Development and Function 2 Carbohydrate Metabolism, Cell 2 1 Cellular Compromise, Cellular 3 1 Death, Cellular Assembly and Development, Connective Organization Tissue Development and Function 3 Cardiovascular System 2 1 Gene Expression, 3 1 Development and Function, Cardiovascular Disease, Cellular Organ Morphology, Organismal Growth and Proliferation Injury and Abnormalities 4 Cell Death, Cellular Function 2 1 Gene Expression, 3 1 and Maintenance, Reproductive Cardiovascular Disease, Cellular System Development and Growth and Proliferation Function 5 RNA Damage and Repair, 2 1 Developmental Disorder, Genetic Disorder

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Hypomethylated and Upregulated 1 Cellular Movement, 23 14 Cellular Growth and 20 10 Cardiovascular System Proliferation, Tumor Development and Function, Morphology, Cell Death Cancer 2 Cellular Development, Cell 21 13 Cellular Movement, Cell Cycle, 11 6 Cycle, Cancer Cell-To-Cell Signaling and Interaction 3 Cellular Movement, Skeletal 19 12 Cell-To-Cell Signaling and 2 1 and Muscular System Interaction, Molecular Development and Function, Cell Transport, Small Molecule Death Biochemistry

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

6.4.6 Validation of Gene Expression by qRT-PCR of Selected Genes Identified from Illumina HumanHT-12 v4.0 BeadChip

We performed qRT-PCR to validate the gene expression results of selected genes which was inversely regulated by CpG promoter methylation changes discussed in Section 6.4.5. We selected genes based on a greater magnitude of fold change in gene expression identified from microarray and relevant biological pathways of interest. As presented in Table 6.31, the direction of change in gene expression in response to GGH modulation was significantly consistent between Illumina HumanHT-12 v4.0 BeadChip and qRT-PCR analyses in HCT116 and MDA-MB-435 cell lines (P < 0.05), although the magnitude of change was different. Generally, the direction and magnitude of fold change in gene expression detected by microarray and qRT-PCR were consistent in genes with the greater fold changes, while genes with the lesser fold changes were unlikely to have a similar magnitude of change between two analyses. It is presumed that difference in transcript(s) between the microarray probe set and qRT-PCR probes might influence different hybridization kinetics of the probe sets for gene (438). Overall, results determined by qRT-PCR were greater than fold change assessed for the same genes by microarray analysis consistent with the previous study (439).

184

Table 6. 31 Comparison of fold changes in gene expression detected by Illumina HumanHT-12 v4.0 BeadChip and qRT-PCR analyses in the GGH-modulated HCT116 and MDA-MB-435 cell lines

Gene Microarray qRT-PCR Description Symbol Fold Change Relative Expression Fold Hypermethylated & Downregulated HCT116 GGH Overexpression ZNF91* zinc finger protein 91 -1.70 0.16 (0.12-0.21) HLA-DRA major histocompatibility complex, class II, -1.37 0.55 (0.47-0.65) DR alpha GGH Inhibition ALDH1A3 aldehyde dehydrogenase 1 family, member -1.34 0.55 (0.45-0.68) A3 MDA-MB-435 GGH Overexpression TYR tyrosinase (oculocutaneous albinism IA) -31.95 0.0020 (0.0019-0.0021) APOD* apolipoprotein D -5.41 0.033 (0.026-0.043) MSRB2 methionine sulfoxide reductase B2 -3.20 0.06 (0.05-0.07) GGH Inhibition PCOLCE* procollagen C-endopeptidase enhancer -1.69 0.20 (0.19-0.21) IL11RA interleukin 11 receptor, alpha -1.63 0.25 (0.18-0.34) Hypomethylated & Upregulated HCT116 GGH Overexpression FGFBP1 fibroblast growth factor binding protein 1 4.08 10.43 (9.88-11.01) GGH Inhibition CRABP2 cellular retinoic acid binding protein 2 1.72 2.16 (1.85-2.53) AKR1C3* aldo-keto reductase family 1, member C3 1.47 7.01 (6.10-8.05) MMP7* matrix metallopeptidase 7 1.44 3.60 (3.28-3.95) MDA-MB-435 GGH Overexpression S100A4 S100 calcium binding protein A4 13.13 30.89 (29.20-32.68) NNMT nicotinamide N-methyltransferase 4.80 6.02 (4.51-8.03) IGFBP7 insulin-like growth factor binding protein 7 3.66 3.01 (2.43-3.72) GGH Inhibition BCHE butyrylcholinesterase 2.77 5.38 (4.65-6.21) CDKN1A cyclin-dependent kinase inhibitor 1A 2.57 4.93 (4.48-5.43) CCND1* cyclin D1 1.65 3.23 (2.90-3.59)

*, β-actin used for housekeeping gene; P < 0.05.

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6.4.7 Pathway-Specific Gene Expression Analysis

The lists of genes involved in folate biosynthesis, one-carbon pool by folate, cell cycle (G1/S and G2/M), and apoptosis were referred to the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/pathway.html) and the Ingenuity Knowledge Base. Among genes differentially regulated (> 1.3 or < -1.3 fold change vs. corresponding control) in response to GGH modulation in HCT116 and MDA-MB-435 cells, the lists of genes involved in specific pathways are presented in Tables 6.32 to 6.34. We identified genes that were commonly up- or downregulated in HCT116 and MDA-MB-435 cells and genes regulated in opposite directions between GGH overexpression and inhibition. Furthermore, genes that were either hypermethylated and downregulated or hypomethylated and upregulated in each system were identified.

6.4.7.1 Folate Biosynthesis and One-Carbon Pool by Folate Pathway

In MDA-MB-435 cells, NNMT, nicotinamide N-methyltransferase, was upregulated (fold change 4.8) in response to GGH overexpression, while it was downregulated (fold change -3.24) in response to GGH inhibition (Table 6.32). In the GGH-overexpressed MDA-MB-435 cells, increased expression of NNMT were related with hypomethylation (β-value difference -0.37; Table 6.28; Appendix 8.3), whereas ABCC5, ATP-binding cassette, sub-family C, member 5, was downregulated (fold change -1.34; Table 6.32) and hypermethylated (β-value difference 0.21; Appendix 8.3).

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Table 6. 32 List of differentially expressed genes involved in folate biosynthesis and one- carbon pool by folate pathways in the GGH-modulated HCT116 and MDA-MB-435 cells

Gene Fold Change Description Probe ID Pathway Symbol (vs. Control)

GGH Overexpression in HCT116 COMT 1.35 catechol-O-methyltransferase ILMN_1730084 folate

GGH Overexpression in MDA-MB-435 NNMT*† 4.80 nicotinamide N-methyltransferase ILMN_1715508 folate DPYD 1.51 dihydropyrimidine dehydrogenase ILMN_1795715 folate GSTT1 -2.72 glutathione S-transferase theta 1 ILMN_1730054 folate ADA -1.51 ILMN_1803686 folate ABCC5* -1.34 ATP-binding cassette, sub-family C ILMN_1706531 folate (CFTR/MRP), member 5 SLC25A32 -1.33 solute carrier family 25, member 32 ILMN_1683212 folate MTFMT -1.66 mitochondrial methionyl-tRNA formyltransferase ILMN_1672884 one-carbon

GGH Inhibition in MDA-MB-435 NNMT† -3.24 nicotinamide N-methyltransferase ILMN_1715508 folate ADA -1.31 adenosine deaminase ILMN_1803686 folate

*, genes whose expression was regulated by DNA methylation; †, genes expressed in opposite directions between GGH overexpression and inhibition in the same cell line.

6.4.7.2 Cell Cycle Pathway

In MDA-MB-435 cells, CDK2 (cyclin-dependent kinase 2) was downregulated (fold change - 1.76) in response to GGH overexpression, and upregulated (fold change 1.40) in response to GGH inhibition (Table 6.33). CDKN1A (cyclin-dependent kinase inhibitor 1A [p21, Cip1]) was upregulated in both the GGH-inhibited HCT116 and MDA-MB-435 cells (Table 6.33). Furthermore, the CDKN1A and CCND1 (cyclin D1) were related to hypomethylation and upregulation in the GGH-inhibited MDA-MB-435 cells (β-value difference -0.29 for CDKN1A and -0.23 for CCND1; Appendix 8.4; Tables 6.29 and 6.33). CDKN1A and CCND1 were also upregulated in the GGH-overexpressed MDA-MB-435 cells (Table 6.33). Increased expression of HDAC9 (histone deacetylase 9) was related to hypomethylation (β-value difference -0.29; Appendix 8.3) in the GGH-overexpressed MDA-MB-435 cells.

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Table 6. 33 List of differentially expressed genes involved in the cell cycle pathway in the GGH-modulated HCT116 and MDA-MB-435 cells

Gene Fold Change Description Probe ID Pathway Symbol (vs. Control)

GGH Overexpression in HCT116 CDK6 -2.05 cyclin-dependent kinase 6 ILMN_1802615 G1/S GGH Inhibition in HCT116 CDKN1A§ 1.63 cyclin-dependent kinase inhibitor 1A (p21, Cip1) ILMN_1784602 G1/S, G2/M CDK6 -1.67 cyclin-dependent kinase 6 ILMN_1802615 G1/S GGH Overexpression in MDA-MB-435 CDKN1A 2.29 cyclin-dependent kinase inhibitor 1A (p21, Cip1) ILMN_1784602 G1/S, G2/M CCND1 2.07 cyclin D1 ILMN_1688480 G1/S HDAC9* 1.60 histone deacetylase 9 ILMN_1781173 G1/S CDKN2B 1.48 cyclin-dependent kinase inhibitor 2B (p15, ILMN_2376723 G1/S inhibits CDK4) MAX 1.36 MYC associated factor X ILMN_1706546 G1/S CDK2† -1.76 cyclin-dependent kinase 2 ILMN_1665559 G1/S PRKCZ 1.77 protein kinase C, zeta ILMN_2386982 G2/M PLK1 1.48 polo-like kinase 1 (Drosophila) ILMN_1736176 G2/M CCNB1 1.34 cyclin B1 ILMN_1712803 G2/M KAT2B -2.16 K(lysine) acetyltransferase 2B ILMN_3243142 G2/M YWHAZ -1.60 tyrosine 3-monooxygenase/ 5- ILMN_1801928 G2/M monooxygenase activation protein, zeta polypeptide -1.57 ILMN_1669286 PRKDC -1.51 protein kinase, DNA-activated, catalytic ILMN_2334121 G2/M polypeptide -1.36 ILMN_2253648 GGH Inhibition in MDA-MB-435 CDKN1A§* 2.57 cyclin-dependent kinase inhibitor 1A (p21, Cip1) ILMN_1784602 G1/S, G2/M CCND1* 1.65 cyclin D1 ILMN_1688480 G1/S SMAD3 1.60 SMAD family member 3 ILMN_1682738 G1/S GSK3B 1.44 glycogen synthase kinase 3 beta ILMN_1779376 G1/S CDK2† 1.40 cyclin-dependent kinase 2 ILMN_1665559 G1/S CUL1 1.39 cullin 1 ILMN_1749629 G1/S, G2/M CDKN2B 1.38 cyclin-dependent kinase inhibitor 2B (p15, ILMN_2376723 G1/S inhibits CDK4) CCNE1 1.36 cyclin E1 ILMN_2374425 G1/S HDAC4 -1.61 histone deacetylase 4 ILMN_1764396 G1/S CDKN1B -1.37 cyclin-dependent kinase inhibitor 1B (p27, Kip1) ILMN_2196347 G1/S

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CHEK1 1.34 CHK1 checkpoint homolog (S. pombe) ILMN_1664630 G2/M TOP2A -1.42 topoisomerase (DNA) II alpha 170kDa ILMN_1686097 G2/M TOP2B -1.36 topoisomerase (DNA) II beta 180kDa ILMN_1777663 G2/M

*, genes whose expression was regulated by DNA methylation; †, genes expressed in opposite directions between GGH overexpression and inhibition in the same cell line; §, genes differentially expressed in both the GGH-modulated HCT116 and MDA-MB-435 cells.

6.4.7.3 Apoptosis Pathway

We observed that decreased expression of GAS2 (growth arrest-specific 2), expression of which was inversely regulated by CpG promoter methylation in the GGH-overexpressed MDA-MB- 435 cells (fold change -1.32, Table 6.34, Appendix 8.3; β-value difference 0.32, Appendix 8.3).

Table 6. 34 List of differentially expressed genes involved in the apoptosis pathway in the GGH-modulated HCT116 and MDA-MB-435 cells

Gene Fold Change Description Probe ID Pathway Symbol (vs. Control)

GGH Overexpression in HCT116 PRKCA -1.38 protein kinase C, alpha ILMN_1771800 apoptosis GGH Overexpression in MDA-MB-435 PRKCA 1.74 protein kinase C, alpha ILMN_1771800 apoptosis BCL2A1 1.54 BCL2-related protein A1 ILMN_1769229 apoptosis MCL1 1.41 myeloid cell leukemia sequence 1 (BCL2-related) ILMN_1803988 apoptosis CASP9 1.30 caspase 9, apoptosis-related cysteine peptidase ILMN_1718070 apoptosis CYCS -1.34 cytochrome c, somatic, nuclear gene encoding ILMN_1730416 apoptosis mitochondrial protein GAS2* -1.32 growth arrest-specific 2 ILMN_1804569 apoptosis BCL2 -1.31 B-cell CLL/lymphoma 2, nuclear gene encoding ILMN_1801119 apoptosis mitochondrial protein GGH Inhibition in MDA-MB-435 BCL2L1 1.48 BCL2-like 1, nuclear gene encoding ILMN_1654118 apoptosis mitochondrial protein BCL2A1 1.40 BCL2-related protein A1 ILMN_1769229 apoptosis MCL1 1.38 myeloid cell leukemia sequence 1 (BCL2-related) ILMN_1803988 apoptosis

*, genes whose expression was regulated by DNA methylation.

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6.4.8 GGH-Specific Gene Expression Analysis

To investigate genes associated with the function of GGH, we identified genes differentially expressed in the opposite direction between GGH overexpression and inhibition.

6.4.8.1 GGH-Specific Gene Expression in HCT116 Cells

The following venn diagrams show the number of genes differentially expressed in opposite directions in the GGH-modulated HCT116 cells (Figure 6.14).

Figure 6. 14 Number of genes differentially expressed in the opposite direction between GGH overexpression and inhibition in the GGH-modulated HCT116 colon cancer cells

Twenty-eight genes that were downregulated in response to GGH overexpression and upregulated in response to GGH inhibition were associated with cellular movement, cell death, cellular growth and proliferation, cell-to-cell signaling and interaction, and RNA post- transcriptional modification (Figure 6.14; Table 6.35). Eleven genes were upregulated in response to GGH overexpression and downregulated in response to GGH inhibition, and these genes were involved in carbohydrate metabolism, cellular function and maintenance, small molecule biochemistry, lipid metabolism, and molecular transport (Figure 6.14; Table 6.35). The list of top networks generated by mapping the focus genes associated with the GGH-specific altered expression in the GGH-modulated HCT116 cells is presented in Table 6.36. The list of genes associated with the GGH-specific altered expression in the GGH-modulated HCT116 cells is presented in Appendix 10, and the top ten genes are shown in Table 6.37.

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Table 6. 35 The top molecular and cellular functions associated with the GGH-specific gene expression in the GGH-modulated HCT116 colon cancer cells

Downregulated in GGH Overexpression and Upregulated in GGH Overexpression and

Upregulated in GGH Inhibition Downregulated in GGH Inhibition No. of No. of Category P-value Category P-value Genes Genes Cellular Movement 1.69E-03 - 3 Carbohydrate Metabolism 7.53E-05 - 2 4.30E-02 1.93E-02 Cell Death 2.59E-03 - 7 Cellular Function and 7.53E-05 - 3 4.83E-02 Maintenance 1.45E-02 Cellular Growth and 3.37E-03 - 4 Small Molecule 7.53E-05 - 4 Proliferation 1.89E-02 Biochemistry 4.87E-02 Cell-To-Cell Signaling and 6.73E-03 - 2 Lipid Metabolism 1.93E-04 - 2 Interaction 4.94E-02 4.58E-02 RNA Post-Transcriptional 7.00E-03 - 3 Molecular Transport 1.93E-04 - 2 Modification 2.17E-02 3.94E-02

Table 6. 36 The top networks matched by the genes with the GGH-specific altered expression in the GGH-modulated HCT116 colon cancer cells

Downregulated in GGH Overexpression Upregulated in GGH Overexpression and

and Upregulated in GGH Inhibition Downregulated in GGH Inhibition Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cellular Development, 25 12 Cellular Movement, Cellular 14 6 Hematological System Development, Cellular Growth Development and Function, and Proliferation Hematopoiesis 2 Developmental Disorder, 6 4 Cellular Development, Cellular 3 1 Hematological Disease, Growth and Proliferation, Hereditary Disorder Embryonic Development 3 Developmental Disorder, 2 1 Hereditary Disorder, Metabolic Disease 4 Ophthalmic Disease, 2 1 Connective Tissue Disorders, Gastrointestinal Disease 5 Cellular Movement, Hair and 2 1 Skin Development and Function, Embryonic Development

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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Table 6. 37 List of the top genes associated with the GGH-specific altered expression in the GGH-modulated HCT116 colon cancer cells

Fold Change (vs. Control) Gene Description Accession Symbol GGH GGH Overexpression Inhibition Downregulated in GGH Overexpression and Upregulated in GGH Inhibition

POLE4 -6.53 1.59 polymerase (DNA-directed), epsilon 4 (p12 NM_019896.2 subunit) PVRL3* -3.86 1.41 poliovirus receptor-related 3 NM_015480.1 TNFRSF6B* -2.61 2.49 tumor necrosis factor receptor superfamily, NM_032945.2 member 6b, decoy PDE4B -1.77 3.24 phosphodiesterase 4B, cAMP-specific NM_002600.3 (phosphodiesterase E4 dunce homolog, Drosophila) TRIM33 -1.69 1.33 tripartite motif-containing 33 NM_015906.3 PYGL -1.68 1.37 phosphorylase, glycogen, liver NM_002863.3 ALG6 -1.59 1.66 asparagine-linked glycosylation 6 homolog (S. NM_013339.2 cerevisiae, alpha-1,3-glucosyltransferase) MTAP -1.59 1.36 methylthioadenosine phosphorylase NM_002451.3 MCOLN2 -1.54 1.51 mucolipin 2 NM_153259.2 SACS -1.52 1.52 spastic ataxia of Charlevoix-Saguenay (sacsin) NM_014363.3

Upregulated in GGH Overexpression and Downregulated in GGH Inhibition ANXA10 1.62 -6.85 annexin A10 NM_007193.3 TACSTD2 1.37 -2.38 tumor-associated calcium signal transducer 2 NM_002353.1 TMEM200A 1.67 -1.89 transmembrane protein 200A NM_052913.2 UPP1 1.90 -1.79 uridine phosphorylase 1 NM_003364.2 PBX1 1.68 -1.73 pre-B-cell leukemia homeobox 1 NM_002585.1 PHF19 1.37 -1.62 PHD finger protein 19 NM_001009936.1 RERG 2.97 -1.49 RAS-like, estrogen-regulated, growth inhibitor NM_032918.1 IRS1 1.38 -1.47 insulin receptor substrate 1 NM_005544.1 PPARG 1.37 -1.39 peroxisome proliferator-activated receptor NM_015869.4 gamma RRP7A 1.32 -1.36 ribosomal RNA processing 7 homolog A (S. NM_015703.3 cerevisiae)

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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6.4.8.2 GGH-Specific Gene Expression in MDA-MB-435 Cells

In the MDA-MB-435 cell line, 80 genes were downregulated in response to GGH overexpression and upregulated in response to GGH inhibition, and these genes were involved in cellular compromise, cell death, cell morphology, cellular movement, and cellular development (Figure 6.15; Table 6.38). One hundred thirty-three genes that were upregulated in response to GGH overexpression and downregulated in response to GGH inhibition were associated with cellular compromise, cell cycle, cellular assembly and organization, cellular function and maintenance, and cellular movement (Figure 6.15; Table 6.38). The list of top networks generated by mapping the focus genes associated with the GGH-specific altered expression in the GGH- modulated MDA-MB-435 cells is presented in Table 6.39. The list of genes associated with the GGH-specific altered expression in the GGH-modulated MDA-MB-435 cells is presented in Appendix 10, and the top ten genes are shown in Table 6.40.

Figure 6. 15 Number of genes differentially expressed in the opposite direction between GGH overexpression and inhibition in the GGH-modulated MDA-MB-435 breast cancer cells

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Table 6. 38 The top molecular and cellular functions associated with the GGH-specific gene expression in the GGH-modulated MDA-MB-435 breast cancer cells

Downregulated in GGH Overexpression and Upregulated in GGH Overexpression and

Upregulated in GGH Inhibition Downregulated in GGH Inhibition No. of No. of Category P-value Category P-value Genes Genes Cellular Compromise 4.04E-04 - 4 Cellular Compromise 7.00E-05 - 4 2.21E-02 1.53E-02 Cell Death 8.54E-04 - 22 Cell Cycle 3.48E-04 - 4 4.69E-02 4.53E-02 Cell Morphology 8.59E-04 - 11 Cellular Assembly and 2.54E-03 - 8 4.79E-02 Organization 4.53E-02 Cellular Movement 1.46E-03 - 12 Cellular Function and 2.54E-03 - 7 4.87E-02 Maintenance 4.53E-02 Cellular Development 2.53E-03 - 15 Cellular Movement 3.39E-03 - 20 4.55E-02 3.79E-02

Table 6. 39 The top networks matched by the genes with the GGH-specific altered expression in the GGH-modulated MDA-MB-435 breast cancer cells

Downregulated in GGH Overexpression Upregulated in GGH Overexpression and

and Upregulated in GGH Inhibition Downregulated in GGH Inhibition Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cellular Function and 22 13 Dermatological Diseases and 42 24 Maintenance, Cell Signaling, Conditions, Immunological Small Molecule Biochemistry Disease, Inflammatory Disease 2 Cancer, Cell Death, Cell Cycle 22 13 Tissue Morphology, Cell Cycle, 24 16 Cell Death 3 Cellular Movement, Cell Death, 20 12 Cell-To-Cell Signaling and 20 14 Cancer Interaction, Cellular Growth and Proliferation, Skeletal and Muscular System Development and Function 4 Dermatological Diseases and 2 1 Developmental Disorder, 16 12 Conditions, Hereditary Hematological Disease, Disorder, Cell Cycle Hereditary Disorder 5 Developmental Disorder, 2 1 Cell Morphology, Connective 16 12 Hereditary Disorder, Infectious Tissue Development and Disease Function, Skeletal and Muscular System Development and Function

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

194

Table 6. 40 List of the top genes associated with the GGH-specific altered expression in the GGH-modulated MDA-MB-435 breast cancer cells

Fold Change (vs. Control) Gene Description Accession Symbol GGH GGH Overexpression Inhibition

Downregulated in GGH Overexpression and Upregulated in GGH Inhibition

SPP1* -4.51 5.88 secreted phosphoprotein 1 NM_001040058.1 PRSS7* -1.94 4.66 protease, serine, 7 (enterokinase) NM_002772.1 TYR -31.95 4.07 tyrosinase (oculocutaneous albinism IA) NM_000372.4 CYB5R2 -1.34 3.05 cytochrome b5 reductase 2 NM_016229.3 BCHE* -4.82 2.31 butyrylcholinesterase NM_000055.2 ADM -2.63 2.31 adrenomedullin NM_001124.1 TMEM166 -3.05 2.07 transmembrane protein 166 NM_032181.1 ORC5L -1.35 2.07 origin recognition complex, subunit 5-like NM_002553.2 (yeast) PNLIPRP3 -1.34 2.05 pancreatic lipase-related protein 3 NM_001011709.1 DYNC1I1 -5.83 2.03 dynein, cytoplasmic 1, intermediate chain 1 NM_004411.3

Upregulated in GGH Overexpression and Downregulated in GGH Inhibition

CTHRC1* 1.77 -4.44 collagen triple helix repeat containing 1 NM_138455.2 NNMT 4.80 -3.24 nicotinamide N-methyltransferase NM_006169.2 HLA-DOA 1.44 -3.09 major histocompatibility complex, class II, NM_002119.3 DO alpha FSCN1 3.31 -3.03 fascin homolog 1, actin-bundling protein NM_003088.2 (Strongylocentrotus purpuratus) CDC42EP5 3.76 -2.53 CDC42 effector protein (Rho GTPase NM_145057.2 binding) 5 CHN1 2.79 -2.30 chimerin (chimaerin) 1 NM_001025201.1 SLC2A3 1.94 -2.24 solute carrier family 2 (facilitated glucose NM_006931.1 transporter), member 3 HLA-DRB6 2.91 -2.20 major histocompatibility complex, class II, NR_001298.1 DR beta 6 (pseudogene) HLA-DQA1 6.26 -2.19 PREDICTED: major histocompatibility XM_936128.2 complex, class II, DQ alpha 1, transcript variant 10 PHF21A 1.75 -2.11 PHD finger protein 21A NM_016621.2

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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6.5 Discussion

We investigated the effects of GGH modulation on global and gene-specific methylation and gene expression using an in vitro model of GGH overexpression and inhibition in HCT116 and MDA-MB-435 cells with predictable functional consequences. In both HCT116 and MDA-MB- 435 cells, GGH overexpression was associated with significantly lower global DNA methylation and lower DNMT activity compared with controls expressing endogenous GGH, which is likely related to the lower total intracellular folate concentrations and lower content of long-chain folylpolyglutamates in response to GGH overexpression. In contrast, GGH inhibition showed significantly higher global DNA methylation and higher DNMT activity compared with controls expressing endogenous GGH in both cell lines. This finding is likely accounted for by the observed higher total intracellular folate concentrations and higher content of long-chain folylpolyglutamates in response to GGH inhibition.

We interrogated the DNA methylation status of 27,578 individual CpG sites located at promoter regions of 14,495 genes using an epigenomic approach (360). In both cell lines, GGH inhibition demonstrated greater promoter CpG methylation changes compared with GGH overexpression, and MDA-MB-435 cells revealed more promoter CpG methylation alterations in response to GGH modulation compared with HCT116 cells. Functional analysis revealed that several molecular and cellular functions including cell-to-cell signaling and interaction and molecular transport were affected by GGH modulation in HCT116 and MDA-MB-435 cells.

We also observed that the proportion of differentially methylated CpG loci was generally greater in non-CpG islands compared with CpG islands. While the association of promoter CpG island methylation with gene silencing is well established, little is known about methylation of non- CpG island promoters. Recent studies have shown that DNA methylation is also important for the regulation of non-CpG island promoters. Tissue-specific expression of MASPIN, which does not contain a CpG island within its promoter, was regulated by DNA methylation (440). Similarly, methylation of the non-CpG island Oct-4 promoter strongly influences its expression level (441). More recently, it has been suggested that DNA methylation can directly silence genes with non-CpG island promoters and contribute to the establishment of tissue-specific methylation patterns (325). The epigenetic signatures of DNA methylation, histone marks and

196 nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis (325).

We also interrogated the effect of GGH modulation on the expression of 31,335 annotated genes including 47,231 probes using a genomic approach. Similar to methylation profiles, MDA-MB- 435 cells demonstrated greater altered expression of genes in response to GGH modulation compared with HCT116 cells. In the GGH overexpression system, we identified 152 genes (0.5%; 91 down- and 61 upregulated) and 1383 genes (4.4%; 628 down- and 755 upregulated) that are differentially expressed in HCT116 and MDA-MB-435 cells, respectively, and these genes were primarily involved in cellular movement and cell death. In the GGH inhibition system, 321 genes (1.0%; 139 down- and 182 upregulated) and 859 genes (2.7%; 402 down- and 457 upregulated) associated with cell death, cellular development, cellular growth and proliferation, and cellular movement were differentially expressed in HCT116 and MDA-MB- 435 cells, respectively.

Promoter CpG DNA methylation is generally inversely related to gene expression. More specifically, aberrant promoter CpG DNA hypermethylation is known to be related with transcriptional gene silencing of tumor suppressor and mismatch repair genes (298, 313), while a correlation between hypomethylation of promoter regions and transcriptional activation of tumor-promoting genes has been described (442, 443). Therefore, we performed an integrated analysis of differentially methylated and expressed genes in response to GGH modulation in order to identify genes whose expressions may be regulated by DNA methylation. Overall, only a small number of genes were associated with the inverse relationship between promoter methylation and gene expression. This observation is similar to reports from other studies (444, 445). It appears that the genetic and/or other epigenetic mechanisms such as histone modification, chromatin remodeling, and miRNA might also affect gene expression.

Interestingly, in response to GGH modulation, upregulated genes related with hypomethylation were more frequently observed compared to downregulated genes by hypermethylation except for HCT116 cells overexpressing GGH. This observation is in agreement with a recent study demonstrating that activation of growth-promoting genes related with promoter hypomethylation was more frequently observed compared with gene inactivation by promoter hypermethylation in

197 antiestrogen-resistant cells (446). One of the hypermethylated and downregulated genes in the GGH-inhibited HCT116 cells was IGFBP3, which binds insulin-like growth factor 1 (IGF1) blocking the mitogenic and anti-apoptotic actions of IGF1. Recent studies suggest that IGFBP3 plays a role in growth inhibitory and proapoptotic actions that are independent of binding IGF1 (447, 448). Increased IGFBP-3 levels were associated with improved clinical outcomes (449, 450), and a loss of IGFBP3 expression by promoter hypermethylation reduced sensitivity to cisplatin in NSCLC (429).

In MDA-MB-435 cells, CDC42EP5, CDC42 effector protein (Rho GTPase binding) 5, was hypomethylated (β-value difference -0.58) and upregulated (fold change 3.76) in GGH overexpression, while it was hypermethylated (β-value difference 0.24) and downregulated (fold change -2.53) in GGH inhibition. CDC42EP5 may be involved in the organization of the actin cytoskeleton and act downstream of CDC42 to induce actin filament assembly leading to cell shape changes (451). As detected by microarray gene expression profiling and confirmed by qRT-PCR, TYR is one of the most downregulated (fold change -31.95) and hypermethylated (β- value difference 0.26) genes in the GGH-overexpressed MDA-MB-435 cells, and it was upregulated (fold change 4.07) in the GGH-inhibited MDA-MB-435 cells. TYR encodes tyrosinase, a melanosomal enzyme that catalyzes the rate-limiting steps of melanin biosynthesis (452).

In the GGH-overexpressed MDA-MB-435 cells, we identified 5 upregulated and hypomethylated genes that were downregulated in the GGH-inhibited MDA-MB-435 cells. These genes include S100A4, NNMT, COL8A1, CHN1, and HLA-DPA1. In particular, S100A4 encodes a member of the S100 family of calcium-binding proteins. S100 family members have a wide range of intracellular functions, including the regulation of homeostasis, protein phosphorylation, cytoskeletal rearrangements, and transcriptional activity, and extracellular functions such as the regulation of cell proliferation and activation, apoptosis, and chemotaxis (453, 454). High S100A4 levels were associated with a poor clinical response to infliximab in patients with RA (455). NNMT is known to encode nicotinamide N-methyltransferase, an enzyme which catabolizes nicotinamide and other pyridine compounds in a reaction that uses the methyl group generated during the conversion of SAM to SAH, involved in the biotransformation of many drugs and xenobiotic compounds (456). The NNMT gene, located in the region 11q23, is a major

198 determinant of Hcy (457). Recent finding found that downregulation of NNMT inhibited proliferation in KB cancer cells, suggesting NNMT might be a target for therapeutics and could alter the efficacy of standard chemotherapeutic drugs (458).

Furthermore, among genes involved in folate biosynthesis and one-carbon metabolism, we found upregulation of DPYD and downregulation of GSTT1, ABCC5, SLC25A32, and MTFMT in the GGH-overexpressed MDA-MB-435 cells. DPYD encodes dihydropyrimidine dehydrogenase that is the rate-limiting enzyme involved in 5FU metabolism and a major determinant of 5FU efficacy (233, 234). DPYD overexpression in cancer cell lines is associated with 5FU resistance (237), and high DPYD mRNA expression in colorectal tumors has been shown to correlate with resistance to 5FU (238). In contrast, in addition to low GGH expression and high 5,10 methyleneTHF in CIMP+ tumors, methylation-induced silencing of DPYD may also be associated with a good response of CIMP+ to 5FU (186). Thus, it appears that the observed upregulation of DPYD in the GGH-overexpressed MDA-MB-435 cells might be associated with 5FU resistance. Indeed, we have reported that GGH overexpression decreased chemosensitivity of MDA-MB-435 cells to 5FU by decreasing the relative intracellular concentration of long- chain 5,10-methyleneTHF-polyglutamates, resulting in less efficient formation and stabilization of the inhibitory 5,10-methyleneTHF-TS-FdUMP ternary complex as described in Chapter 4.

GSTT1 encodes the phase II metabolizing enzyme glutathione s-transferase (GST) theta. Evidence indicates that GST expression plays an important role in determining the cytotoxicity of chemotherapeutic drugs, including alkylating agents and intercalating agents (459). Children who lacked GSTT1 had greater toxicity and reduced survival after chemotherapy for acute myeloid leukemia (AML) compared with children with at least one GSTT1 allele (460). Absence of GSTT1 may reduce or delay metabolism of the chemotherapy drugs used for AML. It is possible that GSTT1 genotype influences the production or excretion of drug metabolites that contribute to toxicity to a greater degree than it influences metabolites that have an antileukemic effect (460).

MRP5 encoded by the ABCC5 gene is known to relate with resistance to antifolates and 5FU. MRP5 can efflux mono- and diglutamate forms of MTX and transport 5FdUMP, a metabolite of 5FU (12, 14, 68, 296). MRPs contribute to drug resistance or increase drug efficacy depending on polyglutamylation of antifolates and intracellular folate concentrations (11). In addition, the

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SLC25A32 gene encodes a folate transporter that shuttles folate from the cytoplasm into the mitochondria (461). Evidence suggests that a reduced, monoglutamylated form of cytoplasmic folate (probably THF or 5-formylTHF) are transported to the mitochondria by the mitochondrial folate transporter (MFT), followed by mitochondrial FPGS induced polyglutamylation resulting in mitochondrial folate accumulation (99). Taken together, downregulation of ABCC5 and SLC25A32 supports the role of MRP5 and MFT in the modulation of the intracellular folate levels as well as cellular folate homeostasis since we have previously found that GGH overexpression decreased total intracellular folate concentrations in MDA-MB-435 cells (Chapter 4).

In the GGH-inhibited MDA-MB-435 cells, BCHE, which encodes butyrylcholinesterase, was associated with upregulation and hypomethylation, and it was downregulated in the GGH- overexpressed MDA-MB-435 cells. We also found CDKN1A and CCND1, genes involved in cell cycle, were upregulated and hypomethylated. CDKN1A encodes a cyclin-dependent kinase inhibitor, p21cip1, which inhibits G and S phase progression, thereby allowing time for DNA repair in response to cell injury (462). CDKN1A expression is tightly controlled by TP53, through which it mediates the p53-dependent cell cycle G1 phase arrest in response to different stress stimuli (375). CCND1 (cyclin D1) is a downstream effector of EGFR signaling that regulates cell cycle. CCND1 is found to be associated with DHFR and TS regulation at the transcription levels. The transcription factors -1 and DP-1 are released by the action of CCND1, a protein involved in the retinoblastoma protein phosphorylation, inducing an increase in the transcription levels of DHFR and TS (463). Furthermore, a recent study suggests that CCND1 can lead to an increase in DHFR and TS expression, and reduction of sensitivity to MTX (464). As for MDA-MD-435 cells, although CDKN1A and CCND1 were also upregulated in response to GGH overexpression, CDK2 (cyclin-dependent kinase 2) was down- and upregulated in response to GGH overexpression and inhibition, respectively. CDK2 maintains a balance of S- phase regulatory proteins and thereby coordinates subsequent p53-independent G2/M checkpoint activation (465). CDK2 expression was downregulated in the 5FU resistant cell lines, suggesting that decreased CDK2 activity may delay the transition of resistant cells from G1 into S-phase (466, 467). Delayed S-phase entry and/or reduced S-phase traverse may provide resistant cells with enough time to repair 5FU-induced damage before progressing to G2-M phase. Thus, 5FU resistance may be, at least partially, reversed by specific targeting of the G1-S checkpoint arrest

200 in the resistant cells (467). Accordingly, downregulation of CDK2 in the GGH-overexpressed MDA-MB-435 cells is likely to be associated with decreased chemosensitivity to 5FU as presented in Chapter 4. Restoration of the G1 checkpoint by targeting CDK2 is currently one of the major strategies for anticancer drug development (468). In addition to cell cycle progression, CDK2 is activated by anticancer drugs such as MTX and docetaxel, resulting in apoptosis (469). HDAC9, which encodes histone deacetylase 9, was upregulated and hypomethylated in the GGH-overexpressed MDA-MB-435 cells. The upregulation of HDAC9 has been shown to correlate with reduced overall disease-free survival and poor prognosis in children with ALL or medulloblastoma (470, 471).

We also investigated genes commonly differentially methylated and/or expressed in response to GGH modulation between HCT116 and MDA-MB-435 cell lines. There were 61 hyper- and 54 hypomethylated genes common in both cell lines in response to GGH overexpression, whereas 117 hyper- and 129 hypomethylated genes were common to both cell lines in GGH inhibition. In addition, 9 down- and 10 upregulated genes were common in GGH overexpression, while 11 down- and 12 upregulated genes were common in GGH inhibition. There were no common genes, expression of which resulted from CpG promoter methylation changes in response to GGH modulation between HCT116 and MDA-MB-435 cell lines. As a result of the investigation into genes differentially expressed in the opposite direction between GGH overexpression and inhibition, no common genes were identified in both HCT116 and MDA-MB-435 cell lines.

In summary, our results were consistent with our a priori hypothesis that the GGH modulation- induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates would affect global DNA methylation and DNMT activity as well as the degree of changes in CpG promoter DNA methylation and gene expression. We demonstrated that GGH overexpression was associated with decreased global DNA methylation and DNMT activity, while GGH inhibition showed increased global DNA methylation and DNMT activity. Furthermore, we showed that GGH modulation influenced differential gene expression and CpG promoter DNA methylation involved in important biological pathways, and some of the observed altered gene expression appeared to be regulated by DNA methylation. In the GGH- overexpressed MDA-MB-435 cells, we identified several differentially expressed genes involved in folate biosynthesis, one-carbon pool by folate, and cell cycle, which might be associated with

201 the observed decreases in total intracellular folate concentrations and 5FU efficacy in response to GGH overexpression. The potential role of GGH modulation in DNA methylation and the effect on chemosensitivity needs further exploration.

CHAPTER 7: STUDY 4 – THE EFFECT OF FPGS MODULATION ON GLOBAL AND GENE-SPECIFIC DNA METHYLATION AND GENE EXPRESSION IN HUMAN COLON AND BREAST CANCER CELLS

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7.1 Abstract

Background: Folate mediates the transfer of one-carbon units for the generation of SAM, the primary methyl group donor for most biological methylation reactions including DNA methylation, which is catalyzed by DNMT. Both genomic DNA hypomethylation and gene- specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis. DNA methylation and DNMT are also potential therapeutic targets and may modify the effect of specific chemotherapeutic agents. FPGS facilitates intracellular retention of folate by polyglutamylation, thereby playing a critical role in folate homeostasis. We investigated whether FPGS modulation would affect global and gene-specific methylation and gene expression profiles in human HCT116 colon and MDA-MB-435 breast cancer cells.

Methods: An in vitro model of FPGS overexpression/inhibition in HCT116 was generated by transfecting the cells with the sense or antisense FPGS cDNA, respectively. An in vitro model of FPGS overexpression/inhibition in MDA-MB-435 cells was generated by transfecting cells with the sense FPGS cDNA or FPGS-targeted siRNA, respectively. Global DNA methylation and DNMT activity were determined. Illumina HT-12 and Infinium Methylation assays were used, and functional analysis was performed by Ingenuity Pathway Analysis. We validated mRNA expression of selected genes, expression of which was regulated by DNA methylation, by qRT- PCR.

Results: We hypothesized that FPGS overexpression would increase, whereas FPGS inhibition would decrease, global DNA methylation and DNMT activity due to the observed intracellular folate concentrations and content of long-chain folylpolyglutamates. FPGS overexpression was associated with lower global DNA methylation and DNMT activity compared with controls in HCT116 cells, while it was associated with higher global DNA methylation and DNMT activity than controls in MDA-MB-435 cells. FPGS inhibition was associated with higher global DNA methylation compared with controls in HCT116 cells, whereas it did not affect global DNA methylation in MDA-MB-435 cells. FPGS inhibition was associated with lower DNMT activity compared with controls in both cell lines. In HCT116 cells, we identified 2897 differentially expressed genes (most commonly involved pathways: cell cycle and DNA replication, recombination, and repair) associated with FPGS overexpression, and 359 differentially

204 expressed genes (most commonly involved pathways: cell death and cell cycle) associated with FPGS inhibition; 864 and 626 genes showed altered CpG promoter methylation associated with FPGS overexpression and inhibition, respectively; and an integrated analysis revealed 65 and 6 genes, expression of which was regulated by CpG promoter methylation changes associated with FPGS overexpression and inhibition, respectively. In MDA-MB-435 cells, we identified 1502 differentially expressed genes (most commonly involved pathways: cell death and cellular movement) associated with FPGS overexpression, and 829 differentially expressed genes (most commonly involved pathways: cell death and cellular assembly and organization) associated with FPGS inhibition; 2239 and 2024 genes showed altered CpG promoter methylation associated with FPGS overexpression and inhibition, respectively; and an integrated analysis revealed 95 and 43 genes, expression of which was regulated by CpG promoter methylation changes associated with FPGS overexpression and inhibition, respectively.

Conclusions: Our data indicate that FPGS modulation can affect global DNA methylation and DNMT activity as well as differential gene expression and CpG promoter DNA methylation involved in important biological pathways, and some of the observed altered gene expression appear to be regulated by DNA methylation.

7.2 Introduction

Folate mediates the transfer of one-carbon units necessary for nucleotide biosynthesis, and hence is an essential factor for DNA synthesis (1, 29). Intracellular folate is converted to polyglutamates by FPGS, while GGH removes the terminal glutamates, thereby facilitating the export of folate (1). Polyglutamylated folates are better retained intracellularly and are better substrates for intracellular folate-dependent enzymes than monoglutamates (2). We have previously developed an appropriate in vitro model of FPGS overexpression and inhibition in HCT116 and MDA-MB-435 cells with predictable functional consequences: FPGS overexpression is associated with higher, whereas FPGS inhibition demonstrates lower, concentrations of total intracellular folate and content of long-chain folylpolyglutamates compared with cotrols expressing endogenous FPGS (4, 17). It has also been reported that FPGS modulation affects the chemosensitivity of cancer cells to 5FU by inducing changes in the

205 intracellular retention of specific folate cofactor such as 5,10-methyleneTHF and to MTX by inducing changes in the intracellular retention of MTX, respectively (4, 17).

Furthermore, folate mediates the transfer of one-carbon units for the generation of SAM, the primary methyl group donor for most biological methylation reactions including DNA methylation, which is catalyzed by DNMT (1, 29). Polyglutamylation is also important in DNA methylation as polyglutamylated folates are better substrates for folate-dependent enzymes such as MTHFR and MS that are involved in the generation of SAM, which is a substrate for DNA methylation mediated by DNMT (2, 28). And hence, FPGS modulation may affect DNA methylation at global and gene-specific levels with consequent functional ramifications. Both genomic DNA hypomethylation and gene-specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis (29). DNA methylation and DNMT are also potential therapeutic targets and may modify the effect of specific chemotherapeutic agents (30), suggesting that the FPGS-modulated DNA methylation changes might influence chemosensitivity to chemotherapeutic agents. Therefore, the objective of this study was to investigate whether FPGS modulation would affect global and gene-specific DNA methylation and DNMT activity, and to interrogate the effect of FPGS modulation on gene expression in HCT116 and MDA-MB-435 cell lines.

We hypothesized that (1) FPGS overexpression would increase global DNA methylation and DNMT activity, whereas FPGS inhibition would decrease global DNA methylation and DNMTactivity; and (2) FPGS overexpression and inhibition would further affect the degree of promoter methylation and the differential expression of genes.

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7.3 Materials and Methods

7.3.1 Cell Lines and Culture

Cells were maintained in RPMI-1640 medium as described in Section 4.3.1.

7.3.2 Construction and Transfection of FPGS Expression Vectors

The full length human FPGS cDNA (472) was provided by Dr A. Bognar (University of Toronto, Toronto, Canada). The full length human FPGS cDNA was subcloned into the EcoRI site of the eukaryotic expression vector pIRESneo (Clontech, Palo Alto, CA) containing a CMV promoter and a neomycin resistance gene expression cassette in the sense and antisense orientation to generate the sense and antisense FPGS expression vectors, respectively, as described (17). The FPGS-targeted siRNA was designed according to the manufacturer’s protocol (QIAGEN, Canada) and ligated into the vector between the BamH1 and HindIII restriction sites of the pSilencer neo siRNA expression vector (Ambion, Austin, TX). The oligonucleotides were designed encoding the desired siRNA strand: GACGGGATTCTTTAGCTCT (forward) and AGAGCTAAAGAATCCCGTC (reverse), and AGAGCTAAAGAATCCCGTC (forward) and GACGGGATTCTTTAGCTCT (reverse) (4).

The pIRESneo vector containing the sense or antisense FPGS cDNA was stably transfected into HCT116 cells using Lipofectin (Invitrogen) according to the manufacturer’s protocol. In a separate transfection, HCT116 cells were stably transfected with empty pIRESneo vector as corresponding control expressing endogenous FPGS (17). Similarly, MDA-MB-435 cells were transfected with the sense FPGS cDNA or FPGS-targeted siRNA, respectively, to generate an in vitro model of FPGS overexpression and inhibition. In separate transfection, MDA-MB-435 cells were stably transfected with empty pIRESneo and pSilencer vectors as corresponding controls expressing endogenous FPGS for the FPGS overexpression and FPGS inhibition systems, respectively (4).

Transfected cells were incubated with 500 μg/ml of neomycin (Invitrogen) to select for cells that expressed the various constructs. After a population of cells was selected, individual clonal cell

207 lines were isolated and expanded. Cells were maintained in complete medium supplemented with 500 μg/ml of neomycin. Several (> 10) clones expressing the sense and antisense FPGS cDNA/FPGS-targeted siRNA and empty vectors were screened at random, and two independent clones of each construct were selected for further analyses. Data from three experiments using two independent clones of each construct were similar, and thus, the data from one experiment are presented. In order to obviate the potential confounding effect of different cell passage numbers on DNA methylation, cells expressing the sense FPGS, antisense FPGS or FPGS- targeted siRNA, and corresponding controls with similar passage numbers were used for DNA methylation analysis.

7.3.3 Genomic DNA Isolation and Methylation Analysis

Genomic DNA isolation and methylation were performed as described in Section 6.3.3.

7.3.4 DNA Methyltransferase Enzyme Activity Assay

DNA methyltransferase enzyme activity was determined as described in Section 6.3.4.

7.3.5 Gene-Specific Promoter CpG Island Methylation Analysis

Gene-specific promoter CpG island methylation assay was performed using the Illumina Infinium HumanMethylation 27 BeadChip as described in Section 6.3.5.

7.3.6 Gene Expression Analysis

RNA preparation and gene expression assay was performed using the Illumina HumanHT-12 v4.0 BeadChip as described in Section 6.3.6.

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7.3.7 Integrated Analysis of DNA Methylation and Gene Expression Data Integrated analyses of the Illumina Infinium DNA methylation and gene expression data were performed as described in Section 6.3.7.

7.3.8 Ingenuity Pathway Analysis

The functional analysis was performed using Ingenuity Pathway Analysis (IPA, Ingenuity® Systems, Redwood City, CA; http://www.ingenuity.com) as described in Section 6.3.8.

7.3.9 Quantitative Reverse Transcriptase-Polymerase Chain Reaction

To confirm the data obtained from gene expression analysis using the Illumina HumanHT-12 v4.0 BeadChip, qRT-PCR was performed as described in Section 6.3.9. The information of selected primer sequences is presented in Table 7. 1.

7.3.10 Statistical Analysis

Differences in global DNA methylation, DNMT enzyme activity, and qRT-PCR analysis were analyzed using the Student’s t-test as described in Section 6.3.10. The analysis was performed for comparisons between cells expressing the sense FPGS (Sense) and control (Control) and between cells expressing the antisense FPGS (Antisense) and control (Control) for HCT116 cells. For MDA-MB-435 cells, comparisons between cells expressing the sense FPGS (Sense) and controls (Control-S) and between cells transfected with the FPGS-targeted siRNA (siRNA) and controls (Control-si) were performed. The analyses of gene-specific CpG promoter methylation and gene expression profiles are described in Sections 6.3.5 and 6.3.6 and Appendices 3 and 4.

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Table 7. 1 Primer sequences for genes selected for qRT-PCR

Gene Forward Primer Reverse Primer Symbol

ABCC2 5'-ACCATTACCTTGTCACTGTCCATGA-3' 5'-CCAACGGTGGTCGGAGACA-3'

ALDH1A3 5'-TCTCGACAAAGCCCTGAAGT-3' 5'-GTCCGATGTTTGAGGAAGGA-3'

5'-TGACGGGGTCACCCACACTGTGCCC 5'-CTAGAAGCATTTGCGGTGGACGAT β-actin ATCTA-3' GGAGGG-3'

BRCA1 5'-ACAGCTGTGTGGTGCTTCTGTG-3′ 5′-CATTGTCCTCTGTCCAGGCATC-3′

CD55 5'-TGTGGCCTTCCCCCAGAT-3' 5'-TCCTCGGGAAAACTTGTACGG-3'

CTSL1 5'-GACTCTGAGGAATCCTATCCA-3' 5′-AAGGACTCATGACCTGCATCAA-3'

GAPDH 5'-ACCACAGTCCATGCCATCAC-3' 5'-TCCACCACCCTGTTGCTGTA-3'

GPM6A 5'-TATTGTGGCACTTGCTGGAG-3' 5'-GGCAGGCGTCTTTCACATAG-3'

GSTT1 5'-CACGACTCTGCGGAGAAGCT-3' 5'-TGCAGGGTCACATCCAACTCT-3'

HLA-DPA1 5'-CCAAGAGCCAATCCAGATGCCTG-3' 5'-AGGACGGTGCCCACGATGATG-3'

IRF1 5'-TTCCCTCTTCCACTCGGAGT-3' 5'-GATATCTGGCAGGGAGTTCA-3'

LRP5 5′-ATCGACTGTATCCCCGGGGC-3′ 5′-CACCACGCGCTGGCACACAA-3′

MECOM 5'-TTGCCAAGTAACAGCTTTGCTG-3' 5'-CCAAAGGGTCCGAATGTGACTT-3'

MSH2 5'-TTCATGGCTGAAATGTTGGA-3' 5'-ATGCTAACCCAAATCCATCG-3'

MT1E 5'-GCCCGACCTCCGTCTATAA-3' 5'-AACAAGCAGTCAGGCAGTTG-3'

S100A4 5'-CCACAAGTACTCGGGCAAAG-3' 5'-GTCCCTGTTGCTGTCCAAGT-3'

SOX10 5'-CCAGTACCCGCACCTGCAC-3' 5'-CTTTCGTTCAGCAGCCTCCAG-3'

THBS2 5'-TTGCAAATGGGTGTGACGCGGT-3' 5'-AAGCACCGCACTTTGCTCTGCT-3'

TYR 5'-CTCAAAGCAGCATGCACAAT-3' 5'-GCCCAGATCTTTGGATGAAA-3'

WNT16 5'-AAAGAAATGTTTCCCTGCCC-3' 5'-GACATTTTCCATGGGTTTGC-3'

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7.4 Results

7.4.1 Global DNA Methylation

FPGS overexpression was associated with lower global DNA methylation compared with controls in HCT116 cells (18% lower, P < 0.05; Figure 7.1A), while being associated with higher global DNA methylation than controls in MDA-MB-435 cells (13% higher, P < 0.001; Figure 7.1B). FPGS inhibition was associated with higher global DNA methylation compared with controls in HCT116 cells (12% higher, P < 0.05; Figure 7.1A), whereas it did not affect global DNA methylation in MDA-MB-435 cells (P = 0.588; Figure 7.1C).

7.4.2 DNA Methyltransferase Enzyme Activity

FPGS overexpression showed lower DNMT activity than controls in HCT116 cells (77% lower, P < 0.05; Figure 7.2A), whereas it was associated with higher DNMT activity than controls in MDA-MB-435 cells (115% higher, P < 0.001, Figure 7.2B). FPGS inhibition was associated with lower DNMT activity compared with controls in both cell lines (HCT116, 31% lower, P < 0.05, Figure 7.2A; MDA-MB-435, 64% lower, P < 0.001, Figure 7.2C).

.

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Figure 7. 1 Global DNA methylation in the FPGS-modulated HCT116 colon (A) and MDA-MB-435 breast (B, C) cancer cells. The in vitro methyl acceptance assay produces an inverse relationship between the endogenous DNA methylation status and exogenous [3H- methyl] incorporation into DNA. Control(-S), cells expressing endogenous FPGS; Sense, cells transfected with the sense FPGS cDNA; Antisense, cells transfected with the antisense FPGS cDNA; Control-si, cells expressing endogenous FPGS; siRNA, cells transfected with the FPGS- targeted siRNA. Different letters among each group denote significant difference at P < 0.05. *, P < 0.05 compared with corresponding control. Values are mean ± SD.

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Figure 7. 2 DNA methyltransferase enzyme activity in the FPGS-modulated HCT116 colon (A) and MDA-MB-435 breast (B, C) cancer cells. The assay produces a positive relationship between the endogenous enzyme activity and exogenous [3H-methyl] incorporation into DNA. Control(-S), cells expressing endogenous FPGS; Sense, cells transfected with the sense FPGS cDNA; Antisense, cells transfected with the antisense FPGS cDNA; Control-si, cells expressing endogenous FPGS; siRNA, cells transfected with the FPGS-targeted siRNA. Different letters among each group denote significant difference at P < 0.05. *, P < 0.05 compared with corresponding control. Values are mean ± SD.

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7.4.3 Gene-Specific Promoter CpG Island Methylation

Similar to the results in GGH modulation, scatter plots of DNA methylation β-values in FPGS modulation showed differentially methylated loci between Sense and Control and between Antisense and Control for HCT116 cells, and between Sense and Control-S and between siRNA and Control-si for MDA-MB-435 cells as measured by Illumina Infinium HumanMethylation 27 BeadChip. The presence of more spread in each plot represents higher degree of hyper- or hypomethylation changes. MDA-MB-435 cells showed more CpG methylation alterations in response to FPGS modulation than HCT116 cells (Figure 7.3).

A B

2 R2 = 0.952 R = 0.961

C D

R2 = 0.914 R2 = 0.922

Figure 7. 3 Scatter plots of DNA methylation β-value of FPGS overexpression and inhibition in HCT116 colon (A, B) and MDA-MB-435 breast (C, D) cancer cell lines. Each dot indicates β-value at the particular gene locus. β-Value represents the ratio of the intensity of the methylated bead type to the combined locus intensity (methylated+unmethylated) ranging from 0 to 1. Values close to 0 indicate low levels of DNA methylation, while values close to 1 indicate high levels of DNA methylation. The presence of more spread in each graph represents higher degree of hyper- or hypomethylation changes.

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To illustrate the representative distribution of methylation levels across more than 27,000 CpG sites, Figures 7.4 and 7.5 displayed histograms of β-values in the FPGS-modulated HCT116 and MDA-MB-435 cells. The number of CpG sites methylated at different β-values in the FPGS- modulated HCT116 and MDA-MB-435 cell lines showed a bimodal distribution (Figures 7.4 and 7.5). These histograms demonstrated that MDA-MB-435 cells were associated with more CpG methylation alterations than HCT116 cells in response to FPGS modulation (Figures 7.4 and 7.5). These obervations were consistent with the previously presented results (Figure 7.3).

Figure 7. 4 Number of CpG sites methylated at different β-values in the FPGS- modulated HCT116 colon cancer cells. The histogram indicates the number of CpG sites methylated at different β-values, and represents the distribution of β-values for 27,578 CpG sites in each system.

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Figure 7. 5 Number of CpG sites methylated at different β-values in the FPGS- modulated MDA-MB-435 breast cancer cells. The histogram indicates the number of CpG sites methylated at different β-values, and represents the distribution of β-values for 27,578 CpG sites in each system.

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A two-dimensional hierarchical clustering represented the patterns of Infinium DNA methylation β-value in the FPGS-modulated HCT116 (Figure 7.6) and MDA-MB-435 cell lines (Figure 7.7). It revealed distinctively different DNA methylation alteration profiles between FPGS overexpression and inhibition in both cell lines (Figures 7.6 and 7.7).

Figure 7. 6 Two-dimensional hierarchical clustering of DNA methylation β-value in the FPGS-modulated HCT116 cell line. A color gradient from dark blue to red indicated the low and high methylation, respectively.

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Figure 7. 7 Two-dimensional hierarchical clustering of DNA methylation β-value in the FPGS-modulated MDA-MB-435 cell line. A color gradient from dark blue to red indicated the low and high methylation, respectively.

The number of genes differentially methylated in both cell lines is presented in Table 7.2. We calculated hyper- or hypomethylation by subtracting β-value of corresponding control from β- value of Sense, Antisense or siRNA. We determined 0.2 as β-value difference at 99% confidence based on intra- and inter- assay variations. In the HCT116 cell line, we identified 864 genes that were differentially methylated (446 hypermethylated and 418 hypomethylated) in response to FPGS overexpression, while 626 genes were differentially methylated (247 hypermethylated and

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379 hypomethylated) in response to FPGS inhibition (Table 7.2). With respect to the MDA-MB- 435 cell line, we identified 2239 genes that were differentially methylated (1161 hypermethylated and 1078 hypomethylated) in response to FPGS overexpression, while 2024 genes were differentially methylated (1150 hypermethylated and 874 hypomethylated) in response to FPGS inhibition (Table 7.2). The number of genes differentially methylated when we applied 0.5 as a more stringent criterion for β-value difference is also shown in Table 7.2.

Table 7. 2 Summary of number of genes differentially methylated in the FPGS- modulated HCT116 and MDA-MB-435 cell lines

FPGS Overexpression FPGS Inhibition

Hyper- Hypo- Hyper- Hypo- Total Total methylation methylation methylation methylation

HCT116 β-value difference |0.2| 446 418 864 247 379 626 (3.1%) (2.9%) (6.0%) (1.7%) (2.6%) (4.3%) β-value difference |0.5| 6 6 12 2 6 8 (0.04%) (0.04%) (0.08%) (0.01%) (0.04%) (0.05%)

MDA-MB-435

β-value difference |0.2| 1161 1078 2239 1150 874 2024 (8.0%) (7.4%) (15.4%) (7.9%) (6.0%) (13.9%) β-value difference |0.5| 85 92 177 91 56 147 (0.6%) (0.6%) (1.2%) (0.6%) (0.4%) (1.0%)

The numbers in brackets indicate the percentage of genes differentially methylated relative to total genes targeted in the Infinium assay.

7.4.3.1 Genes Differentially Methylated in the FPGS-Modulated HCT116 Cells

To identify the most relevant biological and disease processes related to differentially methylated genes in each system, a functional analysis was performed using IPA. Some genes were assigned to more than one category. The top five molecular and cellular functions are presented based on significance. Genes with functions relating to cellular assembly and organization, cellular function and maintenance, and cellular movement were found to be differentially methylated in the FPGS-overexpressed HCT116 cells (Table 7.3). Specifically, in the FPGS-overexpressed HCT116 cells, the hypermethylated genes were associated with cellular assembly and

219 organization, cellular function and maintenance, small molecule biochemistry, cell-to-cell signaling and interaction, and cellular movement, while the hypomethylated genes were involved in protein systhesis, cellular assembly and organization, cellular function and maintenance, cellular movement, and cell death (Table 7.3). As for the FPGS-inhibited HCT116 cells, major function categories of the hypermethylated genes included cellular movement, cell morphology, cell-to-cell signaling and interaction, drug metabolism, and molecular transport, whereas those of the hypomethylated genes consisted of antigen presentation, cellular movement, cellular assembly and organization, cell death, and cellular growth and proliferation (Table 7.3).

Table 7. 3 The top molecular and cellular functions associated with differentially methylated genes in the FPGS-modulated HCT116 colon cancer cells

Hypermethylation Hypomethylation No. of No. of Category P-value Category P-value Genes Genes FPGS Overexpression Cellular Assembly and 2.63E-04 - 32 Protein Synthesis 6.33E-04 - 8 Organization 4.72E-02 4.61E-02 Cellular Function and 2.63E-04 - 34 Cellular Assembly and 1.60E-03 - 13 Maintenance 4.72E-02 Organization 4.61E-02 Small Molecule 1.43E-03 - 34 Cellular Function and 1.60E-03 - 14 Biochemistry 4.96E-02 Maintenance 4.61E-02 Cell-To-Cell Signaling and 1.68E-03 - 28 Cellular Movement 2.04E-03 - 22 Interaction 4.85E-02 4.61E-02 Cellular Movement 1.68E-03 - 38 Cell Death 3.15E-03 - 21 4.72E-02 4.74E-02 FPGS Inhibition Cellular Movement 5.29E-04 - 17 Antigen Presentation 1.27E-04 - 19 4.39E-02 4.74E-02 Cell Morphology 3.57E-03 - 18 Cellular Movement 1.27E-04 - 42 4.43E-02 4.85E-02 Cell-To-Cell Signaling and 3.57E-03 - 15 Cellular Assembly and 2.83E-04 - 32 Interaction 3.96E-02 Organization 4.15E-02 Drug Metabolism 3.57E-03 - 5 Cell Death 3.00E-04 - 18 3.96E-02 4.85E-02 Molecular Transport 3.57E-03 - 14 Cellular Growth and 1.85E-03 - 76 3.96E-02 Proliferation 4.33E-02

Next, we performed a functional analysis of a network using IPA to examine further regulatory relationships between differentially methylated genes and biological processes. This network

220 analysis identified the biological functions and/or diseases that were most significant to the genes in the network. The list of top networks in the FPGS-modulated HCT116 cells is presented in Table 7.4.

Table 7. 4 The top networks matched by the genes differentially methylated in the FPGS- modulated HCT116 colon cancer cells Hypermethylation Hypomethylation Focus Focus No Top Functions Score Top Functions Score Genes Genes FPGS Overexpression 1 Cellular Growth and 43 29 Cellular Development, Cellular 31 23 Proliferation, Cell Death, Growth and Proliferation, Molecular Transport Hematological System Development and Function 2 Gastrointestinal Disease, 34 25 Immunological Disease, Cancer, 29 22 Embryonic Development, Hematological Disease Organismal Development 3 RNA Post-Transcriptional 19 17 Cellular Function and 16 15 Modification, Cellular Maintenance, Tissue Development, Hematological Morphology, Organismal Injury System Development and and Abnormalities Function 4 Connective Tissue Disorders, 17 16 Cell Morphology, Cellular 15 14 Inflammatory Disease, Function and Maintenance, Inflammatory Response DNA Replication, Recombination, and Repair 5 Tissue Development, 17 16 Cellular Development, Cellular 13 13 Hematological System Growth and Proliferation, Development and Function, Nervous System Development Immune Cell Trafficking and Function FPGS Inhibition 1 Cell Morphology, Cellular 41 25 Infectious Disease, Cellular 36 25 Development, Hematological Development, Cellular Growth System Development and and Proliferation Function 2 Gene Expression, Lymphoid 20 15 Cellular Development, Cellular 36 25 Tissue Structure and Growth and Proliferation, Cell Development, Tissue Death Morphology 3 Inflammatory Response, Cell 16 13 Connective Tissue Disorders, 15 14 Death, Cellular Development Inflammatory Disease, Skeletal and Muscular Disorders 4 Cell Cycle, Cancer, Hereditary 16 13 Cellular Movement, Cellular 15 14 Disorder Development, Cellular Growth and Proliferation 5 Endocrine System Development 13 11 Cardiovascular Disease, 15 14 and Function, Cellular Function Hematological Disease, and Maintenance, Metabolic Disease Hematological System Development and Function The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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7.4.3.2 Genes Differentially Methylated in the FPGS-Modulated MDA-MB-435 Cells

Functional analysis revealed that in the FPGS-overexpressed MDA-MB-435 cells, the hypermethylated genes were associated with cell-to-cell signaling and interaction, cellular movement, cell death, cellular function and maintenance, and molecular transport, while the hypomethylated genes were involved in cell signaling, molecular transport, vitamin amd mineral metabolism, cellular development, and cellular growth and proliferation (Table 7.5). As for the FPGS-inhibited MDA-MB-435 cells, the hypermethylated genes were related with nucleic acid metabolism, small molecule biochemistry, cell death, cellular function and maintenance, and cellular development, whereas the hypomethylated genes were involved in antigen presentation, cell-to-cell signaling and interaction, cell death, cellular movement, and carbohydrate metabolism (Table 7.5). The list of top networks generated by mapping the focus genes that were differentially methylated in the FPGS-modulated MDA-MB-435 cells is shown in Table 7.6.

Table 7. 5 The top molecular and cellular functions associated with differentially methylated genes in the FPGS-modulated MDA-MB-435 breast cancer cells

Hypermethylation Hypomethylation No. of No. of Category P-value Category P-value Genes Genes FPGS Overexpression Cell-To-Cell Signaling and 5.15E-09 - 124 Cell Signaling 1.54E-10 - 80 Interaction 1.78E-02 3.61E-02 Cellular Movement 2.80E-06 - 94 Molecular Transport 1.54E-10 - 137 1.70E-02 3.62E-02 Cell Death 1.29E-05 - 147 Vitamin and Mineral 1.54E-10 - 64 1.70E-02 Metabolism 1.27E-02 Cellular Function and 1.57E-05 - 86 Cellular Development 1.75E-06 - 99 Maintenance 1.70E-02 3.61E-02 Molecular Transport 1.57E-05 - 97 Cellular Growth and 1.75E-06 - 183 1.70E-02 Proliferation 3.61E-02 FPGS Inhibition Nucleic Acid Metabolism 9.72E-06 - 39 Antigen Presentation 2.99E-06 - 34 2.34E-02 1.88E-02 Small Molecule 9.72E-06 - 89 Cell-To-Cell Signaling 2.99E-06 - 95 Biochemistry 2.82E-02 and Interaction 4.18E-02 Cell Death 1.18E-05 - 139 Cell Death 3.14E-06 - 56 2.22E-02 4.05E-02 Cellular Function and 2.20E-05 - 97 Cellular Movement 6.68E-06 - 82 Maintenance 2.68E-02 4.18E-02 Cellular Development 6.66E-05 - 107 Carbohydrate Metabolism 3.23E-05 - 43 2.85E-02 3.19E-02

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Table 7. 6 The top networks matched by the genes differentially methylated in the FPGS- modulated MDA-MB-435 breast cancer cells Hypermethylation Hypomethylation Focus Focus No Top Functions Score Top Functions Score Genes Genes FPGS Overexpression 1 Cardiovascular System 34 31 Cell-To-Cell Signaling and 36 32 Development and Function, Interaction, Hematological Cell-To-Cell Signaling and System Development and Interaction, Cellular Movement Function, Immune Cell Trafficking 2 Inflammatory Response, 32 30 Cell Signaling, Molecular 32 30 Cellular Growth and Transport, Vitamin and Mineral Proliferation, Hematological Metabolism System Development and Function 3 Cellular Development, Cellular 30 29 Cell-To-Cell Signaling and 30 29 Growth and Proliferation, Interaction, Hematological Tumor Morphology System Development and Function, Immune Cell Trafficking 4 Cellular Development, 28 28 Cell Death, Cell Signaling, 28 28 Embryonic Development, Molecular Transport Nervous System Development and Function 5 Hematological System 23 25 Cellular Development, Cell 27 27 Development and Function, Death, Developmental Disorder Inflammatory Response, Tissue Morphology

FPGS Inhibition 1 Cell-To-Cell Signaling and 30 29 Cancer, Hematological Disease, 33 29 Interaction, Hematological Cellular Movement System Development and Function, Immune Cell Trafficking 2 Cell Signaling, Molecular 30 29 Cell-To-Cell Signaling and 31 28 Transport, Vitamin and Mineral Interaction, Cellular Movement, Metabolism Cell Signaling 3 Cellular Assembly and 30 29 Cellular Movement, 29 27 Organization, Developmental Hematological System Disorder, Hereditary Disorder Development and Function, Immune Cell Trafficking 4 Dermatological Diseases and 28 28 Cell Death, Tumor Morphology, 27 26 Conditions, Inflammatory Cellular Movement Disease, Immunological Disease 5 Nutritional Disease, Nucleic 26 27 Hematological System 26 25 Acid Metabolism, Small Development and Function, Molecule Biochemistry Tissue Morphology, Cell-To- Cell Signaling and Interaction The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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7.4.3.3 Common Genes Differentially Methylated in the FPGS-Modulated HCT116 and MDA-MB-435 Cells

A venn diagram was used to depict the number of genes commonly differentially methylated in the FPGS-modulated HCT116 and MDA-MB-435 cell lines (Figures 7.8 and 7.9). Sixty-four hyper- and 52 hypomethylated genes were common between the HCT116 and MDA-MB-435 cell lines in response to FPGS overexpression (Figure 7.8), whereas 31 hyper- and 38 hypomethylated genes were common between these cell lines in response to FPGS inhibition (Figure 7.9). The proportion of the differentially methylated CpG loci located within CpG islands and non-CpG islands is also presented in Figures 7.8 and 7.9. Generally, the proportion of differentially methylated CpG loci in non-CpG islands was greater than those within CpG islands.

Figure 7. 8 Number of genes commonly differentially methylated in HCT116 and MDA- MB-435 cells and distribution of differentially methylated CpG loci in response to FPGS overexpression

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Figure 7. 9 Number of genes commonly differentially methylated in HCT116 and MDA- MB-435 cells and distribution of differentially methylated CpG loci in response to FPGS inhibition

In the FPGS-overexpressed HCT116 and MDA-MB-435 cell lines, the commonly hypermethylated genes were associated with cell morphology, cellular assembly and organization, cellular function and maintenance, cellular movement, and cellular development, while the commonly hypomethylated genes were related to cell death, cell cycle, cell-to-cell signaling and interaction, cellular compromise, and cellular development (Table 7.7). In the FPGS-inhibited HCT116 and MDA-MB-435 cell lines, the major function categories of the commonly hypermethylated genes included cell cycle, cell-to-cell signaling and interaction, cellular movement, cell morphology, and cellular growth and proliferation, whereas those of the commonly hypomethylated genes consisted of cell cycle, cell death, cell morphology, cellular assembly and organization, and cellular compromise (Table 7.7). The list of top networks

225 generated by mapping the focus genes that were commonly differentially methylated in both the FPGS-modulated HCT116 and MDA-MB-435 cells is presented in Tables 7.8 and 7.9.

Table 7. 7 The top molecular and cellular functions associated with genes with commonly differentially methylated in response to FPGS modulation in both the HCT116 and MDA- MB-435 cells

FPGS Overexpression FPGS Inhibition No. of No. of Category P-value Category P-value Genes Genes Hypermethylated Cell Morphology 2.17E-05 - 8 Cell Cycle 1.61E-03 - 4 4.26E-02 4.44E-02 Cellular Assembly and 2.17E-05 - 8 Cell-To-Cell Signaling 1.61E-03 - 3 Organization 3.88E-02 and Interaction 2.54E-02

Cellular Function and 2.17E-05 - 10 Cellular Movement 1.61E-03 - 2 Maintenance 3.88E-02 2.86E-02 Cellular Movement 4.57E-05 - 9 Cell Morphology 3.21E-03 - 3 4.26E-02 4.93E-02 Cellular Development 3.17E-04 - 6 Cellular Growth and 3.21E-03 - 4 2.34E-02 Proliferation 4.28E-02

Hypomethylated Cell Death 2.83E-04 - 6 Cell Cycle 2.12E-03 - 2 3.83E-02 6.34E-03 Cell Cycle 2.93E-03 - 6 Cell Death 2.12E-03 - 3 4.12E-02 4.56E-02 Cell-To-Cell Signaling and 3.00E-03 - 10 Cell Morphology 2.12E-03 - 4 Interaction 4.98E-02 3.54E-02

Cellular Compromise 3.00E-03 - 2 Cellular Assembly and 2.12E-03 - 3 3.25E-02 Organization 4.77E-02

Cellular Development 3.00E-03 - 3 Cellular Compromise 2.12E-03 - 2 4.69E-02 3.95E-02

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Table 7. 8 The top networks matched by the genes differentially methylated in both the FPGS-overexpressed HCT116 and MDA-MB-435 cells

Hypermethylation Hypomethylation Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cell Death, Developmental 21 12 Endocrine System Disorders, 24 12 Disorder, Hereditary Disorder Cancer, Developmental Disorder 2 Cancer, Endocrine System 15 9 Connective Tissue Development 12 7 Disorders, Reproductive System and Function, Embryonic Disease Development, Organ Development 3 Cellular Movement, Immune 7 5 Cancer, Hematological Disease, 2 1 Cell Trafficking, Cell Death Endocrine System Disorders 4 Cellular Compromise, Energy 5 2 Cell Cycle, Cellular Response to 2 1 Production, Lipid Metabolism Therapeutics, Connective Tissue Development and Function 5 Lipid Metabolism, Small 2 1 Embryonic Development, 2 1 Molecule Biochemistry, Organismal Development, Molecular Transport Tissue Development The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

Table 7. 9 The top networks matched by the genes differentially methylated in both the FPGS-inhibited HCT116 and MDA-MB-435 cells

Hypermethylation Hypomethylation Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cell Cycle, DNA Replication, 12 6 Cellular Movement, Cell Death, 18 9 Recombination, and Repair, Cellular Development Neurological Disease 2 Cellular Movement, Cell 3 1 Cancer, Cardiovascular Disease, 2 1 Signaling, Molecular Transport Cellular Development

3 Tissue Morphology, DNA 3 1 Cell Morphology, Nervous 2 1 Replication, Recombination, System Development and and Repair, Cell Morphology Function, Tissue Morphology 4 Developmental Disorder, 3 1 Organ Morphology, Cell Cycle, 2 1 Hereditary Disorder, Skeletal Cellular Development and Muscular Disorders 5 Organ Morphology, Skeletal 2 1 Cellular Development, 2 1 and Muscular System Connective Tissue Development Development and Function, Cell and Function, Embryonic Signaling Development The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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7.4.4 Gene Expression

A two-dimensional hierarchical clustering showed the patterns of gene expression fold change for 47,231 probes in the FPGS-modulated HCT116 (Figure 7.10) and MDA-MB-435 cell lines (Figure 7.11). It demonstrated distinctively different patterns of gene expression in the FPGS- modulated HCT116 and MDA-MB-435 cell lines (Figures 7.10 and 7.11).

The number of the genes differentially expressed in the FPGS-modulated HCT116 and MDA- MB-435 cells is presented in Table 7.10. We determined the number of genes with a fold change > 1.3 or < -1.3 using an one-way ANOVA with the FDR corrected P-value ≤ 0.05. In the HCT116 cell line, we identified 2897 genes that were differentially expressed (1576 downregulated and 1321 upregulated) associated with FPGS overexpression, while 359 genes were differentially expressed (129 downregulated and 230 upregulated) associated with FPGS inhibition (Table 7.10). With respect to the MDA-MB-435 cell line, we identified 1502 genes that were differentially expressed (840 downregulated and 662 upregulated) associated with FPGS overexpression, whereas 829 genes were differentially expressed (442 downregulated and 387 upregulated) associated with FPGS inhibition (Table 7.10). The top fifty genes most differentially expressed in the FPGS-modulated HCT116 and MDA-MB-435 cell lines are shown in Appendix 11.

Table 7. 10 Summary of number of genes differentially expressed in the FPGS- modulated HCT116 and MDA-MB-435 cell lines

FPGS Overexpression FPGS Inhibition

Down- Up- Down- Up- Total Total regulation regulation regulation regulation

HCT116 FC |1.3| 1576 1601 3177 129 230 359 (5.0%) (5.1%) (10.1%) (0.4%) (0.7%) (1.1%)

FC |1.5| 953 1027 1980 26 77 103

(3.0%) (3.3%) (6.3%) (0.1%) (0.2%) (0.3%)

MDA-MB-435 FC |1.3| 840 942 1782 442 387 829 (2.7%) (3.0%) (5.7%) (1.4%) (1.2%) (2.6%) FC |1.5| 465 600 1065 175 168 343 (1.5%) (1.9%) (3.4%) (0.6%) (0.5%) (1.1%)

FC: fold change; The numbers in brackets indicate the percentage of genes differentially expressed relative to total genes targeted in the Illumina HumanHT-12 v4.0 BeadChip.

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Figure 7. 10 Two-dimensional hierarchical clustering of gene expression fold change in the FPGS-modulated HCT116 cell line. A color gradient from green to red indicated the low and high fold change, respectively.

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Figure 7. 11 Two-dimensional hierarchical clustering of gene expression fold change in the FPGS-modulated MDA-MB-435 cell line. A color gradient from green to red indicated the low and high fold change, respectively.

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7.4.4.1 Genes Differentially Expressed in the FPGS-Modulated HCT116 Cells

As a result of the functional analysis using IPA to identify biological and disease processes most relevant to differentially expressed genes in the FPGS-modulated HCT116 cells, genes involved in cell cycle, cellular assembly and organization, DNA replication, recombination, and repair, RNA post-transcriptional modification, and cell death were identified in FPGS overexpression, while genes associated with cell death, cell cycle, cell morphology, cellular function and maintenance, and cellular compromise were identified in FPGS inhibition (Table 7.11). The list of genes for each top function is shown in Appendix 12. The top ten genes most differentially expressed in the FPGS-modulated HCT116 cells are shown in Tables 7.12 and 7.13.

Assignment of biological processes and subsequent construction of networks was done using IPA. A table with functions, involved genes, and significance scores of top five functions is presented in Table 7.14. As mentioned earlier, the focus genes indicate the number of genes from our data set involved in each of the networks. The functions of network shown the highest score included DNA replication, recombination, and repair, cell cycle, and cellular assembly and organization in the HCT116 cells that overexpressed FPGS, whereas those of the highest-scoring network included cell morphology, cellular function and maintenance, and metabolic disease in the HCT116 cells in which FPGS was inhibited (Table 7.14). The list of genes for each top network in the FPGS-modulated HCT116 is shown in Appendix 13.

Table 7. 11 The top molecular and cellular functions associated with differentially expressed genes in the FPGS-modulated HCT116 colon cancer cells

FPGS Overexpression FPGS Inhibition No. of No. of Category P-value Category P-value Genes Genes Cell Cycle 1.27E-15 - 346 Cell Death 4.02E-09 - 93 3.21E-02 2.39E-02 Cellular Assembly and 1.27E-15 - 181 Cell Cycle 6.78E-07 - 44 Organization 3.21E-02 2.46E-02 DNA Replication, 1.27E-15 - 291 Cell Morphology 7.21E-06 - 28 Recombination, and Repair 3.21E-02 2.46E-02 RNA Post-Transcriptional 6.86E-13 - 87 Cellular Function and 7.21E-06 - 30 Modification 1.99E-02 Maintenance 2.46E-02 Cell Death 3.29E-12 - 527 Cellular Compromise 2.98E-05 - 13 3.21E-02 2.27E-02

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Table 7. 12 List of the top differentially expressed genes in the FPGS-overexpressed HCT116 colon cancer cells

Gene Fold Description Accession Symbol Change Downregulated CCNA2 -4.84 cyclin A2 NM_001237.2 DLGAP5* -4.06 discs, large (Drosophila) homolog-associated protein 5 NM_014750.3 PPIL5 -3.78 peptidylprolyl isomerase (cyclophilin)-like 5 NM_203467.1 KPNA2* -3.66 karyopherin alpha 2 (RAG cohort 1, importin alpha 1) NM_002266.2 HMMR* -3.63 hyaluronan-mediated motility receptor (RHAMM) NM_012485.1 AURKA* -3.62 aurora kinase A NM_198434.1 CBX1 -3.60 chromobox homolog 1 (HP1 beta homolog NM_006807.3 Drosophila ) RRM1 -3.59 M1 polypeptide NM_001033.2 CDC20 -3.54 cell division cycle 20 homolog (S. cerevisiae) NM_001255.2 MAD2L1 -3.53 MAD2 mitotic arrest deficient-like 1 (yeast) NM_002358.2 Upregulated GDF15 15.30 growth differentiation factor 15 NM_004864.1 TNFRSF6B* 11.17 tumor necrosis factor receptor superfamily, member 6b, NM_032945.2 decoy DDIT4 9.12 DNA-damage-inducible transcript 4 NM_019058.2 DDIT3 7.49 DNA-damage-inducible transcript 3 NM_004083.4 TRIB3 7.00 tribbles homolog 3 (Drosophila) NM_021158.3 PCK2* 6.04 phosphoenolpyruvate carboxykinase 2 (mitochondrial) NM_004563.2 RBCK1* 5.87 RanBP-type and C3HC4-type zinc finger containing 1 NM_031229.2 PRIC285 5.13 peroxisomal proliferator-activated receptor A NM_033405.2 interacting complex 285 NDRG1 4.95 N-myc downstream regulated gene 1 NM_006096.2 ASNS* 4.89 asparagine synthetase NM_133436.1

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Table 7. 13 List of the top differentially expressed genes in the FPGS-inhibited HCT116 colon cancer cells

Gene Fold Description Accession Symbol Change Downregulated BMP4* -2.57 bone morphogenetic protein 4 NM_130851.1 DNTTIP2 -2.11 deoxynucleotidyltransferase, terminal, interacting NM_014597.3 protein 2 PKIB -2.01 protein kinase (cAMP-dependent, catalytic) inhibitor NM_032471.4 beta NR2F2 -2.00 nuclear receptor subfamily 2, group F, member 2 NM_021005.2 PER2 -1.97 period homolog 2 (Drosophila) NM_022817.2 RERG -1.73 RAS-like, estrogen-regulated, growth inhibitor NM_032918.1 ROCK2 -1.73 Rho-associated, coiled-coil containing protein kinase 2 NM_004850.3 AXIN2 -1.72 axin 2 (conductin, axil) NM_004655.2 PVRL3* -1.71 poliovirus receptor-related 3 NM_015480.1 DUSP6* -1.71 dual specificity phosphatase 6 NM_022652.2 Upregulated GDF15 3.47 growth differentiation factor 15 NM_004864.1 DDIT4 3.44 DNA-damage-inducible transcript 4 NM_019058.2 CDKN1A* 2.94 cyclin-dependent kinase inhibitor 1A (p21, Cip1) NM_000389.2 LCN2 2.72 lipocalin 2 NM_005564.3 CYP24A1 2.58 cytochrome P450, family 24, subfamily A, polypeptide NM_000782.3 1 ANXA10 2.36 annexin A10 NM_007193.3 IFI27 2.32 interferon, alpha-inducible protein 27 NM_005532.3 IRF9 2.12 interferon regulatory factor 9 NM_006084.4 PHLDB2* 2.11 pleckstrin homology-like domain, family B, member 2 NM_145753.1 IFIT1 2.11 interferon-induced protein with tetratricopeptide NM_001548.3 repeats 1

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Table 7. 14 The top networks matched by the genes differentially expressed in the FPGS- modulated HCT116 colon cancer cells

FPGS Overexpression FPGS Inhibition Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 DNA Replication, 23 34 Cell Morphology, Cellular 45 31 Recombination, and Repair, Function and Maintenance, Cell Cycle, Cellular Assembly Metabolic Disease and Organization 2 Molecular Transport, Small 21 33 Cell Cycle, DNA Replication, 36 27 Molecule Biochemistry, Nucleic Recombination, and Repair, Acid Metabolism Cellular Assembly and Organization 3 DNA Replication, 21 33 Cellular Assembly and 30 24 Recombination, and Repair, Organization, Cellular Function Cellular Assembly and and Maintenance, Cancer Organization, Cell Cycle

4 DNA Replication, 21 33 Inflammatory Disease, 26 22 Recombination, and Repair, Neurological Disease, Skeletal Developmental Disorder, and Muscular Disorders Hematological Disease 5 Molecular Transport, RNA 21 33 DNA Replication, 16 16 Trafficking, Cell Death Recombination, and Repair, Cell Morphology, Connective Tissue Development and Function

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

7.4.4.2 Genes Differentially Expressed in the FPGS-Modulated MDA-MB-435 Cells

A functional characterization identified genes involved in cell death, cellular movement, cellular growth and proliferation, cell cycle, and cell-to-cell signaling and interaction in the FPGS- overexpressed MDA-MB-435 cells, while genes associated with cell death, cellular assembly and organization, cell cycle, cellular compromise, and cellular function and maintenance were identified in the MDA-MB-435 cells in which FPGS was inhibited (Table 7.15). The list of genes for each top function is shown in Appendix 12. The top ten genes most differentially expressed in the FPGS-modulated MDA-MB-435 cells are shown in Tables 7.16 and 7.17.

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As a result of network construction for biological processes using IPA, the functions of network shown the highest score included cancer, reproductive system disease, and nucleic acid metabolism in the FPGS-overexpressed MDA-MB-435 cells, whereas those of the highest- scoring network included cell cycle, dermatological disease and conditions, and lipid metabolism in the FPGS-inhibited MDA-MB-435 cells (Table 7.18). A table with functions, involved genes, and significance scores of top five functions is presented in Table 7.18. The list of genes for each top network in the FPGS-modulated MDA-MB-435 cells is shown in Appendix 13.

Table 7. 15 The top molecular and cellular functions associated with differentially expressed genes in the FPGS-modulated MDA-MB-435 breast cancer cells

FPGS Overexpression FPGS Inhibition No. of No. of Category P-value Category P-value Genes Genes Cell Death 5.08E-11 - 342 Cell Death 3.06E-04 - 117 4.92E-02 4.81E-02 Cellular Movement 4.18E-06 - 171 Cellular Assembly and 1.99E-03 - 27 4.95E-02 Organization 4.81E-02 Cellular Growth and 6.69E-06 - 335 Cell Cycle 2.31E-03 - 13 Proliferation 4.66E-02 4.81E-02 Cell Cycle 2.91E-05 - 134 Cellular Compromise 2.31E-03 - 9 4.97E-02 4.81E-02 Cell-To-Cell Signaling and 7.96E-05 - 58 Cellular Function and 2.31E-03 - 27 Interaction 4.20E-02 Maintenance 4.81E-02

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Table 7. 16 List of the top differentially expressed genes in the FPGS-overexpressed MDA-MB-435 breast cancer cells

Gene Fold Description Accession Symbol Change Downregulated TSPAN7* -22.33 tetraspanin 7 NM_004615.2 TYR -20.19 tyrosinase (oculocutaneous albinism IA) NM_000372.4 APOD -11.66 apolipoprotein D NM_001647.2 TRIM48 -11.38 tripartite motif-containing 48 NM_024114.2 BCHE* -11.02 butyrylcholinesterase NM_000055.1 IGSF11 -10.38 immunoglobulin superfamily, member 11 NM_001015887.1 DCT -9.55 dopachrome tautomerase (dopachrome delta-isomerase, NM_001922.2 tyrosine-related protein 2) CHCHD6 -9.43 coiled-coil-helix-coiled-coil-helix domain containing 6 NM_032343.1 ALDH1A1* -9.30 aldehyde dehydrogenase 1 family, member A1 NM_000689.3 GPM6B* -5.99 glycoprotein M6B NM_001001995.1 Upregulated IL24* 12.91 interleukin 24 NM_006850.2 HLA-DQA1 9.13 PREDICTED: major histocompatibility complex, class XM_936128.2 II, DQ alpha 1, transcript variant 10 C21orf34 8.79 chromosome 21 open reading frame 34 NM_001005734.1 C20orf100 7.51 open reading frame 100 NM_032883.1 CNN3 7.16 calponin 3, acidic NM_001839.2 NNMT 6.82 nicotinamide N-methyltransferase NM_006169.2 SPRR2D* 6.26 small -rich protein 2D NM_006945.3 CDC42EP5 5.99 CDC42 effector protein (Rho GTPase binding) 5 NM_145057.2 AHNAK* 5.96 AHNAK nucleoprotein NM_024060.2 COL13A1* 5.71 collagen, type XIII, alpha 1 NM_080805.2

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Table 7. 17 List of the top differentially expressed genes in the FPGS-inhibited MDA- MB-435 breast cancer cells

Gene Fold Description Accession Symbol Change Downregulated HLA-DQA1 -5.75 PREDICTED: major histocompatibility complex, class XM_936128.2 II, DQ alpha 1, transcript variant 10 THBS2 -5.25 thrombospondin 2 NM_003247.2 AIF1L* -4.57 allograft inflammatory factor 1-like NM_031426.2 C21orf34 -4.48 chromosome 21 open reading frame 34 NM_001005734.1 HLA-DOA -3.95 major histocompatibility complex, class II, DO alpha NM_002119.3 C1S -3.26 complement component 1, s subcomponent NM_001734.2 HLA-DMB -3.17 major histocompatibility complex, class II, DM beta NM_002118.3 CTHRC1 -2.88 collagen triple helix repeat containing 1 NM_138455.2 HLA-DPA1 -2.87 major histocompatibility complex, class II, DP alpha 1 NM_033554.2 HLA-DRB4 -2.82 major histocompatibility complex, class II, DR beta 4 NM_021983.4 Upregulated BCHE* 7.02 butyrylcholinesterase NM_000055.1 HAPLN1* 6.12 hyaluronan and proteoglycan link protein 1 NM_001884.2 CXorf26 5.01 chromosome X open reading frame 26 NM_016500.3 SLFN11 4.08 schlafen family member 11 NM_152270.2 S100A4* 4.04 S100 calcium binding protein A4 NM_019554.2 TRIM48 3.67 tripartite motif-containing 48 NM_024114.2 IL1RAPL1 3.41 interleukin 1 receptor accessory protein-like 1 NM_014271.2 RAB38 3.27 RAB38, member RAS oncogene family NM_022337.1 RENBP 3.13 renin binding protein NM_002910.4 ANKS1A 2.83 ankyrin repeat and sterile alpha motif domain NM_015245.2 containing 1A

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Table 7. 18 The top networks matched by the genes differentially expressed in the FPGS- modulated MDA-MB-435 breast cancer cells

FPGS Overexpression FPGS Inhibition

Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cancer, Reproductive System 31 34 Cell Cycle, Dermatological 41 33 Disease, Nucleic Acid Diseases and Conditions, Lipid Metabolism Metabolism 2 Infectious Disease, Cancer, 27 32 Immunological Disease, 36 31 Dermatological Diseases and Neurological Disease, Skeletal Conditions and Muscular Disorders 3 Cancer, Cardiovascular System 27 32 Cell Death, Cellular Growth and 30 28 Development and Function, Proliferation, Cancer Organismal Development 4 DNA Replication, 27 32 Cellular Growth and 26 26 Recombination, and Repair, Proliferation, Cellular Cancer, Cellular Response to Movement, Neurological Therapeutics Disease 5 Molecular Transport, Small 25 31 Antimicrobial Response, Cell- 26 26 Molecule Biochemistry, To-Cell Signaling and Infectious Disease Interaction, Embryonic Development

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

7.4.4.3 Genes Commonly Differentially Expressed in the FPGS-Modulated HCT116 and MDA-MB-435 Cells

Venn diagrams show the number of genes differentially expressed in both the FPGS-modulated HCT116 and MDA-MB-435 cell lines (Figure 7.12). One hundred seventy-nine down- and 280 upregulated genes were common between HCT116 and MDA-MB-435 cell lines in response to FPGS overexpression (Figure 7.12A), while 8 down- and 12 upregulated genes were common between these cell lines in response to FPGS inhibition (Figure 7.12B).

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A

B

Figure 7. 12 Number of genes commonly differentially expressed in both the HCT116 and MDA-MB-435 cells in FPGS overexpression (A) and FPGS inhibition (B)

In the FPGS-overexpressed HCT116 and MDA-MB-435 cell lines, the commonly downregulated genes were associated with DNA replication, recombination, and repair, cell cycle, cell morphology, cellular function and maintenance, and cell death, whereas the commonly upregulated genes were related to cell death, cellular development, gene expression, cellular growth and proliferation, and cell-to-cell signaling and interaction (Table 7.19). As for the FPGS-inhibited HCT116 and MDA-MB-435 cell lines, the major function categories of the commonly downregulated genes included lipid metabolism, molecular transport, nucleic acid metabolism, small molecule biochemistry, and cell death, while those of genes upregulated in common consisted of cellular movement, cellular compromise, vitamin and mineral metabolism, cell cycle, and molecular transport (Table 7.19). The list of top networks generated by mapping the focus genes that were commonly differentially expressed in both the FPGS-modulated HCT116 and MDA-MB-435 cells is presented in Tables 7.20 and 7.21.

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Table 7. 19 The top molecular and cellular functions associated with genes that are commonly differentially expressed in the FPGS-modulated HCT116 and MDA-MB-435 cells

FPGS Overexpression FPGS Inhibition No. of No. of Category P-value Category P-value Genes Genes Downregulated DNA Replication, 5.54E-09 – 31 Lipid Metabolism 5.12E-04 – 1 Recombination, and Repair 4.26E-02 3.58E-03 Cell Cycle 9.56E-05 – 28 Molecular Transport 5.12E-04 – 1 4.45E-02 3.58E-03 Cell Morphology 1.16E-04 – 3 Nucleic Acid Metabolism 5.12E-04 – 2 1.08E-02 8.67E-03 Cellular Function and 1.16E-04 – 6 Small Molecule 5.12E-04 – 2 Maintenance 4.26E-02 Biochemistry 8.67E-03 Cell Death 4.19E-03 – 14 Cell Death 1.02E-03 – 1 2.53E-02 3.18E-02 Upregulated Cell Death 1.15E-09 – 78 Cellular Movement 2.63E-04 – 2 2.70E-02 2.63E-04 Cellular Development 6.19E-07 – 59 Cellular Compromise 8.77E-04 – 1 2.58E-02 8.77E-04 Gene Expression 2.23E-06 – 55 Vitamin and Mineral 8.77E-04 – 1 2.37E-02 Metabolism 8.77E-04 Cellular Growth and 5.13E-06 – 73 Cell Cycle 2.63E-03 – 1 Proliferation 2.58E-02 2.63E-03 Cell-To-Cell Signaling and 2.95E-05 – 34 Molecular Transport 3.50E-03 – 2 Interaction 2.70E-02 2.34E-02

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Table 7. 20 The top networks matched by the genes differentially expressed in both the FPGS-overexpressed HCT116 and MDA-MB-435 cells

Downregulation Upregulation Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 DNA Replication, 52 29 Dermatological Diseases and 36 26 Recombination, and Repair, Conditions, Immunological Cancer, Gastrointestinal Disease Disease, Inflammatory Disease 2 DNA Replication, 26 18 Cell Death, Cancer, 34 25 Recombination, and Repair, Hematological Disease Cellular Assembly and Organization, Cell Cycle 3 Cellular Development, Cellular 18 14 Cellular Movement, Cellular 30 23 Growth and Proliferation, Growth and Proliferation, Respiratory System Cellular Development Development and Function 4 DNA Replication, 15 12 Cancer, Reproductive System 26 21 Recombination, and Repair, Disease, Cell Death Cell Cycle, Cellular Assembly and Organization 5 Cellular Movement, 15 12 Lipid Metabolism, Molecular 17 16 Hematological System Transport, Small Molecule Development and Function, Biochemistry Immune Cell Trafficking

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

Table 7. 21 The top networks matched by the genes differentially expressed in both the FPGS-inhibited HCT116 and MDA-MB-435 cells

Downregulation Upregulation Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cancer, Hereditary Disorder, 3 1 Cell Cycle, Cancer, Cellular 26 11 Respiratory Disease Development 2 DNA Replication, 2 1 Cellular Function and 3 1 Recombination, and Repair, Maintenance, Cellular Nucleic Acid Metabolism, Movement, Cell-To-Cell Small Molecule Biochemistry Signaling and Interaction 3 Cell Death, Tumor Morphology, 2 1 Cell Morphology

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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7.4.5 Integrated Analysis of Gene Expression and Promoter DNA Methylation Changes

To identify and prioritize differentially expressed genes that are regulated by DNA methylation as a result of FPGS modulation, we performed an integrated analysis of differentially expressed genes and differentially methylated genes in the FPGS-modulated HCT116 and MDA-MB-435 cells. As shown in Figure 7.13, β-value difference and log2-transformed gene expression value difference between Sense and Control(-S), between Antisense and Control, and between siRNA and Control-si are plotted on x-axis and y-axis, respectively. Red data points highlight those genes that are hypermethylated with a β-value difference > 0.2 and show < -1.3 fold change in their expression levels, while green data points indicate those genes that are hypomethylated with a β-value difference < -0.2 and show > 1.3 fold change in their expression levels. The number of genes with altered expression and CpG promoter DNA methylation in the FPGS-modulated HCT116 and MDA-MB-435 cells are summarized in Table 7.22. We detected 71 and 138 genes with the inverse relationship between gene expression and promoter DNA methylation changes in the FPGS-modulated HCT116 and MDA-MB-435 cells, respectively (Figure 7.13; Table 7.22). The list of genes with altered promoter DNA methylation and expression in the FPGS- modulated HCT116 and MDA-MB-435 cell lines is shown in Appendix 14.

Table 7. 22 Summary of number of genes with altered expression and promoter DNA methylation in the FPGS-modulated HCT116 and MDA-MB-435 cell lines

FPGS Overexpression FPGS Inhibition

Hypermethylated & Hypomethylated & Hypermethylated & Hypomethylated & Downregulated/ Upregulated/ Downregulated/ Upregulated/ Downregulated (%) Upregulated (%) Downregulated (%) Upregulated (%) HCT116 34/1576 (2.2%) 31/1321 (2.3%) 2/129 (1.6%) 4/230 (1.7%)

MDA-MB-435 41/840 (4.9%) 54/662 (8.2%) 30/442 (6.8%) 13/387 (3.4%)

The numbers in brackets indicate the percentage of genes whose expression was inversely regulated by promoter DNA methylation relative to genes differentially expressed.

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A B

C D

Figure 7. 13 Integrated analysis of gene expression and promoter DNA methylation changes between Sense and Control(-S) (A, C), between Antisense and Control (B), and between siRNA and Control-si (D) in the FPGS-modulated HCT116 (A, B) and MDA-MB- 435 cells (C, D). Red data points highlight those genes that are hypermethylated with β-value difference > 0.2 and show < -1.3 fold change in their expression levels, while green data points indicate those genes that are hypomethylated with β-value difference < -0.2 and show > 1.3 fold change in their expression levels.

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7.4.5.1 Genes Regulated by DNA Methylation in the FPGS-Modulated HCT116 Cells

To evaluate which biological and disease processes are most relevant to these genes, we performed the functional analysis using IPA. In the FPGS-overexpressed HCT116 cells, hypermethylated and downregulated genes were involved in cell cycle, DNA replication, recombination, and repair, cell death, cell morphology, and cell-to-cell signaling and interaction, while hypomethylated and upregulated genes were associated with cell death, cell cycle, cell-to- cell signaling and interaction, cellular development, and cellular function and maintenance (Table 7.23). As for the HCT116 cells in which FPGS was inhibited, MECOM (ecotropic viral integration site 1, hypermethylated and downregulated) was associated with gene expression and cellular function and maintenance whereas ABCC2 (ATP-binding cassette, sub-family C, member 2) was involved in drug metabolism, molecular transport, lipid metabolism, small molecule biochemistry, and amino acid metabolism (Table 7.23). The list of top genes with altered promoter DNA methylation and expression in the FPGS-modulated HCT116 cells is shown in Tables 7.24 and 7.25.

Table 7. 23 The top molecular and cellular functions associated with genes with altered expression and promoter methylation in the FPGS-modulated HCT116 colon cancer cells FPGS Overexpression FPGS Inhibition No. of No. of Category P-value Category P-value Genes Genes Hypermethylated and Downregulated Cell Cycle 2.95E-06 - 5 Gene Expression 6.47E-03 - 1 4.65E-02 3.21E-02 DNA Replication, 5.46E-05 - 5 Cellular Function and 1.23E-02 - 1 Recombination, and Repair 3.95E-02 Maintenance 1.23E-02 Cell Death 1.53E-03 - 5 4.82E-02 Cell Morphology 1.83E-03 - 3 3.06E-02 Cell-To-Cell Signaling and 1.83E-03 - 1 Interaction 1.83E-03 Hypomethylated and Upregulated Cell Death 1.60E-03 - 7 Drug Metabolism 1.52E-04 - 1 4.84E-02 9.13E-04 Cell Cycle 3.19E-03 - 2 Molecular Transport 1.52E-04 - 1 1.27E-02 1.83E-03 Cell-To-Cell Signaling and 3.19E-03 - 2 Lipid Metabolism 3.04E-04 - 1 Interaction 2.06E-02 7.61E-04 Cellular Development 3.19E-03 - 1 Small Molecule 3.04E-04 - 1 4.08E-02 Biochemistry 9.13E-04 Cellular Function and 3.19E-03 - 2 Amino Acid Metabolism 9.13E-04 - 1 Maintenance 3.19E-03 9.13E-04

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Table 7. 24 List of the top genes with altered promoter methylation and expression in the FPGS-overexpressed HCT116 colon cancer cells

DNA Methylation Gene Expression Gene Entrez Description Symbol β-Value Difference Fold Change Gene ID (vs. Control) (vs. Control) Hypermethylated and Downregulated DEPDC1B* DEP domain containing 1B 0.38 -2.74 55789 MSH2* mutS homolog 2, colon cancer, 0.31 -2.08 4436 nonpolyposis type 1 (E. coli) FANCE Fanconi anemia, complementation 0.45 -1.94 2178 group E ALDH1A3* aldehyde dehydrogenase 1 family, 0.29 -1.93 220 member A3 HNRNPM* heterogeneous nuclear 0.20 -1.92 4670 ribonucleoprotein M BRCA1* breast cancer 1, early onset 0.24 -1.88 672 WEE1 WEE1 homolog (S. pombe) 0.20 -1.78 7465 LIME1 Lck interacting transmembrane 0.23 -1.75 54923 adaptor 1 C16orf35 chromosome 16 open reading frame 35 0.21 -1.73 8131 MNS1 meiosis-specific nuclear structural 1 0.61 -1.69 55329 Hypomethylated and Upregulated H1F0 H1 histone family, member 0 -0.33 3.52 3005 HCP5 HLA complex P5 -0.34 2.62 10866 REEP6 receptor accessory protein 6 -0.35 2.24 92840 ST6GALNAC2 ST6 (alpha-N-acetyl-neuraminyl- -0.22 2.20 10610 2,3-beta-galactosyl-1, 3)-N- acetylgalactosaminide alpha-2,6- sialyltransferase 2 HLA-DMA major histocompatibility complex, -0.26 2.16 3108 class II, DM alpha TRAPPC6A trafficking protein particle complex 6A -0.23 2.12 79090 OAF OAF homolog (Drosophila) -0.26 2.01 220323 SHC1 SHC (Src homology 2 domain -0.22 1.97 6464 containing) transforming protein 1 ARFGAP3 ADP-ribosylation factor GTPase -0.21 1.93 26286 activating protein 3 GPM6A glycoprotein M6A -0.21 1.91 2823

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Table 7. 25 List of the top genes with altered promoter methylation and expression in the FPGS-inhibited HCT116 colon cancer cells

DNA Methylation Gene Expression Gene Entrez Description Symbol β-Value Difference Fold Change Gene ID (vs. Control) (vs. Control) Hypermethylated and Downregulated MECOM ecotropic viral integration site 1 0.27 -1.62 2122 FAM178A chromosome 10 open reading frame 6 0.27 -1.45 55719 Hypomethylated and Upregulated ABCC2 ATP-binding cassette, sub-family C -0.26 1.56 1244 (CFTR/MRP), member 2 SKAP1* src kinase associated phosphoprotein 1 -0.21 1.51 8631 REEP6* receptor accessory protein 6 -0.43 1.37 92840 TTC33 tetratricopeptide repeat domain 33 -0.30 1.34 23548

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

Result of the network analysis for biological processes, obtained through mapping focus genes whose expression was inversely regulated by CpG promoter methylation in the FPGS-modulated HCT116 is shown in Table 7.26. In the HCT116 cells that overexpressed FPGS, the functions of network with the highest score included DNA replication, recombination, and repair, cell cycle, and vitamin and mineral metabolism in hypermethylated and downregulated genes, whereas those of the highest-scoring network in hypomethylated and upregulated genes included inflammatory response, cell death, and cell-to-cell signaling and interaction (Table 7.26). With respect to the FPGS-inhibited HCT116 cells, the functions of top network included cancer, hematological disease, and cell morphology in hypermethylated and downregulated genes, while those of the highest-scoring network in hypomethylated and upregulated genes included cellular movement, hematological system development and function, and immune cell trafficking (Table 7.26). The list of genes for each top network in the FPGS-modulated HCT116 cells is presented in Appendix 15.

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Table 7. 26 The top networks matched by the genes with altered expression and promoter methylation in the FPGS-modulated HCT116 colon cancer cells

FPGS Overexpression FPGS Inhibition

Focus Focus No Top Functions Score Top Functions Score Genes Genes Hypermethylated and Downregulated 1 DNA Replication, 20 10 Cancer, Hematological Disease, 3 1 Recombination, and Repair, Cell Morphology Cell Cycle, Vitamin and Mineral Metabolism 2 Cellular Function and 2 1 Maintenance, Cellular Movement, Cell-To-Cell Signaling and Interaction 3 Tissue Morphology, DNA 2 1 Replication, Recombination, and Repair, Cell Morphology 4 DNA Replication, 2 1 Recombination, and Repair, Digestive System Development and Function, Cell Death 5 Molecular Transport, Nucleic 2 1 Acid Metabolism, Small Molecule Biochemistry

Hypomethylated and Upregulated 1 Inflammatory Response, Cell 18 9 Cellular Movement, 3 1 Death, Cell-To-Cell Signaling Hematological System and Interaction Development and Function, Immune Cell Trafficking 2 Cellular Movement, Infectious 2 1 Lipid Metabolism, Molecular 3 1 Disease, Cellular Development Transport, Small Molecule Biochemistry 3 Cell-To-Cell Signaling and 2 1 Interaction, Cellular Function and Maintenance, Connective Tissue Development and Function 4 Amino Acid Metabolism, Post- 2 1 Translational Modification, Small Molecule Biochemistry 5 Carbohydrate Metabolism, 2 1 Cellular Assembly and Organization, DNA Replication, Recombination, and Repair

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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7.4.5.2 Genes Regulated by DNA Methylation in the FPGS-Modulated MDA- MB-435 Cells

The same set of analyses were performed for genes with altered promoter DNA methylation and expression in the FPGS-modulated MDA-MB-435 cells. In the MDA-MB-435 cells that overexpressed FPGS, hypermethylated and downregulated genes were associated with drug metabolism, molecular transport, cell cycle, cell death, and cellular assembly and organization, while hypomethylated and upregulated genes were involved in cellular movement, cell-to-cell signaling and interaction, cell death, post-translational modification, and cell signaling (Table 7.27). As for the MDA-MB-435 cells in which FPGS is inhibited, hypermethylated and downregulated genes were involved in cellular movement, carbohydrate metabolism, small molecule biochemistry, cellular growth and proliferation, and cell cycle, whereas hypomethylated and upregulated genes were associated with cell cycle, cellular movement, post- translational modification, protein degradation, and protein systhesis (Table 7.27). The list of top genes with altered promoter DNA methylation and expression in the FPGS-modulated MDA- MB-435 cells is shown in Tables 7.28 and 7.29.

Table 7. 27 The top molecular and cellular functions associated with genes with altered expression and promoter methylation in the FPGS-modulated MDA-MB-435 breast cancer cells FPGS Overexpression FPGS Inhibition No. of No. of Category P-value Category P-value Genes Genes Hypermethylated and Downregulated Drug Metabolism 2.60E-04 - 4 Cellular Movement 1.98E-03 - 1 2.44E-02 4.84E-02 Molecular Transport 3.24E-04 - 2 Carbohydrate Metabolism 3.95E-03 - 1 8.20E-03 3.95E-03 Cell Cycle 2.74E-03 - 2 Small Molecule 3.95E-03 - 1 5.47E-03 Biochemistry 3.95E-03 Cell Death 2.74E-03 - 1 Cellular Growth and 5.93E-03 - 4 2.74E-03 Proliferation 4.84E-02 Cellular Assembly and 2.74E-03 - 3 Cell Cycle 7.89E-03 - 2 Organization 1.09E-02 3.31E-02 Hypomethylated and Upregulated Cellular Movement 4.92E-05 - 13 Cell Cycle 8.37E-04 - 1 4.90E-02 4.18E-03 Cell-To-Cell Signaling and 1.18E-04 - 8 Cellular Movement 3.35E-03 - 1 Interaction 3.87E-02 3.35E-03 Cell Death 1.54E-03 - 17 Post-Translational 1.99E-02 - 2 4.58E-02 Modification 3.46E-02 Post-Translational 1.65E-03 - 6 Protein Degradation 1.99E-02 - 1 Modification 4.62E-02 1.99E-02 Cell Signaling 2.52E-03 - 6 Protein Synthesis 1.99E-02 - 1 1.78E-02 1.99E-02

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Table 7. 28 List of the top genes with altered promoter methylation and expression in the FPGS-overexpressed MDA-MB-435 breast cancer cells

DNA Methylation Gene Expression Gene Entrez Description Symbol β-Value Difference Fold Change Gene ID (vs. Control) (vs. Control)

Hypermethylated and Downregulated TSPAN7* tetraspanin 7 0.61 -22.33 7102 TYR tyrosinase (oculocutaneous albinism IA) 0.42 -20.19 7299 GYG2* glycogenin 2 0.24 -4.24 8908 NBL1* neuroblastoma, suppression of 0.37 -2.94 4681 tumorigenicity 1 PLSCR1 phospholipid scramblase 1 0.21 -2.62 5359 BNC2 basonuclin 2 0.35 -2.33 54796 PTGFRN* prostaglandin F2 receptor negative 0.38 -2.25 5738 regulator GSTT1 glutathione S-transferase theta 1 0.47 -2.19 2952 GDPD5 glycerophosphodiester 0.52 -2.09 81544 phosphodiesterase domain containing 5 TMEM51 transmembrane protein 51 0.26 -2.01 55092

Hypomethylated and Upregulated IL24* interleukin 24 -0.23 12.91 11009 CNN3 calponin 3, acidic -0.36 7.16 1266 LAIR2* leukocyte-associated -0.30 5.50 3904 immunoglobulin-like receptor 2 OLFM1 olfactomedin 1 -0.24 4.58 10439 PTPN22* protein tyrosine phosphatase, non- -0.38 3.21 26191 receptor type 22 (lymphoid) MX1 myxovirus (influenza virus) -0.44 3.13 4599 resistance 1, interferon-inducible protein p78 (mouse) HLA-DPA1 major histocompatibility complex, -0.64 2.82 3113 class II, DP alpha 1 S100A4* S100 calcium binding protein A4 -0.37 2.79 6275 CD55 CD55 molecule, decay accelerating -0.35 2.58 1604 factor for complement (Cromer blood group) C15orf52 chromosome 15 open reading frame 52 -0.26 2.53 388115

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Table 7. 29 List of the top genes with altered promoter methylation and expression in the FPGS-inhibited MDA-MB-435 breast cancer cells

DNA Methylation Gene Expression Gene Entrez Description Symbol β-Value Difference Fold Change Gene ID (vs. Control) (vs. Control) Hypermethylated and Downregulated THBS2 thrombospondin 2 0.45 -5.25 7058 C1S complement component 1, 0.21 -3.26 716 s subcomponent HLA-DPA1 major histocompatibility complex, 0.28 -2.87 3113 class II, DP alpha 1 CAP2 CAP, adenylate cyclase-associated 0.20 -2.55 10486 protein, 2 (yeast) CTSL2 cathepsin L2 0.36 -2.48 1515 LRP5 low density lipoprotein receptor- 0.26 -2.02 4041 related protein 5 FAHD1* fumarylacetoacetate hydrolase 0.22 -1.89 81889 domain containing 1 SCARF2 scavenger receptor class F, member 2 0.30 -1.88 91179 CDC42EP5 CDC42 effector protein (Rho 0.30 -1.78 148170 GTPase binding) 5 GBGT1 globoside alpha-1,3-N- 0.33 -1.70 26301 acetylgalactosaminyltransferase 1

Hypomethylated and Upregulated CNN3* calponin 3, acidic -0.62 2.19 1266 MT1E metallothionein 1E -0.24 1.90 4493 LEPREL1 leprecan-like 1 -0.39 1.82 55214 MT1A metallothionein 1A -0.25 1.69 4489 RHEB Ras homolog enriched in brain -0.24 1.66 6009 PIR* pirin (iron-binding nuclear protein) -0.31 1.59 8544 ARHGAP15* Rho GTPase activating protein 15 -0.35 1.52 55843 CTSL1 cathepsin L1 -0.29 1.50 1514 TM4SF18 transmembrane 4 L six family -0.49 1.49 116441 member 18 PDIA5 protein disulfide isomerase family -0.33 1.42 10954 A, member 5

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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The top networks matched by genes with altered promoter methylation and expression in the FPGS-modulated MDA-MB-435 cells are presented in Table 7.30. In the FPGS-overexpressed MDA-MB-435 cells, the functions of network shown the highest score included cell death, cancer, and cellular development in hypermethylated and downregulated genes, while those of the highest-scoring network in hypomethylated and upregulated genes included cellular function and maintenance, hematological system development and function, and cell-mediated immune response (Table 7.30). As for the FPGS-inhibited MDA-MB-435 cells, the functions of network with the highest score in hypermethylated and downregulated genes included cell death, cell cycle, and cancer, whereas those of the highest-scoring network in hypomethylated and upregulated genes included inflammatory disease, inflammatory response, and organ morphology (Table 7.30). The list of genes for each top network in the FPGS-modulated MDA- MB-435 cells is presented in Appendix 15.

Table 7. 30 The top networks matched by the genes with altered expression and promoter methylation in the FPGS-modulated MDA-MB-435 breast cancer cells

FPGS Overexpression FPGS Inhibition

Focus Focus No Top Functions Score Top Functions Score Genes Genes

Hypermethylated and Downregulated

1 Cell Death, Cancer, Cellular 21 11 Cell Death, Cell Cycle, Cancer 27 13 Development

2 Cellular Development, Cellular 10 6 Cellular Assembly and 2 1 Growth and Proliferation, Organization, Cellular Function Organismal Survival and Maintenance, Cell Signaling

3 Cellular Assembly and 2 1 Connective Tissue Development 2 1 Organization, RNA Post- and Function, Skeletal and Transcriptional Modification, Muscular System Development Cellular Growth and and Function, Tissue Proliferation Development

4 Cell Morphology, Cellular 2 1 Immunological Disease, 2 1 Assembly and Organization, Infectious Disease, Cell Cellular Growth and Morphology Proliferation

5 Cellular Assembly and 2 1 Cell-To-Cell Signaling and 2 1 Organization, Cellular Function Interaction, Connective Tissue and Maintenance, Cell Cycle Development and Function, Cellular Movement

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Hypomethylated and Upregulated

1 Cellular Function and 28 15 Inflammatory Disease, 6 2 Maintenance, Hematological Inflammatory Response, Organ System Development and Morphology Function, Cell-mediated Immune Response

2 Cancer, Gastrointestinal 25 14 Carbohydrate Metabolism, 3 1 Disease, Gene Expression Cellular Compromise, Inflammatory Response

3 Cardiac Proliferation, 2 1 Tissue Development, Genetic 3 1 Cardiovascular System Disorder, Metabolic Disease Development and Function, Cell Death

4 Cell Cycle, Cellular Growth and 2 1 Cellular Growth and 3 1 Proliferation, Hematopoiesis Proliferation, Tumor Morphology, Cancer

5 Infectious Disease, Cancer, 2 1 Cell Cycle, Cellular 3 1 Renal and Urological Disease Compromise, Molecular Transport

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

7.4.6 Validation of Gene Expression by qRT-PCR of Selected Genes Identified from Illumina HumanHT-12 v4.0 BeadChip

Selected differentially expressed genes that were regulated by DNA methylation, presented in Section 7.4.5, were further analyzed by qRT-PCR to validate the gene expression results obtained using Illumina HumanHT-12 v4.5 BeadChip. Similar to GGH modulation, genes were selected for qRT-PCR based on a greater magnitude of fold change in gene expression identified from microarray and relevant biological pathways of interest. The direction of change in gene expression in response to FPGS modulation was consistent between Illumina HumanHT-12 v4.0 BeadChip and qRT-PCR analyses in HCT116 and MDA-MB-435 cell lines (P < 0.05), although the magnitude of change was different (Table 7.31). It appears that difference in transcript(s) between the microarray probe set and qRT-PCR probes might influence different hybridization kinetics of the probe sets for gene (438).

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Table 7. 31 Comparison of fold changes in gene expression detected by Illumina HumanHT-12 v4.0 BeadChip and qRT-PCR analyses in the FPGS-modulated HCT116 and MDA-MB-435 cell lines

Gene Microarray q RT-PCR Description Symbol Fold Change Relative Expression Fold Hypermethylated & Downregulated HCT116 FPGS Overexpression MSH2* mutS homolog 2, colon cancer, nonpolyposis type 1 (E.coli) -2.08 0.25 (0.24-0.27) ALDH1A3 aldehyde dehydrogenase 1 family, member A3 -1.93 0.22 (0.17-0.28) BRCA1* breast cancer 1, early onset -1.88 0.26 (0.24-0.29) FPGS Inhibition MECOM ecotropic viral integration site 1 -1.62 0.10 (0.08-0.12) MDA-MB-435 FPGS Overexpression TYR tyrosinase (oculocutaneous albinism IA) -20.19 0.028 (0.023-0.034) GSTT1* glutathione S-transferase theta 1 -2.19 0.17 (0.15-0.18) SOX10 SRY (sex determining region Y)-box 10 -1.83 0.33 (0.30-0.36) FPGS Inhibition THBS2 thrombospondin 2 -5.25 0.05 (0.04-0.07) HLA-DPA1* major histocompatibility complex, class II, DP alpha 1 -2.87 0.18 (0.15-0.22) LRP5 low density lipoprotein receptor-related protein 5 -2.02 0.18 (0.16-0.20) Hypomethylated & Upregulated HCT116 FPGS Overexpression GPM6A* glycoprotein M6A 1.91 2.98 (2.67-3.32) IRF1 interferon regulatory factor 1 1.90 1.62 (1.52-1.72) WNT16* wingless-type MMTV integration site family, 1.65 3.02 (2.86-3.20) member 16 FPGS Inhibition ABCC2 ATP-binding cassette, sub-family C (CFTR/MRP), 1.56 1.92 (1.84-1.99) member 2 MDA-MB-435 FPGS Overexpression HLA-DPA1* major histocompatibility complex, class II, DP alpha 1 2.82 1.94 (1.78-2.12) S100A4 S100 calcium binding protein A4 2.79 4.03 (3.62-4.49) CD55 CD55 molecule, decay accelerating factor for complement 2.58 1.25 (1.17-1.32) FPGS Inhibition MT1E* metallothionein 1E 1.90 1.92 (1.78-2.07) CTSL1* cathepsin L1 1.50 1.82 (1.76-1.88)

*, β-actin used for housekeeping gene; P < 0.05.

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7.4.7 Pathway-Specific Gene Expression Analysis

As previously described, the lists of genes involved in folate biosynthesis, one-carbon pool by folate, cell cycle (G1/S and G2/M), and apoptosis were referred to the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/pathway.html) and the Ingenuity Knowledge Base. Among genes differentially regulated (> 1.3 or < -1.3 fold change) in response to FPGS modulation in HCT116 and MDA-MB-435 cells, the lists of genes involved in specific pathways are presented in Tables 7.32 to 7.34. We identified genes regulated in opposite directions between FPGS overexpression and inhibition, genes that were commonly up- or downregulated in HCT116 and MDA-MB-435 cells, and genes that were either hypermethylated and downregulated or hypomethylated and upregulated in each system.

7.4.7.1 Folate Biosynthesis and One-Carbon Pool by Folate Pathway

In response to FPGS overexpression, MTHFD2 (methylenetetrahydrofolate dehydrogenase [NADP+ dependent] 2) was upregulated in both the HCT116 and MDA-MB-435 cells, while TYMS (thymidylate synthetase), SLC29A1 (solute carrier family 29 [nucleoside transporters], member 1), TK1 (thymidine kinase 1), and MTHMT (mitochondrial methionyl-tRNA formyltransferase) were downregulated in both cell lines (Table 7.32). MTHFD2 was also upregulated in the FPGS-inhibited HCT116 cells.

In MDA-MB-435 cells, NNMT (nicotinamide N-methyltransferase) was upregulated (fold change 6.82) in FPGS overexpression, while it was downregulated (fold change -1.76) in response to FPGS inhibition (Table 7.32). Additionally, in MDA-MB-435 cells, TK1 (thymidine kinase 1) and GSTT1 (glutathione S-transferase theta 1) were downregulated in FPGS overexpression and upregulated in response to FPGS inhibition (Table 7.32).

Decreased expression of GSTT1 in the FPGS-overexpressed MDA-MB-435 cells was associated with hypermethylation (β-value difference 0.47; Table 7.28; Appendix 14.3), whereas increased expression of ABCC2 (ATP-binding cassette, sub-family C, member 2) in the FPGS-inhibited HCT116 cells was related to hypomethylation (β-value difference -0.26; Table 7.25; Appendix 14.2).

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Table 7. 32 List of differentially expressed genes involved in folate biosynthesis and one- carbon pool by folate pathways in the FPGS-modulated HCT116 and MDA-MB-435 cells

Gene Fold change Description Probe ID Pathway Symbol (vs. Control)

FPGS Overexpression in HCT116 ABCC3 4.03 ATP-binding cassette, sub-family C ILMN_1677814 folate (CFTR/MRP), member 3 FOLR1 2.19 folate receptor 1 (adult) ILMN_1661733 folate 1.85 ILMN_2346339 TPMT 1.69 thiopurine S-methyltransferase ILMN_1740185 folate CDA 1.68 cytidine deaminase ILMN_1714592 folate COMT 1.56 catechol-O-methyltransferase ILMN_1730084 folate MTHFD2§ 1.44 methylenetetrahydrofolate dehydrogenase ILMN_2405521 folate, one-carbon (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase 1.48 ILMN_1674706 ABCC2 1.30 ATP-binding cassette, sub-family C ILMN_1676278 folate (CFTR/MRP), member 2 TYMS§ -2.99 thymidylate synthetase ILMN_1806040 folate, one-carbon SLC29A1§ -2.11 solute carrier family 29 (nucleoside ILMN_1723971 folate transporters), member 1 -1.88 ILMN_2338963 TK1§ -1.86 thymidine kinase 1, soluble ILMN_1806037 folate DNMT1 -1.63 DNA (cytosine-5-)-methyltransferase 1 ILMN_1760201 folate DHFR -1.52 dihydrofolate reductase ILMN_1782813 folate MTHFD1 -1.47 methylenetetrahydrofolate dehydrogenase ILMN_1785324 folate, one-carbon (NADP+ dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase SHMT1 -1.42 serine hydroxymethyltransferase 1 (soluble) ILMN_1811933 folate, one-carbon ABCC5 -1.36 ATP-binding cassette, sub-family C ILMN_1651964 folate (CFTR/MRP), member 5 MTFMT§ -1.44 mitochondrial methionyl-tRNA ILMN_1672884 one-carbon formyltransferase FPGS Inhibition in HCT116 ABCC2* 1.56 ATP-binding cassette, sub-family C ILMN_1676278 folate (CFTR/MRP), member 2 MTHFD2 1.35 methylenetetrahydrofolate dehydrogenase ILMN_1674706 folate, one-carbon (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase 1.34 ILMN_2405521 FPGS Overexpression in MDA-MB-435 NNMT† 6.82 nicotinamide N-methyltransferase ILMN_1715508 folate

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MTHFD2§ 1.84 methylenetetrahydrofolate dehydrogenase ILMN_2405521 folate, one-carbon (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase DPYD 1.49 dihydropyrimidine dehydrogenase ILMN_1795715 folate MTHFD1L 1.77 methylenetetrahydrofolate dehydrogenase ILMN_1772521 one-carbon (NADP+ dependent) 1-like SLC29A1§ -2.57 solute carrier family 29 (nucleoside ILMN_2338963 folate transporters), member 1 -2.27 ILMN_1723971 GSTT1*‡ -2.19 glutathione S-transferase theta 1 ILMN_1730054 folate TYMS§ -2.00 thymidylate synthetase ILMN_1806040 folate, one-carbon ABCC5 -1.80 ATP-binding cassette, sub-family C ILMN_1706531 folate (CFTR/MRP), member 5 TPMT -1.59 thiopurine S-methyltransferase ILMN_1740185 folate GSTM1 -1.58 glutathione S-transferase M1 ILMN_1668134 folate SLC25A32 -1.42 solute carrier family 25, member 32 ILMN_1683212 folate TK1‡§ -1.35 thymidine kinase 1, soluble ILMN_1806037 folate ABCC4 -1.31 ATP-binding cassette, sub-family C ILMN_1788457 folate (CFTR/MRP), member 4 MTFMT§ -1.46 mitochondrial methionyl-tRNA ILMN_1672884 one-carbon formyltransferase

FPGS Inhibition in MDA-MB-435 TK1‡ 1.48 thymidine kinase 1, soluble ILMN_1806037 folate GSTT1‡ 1.41 glutathione S-transferase theta 1 ILMN_1730054 folate NNMT† -1.76 nicotinamide N-methyltransferase ILMN_1715508 folate

*, genes whose expression was regulated by DNA methylation; †, genes upregulated in FPGS overexpression and downregulated in FPGS inhibition; ‡, genes downregulated in FPGS overexpression and upregulated in FPGS inhibition; §, genes differentially expressed in both the FPGS-modulated HCT116 and MDA-MB-435 cells.

7.4.7.2 Cell Cycle Pathway

In response to FPGS overexpression, CDKN2B (cyclin-dependent kinase inhibitor 2B [p15, inhibits CDK4]) and GADD45A (growth arrest and DNA-damage-inducible, alpha) were upregulated in both the HCT116 and MDA-MB-435 cells, whereas SKP2 (S-phase kinase- associated protein 2 [p45]), E2F2 (E2F transcription factor 2), CDK2 (cyclin-dependent kinase 2), CUL1 (cullin 1), and PRKDC (protein kinase, DNA-activated, catalytic polypeptide) were downregulated in both cell lines (Table 7.33).

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In HCT116 cells, CDC25C (cell division cycle 25 homolog C) was downregulated in response to FPGS overexpression and upregulated in response to FPGS inhibition (Table 7.33). As for MDA-MB-435 cells, GADD45A (growth arrest and DNA-damage-inducible, alpha) and CCND1 (cyclin D1) were upregulated in response to FPGS overexpression and downregulated in response to FPGS inhibition (Table 7.33).

In the FPGS-overexpressed HCT116 cells, decreased expressions of BRCA1 (breast cancer 1, early onset) and WEE1 (WEE1 homolog) were related to hypermethylation (β-value difference 0.24 for BRCA1 and 0.20 for WEE1; Appendix 14.1; Table 7.24).

Table 7. 33 List of differentially expressed genes involved in the cell cycle pathway in the FPGS-modulated HCT116 and MDA-MB-435 cells

Gene Fold change Description Probe ID Pathway Symbol (vs. Control) FPGS Overexpression in HCT116 CDKN1A 2.79 cyclin-dependent kinase inhibitor 1A (p21, Cip1) ILMN_1784602 G1/S, G2/M

CDKN2B§ 2.17 cyclin-dependent kinase inhibitor 2B (p15, inhibits ILMN_2376723 G1/S CDK4) 1.54 ILMN_1723198 HDAC4 1.46 histone deacetylase 4 ILMN_1764396 G1/S E2F5 1.33 E2F transcription factor 5, p130-binding ILMN_1782551 G1/S

SKP2§ -2.83 S-phase kinase-associated protein 2 (p45) ILMN_1665538 G1/S, G2/M -2.84 ILMN_1791002 -1.31 ILMN_1801391 SUV39H1 -2.55 suppressor of variegation 3-9 homolog 1 (Drosophila) ILMN_1781479 G1/S CDC25A -2.30 cell division cycle 25 homolog A (S. pombe) ILMN_1711005 G1/S E2F2§ -2.27 E2F transcription factor 2 ILMN_1777233 G1/S CCNE2 -2.10 cyclin E2 ILMN_2412384 G1/S PA2G4 -1.83 proliferation-associated 2G4, 38kDa ILMN_1728984 G1/S CDK6 -1.74 cyclin-dependent kinase 6 ILMN_1802615 G1/S CCNE1 -1.54 cyclin E1 ILMN_2374425 G1/S CDK2§ -1.52 cyclin-dependent kinase 2 ILMN_1665559 G1/S CUL1§ -1.49 cullin 1 ILMN_1749629 G1/S, G2/M TFDP1 -1.37 transcription factor Dp-1 ILMN_1661717 G1/S GADD45A§ 3.29 growth arrest and DNA-damage-inducible, alpha ILMN_2052208 G2/M

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3.18 ILMN_1694075 KAT2B 1.43 K(lysine) acetyltransferase 2B ILMN_3243142 G2/M CCNB1 -3.06 cyclin B1 ILMN_1712803 G2/M CCNB2 -2.80 cyclin B2 ILMN_1801939 G2/M TOP2A -2.58 topoisomerase (DNA) II alpha 170kDa ILMN_1686097 G2/M CHEK1 -2.50 CHK1 checkpoint homolog (S. pombe) ILMN_1664630 G2/M CKS2 -2.30 CDC28 protein kinase regulatory subunit 2 ILMN_1756326 G2/M -2.28 ILMN_2072296 BRCA1* -1.88 breast cancer 1, early onset ILMN_1738027 G2/M -1.70 ILMN_2311089 PKMYT1 -1.82 protein kinase, membrane associated ILMN_1766658 G2/M tyrosine/threonine 1 -1.58 ILMN_2401436 WEE1* -1.78 WEE1 homolog (S. pombe) ILMN_1778561 G2/M PLK1 -1.65 polo-like kinase 1 (Drosophila) ILMN_1736176 G2/M CDC25C‡ -1.55 cell division cycle 25 homolog C (S. pombe) ILMN_2407619 G2/M PRKDC§ -1.53 protein kinase, DNA-activated, catalytic polypeptide ILMN_2253648 G2/M CHEK2 -1.52 CHK2 checkpoint homolog (S. pombe) ILMN_2395240 G2/M -1.48 ILMN_2395236 YWHAH -1.46 tyrosine 3-monooxygenase/tryptophan 5- ILMN_1728512 G2/M monooxygenase activation protein, eta polypeptide YWHAQ -1.38 tyrosine 3-monooxygenase/tryptophan 5- ILMN_1674385 G2/M monooxygenase activation protein, theta polypeptide FPGS Inhibition in HCT116 CDKN1A 2.94 cyclin-dependent kinase inhibitor 1A (p21, Cip1) ILMN_1784602 G1/S, G2/M 1.58 ILMN_1787212 CDC25A -1.34 cell division cycle 25 homolog A (S. pombe) ILMN_1711005 G1/S CCNE2 -1.33 cyclin E2 ILMN_2412384 G1/S CDC25C‡ 1.39 cell division cycle 25 homolog C (S. pombe) ILMN_2407619 G2/M GADD45A 1.37 growth arrest and DNA-damage-inducible, alpha ILMN_2052208 G2/M BRCA1 -1.36 breast cancer 1, early onset ILMN_1738027 G2/M

FPGS Overexpression in MDA-MB-435 HDAC9 2.59 histone deacetylase 9 ILMN_1781173 G1/S 1.54 ILMN_1803563 CCND1† 2.26 cyclin D1 ILMN_1688480 G1/S CDKN2A 1.63 cyclin-dependent kinase inhibitor 2A (melanoma, ILMN_1744295 G1/S, G2/M p16, inhibits CDK4) CDKN2B§ 1.60 cyclin-dependent kinase inhibitor 2B (p15, inhibits ILMN_2376723 G1/S CDK4) CDKN1B 1.45 cyclin-dependent kinase inhibitor 1B (p27, Kip1) ILMN_2196347 G1/S E2F2§ -2.01 E2F transcription factor 2 ILMN_1777233 G1/S

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CDK2§ -1.98 cyclin-dependent kinase 2 ILMN_1665559 G1/S -1.31 ILMN_1653443 CUL1§ -1.88 cullin 1 ILMN_1749629 G1/S, G2/M SKP2§ -1.88 S-phase kinase-associated protein 2 (p45) ILMN_1665538 G1/S, G2/M E2F3 -1.79 E2F transcription factor 3 ILMN_1669502 G1/S HDAC1 -1.51 histone deacetylase 1 ILMN_1727458 G1/S CCND3 -1.48 cyclin D3 ILMN_1668721 G1/S RBL1 -1.36 retinoblastoma-like 1 (p107) ILMN_1782745 G1/S GADD45A†§ 3.28 growth arrest and DNA-damage-inducible, alpha ILMN_2052208 G2/M 1.67 ILMN_1694075 PRKCZ 1.94 protein kinase C, zeta ILMN_2386982 G2/M CDK7 1.65 cyclin-dependent kinase 7 (MO15 homolog, Xenopus ILMN_1778917 G2/M laevis, cdk-activating kinase) CKS2 1.59 CDC28 protein kinase regulatory subunit 2 ILMN_1756326 G2/M 1.37 ILMN_2072296 KAT2B -2.11 K(lysine) acetyltransferase 2B ILMN_3243142 G2/M PRKDC§ -1.50 protein kinase, DNA-activated, catalytic polypeptide ILMN_2253648 G2/M -1.81 ILMN_2334121 YWHAZ -1.41 tyrosine 3-monooxygenase/tryptophan 5- ILMN_1801928 G2/M monooxygenase activation protein, zeta polypeptide

FPGS Inhibition in MDA-MB-435 E2F5 1.58 E2F transcription factor 5, p130-binding ILMN_1782551 G1/S CCND1† -1.42 cyclin D1 ILMN_1688480 G1/S GADD45A† -1.48 growth arrest and DNA-damage-inducible, alpha ILMN_2052208 G2/M

*, genes whose expression was regulated by DNA methylation; †, genes upregulated in FPGS overexpression and downregulated in FPGS inhibition; ‡, genes downregulated in FPGS overexpression and upregulated in FPGS inhibition; §, genes differentially expressed in both the FPGS-modulated HCT116 and MDA-MB-435 cells.

7.4.7.3 Apoptosis Pathway

In response to FPGS overexpression, BCL2L1 (BCL2-like 1) was upregulated in both the HCT116 (fold change 1.79) and MDA-MB-435 (fold change 1.39) cells (Table 7.34). It was also upregulated in the FPGS-inhibited MDA-MB-435 cells (Table 7.34).

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Table 7. 34 List of differentially expressed genes involved in the apoptosis pathway in the FPGS-modulated HCT116 and MDA-MB-435 cells

Gene Fold change Description Probe ID Pathway Symbol (vs. Control) FPGS Overexpression in HCT116 MAPK3 1.79 mitogen-activated protein kinase 3 ILMN_1667260 apoptosis BCL2L1§ 1.79 BCL2-like 1 ILMN_1654118 apoptosis PRKCA 1.42 protein kinase C, alpha ILMN_1771800 apoptosis NFKBIB -1.70 nuclear factor of kappa light polypeptide gene enhancer ILMN_1690473 apoptosis in B-cells inhibitor, beta PARP1 -1.54 poly (ADP-ribose) polymerase family, member 1 ILMN_1686871 apoptosis MAPK1 -1.53 mitogen-activated protein kinase 1 ILMN_2235283 apoptosis MAP2K4 -1.44 mitogen-activated protein kinase kinase 4 ILMN_2100689 apoptosis ACIN1 -1.41 apoptotic chromatin condensation inducer 1 ILMN_1699636 apoptosis CASP3 -1.39 caspase 3, apoptosis-related cysteine peptidase ILMN_2388155 apoptosis RAF1 -1.36 v-raf-1 murine leukemia viral oncogene homolog 1 ILMN_1813489 apoptosis CASP6 -1.35 caspase 6, apoptosis-related cysteine peptidase ILMN_1659350 apoptosis AIFM1 -1.33 apoptosis-inducing factor, mitochondrion-associated, 1 ILMN_1668408 apoptosis ROCK1 -1.31 Rho-associated, coiled-coil containing protein kinase 1 ILMN_1808768 apoptosis FPGS Inhibition in HCT116 MAPK3 1.47 mitogen-activated protein kinase 3 ILMN_1667260 apoptosis FAS 1.44 Fas (TNF receptor superfamily, member 6) ILMN_2319077 apoptosis CASP2 -1.36 caspase 2, apoptosis-related cysteine peptidase ILMN_2410540 apoptosis BID -1.33 BH3 interacting domain death agonist ILMN_2372413 apoptosis FPGS Overexpression in MDA-MB-435 BCL2A1 1.84 BCL2-related protein A1 ILMN_1769229 apoptosis BAD 1.76 BCL2-antagonist of cell death ILMN_1738652 apoptosis CASP9 1.50 caspase 9, apoptosis-related cysteine peptidase ILMN_1718070 apoptosis BCL2L1§ 1.39 BCL2-like 1 ILMN_1654118 apoptosis NFKBIB 1.33 nuclear factor of kappa light polypeptide gene enhancer ILMN_1690473 apoptosis in B-cells inhibitor, beta CASP2 -1.86 caspase 2, apoptosis-related cysteine peptidase ILMN_2410540 apoptosis CYCS -1.44 cytochrome c, somatic ILMN_1730416 apoptosis GAS2 -1.34 growth arrest-specific 2 ILMN_1804569 apoptosis

FPGS Inhibition in MDA-MB-435 BCL2L1 1.38 BCL2-like 1 ILMN_1654118 apoptosis BID -1.44 BH3 interacting domain death agonist ILMN_1763386 apoptosis

§, genes differentially expressed in both the FPGS-modulated HCT116 and MDA-MB-435 cells.

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7.4.8 FPGS-Specific Gene Expression Analysis

To investigate genes associated with the function of FPGS, we identified genes differentially expressed in the opposite direction between FPGS overexpression and inhibition.

7.4.8.1 FPGS-Specific Gene Expression in HCT116 Cells

Venn diagrams show the number of genes differentially expressed in opposite directions in the FPGS-modulated HCT116 cells (Figure 7.14).

Figure 7. 14 Number of genes differentially expressed in the opposite direction between FPGS overexpression and inhibition in the FPGS-modulated HCT116 colon cancer cells

Twenty-four genes were upregulated in response to FPGS overexpression and downregulated in response to FPGS inhibition, and these genes were associated with gene expression, cell-to-cell signaling and interaction, cell morphology, cellular assembly and organization, and cell death (Figure 7.14; Table 7.35). Twenty-one genes that were downregulated in response to FPGS overexpression and upregulated in response to FPGS inhibition were involved in cell cycle, cellular compromise, lipid metabolism, small molecule biochemistry, and vitamin and mineral metabolism (Figure 7.14; Table 7.35). The list of top networks generated by mapping the focus genes associated with the FPGS-specific altered expression in the FPGS-modulated HCT116 cells is presented in Table 7.36. The list of genes associated with the FPGS-specific altered expression in the FPGS-modulated HCT116 cells is presented in Appendix 16, and the top ten genes are shown in Table 7.37.

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Table 7. 35 The top molecular and cellular functions associated with the FPGS-specific gene expression in the FPGS-modulated HCT116 colon cancer cells

Upregulated in FPGS Overexpression and Downregulated in FPGS Overexpression and

Downregulated in FPGS Inhibition Upregulated in FPGS Inhibition No. of No. of Category P-value Category P-value Genes Genes Gene Expression 3.40E-04 - 9 Cell Cycle 1.08E-03 - 2 2.87E-02 3.09E-02 Cell-To-Cell Signaling and 3.60E-04 - 4 Cellular Compromise 1.08E-03 - 2 Interaction 3.07E-02 3.40E-02 Cell Morphology 1.48E-03 - 2 Lipid Metabolism 1.08E-03 - 2 4.08E-02 3.72E-02 Cellular Assembly and 1.48E-03 - 3 Small Molecule 1.08E-03 - 3 Organization 4.08E-02 Biochemistry 3.72E-02 Cell Death 2.97E-03 - 6 Vitamin and Mineral 1.08E-03 - 1 4.63E-02 Metabolism 4.31E-03

Table 7. 36 The top networks matched by the genes with the FPGS-specific altered expression in the FPGS-modulated HCT116 colon cancer cells

Upregulated in FPGS Overexpression and Downregulated in FPGS Overexpression

Downregulated in FPGS Inhibition and Upregulated in FPGS Inhibition Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cell Death, Dermatological 23 11 Inflammatory Response, Cell- 8 4 Diseases and Conditions, To-Cell Signaling and Cellular Growth and Interaction, Hematological Proliferation System Development and Function 2 Cell Death, Cancer, Cell Cycle 15 8 Cancer, Endocrine System 3 1 Disorders, Reproductive System Disease 3 Cellular Development, Cellular 3 1 Growth and Proliferation, Cell Death 4 Cellular Development, 3 1 Embryonic Development, Organ Development

The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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Table 7. 37 List of the top genes associated with the FPGS-specific altered expression in the FPGS-modulated HCT116 colon cancer cells

Fold Change (vs. Control) Gene Description Accession Symbol FPGS FPGS Overexpression Inhibition

Upregulated in FPGS Overexpression and Downregulated in FPGS Inhibition

DUSP6* 2.63 -1.71 dual specificity phosphatase 6 NM_022652.2 ENC1 1.41 -1.64 ectodermal-neural cortex (with BTB-like NM_003633.1 domain) NFIB 1.42 -1.61 nuclear factor I/B NM_005596.2 PPAP2B 1.33 -1.52 phosphatidic acid phosphatase type 2B NM_003713.3 LGALS3 2.10 -1.48 lectin, galactoside-binding, soluble, 3 NM_002306.1 (galectin 3) LAMB1 2.48 -1.44 laminin, beta 1 NM_002291.1 JARID2 1.35 -1.44 jumonji, AT rich interactive domain 2 NM_004973.2 LOC400986 1.39 -1.44 PREDICTED: protein immuno-reactive with XM_001126815.1 anti-PTH polyclonal antibodies TANK 1.32 -1.44 TRAF family member-associated NFKB NM_004180.2 activator SUSD2 1.57 -1.41 sushi domain containing 2 NM_019601.3

Downregulated in FPGS Overexpression and Upregulated in FPGS Inhibition

ALDH1A3* -1.61 1.85 aldehyde dehydrogenase 1 family, member A3 NM_000693.2 GPR110 -1.58 1.70 G protein-coupled receptor 110 NM_153840.2 IGFBP6 -1.59 1.64 insulin-like growth factor binding protein 6 NM_002178.2 ALDH3A2 -1.34 1.49 aldehyde dehydrogenase 3 family, member A2 NM_001031806.1 DLEU2L -1.31 1.49 deleted in lymphocytic leukemia 2-like NR_002771.1 FBXO6 -1.42 1.48 F-box protein 6 NM_018438.4 PPFIBP1 -1.64 1.43 PTPRF interacting protein, binding protein 1 NM_003622.2 (liprin beta 1) RPL34 -2.46 1.43 ribosomal protein L34 NM_000995.2 CDC25C -1.55 1.39 cell division cycle 25 homolog C (S. pombe) NM_001790.3 RDM1 -1.42 1.37 RAD52 motif 1 NM_001034836.1

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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7.4.8.2 FPGS-Specific Gene Expression in MDA-MB-435 Cells

In the MDA-MB-435 cell line, we identified 191 genes upregulated in response to FPGS overexpression and downregulated in response to FPGS inhibition, and these genes were associated with cell cycle, gene expression, cell death, cellular growth and proliferation, and cell- to-cell signaling and interaction (Figure 7.15; Table 7.38). One hundred twenty-two genes that were downregulated in response to FPGS overexpression and upregulated in response to FPGS inhibition were involved in lipid metabolism, small molecule biochemistry, carbohydrate metabolism, molecular transport, and nucleic acid metabolism (Figure 7.15; Table 7.38). The list of top networks generated by mapping the focus genes associated with the FPGS-specific altered expression in the FPGS-modulated MDA-MB-435 cells is presented in Table 7.39. The list of genes associated with the FPGS-specific altered expression in the FPGS-modulated MDA- MB-435 cells is presented in Appendix 16, and the top ten genes are shown in Table 7.40.

Figure 7. 15 Number of genes differentially expressed in the opposite direction between FPGS overexpression and inhibition in the FPGS-modulated MDA-MB-435 breast cancer cells

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Table 7. 38 The top molecular and cellular functions associated with the FPGS-specific gene expression in the FPGS-modulated MDA-MB-435 breast cancer cells

Upregulated in FPGS Overexpression and Downregulated in FPGS Overexpression and

Downregulated in FPGS Inhibition Upregulated in FPGS Inhibition No. of No. of Category P-value Category P-value Genes Genes Cell Cycle 3.57E-04 - 9 Lipid Metabolism 3.11E-05 - 12 4.38E-02 4.45E-02 Gene Expression 3.67E-04 - 26 Small Molecule 3.11E-05 - 25 4.92E-02 Biochemistry 4.45E-02 Cell Death 7.29E-04 - 50 Carbohydrate Metabolism 1.69E-04 - 15 4.63E-02 2.99E-02 Cellular Growth and 8.22E-04 - 41 Molecular Transport 1.69E-04 - 15 Proliferation 4.80E-02 4.45E-02 Cell-To-Cell Signaling and 1.21E-03 - 13 Nucleic Acid Metabolism 1.69E-04 - 10 Interaction 4.38E-02 4.01E-02

Table 7. 39 The top networks matched by the genes with the FPGS-specific altered expression in the FPGS-modulated MDA-MB-435 breast cancer cells

Upregulated in FPGS Overexpression and Downregulated in FPGS Overexpression

Downregulated in FPGS Inhibition and Upregulated in FPGS Inhibition Focus Focus No Top Functions Score Top Functions Score Genes Genes 1 Cellular Growth and 45 27 Cellular Development, Cellular 27 17 Proliferation, Embryonic Growth and Proliferation, Development, Cellular Function Skeletal and Muscular System and Maintenance Development and Function 2 Cell Death, Hematological 32 21 Cell Signaling, Molecular 18 13 System Development and Transport, Small Molecule Function, Cancer Biochemistry 3 Cancer, Cellular Growth and 21 16 Cancer, Gene Expression, 17 12 Proliferation, Cell Death Reproductive System Disease 4 Cell Cycle, Developmental 20 15 DNA Replication, 15 11 Disorder, Hereditary Disorder Recombination, and Repair, Cell Cycle, Cancer 5 Cell-To-Cell Signaling and 20 15 Cell Cycle, Cellular Assembly 15 11 Interaction, Inflammatory and Organization, DNA Response, Cancer Replication, Recombination, and Repair The score indicates the likelihood of the Focus Genes in a network being found together due to random chance; The Focus Genes indicate the uploaded genes of interest for which information is available in the Ingenuity Knowledge Base.

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Table 7. 40 List of the top genes associated with the FPGS-specific altered expression in the FPGS-modulated MDA-MB-435 breast cancer cells

Fold Change (vs. Control) Gene Description Accession Symbol FPGS FPGS Overexpression Inhibition

Upregulated in FPGS Overexpression and Downregulated in FPGS Inhibition

HLA-DQA1 9.13 -5.75 PREDICTED: major histocompatibility complex, XM_936128.2 class II, DQ alpha 1, transcript variant 10 THBS2 2.13 -5.25 thrombospondin 2 NM_003247.2 C21orf34* 8.79 -4.48 chromosome 21 open reading frame 34 NM_001005734.1 HLA-DOA 4.12 -3.95 major histocompatibility complex, class II, DO NM_002119.3 alpha C1S 1.59 -3.26 complement component 1, s subcomponent NM_001734.2 HLA-DMB 3.19 -3.17 major histocompatibility complex, class II, DM NM_002118.3 beta CTHRC1* 4.19 -2.88 collagen triple helix repeat containing 1 NM_138455.2 HLA-DPA1 2.82 -2.87 major histocompatibility complex, class II, DP NM_033554.2 alpha 1 HLA-DRB4 4.04 -2.82 major histocompatibility complex, class II, DR NM_021983.4 beta 4 HLA-DPB1 2.31 -2.76 major histocompatibility complex, class II, DP NM_002121.4 beta 1

Downregulated in FPGS Overexpression and Upregulated in FPGS Inhibition

BCHE* -11.02 7.02 butyrylcholinesterase NM_000055.1 TRIM48 -11.38 3.67 tripartite motif-containing 48 NM_024114.2 ANKS1A -1.92 2.83 ankyrin repeat and sterile alpha motif domain NM_015245.2 containing 1A SLITRK4 -1.80 2.60 SLIT and NTRK-like family, member 4 NM_173078.2 ST3GAL5* -2.14 2.20 ST3 beta-galactoside alpha-2,3-sialyltransferase 5 NM_001042437.1 AKR1B1 -1.50 2.04 aldo-keto reductase family 1, member B1 NM_001628.2 (aldose reductase) DYNC1I1 -5.14 1.99 dynein, cytoplasmic 1, intermediate chain 1 NM_004411.3 PLSCR1 -2.62 1.97 phospholipid scramblase 1 NM_021105.1 MIR1974 -1.65 1.97 microRNA 1974 NR_031738.1 PPARGC1A -4.23 1.95 peroxisome proliferator-activated receptor NM_013261.3 gamma, coactivator 1 alpha

An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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7.5 Discussion

We determined the effects of FPGS modulation on global and gene-specific methylation and gene expression in HCT116 and MDA-MB-435 cell lines using an in vitro model of FPGS overexpression and inhibition with predictable functional consequences (4, 17). In contrast to the GGH modulation system, the changes in global DNA methylation and DNMT activity in response to FPGS modulation in HCT116 and MDA-MB-435 cells were not uniformly consistent with the observed intracellular total and long-chain folylpolyglutamates concentrations (4, 17). Our a prioi hypothesis was that FPGS overexpression would increase global DNA methylation and DNMT activity due to the observed increased intracellular folate concentrations and higher content of long-chain folylpolyglutamates, while FPGS inhibition would decrease global DNA methylation and DNMT activity due to the observed decreased intracellular folate concentrations and lower content of long-chain folylpolyglutamates. Interestingly, however, it appears that the observed global DNA methylation alterations in response to FPGS modulation may be accounted for by the observed changes in GGH mRNA expression and GGH activity in the FPGS modulation system (4, 17). We reported that HCT116 cells expressing the sense FPGS had significantly higher, whereas those expressing the antisense FPGS had significantly lower, GGH mRNA expression compared with those expressing endogenous FPGS (17). In contrast, we showed that MDA-MB-435 cells overexpressing FPGS were associated with lower GGH activity compared to control, while GGH activity did not significantly differ between those in which FPGS is inhibited and those expressing endogenous FPGS (4). In the GGH modulation system in both HCT116 and MDA-MB-435 cell lines, GGH overexpression with higher GGH protein expression/activity (Chapter 4) was associated with lower global DNA methylation than corresponding controls (Chapter 6), while GGH inhibition with lower GGH protein expression/activity (Chapter 4) was associated with higher global DNA methylation compared with corresponding controls (Chapter 6). This inverse relationship between GGH activity and global DNA methylation in the GGH modulation system was also observed in the FPGS modulation system. In HCT116 cells, FPGS overexpression associated with higher GGH mRNA expression showed lower, whereas FPGS inhibition associated with lower GGH mRNA expression showed higher, global DNA methylation compared to controls. Similarly, in MDA- MB-435 cells, FPGS overexpression with lower GGH activity was associated with higher global DNA methylation, while FPGS inhibition with no significant difference in GGH activity did not

267 affect global DNA methylation status. The findings of the current study are in agreement with a recent report which showed that CIMP+ (CpG island methylator phenotype) was associated with low GGH mRNA expression compared with CIMP- in primary CRC whereas FPGS levels were not significantly different between CIMP+ and CIMP− tumors (187). Taken together, GGH may have a more significant effect on global DNA methylation compared to FPGS. Overall, most of the observed DNMT activity was consistent with our expectation except for the FPGS- overexpressed HCT116 cells. These data collectively suggest that the effects of FPGS modulation on global DNA methylation and DNMT activity cannot be solely explained by the observed intracellular folate concentrations and content of long-chain folylpolyglutamates. Moreover, these findings suggest that the effect of FPGS modulation on global DNA methylation and DNMT activity may be cell-specific.

We investigated whether FPGS modulation would affect gene-specific methylation using an epigenomic approach to interrogate the DNA methylation status of 27,578 individual CpG sites located in the promoter regions of 14,495 genes (360). Similar to GGH modulation, MDA-MB- 435 cells revealed more promoter CpG methylation alterations in response to FPGS modulation compared with HCT116 cells. In both cell lines, FPGS overexpression showed more promoter CpG methylation changes than FPGS inhibition. This is in contrast to the observations we made for the GGH modulation system; in both cell lines, GGH inhibition exhibited more alterations in promoter CpG methylation compared with GGH overexpression (Chapter 6). Functional analysis of differentially methylated genes revealed that several molecular and cellular functions including cellular function and maintenance and cellular movement were affected by FPGS overexpression, while several genes associated with cellular movement and cell death were differentially methylated in response to FPGS inhibition in HCT116 and MDA-MB-435 cells. Similar to the observation in the GGH modulation system, we found that the proportion of differentially methylated CpG loci in non-CpG islands was greater compared with those in CpG islands. As explained in the discussion in Chapter 6, DNA methylation is important for the regulation of non-CpG island promoters as well as CpG island promoters (440, 441). It has recently been suggested that DNA methylation can directly silence genes with non-CpG island promoters and contribute to the establishment of tissue-specific methylation patterns since the epigenetic signatures of DNA methylation, histone marks and nucleosome occupancy of non- CpG island promoters are almost identical to CpG island promoters (325).

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We investigated whether FPGS modulation would affect gene expression by examining 31,335 annotated genes including 47,231 probes. Overall, FPGS overexpression revealed more alterations in gene expression compared with FPGS inhibition. Functional analyses of differentially expressed genes showed that 2897 genes (9.2%; 1576 down- and 1321 upregulated) and 1502 genes (4.8%; 840 down- and 662 upregulated) involved in cell cycle and cell death were differentially expressed in response to FPGS overexpression in HCT116 and MDA-MB- 435 cells, respectively. In the FPGS inhibition system, 359 genes (1.1%; 129 down- and 230 upregulated) and 829 genes (2.6%; 442 down- and 387 upregulated) associated with cell death, cell cycle, cellular function and maintenance, and cellular compromise were differentially expressed in HCT116 and MDA-MB-435 cells, respectively.

In addition, we performed the integrated analysis of differentially methylated and expressed genes in response to FPGS modulation in order to identify genes that are regulated by DNA methylation. We further confirmed mRNA expression of 18 selected genes demonstrating the inverse relationship between methylation and expression in response to FPGS modulation using qRT-PCR. In all selected genes, the direction of gene expression change was consistent with that obtained from microarray. Similar to GGH modulation, overall, only a small number of genes were associated with the inverse relationship between promoter methylation and gene expression in response to FPGS modulation, which is consistent with the observations from other studies (444, 445). It appears that the genetic and/or other epigenetic mechanisms such as histone modification, chromatin remodeling, and miRNA likely affect gene expression.

One of the hypermethylated and downregulated genes in the FPGS-overexpressed HCT116 cells was ALDH1A3 (β-value difference 0.29, fold change -1.93). ALDH1A3 was upregulated (fold change 1.85) in the FPGS-inhibited HCT116 cells, while it was hypermethylated and downregulated (β-value difference 0.47, fold change -1.34) in the GGH-inhibited HCT116 cells. ALDH1A3 encodes aldehyde dehydrogenase 1 family, member A3, which may be involved in the detoxification of aldehydes generated by alcohol metabolism and lipid peroxidation and in the oxidation of retinal to retinoic acid (473). A recent study found that ALDH1A3 upregulation could be associated with more aggressive metastatic breast cancer, suggesting the possible role of ALDH1A3 as a prognostic marker and a therapeutic target in breast cancer (474).

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In MDA-MB-435 cells, HLA-DPA1 (major histocompatibility complex, class II, DP alpha 1) was upregulated and hypomethylated (fold change 2.82, β-value difference -0.64) in response to FPGS overexpression, while it was downregulated and hypermethylated (fold change -2.87, β- value difference 0.28) in response to FPGS inhibition. HLA-DPA1 is expressed on antigen presenting cells, such as dendritic cells, B-cells and macrophages. The expression of HLA-DPA1 was also detected on intestinal epithelial cells of noninflamed small intestine and to a lesser extent large intestine (475). It has been shown that alleles containing HLA-DPA1 play an important role in autoimmune diseases such as Crohn’s disease (475) and parasitic infections such as hepatosplenic schistosomiasis japonica (476). In addition, JAM3 (junctional adhesion molecule 3) was associated with upregulation and hypomethylation (fold change 2.04, β-value difference -0.27) in MDA-MB-435 cells overexpressing FPGS, whereas it was downregulated (fold change -2.47) in the FPGS-inhibited MDA-MB-435 cells. JAM3 is known to serve as the major heterotypic adhesion receptor responsible for cell-to-cell adhesion between platelets and leukocytes (477). A more recent study demonstrated that the homophilic interaction of JAM3 can mediate tumor cell-endothelial cell interactions and may thereby be involved in the process of tumor cell metastasis (478). JAM3 regulates both inflammation- and angiogenesis-related endothelial permeability (479). Furthermore, THBS2 (thrombospondin 2) was downregulated and hypermethylated (fold change -5.25, β-value difference 0.45) in MDA-MB-435 cells in which FPGS is inhibited, while it was upregulated (fold change 2.13) in the FPGS-overexpressed MDA-MB-435 cells. The THBS2 gene encodes thrombospondin 2 that mediates cell-to-cell and cell-to-matrix interactions and functions as a potent inhibitor of tumor growth and angiogenesis. It has been suggested that post-transcriptional inactivation of thrombospondin-2 by aberrant DNA methylation may participate in angiogenesis of malignant ovarian tumors through THBS2 downregulation (480). There were no common genes between HCT116 and MDA-MB-435 cells, of which expression was regulated by CpG promoter methylation changes associated with FPGS modulation.

Interestingly, several differentially expressed genes involved in folate biosynthesis and cell cycle were identified in response to FPGS overexpression in both HCT116 and MDA-MB-435 cells. With regards to genes involved in folate biosynthesis, we identified that TYMS, TK1, ABCC5, and SLC29A1 were downregulated in both cell lines overexpressing FPGS, while ABCC2 was upregulated and hypomethylated in the FPGS-inhibited HCT116 cells. The expression and/or

270 activity of TS encoded by the TYMS gene is known to be a major determinant of 5FU efficacy (233, 234). TS overexpression has been shown to be associated with 5FU resistance in CRC (235, 236). The 5FU resistant HCT116 cell line is associated with cellular phenotypes such as reduced apoptosis and more aggressive growth as well as upregulation of TS compared with parental HCT116 cell line (239). Furthermore, TK1 encodes thymidine kinase 1 that plays a dual role in both 5FU activation through conversion from 5FdUR to 5FdUMP and salvage pathways with decreased thymidine pools (481). Accordingly, low TK activities have been associated with a poor response to 5FU treatment due to its role in 5FU activation (467, 482-484), whereas other studies have shown that, conversely, high TK levels contributed to a potential mechanism of resistance to 5FU as thymidylate can be salvaged from thymidine through the action of TK, thereby alleviating the effects of TS deficiency (279, 485-488). A recent study suggests that TK is not a limiting step in the activation of 5FU and that, conversely, high TK levels in tumor cells are a factor of diminished sensitivity to 5FU-based treatment, probably by triggering the pyrimidine salvage pathway in response to TS inhibition (489). Taken together, the observed downregulation of TYMS and TK1 might be associated with increased chemosensitivity to 5FU in response to FPGS overexpression. In the FPGS-inhibited MDA-MB-435 cells, we observed that TK1 was upregulated, which might be likely associated with decreased chemosensitivity to 5FU (4).

It has been shown that MRP5 encoded by the ABCC5 gene can efflux mono- and diglutamate forms of MTX and transport 5FdUMP, a metabolite of 5FU (12, 14, 68, 296), thereby contributing to drug resistance or increased drug efficacy depending on polyglutamylation of antifolates and intracellular folate concentrations (11). MRP5 confers resistance to a number of anticancer agents including 5FU, MTX, and MTA in human kidney cells (296). MRP5 are upregulated in 5FU resistant Capan-1 pancreatic carcinoma cells, while siRNA-induced silencing of MRP5 results in enhanced sensitivity of these cells to 5FU (490). Furthermore, MRP2 encoded by the ABCC2 gene is one of the most important efflux transporters and can transport a large number of drugs including MTX and their conjugates. MRP2 is highly polymorphic and ABCC2 genetic variant affected the impaired MTX elimination in vivo, with pharmacokinetic and toxic implications (491). An in vitro study reported that ABCC2 was expressed at higher levels in tamoxifen-resistant breast cancer cells, suggesting that active metabolites of tamoxifen were transported by ABCC2 from breast cancer cells (492). Accumulating evidence suggests that

271 loss of MRP2 and MRP5 is an important determinant of the expansion of intracellular folate pools, whereas overexpression of one of these ATP-dependent efflux transporters may result in decreased intracellular THF cofactor pools (14). Therefore, downregulation of MRP5 would lead to an increased cellular folate accumulation as well as increased sensitivity to antifolates and 5FU, which are consistent with observations in response to FPGS overexpression in previous studies (4, 17). It seems that upregulation of ABCC2 in the FPGS-inhibited HCT116 cells might affect chemosensitivity to MTX. In addition, SLC29A1 encodes equilibrative nucleoside transporter 1 (ENT1) that mediates nucleoside influx and efflux and exhibits the highest affinity for adenosine. High mRNA expression of ENT1 was associated with a poor response to 5FU in CRC (493) and pancreatic cancer cells (494), suggesting that there is a possibility that better supplies of nucleosides and nucleobases through high ENT1 expression might interfere with the 5FU function that prevents de novo DNA synthesis. Thus, it is likely that decreased expression of SLC29A1 in response to FPGS overexpression might contribute to increased chemosensitivity to 5FU. Also, in FPGS overexpression, we identified downregulated DNMT1 in HCT116 cells and downregulated GGH in MDA-MB-435 cells, respectively, which were consistent with our previous observations (Section 7.4.2 and (4)).

In addition to genes involved in folate biosynthesis, several genes related to the cell cycle were differentially regulated in response to FPGS overexpression in both cell lines; upregulation of CDKN2B and downregulation of PRKDC, SKP2, CUL1, and E2F2 were identified. CDKN2B encodes cyclin-dependent kinase 4 inhibitor B, also known as multiple tumor suppressor 2 (MTS2) or p15INK4B, which forms a complex with CDK4 or CDK6, and prevents the activation of the CDK kinases by cyclin D. Thus, MTS2 functions as a cell growth regulator that inhibits cell cycle G1 progression (495). Inactivation of MTS2 was associated with tumor progression in NSCLC (496) and poor prognosis in T-cell lymphoma patients (497). Inactivation of MTS2 is a potential indicator for a poor response to cisplatin-based chemotherapy and consequently a poor prognosis in advanced ovarian cancer patients (498). Therefore, it is presumed that upregulation of CDKN2B might be associated with a better response to chemotherapy in response to FPGS overexpression. PRKDC encodes the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) that functions with the Ku70/Ku80 heterodimer protein in DNA double strand break repair and recombination (499). In response to DNA damage, PRKDC has been shown to be an upstream regulator of p53-induced apoptosis (500). Lower levels of PRKDC was found in

272 cisplain resistant HeLa/CDDP cells, which might be caused by decreased p53-induced apoptosis through the PRKDC regulated pathway (501). However, it has been shown that increased expression of DNA-PK could confer decreased sensitivity to DNA-damaging anticancer drugs, whereas low DNA-PK activity showed an increased sensitivity (502-506). It was reported that overactivity of the Ku-mediated repair, which resulted in repair infidelity, is a candidate mechanism for chromosomal instability leading to the metastasis of cancer cells (507). Therefore, it seems likely that the enhanced DNA-PK activity could be associated with aberrant use of DNA repair, which may contribute to cancer progression and metastatic potency as well as induction of therapy resistance (506). Overall, it is likely that downregulation of PRKDC might be associated with increased sensitivity to DNA-damaging anticancer drugs such as 5FU and antifolates in response to FPGS overexpression. The SKP2 gene encodes S-phase kinase-associated protein 2 (SKP2) that functions as a novel oncogene by modulating the degradation of phosphorylated Kip1 p27 , an important cell-cycle regulator at the G1/S transition. SKP2 protein, part of SKP1- CUL1-F-box (SCF) complexes, is considered a negative regulator of the cell-cycle inhibitor, Kip1 p27 , and positively regulates the G1/S transition (508). Targeting glycolysis by an SKP2 deficiency sensitizes Her2-positive breast tumors to herceptin treatment, highlighting the value of SKP2 targeting in clinical cancer therapy (509). Overexpression of SKP2 is associated with a resistance to preoperative -based chemotherapy in primary breast cancer (510). Similarly, high expression of SKP2 was associated with a poor therapeutic response and adverse outcomes in rectal cancer patients treated with neoadjuvant CRT and 5FU. Suppression of SKP2 expression attenuated the viability of CRC cells and promoted the cytotoxicity of 5FU alone or in the presence of concurrent irradiation (511). Therefore, we speculate that downregulation of SKP2 might be associated with increased sensitivity to 5FU in response to FPGS overexpression. As with SKP2, Cullin 1(CUL1) encoded by the CUL1 gene is an essential component of the SCF complex, which mediates the ubiquitination of proteins involved in cell cycle progression, signal transduction and transcription (512). This enzyme has recently been reported to be associated with tumour progression and poor clinical outcome for several different types of tumours. Increased CUL1 expression is found in melanoma (513, 514). High CUL1 expression was also significantly associated with high-grade breast tumours and poor prognosis, suggesting that it may play a role in breast tumour progression (515). Moreover, high expression of CUL1 is associated with lymph node metastasis and poor survival in gastric cancer, while CUL1 knockdown inhibits cell growth by upregulating p27 expression, and decreases cell adhesion

273 ability (516). It has been reported that SKP2 overexpression may be involved in carcinogenesis and progression of human gastric carcinoma in vivo, possibly via p27 (517). Therefore, CUL1 may cooperate with SKP2 to regulate gastric cancer cell cycle progression from G1 phase to S phase through SKP2-dependent p27 degradation (516). Taken together, downregulation of CUL1 and SKP2 in response to FPGS overexpression might contribute to a better response to chemotherapeutic drugs possibly via the upregulation of p27. Furthermore, E2F2 encoded by the E2F2 gene is one of the activators of the E2F family and a transcription factor that plays a critical role in the G1 to S phase transition by attracting numerous upstream signals (518). E2F2 is known to associate with tumor progression in breast cancer although the mechanism by which E2F2 influences breast cancer progression has not been elucidated (519).

There were two downregulated genes involved in cell cycle G2/M, BRCA1 and WEE1, expression of which was regulated by CpG promoter methylation changes associated with FPGS overexpression in HCT116 cells. BRCA1 (breast cancer susceptibility gene 1) is a tumor suppressor gene that regulates DNA repair, cell cycle checkpoint control, protein ubiquitinylation, chromatin remodeling and transcriptional regulation (520). BRCA1 is directly involved in DNA double-strand break repair through its interactions with RAD51 and the RAD50/MRE11/Nibrin complex (521, 522). BRCA1 is also associated with transcriptional regulation, interacting with RNA Pol II holoenzyme (523, 524) and with transcription factors such as p53 (525, 526). BRCA1 is mutated in 50% of hereditary breast cancer cases and 80% of families predisposed to breast and ovarian cancer (527, 528). It has been shown that methylation within the promoter region of BRCA1 occurs in some cases of sporadic breast and ovarian cancer (529, 530). BRCA1 gene promoter hypermethylation was associated with the functional inactivation of BRCA1 in sporadic ovarian cancer (531). Epigenetic downregulation of BRCA1 has been reported in approximately 30% of sporadic breast cancers and 70% of ovarian cancers (532). Low levels of BRCA1 mRNA were associated with longer survival in lung cancer patients following cisplatin and gemcitabine (533) and ovarian cancer patients treated with platinum-based chemotherapy (534). In a retrospective study of locally advanced bladder cancer patients treated with cisplatin- based chemotherapy, those with low or intermediate levels of BRCA1 mRNA exhibited significantly improved response and survival compared with those with a high level of BRCA1 mRNA (535). Preclinical studies suggest that BRCA1 can regulate differential sensitivity to chemotherapeutic agents; in breast cancer cells, inhibition of BRCA1 results in an increased

274 sensitivity to DNA-damage-based chemotherapy but resistance to antimicrotubule agents (536), whereas overexpression of BRCA1 increases resistance to cisplatin and, in contrast, sensitivity to antimicrotubule agents (537, 538). Interestingly, BRCA1 failed to modulate resistance or sensitivity to 5FU in breast cancer cells, perhaps reflecting the distinct mode of action of antimetabolites (538). Overall, evidence has reported that BRCA1 overexpression in human breast and ovarian cancer cell lines led to increased resistance to DNA-damaging agents including cisplatin (539-541), while BRCA1 inhibition results in increased chromosome damage and chemosensitivity to irofulven and cisplatin in ovarian and breast cancer cells (542, 543). Furthermore, WEE1 encodes a tyrosine kinase that plays a critical role in cell cycle regulation by inactivating the cell cycle kinase CDK1 by phosphorylation on Tyr15 (544). This inactivation is important in preventing entry into mitosis until DNA damage is repaired, suggesting that WEE1 has an important function in the G2 checkpoint (545). WEE1 levels rise during S and G2 phase due to increased synthesis, while it falls during the M phase due to decreased synthesis and proteolytic degradation (544). It has been shown that loss or inhibition of WEE1 in cancer cells including breast cancer cells resulted in DNA damage, S phase arrest, cell death, and a significant decrease in cell proliferation (546-548). A growing body of evidence suggests that inhibition of WEE1 can enhance the effects of radiation- or chemotherapy-induced DNA damage in colon cancer, lung cancer, pancreatic cancer, cervical cancer, and melanoma (545, 549-553). In human colon cancer cells, WEE1 inhibition by MK-1775, a WEE1 inhibitor, also potentiated the antitumor efficacy of DNA-damaging agents, including 5FU (554). Taken together, downregulation of BRCA1 and WEE1 seems to be associated with an increased efficacy of chemotherapeutic drugs, including 5FU in response to FPGS overexpression in HCT116 cells.

We also investigated genes commonly differentially methylated and/or expressed in response to FPGS modulation between HCT116 and MDA-MB-435 cell lines. There were 64 hyper- and 52 hypomethylated genes common to both cell lines in response to FPGS overexpression, whereas 31 hyper- and 38 hypomethylated genes were common between both cell lines in response to FPGS inhibition. In addition, 179 down- and 280 upregulated genes were common in FPGS overexpression, while 8 down- and 12 upregulated genes were common in FPGS inhibition. There were no common genes, of which expression resulted from CpG promoter methylation changes in response to FPGS modulation between HCT116 and MDA-MB-435 cell lines. Notably, OSBPL5 (oxysterol-binding protein-related protein 5) was upregulated and

275 downregulated in FPGS overexpression and FPGS inhibition, respectively, in both HCT116 and MDA-MB-435 cell lines. The OSBPL5 gene encodes a member of the oxysterol-binding , a group of intracellular lipid receptors that play a key role in the maintenance of cholesterol balance (555), and little is known about the function of this gene.

In conclusion, our data indicate that the global DNA methylation pattern in response to FPGS modulation in HCT116 and MDA-MB-435 cells may be accounted for by GGH mRNA expression levels/GGH activity changes induced by FPGS modulation. It appears that the effects of FPGS modulation on global DNA methylation and DNMT activity cannot be solely explained by the observed intracellular folate concentrations and content of long-chain folylpolyglutamates, and that it may be cell-specific. In addition, FPGS modulation can affect differential gene expression and CpG promoter DNA methylation involved in important biological pathways, and some of the observed altered gene expression appear to be regulated by DNA methylation. MDA-MB-435 cells revealed more alterations in promoter methylation and combined analysis of methylation and expression compared with HCT116 cells. Overall, FPGS overexpression demonstrated more alterations in promoter methylation, gene expression, and combined analysis of methylation and expression compared with FPGS inhibition. In both the FPGS-overexpressed HCT116 and MDA-MB-435 cell lines, we identified several differentially expressed genes involved in folate biosynthesis and cell cycle, which might be associated with the increased chemosensitivity to 5FU and antifolates in response to FPGS overexpression. The potential role of FPGS modulation in DNA methylation and the effect on chemosensitivity needs further exploration.

CHAPTER 8: OVERALL DISCUSSION AND FUTURE DIRECTIONS

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8.1 Overall Discussion

The objectives of this work were to characterize the pharmacogenetic role of GGH involved in cellular retention and export of folates and antifolates in modulating the chemosensitivity of HCT116 and MDA-MB-435 cell lines to chemotherapeutic agents using in vitro and in vivo systems (Study 1 and Study 2), and to investigate the effects of GGH and FPGS modulation on global and gene-specific DNA methylation and gene expression (Study 3 and Study 4).

Study 1 and Study 2: Polyglutamylated folates and antifolates are retained in cells longer and are better substrates than their monoglutamate counterparts for intracellular folate-dependent enzymes in one-carbon transfer reactions including DNA synthesis (2). Polyglutamylation of antifolates such as MTX would increase its affinity for enzymes in the thymidyalte and purine biosynthetic pathways and thus would enhance the chemotherapeutic effect of MTX (2, 3). Polyglutamylation may also affect cytotoxic sensitivity of cancer cells to fluorinated pyrimidine such as 5FU because polyglutamylated intracellular 5,10-methyleneTHF would allow more efficient formation and stabilization of the inhibitory ternary complex involving TS, 5,10- methyleneTHF and a 5FU metabolite (122). The lysosomal enzyme GGH removes the terminal glutamates and hence, is critical for the hydrolysis and export of intracellular polyglutamylated folates and antifolates (1). We hypothesized that GGH overexpression and inhibition would therefore affect chemosensitivity of cancer cells to antifolates, cytotoxicity of which depends on their polyglutamylation, and to 5FU, cytotoxicity of which depends in part on polyglutamylation of 5,10-methyleneTHF for the formation and stabilization of the ternary inhibitory complex involving TS. We also hypothesized that exogenous folate concentrations would further influence 5FU- and antifolate-induced chemotherapy in addition to GGH modulation.

We generated an appropriate in vitro model of GGH overexpression and inhibition in human HCT116 colon and MDA-MB-435 breast cancer cells with predictable functional consequences. GGH overexpression was associated with higher GGH protein expression and activity, lower total intracellular folate concentrations, lower content of long-chain folylpolyglutamates, and lower TS activity and DHFR protein expression and activity. In contrast, GGH inhibition showed lower GGH protein expression and activity, higher concentrations of total intracellular folate, higher content of long-chain folylpolyglutamates, and increased TS activity and DHFR protein

278 expression and activity. Generally, most of the observed functional consequences of GGH overexpression and inhibition were consistent with the known biological function of GGH and provided an appropriate in vitro model to test the effects of folate and GGH modulation on chemosensitivity of colon and breast cancer cells to 5FU and antifolates. Our data suggest, for the first time, that GGH modulation significantly influences chemosensitivity of cancer cells to 5FU and antifolates, and exogenous folate levels further affect chemosensitivity to these chemotherapeutic agents. However, other functional changes in response to GGH modulation such as changes in total intracellular folate pools, polyglutamylation of other intracellular folate cofactors, and TS and DHFR activity appear to counteract the effect of the GGH modulation- induced changes in polyglutamylation of 5,10-methyleneTHF and antifolates in a cell-specific manner. Overall, GGH modulation demonstrated changes in chemosensitivity of colon and breast cancer cells to 5FU in the expected direction based on the GGH modulation-induced changes in polyglutamylation of 5,10-methyleneTHF only when LV, a precursor for 5,10-methyleneTHF, was supplied exogenously. In general, the GGH modulation-induced changes in MTX- polyglutamylation might be the primary determinant of chemosensitivity of colon and breast cancer cells to MTX in the GGH overexpression system but not in the GGH inhibition system. Our data indicate that FA supplementation might be associated with resistance to 5FU and MTX. The effects of exogenous folate levels and GGH overexpression on 5FU efficacy in HCT116 cells were also validated in xenograft models using nude mice.

Intracellular folate and antifolate accumulation and metabolism, including polyglutamylation, are affected by multiple enzymes in the highly complex folate metabolic pathway (144). Furthermore, changes in a single enzyme such as GGH induce a cascade of adaptive and compensatory changes in other enzymes in order to maintain folate and antifolate homeostasis (1). In addition to changes in polyglutamylation of 5,10-methyleneTHF and antifolates, GGH modulation might have also induced changes in drug uptake into and accumulation in the cells; the forward polyglutamylation reaction by FPGS; intracellular concentration and polyglutamylation of other folate cofactors; intracellular distribution of the various folate species between cytoplasm, lysosome, and mitochondria; and/or transport mechanisms of folates and antifolates into the lysosomes where GGH resides and out of the cells by various ATP-binding cassette transporters (144). These adaptive and compensatory changes have likely played a role in modifying the effects of GGH modulation on chemosensitivity of cancer cells to antifolates

279 and 5FU. Some of the differences between the two cell lines are likely caused by differences in this complexity.

Study 3 and Study 4: Folate mediates the transfer of one-carbon units involved in nucleotide biosynthesis, the methionine cycle, and biological methylation reactions (1, 29). As an essential cofactor for de novo nucleotide biosynthesis, folate plays an important role in DNA synthesis, stability, integrity, and repair (1, 36). Furthermore, folate provides SAM, the primary methyl group donor for most biological methylations including DNA methylation, which is catalyzed by DNMT (1, 27). Polyglutamylation is also important in DNA methylation as polyglutamylated folates are better substrates for folate-dependent enzymes such as MTHFR and MS that are involved in the generation of SAM, which is a substrate for DNA methylation mediated by DNMT (2, 28). And hence, GGH and FPGS modulation may affect DNA methylation at global and gene-specific levels with consequent functional ramifications. Both genomic DNA hypomethylation and gene-specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis (29). DNA methylation and DNMT are also potential therapeutic targets and may modify the effect of specific chemotherapeutic agents (30), suggesting the GGH- and FPGS-modulated DNA methylation changes might influence chemosensitivity to chemotherapeutic agents. We hypothesized that GGH overexpression/FPGS inhibition would decrease, whereas GGH inhibition/FPGS overexpression would increase, global DNA methylation and DNMT activity. We also expected that GGH and FPGS modulation would further affect the degree of CpG promoter DNA methylation and the differential expression of genes.

Global DNA methylation and DNMT activity were decreased in response to GGH overexpression, and increased in the GGH-inhibited system, as expected. Interestingly, however, the effects of FPGS modulation on global DNA methylation were not consistent with our a priori hypothesis. It appears that the changes in global DNA methylation were mainly related to the altered GGH mRNA expression and GGH activity in response to FPGS modulation (4, 17). In contrast, the observed changes in DNMT activity were mostly consistent with our expectation. Taken together, GGH may have a more significant effect on global DNA methylation compared to FPGS. Furthermore, we have shown that GGH and FPGS modulation can affect differential gene expression and CpG promoter DNA methylation involved in important biological pathways,

280 and some of the observed altered gene expression appear to be regulated by DNA methylation. In the GGH-overexpressed MDA-MB-435 cells, we identified several differentially expressed genes involved in folate biosynthesis, one-carbon pool by folate, and cell cycle, which might be associated with the observed decreases in total intracellular folate concentrations and 5FU efficacy in response to GGH overexpression. Specifically, in both the FPGS-overexpressed HCT116 and MDA-MB-435 cell lines, we identified several differentially expressed genes involved in folate biosynthesis and cell cycle, which might be associated with the increased chemosensitivity to 5FU and antifolates in response to FPGS overexpression.

We also investigated genes affected by both GGH and FPGS based on the effects of these enzymes on intracellular polyglutamylation status: we identified genes commonly down- or upregulated between GGH overexpression and FPGS inhibition and between GGH inhibition and FPGS overexpression in HCT116 and MDA-MB-435 cell lines. The number of genes, expression of which was associated with both GGH and FPGS, is presented in Figure 8.1, and more detailed results are shown in Appendix 17. In the MDA-MB-435 cell line, we identified two common genes, ITIH5L and HNRNPU, between genes upregulated in both GGH overexpression and FPGS inhibition and genes downregulated in both GGH inhibition and FPGS overexpression (Figure 8.1). The function of ITIH5L, inter-alpha (globulin) inhibitor H5-like, is largely unknown. HNRNPU encodes heterogeneous nuclear ribonucleoprotein U that functions as a DNA- and RNA-binding protein (556). It is known that HNRNPU is required for cell viability and also functions as a global splicing regulator (557).

Figure 8. 1 Number of genes whose altered expression was affected by both GGH and FPGS in HCT116 (A) and MDA-MB-435 (B) cells

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Taken together, notwithstanding the complexity of the GGH modulation-induced changes in folate and antifolate accumulation and metabolism, GGH status in combination with exogenous folate concentrations may be an important clinical determinant of chemosensitivity of colon and breast cancer cells to 5FU- and MTX-based chemotherapy, which needs further exploration. The potential role of GGH and FPGS modulation in DNA methylation and the effect on chemosensitivity warrants further investigation. Our research will help to identify and characterize novel targets of chemotherapy and lead to clinical studies testing genetic variants on treatment response, prognosis, survival and toxicity in patients with colon and breast cancer receiving 5FU and/or MTX chemotherapy. Furthermore, these studies will help to develop novel anticancer therapies based on SNPs and variants of genes involved in folate metabolism.

8.2 Future Directions

In the present study, we developed an in vitro model of GGH overexpression and inhibition in HCT116 colon and MDA-MB-435 breast cancer cells with predictable functional consequences, and provided an in vitro model to test effects of folate and GGH modulation on chemosensitivity of colon and breast cancer cells to 5FU and antifolates (Study 1 and Study 2). However, we found that differences in in vitro chemosensitivity observed between the GGH-altered cells and cells expressing endogenous levels of GGH activity at different folate concentrations were generally modest in a cell-specific manner. Although we demonstrated that concentrations of intracellular total folate and long-chain polyglutamates reflected different levels of 5-MTHF in media on Day 3 for the in vitro chemosensitivity test (Tables 5.1 and 5.2, Chapter 5), we would likely observe a more significant magnitude of change in chemosensitivity between the GGH- modulated and corresponding control cells by culturing cells at 50 nM or 100 nM 5-MTHF at least one week prior to an in vitro chemosensitivity test. In addition, further studies will help to provide a mechanistic understanding of the effects of folate and GGH modulation on 5FU- and antifolate-based chemotherapy: (1) measurement of TS and DHFR activities at 100 nM and 50 nM 5-MTHF; (2) measurement of FPGS activity at three different folate concentrations; and (3) determination of polyglutamylation and distribution of 5,10-methyleneTHF and classic antifolates (MTX and MTA) at three different folate concentrations. Gaining knowledge of the

282 activities of TS and DHFR would reveal whether 5FU and antifolates were inhibiting their target enzymes as well as unveil how the cells would compensate for the alterations in GGH activity. Determining FPGS activity would reveal whether altered GGH activity might influence FPGS activity. Also, determining the polyglutamylation and distribution of 5,10-methyleneTHF and classic antifolates (MTX and MTA) would eludicate whether increased GGH activity translates into a decreased concentration of polyglutamates of 5,10-methyleneTHF and MTX/MTA and vice versa. This information could then be used to either support or refute the proposed mechanism for 5FU and MTX/MTA cytotoxicity involving polyglutamylated forms of 5,10- methyleneTHF stabilizing the temary complex and MTX/MTA, respectively. Future experiments investigating the effect of GGH modulation on chemosensitivity to chemotherapeutic agents in other colon and breast cancer cell lines as well as other types of cancer cell lines will help to determine whether GGH would be a critical determinant for tailored cancer therapy across cancer cells from various sites.

In addition, introducing the in vitro model into a mouse model more closely represents physiologic effects as compared with tissue culture environments. Results from in vivo studies will confirm the in vitro chemosensitivity data and enhance the understanding of the role of GGH modulation in determining drug efficacy in antifolate- and 5FU-based chemotherapy.

As a result of genome-wide DNA methylation and gene expression profiling in HCT116 and MDA-MB-435 cells, we identified several genes with altered expression, involved in important biological pathways, which were regulated by DNA methylation. Furthermore, these alterations in gene expression might influence chemosensitivity to 5FU and antifolates in response to GGH and FPGS modulation (Study 3 and Study 4). Identification of differentially expressed genes involved in several biological pathways such as folate pathways including folate/antifolate transporters and MRPs/BCRP, cell cycle, apoptosis, cell proliferation, and signal transduction in response to GGH modulation at different folate concentrations will help to elucidate the possible mechanism of the GGH modulation-induced changes in chemosensitivity to 5FU and antifolates at each different folate level. In order to identify epigenetically altered genes affecting chemosensitivity to 5FU and antifolates in response to GGH and FPGS modulation, further studies need to be performed by investigating whether downregulated and hypermethylated

283 genes would regain their expression after treatment with demethylating agents such as 5-aza-CR and 5-aza-CdR.

Also, further preclinical and clinical studies interrogating specific genes in response to GGH modulation are warranted to elucidate the possible mechanism of the GGH modulation-induced chemosensitivity to antifolates and 5FU. Elucidation of the pharmocogenetic ramifications will provide a framework for developing rational, effective and safe tailored chemotherapy.

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APPENDICES

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Appendix 1 Cell survival

Cell survival graphs were constructed based on the values obtained from optical density (OD) measurements. After 72 hours drug exposure, plates containing cells treated with and without chemotherapeutic agents were fixed and stained with SRB. The SRB assay is a rapid and sensitive method for measuring drug-induced cytotoxicity in attached cultures in 96-well microtiter plates. After staining with SRB, cells were then washed and SRB was solubilized and measured. OD values of SRB concentration could be used to determine cell concentration because OD vs. dye concentration experiments previously determined the relationship to be linear. Furthermore, cell protein concentration measured by the Lowry and the Bradford Coomassie methods both correlated linearly with OD values and number of cells, demonstrating that OD of SRB concentration correlate with cell number. Therefore, the various OD values obtained in this experiment were used in the equation shown in section 3.5.9.

(OD drug / OD start drug exposure) - 1 Survival (%) = × 100 (OD no drug / OD start drug exposure) - 1

Where OD drug is the OD of cells that were treated with drug, OD start drug exposure is the OD of control, and OD no drug is the OD of wells that were not treated with drug.

Using the above equation, a survival percentage was calculated for each concentration of drug tested and values were plotted on a Survival vs. Drug Concentration graph.

Statistical significance determination:

Due to the S-shaped survival vs. drug dose curve, survival and concentration values were logit transformed according to the formula:

[ logit (p) = ln (p/[1 - p]) ]

328

Ordinary least squares regression was used to model the effect of log (dose) of chemotherapy and cell type (Control-S vs. Sense, Control-si vs. siRNA) on the logit transformed proportion of cells that survived at each dose. The interaction between cell type and log (dose) was included in the model to test the hypothesis that cell types were differentially sensitive to chemotherapy.

IC50 calculation:

IC50 doses and their 95% confidence intervals were calculated on the log scale from the regression results and then back transformed to the original scale for reporting.

329

Appendix 2 Diet composition

Appendix 2. 1 Experimental diet L-amino acid and nutrient composition

Control Diet Supplemented Diet Nutrient : 2 mg FA/kg (g/kg) : 5 mg FA/kg (g/kg) L-Alanine 3.5 3.5 L- (free-base) 11.2 11.2 L-Asparagine 6.0 6.0 L-Aspartic acid 3.5 3.5 L-Cystine 3.5 3.5 L-Glutamic acid 35.0 35.0 Glycine 23.3 23.3 L-Histidine (free-base) 3.3 3.3 L-Isoleucine 8.2 8.2 L-Leucine 11.1 11.1 L-Lysine HCl 14.4 14.4 L-Methionine 8.2 8.2 L- 11.6 11.6 L-Proline 3.5 3.5 L-Serine 3.5 3.5 L-Threonine 8.2 8.2 L-Tryptophan 1.74 1.74 L-Tyrosine 3.5 3.5 L-Valine 8.2 8.2 Total L-Amino Acid 171.44 171.44

Dextrin 407 407.4 Sucrose 193 193 Cellulose 50 50 Corn oil (Stab. 0.015% BHT) 100 100 Salt Mix (#210006) 57.96 57.96 Vitamin Mix (#317756 folate free) 10 10

330

Choline chloride 2 2 Sodium bicarbonate 6.6 6.6 Folic Acid/Sucrose Premix (1 mg/g) 2.0 - Folic Acid/Sucrose Premix (5 mg/g) - 1.6

Total 1000 1000

Appendix 2. 2 Tufts folate deficient mineral and salt mix used in L-amino acid defined diets (Salt Mix #210006)

Minerals g/kg

Calcium carbonate 14.6 Calcium phosphate, dibasic 0.17 Sodium chloride 12.37 Potassium phosphate, dibasic 17.16 Magnesium sulfate, anhydrous 2.45 Manganese sulfate, monohydrate 0.18 Ferric citrate 0.62 Zinc carbonate 0.054 Cupric carbonate 0.054 Potassium iodide 0.00058 Sodium selenite 0.00058 Chromium potassium sulfate 0.019 Sodium fluoride 0.0023 Molybdic acid, ammonium salt 0.0012 Sucrose 10.27534

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Appendix 2. 3 Tufts folate deficient vitamin mix used in L-amino acid defined diets (#317756 folate free)

Vitamins g/kg

Thiamin HCl 0.006 Riboflavin 0.006 Pyridoxine HCl 0.007 Nicotinic acid 0.03 Calcium pantothenate 0.016 Cyanocobalamin 0.00005 palmitate (500,000 IU/g) 0.008

Vitamin D3 (400,000 IU/g) 0.0025 acetate (500 IU/g) 0.1 Menadioine sodium bisulfate 0.0008 Biotin 0.00002 Sucrose 9.82363

332

Appendix 3 Gene-specific promoter CpG island methylation analysis

DNA Methylation Assay:

The Illumina Infinium HumanMethylation27 BeadChip was used to interrogate the DNA methylation status of 27,578 individual CpG sites located at promoter regions of 14,495 genes (360). Briefly, 1 µg of genomic DNA was bisulfite-converted using the EZ-96 DNA Methylation Kit (Zymo Research) according to the manufacturer’s instructions. Unmethylated cytosines are deaminated to uracil in the presence of bisulfite, while methylated cytosines are refractory to the effects of bisulfite and remain cytosine. The bisulfite conversion included a thermocycling program with a short denaturation step (16 cycles of 95ºC for 30 seconds followed by 50ºC for 1 hour). The amount of bisulfite-converted DNA and completeness of bisulfite conversion was assessed using a panel of MethyLight-based quality control (QC) reactions as previously described (433). All of the samples passed our QC tests and entered into the Infinium DNA methylation assay pipeline for whole-genome amplification (WGA) and enzymatical fragmentation. The bisulfite-converted WGA-DNA samples were purified and applied to the BeadChips. During hybridization, the WGA-DNA molecules annealed to locus-specific DNA oligomers linked to individual bead types. The two bead types corresponded to each CpG locus - one to the methylated (C) and the other to the unmethylated (T) state. Allele-specific primer annealing was followed by single-base extension using DNP- and Biotin-labeled ddNTPs. Both bead types for the same CpG locus incorporated the same type of labeled nucleotide, determined by the base preceding the interrogated “C” in the CpG locus, and therefore would be detected in the same color channel (Figure 1). After extension, the array was fluorescently stained, scanned, and the intensities of the unmethylated and methylated bead types measured. A measure of the level of DNA methylation at each CpG site was scored as beta (β) values using BeadStudio software. DNA methylation β-values represent the ratio of the intensity of the methylated bead type to the combined locus intensity ranging from 0 to 1. Values close to 0 indicate low levels of DNA methylation, while values close to 1 indicate high levels of DNA methylation (360). The detection P-values measure the difference of the signal intensities at the interrogated CpG site compared with those from a set of 16 negative control probes embedded in the assay. We identified all data points with a detection P-value > 0.05 as not statistically significantly different

333 from background measurements, and therefore not trustworthy measures of DNA methylation. These data points were replaced by “NA” values as previously described (434). The assay probe sequences and detailed information on each interrogated CpG site and the associated genomic characteristics on the HumanMethylation27 BeadChip can be obtained at http://www.illumina.com.

Unmethylated DNA Locus Methylated DNA Locus

Figure 1 The infinium assay for methylation. The Infinium assay for methylation detects methylation status at individual CpG loci by typing bisulfite-converted DNA. Methylation protects C from conversion (left), whereas unmethylated C is converted to T (right). A pair of bead-bound probes is used identification CpG island methylator to detect the presence of T or C by hybridization followed by single-base extension with a labeled nucleotide (Weisenberger DJ et al. (2008) Illumina Application Note http://www.illumina.com/documents/products/appnotes/ appnote_dna_methylation_analysis_infinium.pdf).

Data Filtering and Normalization:

We masked data point as “NA” for probes that contain single-nucleotide polymorphisms (SNPs) (dbSNP NCBI build 130/hg18) within the five base pairs from the interrogated CpG site or that overlap with a repetitive element that covers the targeted CpG dinucleotide. Furthermore, we replaced data points with “NA” for probes that are not uniquely aligned to the human genome (NCBI build 36/hg18) at 20 nucleotides at the 3' terminus of the probe sequence, and those that overlap with regions of insertions and deletions in the human genome. We also identified all data points with a detection P-value > 0.05 and replaced those with “NA” values.

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Appendix 4 Gene expression analysis

RNA Isolation:

RNAs from the GGH-modulated HCT116 and MDA-MB-435 cells were isolated using the RNeasy Microarray Tissue Mini Kit (Qiagen, Cat#: 73304) according to the manufacturer’s protocol. The kit integrates phenol/guanidine-based sample lysis and silica-membrane purification of total RNA. After washing the cells with 10 mL of PBS three times, cells were lysed by adding 1 mL of QIAzol lysis reagent to a 100 mm culture dish, scraped and transferred into a new eppendorf tube. The cell lysate was incubated for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complex. Following the incubation, 200 μL of chloroform was added, the sample was shaken vigorously for 15 seconds, and incubated again for 2-3 minutes. The sample was then centrifuged at 12,000 × g for 15 minutes at 4ºC, the upper aqueous phase was collected. One volume of 70% ethanol was added to provide appropriate binding conditions, and the sample was mixed thoroughly by pipetting up and down. The sample (up to 700 μL) was transferred to an RNeasy mini spin column, where was able to bind to the membrane, centrifuged at 10,000 rpm for 15 seconds, and the flow-through was discarded. After repeating washing, 350 μL of Buffer RW1 was added to the RNeasy spin column, centrifuged at 10,000 rpm for 15 seconds to wash the membrane, and the flow-through was discarded. Ten microliters of DNase 1 stock solution (Qiagen) was added to 70 μL of Buffer RDD, gently inverted, and centrifuged briefly. The DNase 1 incubation mix was added to the RNeasy spin column, and placed for 15 minutes. Following washing the membrane, RNA was eluted in 60 μL of RNase-free water (Sigma), and kept on ice. The RNA concentration was measured using a spectrophotometer. All samples had a ratio of A260:A280 nm between 1.9 and 2.1, indicating pure RNA. The purified RNA was stored at -20ºC for further analysis.

RNA Concentration and Integrity:

Total RNA was assessed for the RNA quality verification and microarray hybridization. The Agilent 2100 Bioanalyzer (Agilent Technologies), a microfluidics-based platform, was used for sizing, quantification and quality of RNA. Briefly, 1 μL of the RNA sample was loaded onto each well of the chip, the sample’s components were electrophoretically separated, detected by

335 their fluorescence, and translated into eletropherograms (peaks) and gel-like images (bands). An RNA ladder (a mixture of RNA of known concentration) was run in conjunction with the samples to calculate the concentration of the RNA in the sample. The RNA Integrity Number (RIN) score was generated on the Agilent software. For the microarray analysis, the RNA quality for all of the samples had a RIN score ≥ 7.

RNA Amplification:

The Illumina® TotalPrepTM-96 RNA Amplification Kit (Ambion, Lot#: 1008019) was used for generating biotinylated, amplified cRNA for hybridization with Illumina HumanHT-12 v4.0 Expression BeadChip according to the manufacturer’s protocol.

For the first strand cDNA synthesis, the procedure began with reverse transcription with an oligo (dT) primer bearing a T7promoter using ArrayScript™ reverse transcriptase. Two hundred nanograms of RNA was brought up to a final volume of 11 μL with nuclease-free water. Nine microliters of reverse transcription master mix (1 μL of T7 Oligo primer, 2 μL of 10× first strand buffer, 4 μL of dNTP mix, 1 μL of RNase inhibitor, and 1 μL of ArrayScriptTM reverse transcriptase) was added to the RNA sample, and incubated for 2 hours at 42ºC.

The cDNA then underwent the second strand synthesis and purification to become a template for in vitro transcription with T7 RNA Polymerase. Briefly, for the second strand cDNA synthesis, 80 μL of second strand master mix (63 μL of nuclease-free water, 10 μL of 10× second strand buffer, 4 μL of dNTP mix, 2 μL of DNA polymerase, and 1 μL of RNase H) was added to the sample, and incubated for 2 hours at 16ºC . For the cDNA purification, cDNA was captured by magnetic beads by adding 180 μL of cDNAPure and shaking for 2 minutes. After washing twice with 150 μL of cDNA wash buffer, the cDNA was eluted from the magnetic beads by adding 20 μL of 55ºC nuclease-free water, followed by transferring 17.5 μL of the purified cDNA to wells of a new PCR plate.

For in vitro transcription (IVT) to synthesize cRNA, 7.5 μL of IVT master mix (2.5 μL of T7 10× reaction buffer, 2.5 μL of T7 enzyme mix, and 2.5 μL of biotin-NTP mix) was added to each sample, and incubated for 14 hours at 37ºC. For cRNA purification, 70 μL of cRNA binding mix and 95 μL of ethanol were added to each sample, and shaken for 2 minutes after transferring each

336 sample to U-Bottom plate. The RNA binding beads were captured, and washed twice with 100 μL of cRNA wash solution. The purified cRNA from the RNA binding beads was eluted with 100 μL of 55ºC cRNA elution buffer. The cRNA yield was quantified by NanoDrop (NanoDrop Technologies).

Hybridization:

A total of 750 ng of purified biotinylated cRNA generated from the samples were randomized in triplicate and used to hybridize onto the Illumina HumanHT-12 v4.0 BeadChip. Each array on this BeadChip targets 31,335 annotated genes and includes 47,231 probes designed to cover content from NCBI RefSeq Release 38 (November 7, 2009), as well as legacy UniGene content. Twelve samples are hybridized to one slide for higher throughput and reduced sample-to-sample variability since gaskets separate each array.

The BeadChip was incubated at 58°C, with rotation speed 5 for 18 hours for hybridization. The BeadChip was washed with 500 mL of 1X High temp wash buffer at 55°C, 250 mL of E1BC buffer, and 250 mL of 100% ethanol to remove any unhybridized RNA and non-specific RNA, and then blocked with 4 mL of E1 buffer and stained with streptavidin-Cy3 (GE Healthcare Bio- Sciences) according to the Illumina protocol. After washing and staining, the BeadChip was scanned on the iScan (Illumina), a laser based imaging system with 2 lasers (red: Cy5 and green: Cy3) for detecting fluorescence information on BeadChips. Although the iScan can scan both red and green intensities, the Illumina gene expression array is single colour array; therefore, scans with green laser. The intensities files were quantified in GenomeStudio® (Illumina, version 2010.2) to generate raw intensities files without normalization algorithims. All samples passed Illumina's sample dependent and independent QC metrics. Raw intensities data files need to be normalized to adjust samples signals in order to minimize effects of variation arising from non- biological factors. For our study, the data normalization was performed using GeneSpring (Agilent Technologies) prior to data analysis.

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Appendix 5 The top 50 genes with most differentially altered expression in response to GGH modulation

Appendix 5. 1 The top 50 genes with most differentially downregulated compared with controls in the GGH-overexpressed HCT116 colon cancer cells

Gene Fold Description Accession Probe ID Symbol Change POLE4 -6.53 polymerase (DNA-directed), epsilon 4 (p12 subunit) NM_019896.2 ILMN_1660063 PVRL3 -3.86 poliovirus receptor-related 3 NM_015480.1 ILMN_2188521 NAP1L1 -3.70 nucleosome assembly protein 1-like 1 NM_139207.1 ILMN_1705876 PVRL3 -3.25 poliovirus receptor-related 3 NM_015480.1 ILMN_1727633 PHLDB2 -2.74 pleckstrin homology-like domain, family B, member 2 NM_145753.1 ILMN_2179778 TNFRSF6B -2.61 tumor necrosis factor receptor superfamily, member 6b, decoy NM_032945.2 ILMN_2331231 AKAP12 -2.44 A kinase (PRKA) anchor protein (gravin) 12 NM_144497.1 ILMN_1684836 AKAP12 -2.44 A kinase (PRKA) anchor protein (gravin) 12 NM_144497.1 ILMN_1686846 AKAP12 -2.31 A kinase (PRKA) anchor protein (gravin) 12 NM_005100.2 ILMN_2308950 SUSD2 -2.27 sushi domain containing 2 NM_019601.3 ILMN_1693270 BMP4 -2.19 bone morphogenetic protein 4 NM_130851.1 ILMN_1740900 CDK6 -2.05 cyclin-dependent kinase 6 NM_001259.5 ILMN_1802615 RND3 -1.99 Rho family GTPase 3 NM_005168.3 ILMN_1759513 NR2F2 -1.89 nuclear receptor subfamily 2, group F, member 2 NM_021005.2 ILMN_2094360 TCEA1 -1.86 transcription elongation factor A (SII), 1 NM_201437.1 ILMN_2357770 BAMBI -1.86 BMP and activin membrane-bound inhibitor homolog (Xenopus NM_012342.2 ILMN_1691410 laevis) RFC1 -1.85 replication factor C (activator 1) 1, 145kDa NM_002913.3 ILMN_2217935 TNFRSF6B -1.79 tumor necrosis factor receptor superfamily, member 6b, decoy NM_032945.2 ILMN_2331232 PHLDB2 -1.79 pleckstrin homology-like domain, family B, member 2 NM_145753.1 ILMN_1719792 PDE4B -1.77 phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 NM_002600.3 ILMN_2340259 dunce homolog, Drosophila) PDLIM1 -1.77 PDZ and LIM domain 1 NM_020992.2 ILMN_1788955 PIP4K2A -1.71 phosphatidylinositol-5-phosphate 4-kinase, type II, alpha NM_005028.4 ILMN_3236637 MNS1 -1.70 meiosis-specific nuclear structural 1 NM_018365.1 ILMN_2157240 ZNF91 -1.70 zinc finger protein 91 NM_003430.2 ILMN_1802053 SLC2A3 -1.70 solute carrier family 2 (facilitated glucose transporter), member 3 NM_006931.1 ILMN_1775708 CNTNAP2 -1.69 contactin associated protein-like 2 NM_014141.4 ILMN_1690223 TRIM33 -1.69 tripartite motif-containing 33 NM_015906.3 ILMN_1682316 PYGL -1.68 phosphorylase, glycogen, liver NM_002863.3 ILMN_1696187 TCEA1 -1.68 transcription elongation factor A (SII), 1 NM_006756.2 ILMN_1709851 BDNF -1.65 brain-derived neurotrophic factor NM_001709.3 ILMN_1751276 FAM111A -1.65 family with sequence similarity 111, member A NM_022074.2 ILMN_2410038 BMP4 -1.64 bone morphogenetic protein 4 NM_001202.2 ILMN_1709734 GLIS3 -1.64 GLIS family zinc finger 3 NM_152629.3 ILMN_2402600 MACF1 -1.62 microtubule-actin crosslinking factor 1 NM_012090.3 ILMN_2301624 ALG6 -1.59 asparagine-linked glycosylation 6 homolog (S. cerevisiae, alpha- NM_013339.2 ILMN_1771411 1,3-glucosyltransferase) MTAP -1.59 methylthioadenosine phosphorylase NM_002451.3 ILMN_1753639 EPS8 -1.58 epidermal growth factor receptor pathway substrate 8 NM_004447.4 ILMN_1651699 SPAG9 -1.55 sperm associated antigen 9 NM_003971.3 ILMN_2263718 MCOLN2 -1.54 mucolipin 2 NM_153259.2 ILMN_1660462

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SACS -1.52 spastic ataxia of Charlevoix-Saguenay (sacsin) NM_014363.3 ILMN_2131523 JAK1 -1.52 Janus kinase 1 NM_002227.2 ILMN_1793384 PDGFC -1.51 platelet derived growth factor C NM_016205.1 ILMN_1683023 MRPS31 -1.50 mitochondrial ribosomal protein S31 NM_005830.2 ILMN_1654552 AP1S2 -1.50 adaptor-related protein complex 1, sigma 2 subunit NM_003916.3 ILMN_2120273 TNFRSF6B -1.49 tumor necrosis factor receptor superfamily, member 6b, decoy NM_003823.2 ILMN_1661825 ADAM19 -1.48 ADAM metallopeptidase domain 19 (meltrin beta) NM_033274.2 ILMN_1713751 ADAM10 -1.48 ADAM metallopeptidase domain 10 NM_001110.2 ILMN_1718946 ABHD10 -1.48 abhydrolase domain containing 10 NM_018394.1 ILMN_1770031 RPS23 -1.45 ribosomal protein S23 NM_001025.4 ILMN_1772459

Appendix 5. 2 The top 50 genes with most differentially upregulated compared with controls in the GGH-overexpressed HCT116 colon cancer cells

Gene Fold Description Accession Probe ID Symbol Change FGFBP1 4.08 fibroblast growth factor binding protein 1 NM_005130.3 ILMN_1785404 RERG 2.97 RAS-like, estrogen-regulated, growth inhibitor NM_032918.1 ILMN_1746359 GDF15 2.44 growth differentiation factor 15 NM_004864.1 ILMN_2188862 SCG2 2.21 secretogranin II (chromogranin C) NM_003469.3 ILMN_1703178 LCN2 1.93 lipocalin 2 NM_005564.3 ILMN_1692223 UPP1 1.90 uridine phosphorylase 1 NM_003364.2 ILMN_1798256 PLAU 1.89 plasminogen activator, urokinase NM_002658.2 ILMN_1656057 GAS6 1.86 growth arrest-specific 6 NM_000820.1 ILMN_1779558 HIST1H2BK 1.81 histone cluster 1, H2bk NM_080593.1 ILMN_1796179 PBX1 1.68 pre-B-cell leukemia homeobox 1 NM_002585.1 ILMN_1784678 TMEM200A 1.67 transmembrane protein 200A NM_052913.2 ILMN_1725387 C12orf47 1.65 chromosome 12 open reading frame 47 XR_017973.1 ILMN_1798957 ANXA10 1.62 annexin A10 NM_007193.3 ILMN_1699421 KLK6 1.62 kallikrein-related peptidase 6 NM_001012964.1 ILMN_1780255 SFTA1P 1.60 surfactant associated 1 (pseudogene) NR_027082.1 ILMN_3310065 CTH 1.60 cystathionase (cystathionine gamma-) NM_153742.3 ILMN_2305112 SYTL2 1.59 synaptotagmin-like 2 NM_206929.1 ILMN_2336609 HMGCS1 1.58 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) NM_002130.6 ILMN_1797728 SCAND1 1.54 SCAN domain containing 1 NM_016558.2 ILMN_1794230 ALDH2 1.53 aldehyde dehydrogenase 2 family (mitochondrial) NM_000690.2 ILMN_1793859 SYTL2 1.52 synaptotagmin-like 2 NM_206928.1 ILMN_1682929 HRASLS3 1.51 HRAS-like suppressor 3 NM_007069.2 ILMN_1667711 NAPRT1 1.49 nicotinate phosphoribosyltransferase domain containing 1 NM_145201.3 ILMN_1710752 TSC22D1 1.49 TSC22 domain family, member 1 NM_006022.2 ILMN_1692177 DHRS11 1.48 dehydrogenase/reductase (SDR family) member 11 NM_024308.3 ILMN_1756701 HIST1H2BD 1.48 histone cluster 1, H2bd NM_138720.1 ILMN_1651496 NFIB 1.47 nuclear factor I/B NM_005596.2 ILMN_1778991 PHLDA1 1.45 pleckstrin homology-like domain, family A, member 1 NM_007350.3 ILMN_1687978 MAPKAPK5 1.44 mitogen-activated protein kinase-activated protein kinase 5 NM_139078.1 ILMN_2322935 G6PC3 1.44 glucose 6 phosphatase, catalytic, 3 NM_138387.2 ILMN_2127477 ELMO3 1.43 engulfment and cell motility 3 NM_024712.3 ILMN_1752665

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GCAT 1.43 glycine C-acetyltransferase (2-amino-3-ketobutyrate coenzyme NM_014291.2 ILMN_1724437 A ligase) PROM2 1.42 prominin 2 NM_144707.1 ILMN_1761946 C21orf70 1.41 chromosome 21 open reading frame 70 NM_058190.2 ILMN_2104924 TMC6 1.41 transmembrane channel-like 6 NM_007267.5 ILMN_1794677 SERINC2 1.41 serine incorporator 2 NM_178865.3 ILMN_2111932 MAPKAPK5 1.39 mitogen-activated protein kinase-activated protein kinase 5 NM_139078.1 ILMN_1699082 LOC389816 1.39 cytokeratin associated protein NM_001013653.1 ILMN_2249018 LOC152195 1.39 hypothetical protein LOC152195 NM_194289.1 ILMN_1716468 CA2 1.39 carbonic anhydrase II NM_000067.1 ILMN_1662795 ACOX2 1.38 acyl-Coenzyme A oxidase 2, branched chain NM_003500.2 ILMN_1685703 TRNP1 1.38 TMF1-regulated nuclear protein 1 NM_001013642.2 ILMN_1695946 LPCAT3 1.38 lysophosphatidylcholine acyltransferase 3 NM_005768.5 ILMN_1805225 IRS1 1.38 insulin receptor substrate 1 NM_005544.1 ILMN_1759232 RBBP9 1.38 retinoblastoma binding protein 9 NM_006606.2 ILMN_1786050 FASN 1.37 fatty acid synthase NM_004104.4 ILMN_1784871 PHF19 1.37 PHD finger protein 19 NM_001009936.1 ILMN_1756676 TACSTD2 1.37 tumor-associated calcium signal transducer 2 NM_002353.1 ILMN_1739001 PPARG 1.37 peroxisome proliferator-activated receptor gamma NM_015869.4 ILMN_1800225 CA2 1.36 carbonic anhydrase II NM_000067.1 ILMN_2199439

Appendix 5. 3 The top 50 genes with most differentially downregulated compared with controls in the GGH-inhibited HCT116 colon cancer cells

Gene Fold Description Accession Probe ID Symbol Change ANXA10 -6.85 annexin A10 NM_007193.3 ILMN_1699421 ZBED2 -3.48 zinc finger, BED-type containing 2 NM_024508.3 ILMN_1651365 DHRS2 -3.33 dehydrogenase/reductase (SDR family) member 2 NM_182908.3 ILMN_2384857 KRT80 -2.76 keratin 80 NM_182507.2 ILMN_1705814 TACSTD2 -2.38 tumor-associated calcium signal transducer 2 NM_002353.1 ILMN_1739001 PHLDB2 -2.23 pleckstrin homology-like domain, family B, member 2 NM_145753.1 ILMN_2179778 NR4A2 -2.22 nuclear receptor subfamily 4, group A, member 2 NM_006186.2 ILMN_1782305 PHLDB2 -2.09 pleckstrin homology-like domain, family B, member 2 NM_145753.1 ILMN_1719792 DHRS2 -2.08 dehydrogenase/reductase (SDR family) member 2 NM_182908.3 ILMN_1725726 NCAPD3 -2.07 non-SMC condensin II complex, subunit D3 NM_015261.2 ILMN_1683441 HYOU1 -2.01 hypoxia up-regulated 1 NM_006389.2 ILMN_1673649 APPL1 -1.99 adaptor protein, phosphotyrosine interaction, PH domain and NM_012096.2 ILMN_1763730 leucine zipper containing 1 TMEM200A -1.89 transmembrane protein 200A NM_052913.2 ILMN_1725387 TFPI -1.85 tissue factor pathway inhibitor (lipoprotein-associated NM_006287.4 ILMN_1662619 coagulation inhibitor) IGFBP3 -1.84 insulin-like growth factor binding protein 3 NM_000598.4 ILMN_1746085 MPP7 -1.82 membrane protein, palmitoylated 7 (MAGUK p55 subfamily NM_173496.3 ILMN_1721774 member 7) SLC2A3 -1.82 solute carrier family 2 (facilitated glucose transporter), member NM_006931.1 ILMN_1775708 3 UPP1 -1.79 uridine phosphorylase 1 NM_003364.2 ILMN_1798256 AHR -1.77 aryl hydrocarbon receptor NM_001621.3 ILMN_2162799

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TSHZ1 -1.77 teashirt zinc finger homeobox 1 NM_005786.4 ILMN_1718907 TMPO -1.76 thymopoietin NM_003276.1 ILMN_1677747 TFPI -1.74 tissue factor pathway inhibitor (lipoprotein-associated NM_006287.4 ILMN_1707124 coagulation inhibitor) ESAM -1.73 endothelial cell adhesion molecule NM_138961.1 ILMN_1668092 PBX1 -1.73 pre-B-cell leukemia homeobox 1 NM_002585.1 ILMN_1784678 UCA1 -1.72 urothelial cancer associated 1 (non-protein coding) NR_015379.2 ILMN_3239254 STC1 -1.71 stanniocalcin 1 NM_003155.2 ILMN_1758164 AKAP12 -1.70 A kinase (PRKA) anchor protein (gravin) 12 NM_144497.1 ILMN_1686846 ATG12 -1.68 ATG12 autophagy related 12 homolog (S. cerevisiae) NM_004707.2 ILMN_2188204 HYOU1 -1.68 hypoxia up-regulated 1 NM_006389.2 ILMN_2141790 DDIT3 -1.67 DNA-damage-inducible transcript 3 NM_004083.4 ILMN_1676984 CDK6 -1.67 cyclin-dependent kinase 6 NM_001259.5 ILMN_1802615 BMP4 -1.67 bone morphogenetic protein 4 NM_001202.2 ILMN_1709734 ZNF443 -1.67 zinc finger protein 443 NM_005815.2 ILMN_2104967 CD163L1 -1.66 CD163 molecule-like 1 NM_174941.4 ILMN_3242540 FBXO5 -1.66 F-box protein 5 NM_012177.2 ILMN_1710676 KIAA1731 -1.64 KIAA1731 NM_033395.1 ILMN_3235104 IGFBP6 -1.64 insulin-like growth factor binding protein 6 NM_002178.2 ILMN_1669362 CENTG2 -1.63 centaurin, gamma 2 NM_014914.2 ILMN_1800530 DCLK1 -1.63 doublecortin-like kinase 1 NM_004734.2 ILMN_2165354 PHF19 -1.62 PHD finger protein 19 NM_001009936.1 ILMN_1756676 CKLF -1.62 chemokine-like factor NM_001040139.1 ILMN_2414027 CYP24A1 -1.60 cytochrome P450, family 24, subfamily A, polypeptide 1 NM_000782.3 ILMN_1685663 RIOK3 -1.59 RIO kinase 3 (yeast) NM_003831.2 ILMN_2404135 KIAA0101 -1.58 KIAA0101 NM_014736.4 ILMN_2285996 TOPBP1 -1.58 topoisomerase (DNA) II binding protein 1 NM_007027.2 ILMN_1684929 PLEKHB2 -1.57 pleckstrin homology domain containing, family B (evectins) NM_001031706.1 ILMN_1698323 member 2 TMEM136 -1.56 transmembrane protein 136 NM_174926.1 ILMN_1815346 MCM2 -1.56 minichromosome maintenance complex component 2 NM_004526.2 ILMN_1681503

Appendix 5. 4 The top 50 genes with most differentially upregulated compared with controls in the GGH-inhibited HCT116 colon cancer cells

Gene Fold Description Accession Probe ID Symbol Change IFI27 3.91 interferon, alpha-inducible protein 27 NM_005532.3 ILMN_2058782 PDE4B 3.24 phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 NM_002600.3 ILMN_2340259 dunce homolog, Drosophila) CA2 3.08 carbonic anhydrase II NM_000067.1 ILMN_1662795 SNTB1 2.75 syntrophin, beta 1 (dystrophin-associated protein A1, 59kDa, NM_021021.2 ILMN_1793410 basic component 1) CA2 2.60 carbonic anhydrase II NM_000067.1 ILMN_2199439 TNFRSF6B 2.49 tumor necrosis factor receptor superfamily, member 6b, decoy NM_032945.2 ILMN_2331231 CALB2 2.47 calbindin 2 NM_007088.2 ILMN_1748840 ABLIM1 2.37 actin binding LIM protein 1 NM_006720.3 ILMN_1785424 SUMO3 2.36 SMT3 suppressor of mif two 3 homolog 3 NM_006936.2 ILMN_1725642 TNFRSF6B 2.18 tumor necrosis factor receptor superfamily, member 6b, decoy NM_003823.2 ILMN_1661825

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TNFRSF6B 2.16 tumor necrosis factor receptor superfamily, member 6b, decoy NM_032945.2 ILMN_2331232 KCNS3 2.13 potassium voltage-gated channel, delayed-rectifier, subfamily S, NM_002252.3 ILMN_2175114 member 3 FAT1 2.04 FAT tumor suppressor homolog 1 (Drosophila) NM_005245.3 ILMN_3247578 SLC1A3 2.01 solute carrier family 1 (glial high affinity glutamate transporter), NM_004172.3 ILMN_1738552 member 3 HIST1H2BD 1.99 histone cluster 1, H2bd NM_138720.1 ILMN_1651496 LEMD1 1.91 LEM domain containing 1 NM_001001552.3 ILMN_1785444 PRKCD 1.91 protein kinase C, delta NM_006254.3 ILMN_1801105 NOV 1.90 nephroblastoma overexpressed gene NM_002514.2 ILMN_1787186 HIST1H2BK 1.87 histone cluster 1, H2bk NM_080593.1 ILMN_1796179 ACOX2 1.86 acyl-Coenzyme A oxidase 2, branched chain NM_003500.2 ILMN_1685703 CD33 1.83 CD33 molecule NM_001772.3 ILMN_1747622 C21orf33 1.81 chromosome 21 open reading frame 33 NM_198155.2 ILMN_1737588 CHPF 1.81 chondroitin polymerizing factor NM_024536.4 ILMN_1731353 COL6A1 1.79 collagen, type VI, alpha 1 NM_001848.2 ILMN_1732151 SERPINB5 1.78 serpin peptidase inhibitor, clade B (ovalbumin), member 5 NM_002639.3 ILMN_1793888 CLDND1 1.77 claudin domain containing 1 NM_001040181.1 ILMN_1710326 ABHD10 1.77 abhydrolase domain containing 10 NM_018394.1 ILMN_1770031 FAT1 1.76 FAT tumor suppressor homolog 1 (Drosophila) NM_005245.3 ILMN_1754795 C21orf33 1.74 chromosome 21 open reading frame 33 NM_004649.5 ILMN_1682812 RINL 1.74 Ras and Rab interactor-like NM_198445.2 ILMN_1790962 VPS41 1.72 vacuolar protein sorting 41 (yeast) NM_014396.2 ILMN_1703379 ADARB1 1.72 adenosine deaminase, RNA-specific, B1 (RED1 homolog rat) NM_001112.2 ILMN_1679797 SRPX 1.72 sushi-repeat-containing protein, X-linked NM_006307.3 ILMN_1709486 CRABP2 1.72 cellular retinoic acid binding protein 2 NM_001878.2 ILMN_1690170 CLDND1 1.71 claudin domain containing 1 NM_001040181.1 ILMN_2352563 LRRC26 1.71 leucine rich repeat containing 26 NM_001013653.2 ILMN_1680757 OSR1 1.69 odd-skipped related 1 (Drosophila) NM_145260.2 ILMN_2197128 CPOX 1.68 coproporphyrinogen oxidase NM_000097.4 ILMN_3240389 MRPS6 1.68 mitochondrial ribosomal protein S6 NM_032476.2 ILMN_1723874 PIGP 1.67 phosphatidylinositol glycan anchor biosynthesis, class P NM_153682.2 ILMN_1774949 UGDH 1.66 UDP-glucose dehydrogenase NM_003359.2 ILMN_1729563 SCARNA16 1.66 small Cajal body-specific RNA 16 NR_003013.1 ILMN_3237446 TSPAN7 1.66 tetraspanin 7 NM_004615.2 ILMN_2120695 ALG6 1.66 asparagine-linked glycosylation 6 homolog (S. cerevisiae, alpha- NM_013339.2 ILMN_1771411 1,3-glucosyltransferase) LAMP1 1.66 lysosomal-associated membrane protein 1 NM_005561.2 ILMN_1782292 ABLIM1 1.65 actin binding LIM protein 1 NM_001003407.1 ILMN_2396672 TMEM50B 1.64 transmembrane protein 50B NM_006134.5 ILMN_2047599 GPR110 1.64 G protein-coupled receptor 110 NM_153840.2 ILMN_2241124 CDKN1A 1.63 cyclin-dependent kinase inhibitor 1A (p21, Cip1) NM_000389.2 ILMN_1784602 MCCC1 1.63 methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) NM_020166.3 ILMN_1760174

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Appendix 5. 5 The top 50 genes with most differentially downregulated compared with controls in the GGH-overexpressed MDA-MB-435 breast cancer cells

Gene Fold Definition Accession Probe ID Symbol Change TYR -31.95 tyrosinase (oculocutaneous albinism IA) NM_000372.4 ILMN_1788774 TSPAN7 -21.45 tetraspanin 7 NM_004615.2 ILMN_2120695 SNCA -11.04 synuclein, alpha (non A4 component of amyloid precursor) NM_007308.1 ILMN_1701933 IGSF11 -9.74 immunoglobulin superfamily, member 11 NM_001015887.1 ILMN_1753502 ALDH1A1 -9.45 aldehyde dehydrogenase 1 family, member A1 NM_000689.3 ILMN_2096372 DCT -9.42 dopachrome tautomerase (dopachrome delta-isomerase, NM_001922.2 ILMN_1701783 tyrosine-related protein 2) TSPAN7 -9.29 tetraspanin 7 NM_004615.2 ILMN_1809291 CHCHD6 -8.00 coiled-coil-helix-coiled-coil-helix domain containing 6 NM_032343.1 ILMN_1785161 TRIM48 -7.89 tripartite motif-containing 48 NM_024114.2 ILMN_1762021 SNCA -5.90 synuclein, alpha (non A4 component of amyloid precursor) NM_000345.2 ILMN_1766165 DYNC1I1 -5.83 dynein, cytoplasmic 1, intermediate chain 1 NM_004411.3 ILMN_1690397 GPM6B -5.59 glycoprotein M6B NM_001001995.1 ILMN_1704665 APOD -5.41 apolipoprotein D NM_001647.2 ILMN_1780170 C4orf18 -5.16 open reading frame 18 NM_016613.5 ILMN_1761941 NOV -5.12 nephroblastoma overexpressed gene NM_002514.2 ILMN_1787186 CAPN3 -5.07 calpain 3, (p94) NM_173087.1 ILMN_2332691 TUBB4 -4.85 tubulin, beta 4 NM_006087.2 ILMN_1682459 BCHE -4.82 butyrylcholinesterase NM_000055.2 ILMN_1685641 GPM6B -4.75 glycoprotein M6B NM_001001995.1 ILMN_1735438 CAPN3 -4.74 calpain 3, (p94) NM_024344.1 ILMN_1687971 MYO10 -4.68 myosin X NM_012334.1 ILMN_2232712 ALDH1A1 -4.52 aldehyde dehydrogenase 1 family, member A1 NM_000689.3 ILMN_1709348 GHR -4.52 growth hormone receptor NM_000163.2 ILMN_1775814 SPP1 -4.51 secreted phosphoprotein 1 NM_001040058.1 ILMN_2374449 BCHE -4.50 butyrylcholinesterase NM_000055.1 ILMN_2176592 FAM65B -4.46 family with sequence similarity 65, member B NM_015864.2 ILMN_1726597 SPP1 -4.13 secreted phosphoprotein 1 NM_000582.2 ILMN_1651354 PIR -4.08 pirin (iron-binding nuclear protein) NM_001018109.1 ILMN_1761247 ST6GALNAC3 -4.04 ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1, 3)- NM_152996.1 ILMN_2127379 N-acetylgalactosaminide alpha-2,6-sialyltransferase 3 PIR -3.96 pirin (iron-binding nuclear protein) NM_001018109.1 ILMN_2383383 C18orf51 -3.95 chromosome 18 open reading frame 51 . NM_001044369.1 ILMN_2173500 C18orf51 -3.92 chromosome 18 open reading frame 51 NM_001044369.1 ILMN_1670718 CTSL2 -3.78 cathepsin L2 NM_001333.2 ILMN_1748352 RRAGD -3.69 Ras-related GTP binding D NM_021244.3 ILMN_1699772 PLSCR1 -3.63 phospholipid scramblase 1 NM_021105.1 ILMN_1745242 ARHGEF3 -3.59 Rho guanine nucleotide exchange factor (GEF) 3 NM_019555.1 ILMN_1781010 MCOLN2 -3.58 mucolipin 2 NM_153259.2 ILMN_1660462 MIR1978 -3.55 microRNA 1978 NR_031742.1 ILMN_3310491 STK32A -3.46 serine/threonine kinase 32A NM_145001.2 ILMN_1756612 GPR143 -3.44 G protein-coupled receptor 143 NM_000273.1 ILMN_1756261 TDRD7 -3.38 tudor domain containing 7 NM_014290.1 ILMN_1705241 SLC24A5 -3.30 solute carrier family 24, member 5 NM_205850.2 ILMN_1786045 PPARGC1A -3.30 peroxisome proliferator-activated receptor gamma, NM_013261.3 ILMN_1750062

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coactivator 1 alpha MITF -3.22 microphthalmia-associated transcription factor NM_198158.1 ILMN_2304186 MSRB2 -3.20 methionine sulfoxide reductase B2 NM_012228.2 ILMN_1657977 SDCBP -3.20 syndecan binding protein (syntenin) NM_001007067.1 ILMN_2363586 NBL1 -3.20 neuroblastoma, suppression of tumorigenicity 1 NM_005380.4 ILMN_2405009 QPCT -3.16 glutaminyl-peptide cyclotransferase NM_012413.3 ILMN_1741727 SIPA1L2 -3.16 signal-induced proliferation-associated 1 like 2 NM_020808.3 ILMN_1732923

Appendix 5. 6 The top 50 genes with most differentially upregulated compared with controls in the GGH-overexpressed MDA-MB-435 breast cancer cells

Gene Fold Definition Accession Probe ID Symbol Change S100A4 13.13 S100 calcium binding protein A4 NM_019554.2 ILMN_1684306 S100A4 10.51 S100 calcium binding protein A4 NM_019554.2 ILMN_1688780 FST 10.32 follistatin NM_013409.1 ILMN_1700081 FGFRL1 6.83 fibroblast growth factor receptor-like 1 NM_021923.3 ILMN_1795865 PLOD2 6.56 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 NM_000935.2 ILMN_1771599 HLA-DQA1 6.26 PREDICTED: major histocompatibility complex, class II, DQ XM_936128.2 ILMN_1808405 alpha 1, transcript variant 10 CNN3 5.93 calponin 3, acidic NM_001839.2 ILMN_1782439 COL13A1 5.76 collagen, type XIII, alpha 1 NM_080805.2 ILMN_2370624 SERPINA3 5.74 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, NM_001085.4 ILMN_1788874 antitrypsin), member 3 AKR1C3 5.49 aldo-keto reductase family 1, member C3 (3-alpha NM_003739.4 ILMN_1713124 hydroxysteroid dehydrogenase, type II) AHNAK 5.17 AHNAK nucleoprotein NM_024060.2 ILMN_1752159 PARVA 5.04 parvin, alpha NM_018222.3 ILMN_3307892 C20orf100 5.04 chromosome 20 open reading frame 100 NM_032883.1 ILMN_2082209 NNMT 4.80 nicotinamide N-methyltransferase NM_006169.2 ILMN_1715508 HTATIP2 4.75 HIV-1 Tat interactive protein 2, 30kDa NM_006410.3 ILMN_1664303 CRYAB 4.48 crystallin, alpha B NM_001885.1 ILMN_1729216 COL13A1 4.16 collagen, type XIII, alpha 1 NM_080815.2 ILMN_2311052 CPVL 4.11 carboxypeptidase, vitellogenic-like NM_031311.3 ILMN_2400759 KLRC2 4.09 killer cell lectin-like receptor subfamily C, member 2 NM_002260.3 ILMN_2059357 MT1X 4.09 metallothionein 1X NM_005952.2 ILMN_1775170 GNG11 4.08 guanine nucleotide binding protein (G protein), gamma 11 NM_004126.3 ILMN_1782419 APCDD1L 3.97 adenomatosis polyposis coli down-regulated 1-like NM_153360.1 ILMN_1689431 CADM1 3.96 cell adhesion molecule 1 NM_014333.3 ILMN_1680132 COL8A1 3.93 collagen, type VIII, alpha 1 NM_020351.2 ILMN_1685433 RAC2 3.93 ras-related C3 botulinum toxin substrate 2 (rho family, small NM_002872.3 ILMN_1709795 GTP binding protein Rac2) JAM3 3.90 junctional adhesion molecule 3 NM_032801.3 ILMN_1769575 M160 3.88 scavenger receptor cysteine-rich type 1 protein M160 NM_174941.3 ILMN_1802780 CD163L1 3.85 CD163 molecule-like 1 NM_174941.4 ILMN_3242540 PTPN22 3.79 protein tyrosine phosphatase, non-receptor type 22 (lymphoid) NM_015967.3 ILMN_2246328 CDC42EP5 3.76 CDC42 effector protein (Rho GTPase binding) 5 NM_145057.2 ILMN_1774982 CATSPER1 3.75 cation channel, sperm associated 1 NM_053054.2 ILMN_1789394 IGFBP7 3.66 insulin-like growth factor binding protein 7 NM_001553.1 ILMN_2062468

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MT1E 3.63 metallothionein 1E NM_175617.3 ILMN_2173611 FKBP11 3.63 FK506 binding protein 11, 19 kDa NM_016594.1 ILMN_1787345 A2M 3.57 alpha-2-macroglobulin NM_000014.4 ILMN_1745607 CHRDL1 3.55 chordin-like 1 NM_145234.2 ILMN_1678233 HAPLN1 3.53 hyaluronan and proteoglycan link protein 1 NM_001884.2 ILMN_2210519 FAM133A 3.50 family with sequence similarity 133, member A NM_173698.1 ILMN_1781742 CYR61 3.50 cysteine-rich, angiogenic inducer, 61 NM_001554.3 ILMN_2188264 CA5B 3.48 carbonic anhydrase VB, mitochondrial NM_007220.3 ILMN_1672807 CPVL 3.46 carboxypeptidase, vitellogenic-like NM_019029.2 ILMN_1682928 COL8A1 3.39 collagen, type VIII, alpha 1 NM_020351.2 ILMN_2402392 PCOLCE2 3.38 procollagen C-endopeptidase enhancer 2 NM_013363.2 ILMN_1746888 CDC42EP4 3.36 CDC42 effector protein (Rho GTPase binding) 4 NM_012121.4 ILMN_1745223 S100A3 3.35 S100 calcium binding protein A3 NM_002960.1 ILMN_1712545 CHRDL1 3.34 chordin-like 1 NM_145234.2 ILMN_2184231 FSCN1 3.31 fascin homolog 1, actin-bundling protein (Strongylocentrotus NM_003088.2 ILMN_1808707 purpuratus) FAM134B 3.25 family with sequence similarity 134, member B NM_001034850.1 ILMN_2387952 ITGA3 3.24 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 NM_002204.1 ILMN_1685397 receptor) NR4A2 3.22 nuclear receptor subfamily 4, group A, member 2 NM_006186.2 ILMN_1782305

Appendix 5. 7 The top 50 genes with most differentially downregulated compared with controls in the GGH-inhibited MDA-MB-435 breast cancer cells

Gene Fold Definition Accession Probe ID Symbol Change CTHRC1 -4.44 collagen triple helix repeat containing 1 NM_138455.2 ILMN_1725090 NNMT -3.24 nicotinamide N-methyltransferase NM_006169.2 ILMN_1715508 CTHRC1 -3.20 collagen triple helix repeat containing 1 NM_138455.2 ILMN_2117508 HLA-DOA -3.09 major histocompatibility complex, class II, DO alpha NM_002119.3 ILMN_1659075 FSCN1 -3.03 fascin homolog 1, actin-bundling protein (Strongylocentrotus NM_003088.2 ILMN_1808707 purpuratus) CAP2 -2.75 CAP, adenylate cyclase-associated protein, 2 (yeast) NM_006366.2 ILMN_1691237 ARHGEF3 -2.68 Rho guanine nucleotide exchange factor (GEF) 3 NM_019555.1 ILMN_1781010 CDC42EP5 -2.53 CDC42 effector protein (Rho GTPase binding) 5 NM_145057.2 ILMN_1774982 GGH -2.49 gamma-glutamyl hydrolase (conjugase, NM_003878.1 ILMN_1681754 folylpolygammaglutamyl hydrolase) GGH -2.48 gamma-glutamyl hydrolase (conjugase, NM_003878.1 ILMN_2195914 folylpolygammaglutamyl hydrolase). LMCD1 -2.40 LIM and cysteine-rich domains 1 NM_014583.2 ILMN_1754969 CHN1 -2.30 chimerin (chimaerin) 1 NM_001025201.1 ILMN_1678493 SLC2A3 -2.24 solute carrier family 2 (facilitated glucose transporter), NM_006931.1 ILMN_1775708 member 3 HLA-DRB6 -2.20 major histocompatibility complex, class II, DR beta 6 NR_001298.1 ILMN_2066066 (pseudogene) HLA-DQA1 -2.19 PREDICTED: major histocompatibility complex, class II, DQ XM_936128.2 ILMN_1808405 alpha 1, transcript variant 10 AMPH -2.17 amphiphysin NM_001635.2 ILMN_1685834 C1orf24 -2.12 chromosome 1 open reading frame 24 NM_052966.1 ILMN_1667966 FAM129A -2.12 family with sequence similarity 129, member A NM_052966.2 ILMN_1810725

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PHF21A -2.11 PHD finger protein 21A NM_016621.2 ILMN_1699496 C21orf34 -2.11 chromosome 21 open reading frame 34 NM_001005734.1 ILMN_1690703 CYTSB -2.10 cytospin B NM_001033555.1 ILMN_3255061 AIF1L -2.10 allograft inflammatory factor 1-like NM_031426.2 ILMN_3246401 S100A4 -2.10 S100 calcium binding protein A4 NM_019554.2 ILMN_1684306 RIPK5 -2.10 receptor interacting protein kinase 5 NM_199462.1 ILMN_2352023 AHNAK -2.09 AHNAK nucleoprotein NM_024060.2 ILMN_1752159 HLA-DPB1 -2.05 major histocompatibility complex, class II, DP beta 1 NM_002121.4 ILMN_1749070 COL8A1 -2.04 collagen, type VIII, alpha 1 NM_020351.2 ILMN_1685433 CTSL2 -2.04 cathepsin L2 NM_001333.2 ILMN_1748352 NR0B1 -2.02 nuclear receptor subfamily 0, group B, member 1 NM_000475.3 ILMN_1800160 HLA-DMB -2.01 major histocompatibility complex, class II, DM beta NM_002118.3 ILMN_1761733 RILPL1 -2.01 Rab interacting lysosomal protein-like 1 NM_178314.2 ILMN_1805643 HLA-DPA1 -1.99 major histocompatibility complex, class II, DP alpha 1 NM_033554.2 ILMN_1772218 HLA-DMA -1.98 major histocompatibility complex, class II, DM alpha NM_006120.2 ILMN_1695311 CYP27A1 -1.96 cytochrome P450, family 27, subfamily A, polypeptide 1 NM_000784.2 ILMN_1704985 IFITM1 -1.96 interferon induced transmembrane protein 1 (9-27) NM_003641.3 ILMN_1801246 PHLDA1 -1.95 pleckstrin homology-like domain, family A, member 1 NM_007350.3 ILMN_3251550 CROP -1.94 cisplatin resistance-associated overexpressed protein NM_016424.3 ILMN_1728180 TYSND1 -1.93 trypsin domain containing 1 NM_001040273.1 ILMN_1775677 KCNG1 -1.93 potassium voltage-gated channel, subfamily G, member 1 NM_002237.3 ILMN_1673769 HLTF -1.91 helicase-like transcription factor NM_139048.2 ILMN_1673820 MARCKS -1.87 myristoylated alanine-rich protein kinase C substrate NM_002356.5 ILMN_1807042 P2RX7 -1.87 purinergic receptor P2X, ligand-gated ion channel, 7 NM_002562.4 ILMN_1759326 PRR4 -1.86 proline rich 4 (lacrimal) NM_001098538.1 ILMN_1753665 TMEM173 -1.85 transmembrane protein 173 NM_198282.1 ILMN_2145116 HEY2 -1.85 hairy/enhancer-of-split related with YRPW motif 2 NM_012259.1 ILMN_1682034 OGT -1.84 O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N- NM_181673.1 ILMN_1697639 acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) HLA-DRA -1.84 major histocompatibility complex, class II, DR alpha NM_019111.3 ILMN_1689655 AHNAK2 -1.84 AHNAK nucleoprotein 2 NM_138420.2 ILMN_3243156 HLA-DRB3 -1.83 major histocompatibility complex, class II, DR beta 3 NM_022555.3 ILMN_1717261 ZNF627 -1.82 zinc finger protein 627 NM_145295.2 ILMN_2197519

Appendix 5. 8 The top 50 genes with most differentially upregulated compared with controls in the GGH-inhibited MDA-MB-435 breast cancer cells

Gene Fold Definition Accession Probe ID Symbol Change CXorf26 10.42 chromosome X open reading frame 26 NM_016500.3 ILMN_1768176 SPP1 5.88 secreted phosphoprotein 1 NM_001040058.1 ILMN_2374449 SLC30A1 5.55 solute carrier family 30 (zinc transporter), member 1 NM_021194.2 ILMN_1745021 SLC30A1 5.45 solute carrier family 30 (zinc transporter), member 1 NM_021194.2 ILMN_2067852 SPP1 4.90 secreted phosphoprotein 1 NM_000582.2 ILMN_1651354 CCL20 4.88 chemokine (C-C motif) ligand 20 NM_004591.1 ILMN_1657234 PRSS7 4.66 protease, serine, 7 (enterokinase) NM_002772.1 ILMN_1695969 PRSS7 4.41 protease, serine, 7 (enterokinase) NM_002772.1 ILMN_2220845

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IL8 4.15 interleukin 8 NM_000584.2 ILMN_2184373 TYR 4.07 tyrosinase (oculocutaneous albinism IA) NM_000372.4 ILMN_1788774 SLITRK4 3.74 SLIT and NTRK-like family, member 4 NM_173078.2 ILMN_2199768 LAIR2 3.57 leukocyte-associated immunoglobulin-like receptor 2 NM_021270.2 ILMN_2323933 FAM133A 3.45 family with sequence similarity 133, member A NM_173698.1 ILMN_1781742 LAIR2 3.43 leukocyte-associated immunoglobulin-like receptor 2 NM_002288.3 ILMN_1807491 IL8 3.35 interleukin 8 NM_000584.2 ILMN_1666733 A2M 3.19 alpha-2-macroglobulin NM_000014.4 ILMN_1745607 RFTN1 3.11 raftlin, lipid raft linker 1 NM_015150.1 ILMN_1800787 CYB5R2 3.05 cytochrome b5 reductase 2 NM_016229.3 ILMN_1739576 SPRR2E 3.00 small proline-rich protein 2E NM_001024209.2 ILMN_2211018 BCHE 2.77 butyrylcholinesterase NM_000055.1 ILMN_2176592 KIAA1199 2.70 KIAA1199 NM_018689.1 ILMN_1813704 MMP1 2.69 matrix metallopeptidase 1 (interstitial collagenase) NM_002421.2 ILMN_1726448 SPRR2D 2.66 small proline-rich protein 2D NM_006945.3 ILMN_2191967 DCBLD2 2.65 discoidin, CUB and LCCL domain containing 2 NM_080927.3 ILMN_1735499 MYLK 2.60 myosin light chain kinase NM_053032.2 ILMN_1691476 CDKN1A 2.57 cyclin-dependent kinase inhibitor 1A (p21, Cip1) NM_000389.2 ILMN_1784602 UCN2 2.54 urocortin 2 NM_033199.3 ILMN_1652413 ID3 2.52 inhibitor of DNA binding 3, dominant negative helix-loop- NM_002167.2 ILMN_1732296 helix protein FGF13 2.51 fibroblast growth factor 13 NM_004114.2 ILMN_1671777 ID1 2.51 inhibitor of DNA binding 1, dominant negative helix-loop- NM_181353.1 ILMN_1664861 helix protein SLC20A1 2.48 solute carrier family 20 (phosphate transporter), member 1 NM_005415.3 ILMN_1672662 TFF2 2.42 trefoil factor 2 (spasmolytic protein 1) NM_005423.3 ILMN_1663919 CASP1 2.38 caspase 1, apoptosis-related cysteine peptidase (interleukin NM_033294.2 ILMN_2326509 1, beta, convertase) DYNLT3 2.34 dynein, light chain, Tctex-type 3 NM_006520.1 ILMN_1681890 BCHE 2.31 butyrylcholinesterase NM_000055.2 ILMN_1685641 ADM 2.31 adrenomedullin NM_001124.1 ILMN_1708934 GBP1 2.30 guanylate binding protein 1, interferon-inducible, 67kDa NM_002053.1 ILMN_2148785 SLFN11 2.26 schlafen family member 11 NM_152270.2 ILMN_1752520 ID2 2.24 inhibitor of DNA binding 2, dominant negative helix-loop- NM_002166.4 ILMN_2086095 helix protein ENC1 2.22 ectodermal-neural cortex (with BTB-like domain) NM_003633.1 ILMN_1779147 NCRNA00161 2.21 non-protein coding RNA 161 NR_026553.1 ILMN_3297789 HBEGF 2.21 heparin-binding EGF-like growth factor NM_001945.1 ILMN_2121408 MYADM 2.20 myeloid-associated differentiation marker NM_001020820.1 ILMN_2308849 CTGF 2.20 connective tissue growth factor NM_001901.2 ILMN_2115125 MMP3 2.12 matrix metallopeptidase 3 (stromelysin 1, progelatinase) NM_002422.3 ILMN_1784459 PLCXD3 2.12 phosphatidylinositol-specific phospholipase C, X domain NM_001005473.1 ILMN_1798841 containing 3 EMP1 2.09 epithelial membrane protein 1 NM_001423.1 ILMN_1801616 SERPINB8 2.08 serpin peptidase inhibitor, clade B (ovalbumin), member 8 NM_002640.3 ILMN_2397028 TMEM166 2.07 transmembrane protein 166 NM_032181.1 ILMN_1702973 ORC5L 2.07 origin recognition complex, subunit 5-like (yeast) NM_002553.2 ILMN_1688094

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Appendix 6 The list of differentially expressed genes associated with the top functions in response to GGH modulation

Appendix 6. 1 The top molecular and cellular functions associated with differentially expressed genes in the GGH- overexpressed HCT116 colon cancer cells

No. of Category Function Function Annotation P-value Genes Genes Cellular Movement migration migration of brain cells 2.74E-04 BDNF, BMP4 2 Cellular Movement migration migration of mononuclear 7.16E-04 ADAM10, JAK1 (includes EG:16451), PLAU, PPARG, SEMA3A, 7 leukocytes TIMP2 (includes EG:21858), TNFRSF6B Cellular Movement migration migration of monocytes 1.34E-03 JAK1 (includes EG:16451), PPARG, SEMA3A, TIMP2 (includes 4 EG:21858) Cellular Movement migration migration of phagocytes 3.29E-03 JAK1 (includes EG:16451), PLAU, PPARG, SEMA3A, TIMP2 5 (includes EG:21858) Cellular Movement migration migration of endothelial cells 6.19E-03 BMP4, MAPKAPK5, PRKCA, SCG2, TIMP2 (includes EG:21858), 6 TNFRSF6B Cellular Movement migration migration of neurons 7.76E-03 BDNF, BMP4 2 Cellular Movement migration T cell migration 8.75E-03 ADAM10, PLAU, SEMA3A, TNFRSF6B 4 Cellular Movement migration migration of granule cells 9.63E-03 BDNF 1 Cellular Movement migration migration of neocortical 9.63E-03 BMP4 1 neurons Cellular Movement migration migration of cells 1.10E-02 ADAM10, ANKS1A, BDNF, BMP4, IRS1, JAK1 (includes 17 EG:16451), MAPKAPK5, PLAU, PPARG, PPIA, PRKCA, S100A10, SCG2, SEMA3A, SPAG9, TIMP2 (includes EG:21858), TNFRSF6B

Cellular Movement migration migration of melanoma cell 2.76E-02 PRKCA, TIMP2 (includes EG:21858) 2 lines Cellular Movement migration migration of fibrosarcoma cell 3.36E-02 PLAU, TIMP2 (includes EG:21858) 2 lines Cellular Movement migration migration of vascular 4.46E-02 MAPKAPK5, TIMP2 (includes EG:21858), TNFRSF6B 3 endothelial cells Cellular Movement cell movement cell movement of myeloid cells 8.74E-04 JAK1 (includes EG:16451), PLAU, PPARG, PPIA, SCG2, SEMA3A, 8 TIMP2 (includes EG:21858), TNFRSF6B

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Cellular Movement cell movement cell movement of mononuclear 1.02E-03 ADAM10, JAK1 (includes EG:16451), PLAU, PPARG, PPIA, 8 leukocytes SEMA3A, TIMP2 (includes EG:21858), TNFRSF6B Cellular Movement cell movement cell movement of leukocytes 2.81E-03 ADAM10, JAK1 (includes EG:16451), PLAU, PPARG, PPIA, 9 SCG2, SEMA3A, TIMP2 (includes EG:21858), TNFRSF6B Cellular Movement cell movement cell movement of monocytes 3.16E-03 JAK1 (includes EG:16451), PPARG, PPIA, SEMA3A, TIMP2 5 (includes EG:21858) Cellular Movement cell movement cell movement of phagocytes 4.53E-03 JAK1 (includes EG:16451), PLAU, PPARG, PPIA, SEMA3A, 7 TIMP2 (includes EG:21858), TNFRSF6B Cellular Movement cell movement cell movement 9.34E-03 ADAM10, ANKS1A, BDNF, BMP4, EPS8, GDF15, IRS1, JAK1 19 (includes EG:16451), MAPKAPK5, PLAU, PPARG, PPIA, PRKCA, S100A10, SCG2, SEMA3A, SPAG9, TIMP2 (includes EG:21858), TNFRSF6B

Cellular Movement cell movement cell movement of lymphocytes 1.14E-02 ADAM10, PLAU, PPIA, SEMA3A, TNFRSF6B 5 Cellular Movement cell movement cell movement of peripheral 1.27E-02 PPARG, PPIA 2 blood monocytes Cellular Movement cell movement cell movement of brain cancer 2.67E-02 ADAM10, PLAU, SEMA3A 3 cell lines Cellular Movement cell movement cell movement of epithelial cell 2.88E-02 ANKS1A, PLAU, PPIA 3 lines Cellular Movement cell movement cell movement of tumor cell 3.40E-02 ADAM10, ANKS1A, EPS8, GDF15, IRS1, PLAU, PPIA, PRKCA, 11 lines S100A10, SEMA3A, TIMP2 (includes EG:21858) Cellular Movement cell movement cell movement of T 3.45E-02 ADAM10, SEMA3A, TNFRSF6B 3 lymphocytes Cellular Movement cell movement cell movement of embryonic 3.69E-02 ANKS1A, PLAU, PPIA 3 cell lines Cellular Movement chemotaxis chemotaxis of cells 2.81E-03 ADAM10, BDNF, BMP4, PLAU, PPARG, PPIA, SCG2, SEMA3A, 9 TNFRSF6B Cellular Movement chemotaxis chemotaxis of leukocytes 1.47E-02 ADAM10, PLAU, PPARG, PPIA, SCG2, TNFRSF6B 6 Cellular Movement chemotaxis chemotaxis of embryonic cell 1.56E-02 PLAU, PPIA 2 lines Cellular Movement chemotaxis chemotaxis of epithelial cell 1.56E-02 PLAU, PPIA 2 lines Cellular Movement chemotaxis chemotaxis of granule cells 1.92E-02 BDNF 1 Cellular Movement chemotaxis chemotaxis of kidney cell lines 2.04E-02 PLAU, PPIA 2

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Cellular Movement chemotaxis chemotaxis of eosinophils 2.21E-02 PPARG, SCG2 2 Cellular Movement chemotaxis chemotaxis of microglia 4.72E-02 TNFRSF6B 1 Cellular Movement invasion invasion of tumor cell lines 3.02E-03 BDNF, BMP4, GDF15, IRS2, LCN2, PLAU, PPARG, PRKCA, 11 RND3, S100A10, TIMP2 (includes EG:21858) Cellular Movement invasion invasion of gastric cancer cells 9.63E-03 GDF15 1 Cellular Movement invasion invasion of lung cancer cell 1.37E-02 BDNF, PPARG, S100A10 3 lines Cellular Movement invasion invasion of carcinoma cell lines 2.47E-02 BDNF, PPARG, S100A10 3 Cellular Movement invasion invasion of pancreatic cancer 2.95E-02 PLAU, TIMP2 (includes EG:21858) 2 cell lines Cellular Movement invasion invasion of melanoma cell lines 3.78E-02 BMP4, RND3 2 Cellular Movement invasion invasion of blood-derived mast 3.80E-02 TIMP2 (includes EG:21858) 1 cells Cellular Movement invasion invasion of endocrine cell lines 3.80E-02 PRKCA 1 Cellular Movement transmigration transmigration of mononuclear 4.56E-03 ADAM10, PPARG, TIMP2 (includes EG:21858) 3 leukocytes Cellular Movement transmigration transmigration of monocytes 1.71E-02 PPARG, TIMP2 (includes EG:21858) 2 Cellular Movement transmigration transmigration of peripheral 2.86E-02 PPARG 1 blood monocytes Cellular Movement chemokinesis chemokinesis of granule cells 9.63E-03 BDNF 1 Cellular Movement infiltration infiltration by microglia 9.63E-03 TNFRSF6B 1 Cellular Movement infiltration infiltration by T lymphocytes 3.80E-02 TNFRSF6B 1 Cellular Movement chemorepulsion chemorepulsion of brain cancer 2.86E-02 SEMA3A 1 cell lines Cellular Movement intravasation intravasation of cells 2.86E-02 PLAU 1 Cell Death necrosis necrosis 9.04E-04 AKAP12, ALDH2, BDNF, BMP4, CDK6, COMT, CRABP2, 29 DHCR24, FASN, GAS6, GDF15, IRS1, JAK1 (includes EG:16451), LCN2, LINGO1, PDCD6IP, PHLDA1, PLA2G16, PLAU, PLK2, PPARG, PPIA, PRKCA, RND3, SAT1, SEMA3A, TNFRSF6B, TOPBP1, UBQLN1

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Cell Death apoptosis apoptosis 1.31E-03 AKAP12, ALDH2, BDNF, BMP4, CDK6, CRABP2, FASN, GAS6, 29 GDF15, HAUS1, HIST1H1C, IRS1, JAK1 (includes EG:16451), LCN2, PDCD6IP, PHLDA1, PLA2G16, PLAU, PLK2, PPARG, PPIA, PRKCA, RND3, SAT1, SCG2, SEMA3A, TNFRSF6B, TOPBP1, UBQLN1

Cell Death apoptosis apoptosis of embryonic cells 1.87E-03 BMP4, TNFRSF6B 2 Cell Death apoptosis apoptosis of tumor cell lines 3.24E-03 AKAP12, BDNF, BMP4, CDK6, CRABP2, FASN, GDF15, JAK1 20 (includes EG:16451), LCN2, PDCD6IP, PLA2G16, PLAU, PLK2, PPARG, PPIA, PRKCA, SAT1, SEMA3A, TNFRSF6B, TOPBP1

Cell Death apoptosis apoptosis of prostate cancer cell 3.28E-03 AKAP12, FASN, GDF15, PLAU, PPIA, PRKCA 6 lines Cell Death apoptosis apoptosis of limb bud cells 9.63E-03 BMP4 1 Cell Death apoptosis apoptosis of neuroglia 1.14E-02 GAS6, PRKCA 2 Cell Death apoptosis apoptosis of stromal fibroblast 1.92E-02 BMP4 1 cells Cell Death apoptosis apoptosis of cytotrophoblastic 3.80E-02 TNFRSF6B 1 cells Cell Death apoptosis apoptosis of endocrine cell 3.80E-02 LCN2 1 lines Cell Death apoptosis apoptosis of brain cancer cell 4.46E-02 BMP4, SEMA3A, TNFRSF6B 3 lines Cell Death apoptosis apoptosis of oligodendrocytes 4.72E-02 GAS6 1 Cell Death cell death cell death 1.44E-03 AKAP12, ALDH2, BDNF, BMP4, CA2, CDK6, COMT, CRABP2, 37 DHCR24, FASN, GAS6, GDF15, HAUS1, HIST1H1C, IRS1, IRS2, JAK1 (includes EG:16451), LCN2, LINGO1, PBX1, PDCD6IP, PHLDA1, PLA2G16, PLAU, PLK2, PPARG, PPIA, PRKACB, PRKCA, RND3, SAT1, SCG2, SEMA3A, TIMP2 (includes EG:21858), TNFRSF6B, TOPBP1, UBQLN1

Cell Death cell death cell death of tumor cell lines 4.70E-03 AKAP12, BDNF, BMP4, CDK6, CRABP2, DHCR24, FASN, 22 GDF15, JAK1 (includes EG:16451), LCN2, PDCD6IP, PLA2G16, PLAU, PLK2, PPARG, PPIA, PRKCA, SAT1, SEMA3A, TNFRSF6B, TOPBP1, UBQLN1

351

Cell Death cell death cell death of lymphoblastoid 9.63E-03 COMT 1 cells Cell Death cell death cell death of brain cancer cell 1.98E-02 BMP4, DHCR24, SEMA3A, TNFRSF6B 4 lines Cell Death cell death cell death of colon cancer cell 3.11E-02 FASN, GDF15, PPARG, PPIA, TNFRSF6B, TOPBP1 6 lines Cell Death survival cell survival 7.21E-03 BDNF, BMP4, CA2, CDK6, GAS6, GDF15, IRS2, JAK1 (includes 16 EG:16451), LINGO1, PBX1, PLAU, PLK2, PRKACB, PRKCA, TIMP2 (includes EG:21858), TOPBP1 Cell Death survival survival of dopaminergic 1.92E-02 LINGO1 1 neurons Cell Death survival survival of oligodendrocytes 2.86E-02 GAS6 1 Cell Death survival survival of retinal ganglion 2.86E-02 BDNF 1 cells Cell Death killing killing of lung cancer cell lines 9.63E-03 PRKCA 1 Cell Death killing killing of brain cancer cell lines 2.86E-02 PLAU 1 Cell Death killing killing of tumor cell lines 4.45E-02 PLAU, PRKCA 2 Cell Death killing killing of endothelial cells 4.72E-02 PLAU 1 Cell Death cell viability cell viability 1.09E-02 BDNF, BMP4, CA2, CDK6, GAS6, GDF15, IRS2, JAK1 (includes 15 EG:16451), LINGO1, PBX1, PLAU, PLK2, PRKACB, PRKCA, TIMP2 (includes EG:21858) Cell Death cell viability cell viability of breast cancer 1.48E-02 CA2, GDF15, IRS2, PBX1 4 cell lines Cell Death cell viability cell viability of tumor cell lines 1.77E-02 BDNF, CA2, CDK6, GDF15, IRS2, JAK1 (includes EG:16451), 12 PBX1, PLAU, PLK2, PRKACB, PRKCA, TIMP2 (includes EG:21858) Cell Death cell viability cell viability of keratinocytes 3.80E-02 PRKCA 1 Cell Death cell viability cell viability of thymocytes 3.80E-02 BMP4 1 Cell Death cell viability cell viability of neurons 4.00E-02 BDNF, LINGO1 2 Cell Death anoikis anoikis of stomach cancer cell 1.92E-02 PRKCA 1 lines Cell Death anoikis anoikis of RPE cells 3.80E-02 PLAU 1 Cell Death cytotoxicity cytotoxicity of ovarian cancer 1.92E-02 PRKCA 1

352

cell lines Cell Death neuroprotection neuroprotection of 4.72E-02 BDNF 1 neuroblastoma cell lines Carbohydrate homeostasis homeostasis of D-glucose 1.02E-03 IRS1, PPARG, PYGL 3 Metabolism Carbohydrate detachment detachment of hyaluronic acid 9.63E-03 ADAM10 1 Metabolism Carbohydrate import import of D-glucose 2.21E-02 IRS1, IRS2 2 Metabolism Carbohydrate synthesis synthesis of glycogen 2.57E-02 IRS1, IRS2 2 Metabolism Carbohydrate synthesis synthesis of D-glucose 2.86E-02 PLAU 1 Metabolism Cellular Function and homeostasis homeostasis of D-glucose 1.02E-03 IRS1, PPARG, PYGL 3 Maintenance Cellular Function and permeability permeability of vascular 4.78E-03 ADAM10, PLAU 2 Maintenance endothelial cells Cellular Function and permeability permeability of microvascular 1.92E-02 PLAU 1 Maintenance endothelial cells Cellular Function and differentiation arrest in differentiation of 9.63E-03 BMP4 1 Maintenance thymocytes Cellular Function and production production of olfactory receptor 9.63E-03 BMP4 1 Maintenance neurons Cellular Function and depolarization depolarization of embryonic 1.92E-02 BDNF 1 Maintenance cell lines Cellular Function and depolarization depolarization of epithelial cell 1.92E-02 BDNF 1 Maintenance lines Cellular Function and depolarization depolarization of kidney cell 1.92E-02 BDNF 1 Maintenance lines Cellular Function and depolarization depolarization of 1.92E-02 BDNF 1 Maintenance neuroblastoma cell lines Cellular Function and import import of D-glucose 2.21E-02 IRS1, IRS2 2 Maintenance Cellular Function and branching branching of axons 2.86E-02 BDNF 1 Maintenance

353

Cellular Function and endocytosis endocytosis by embryonic cell 3.80E-02 PRKCA 1 Maintenance lines Cellular Function and endocytosis endocytosis by epithelial cell 3.80E-02 PRKCA 1 Maintenance lines Cellular Function and formation formation of fibronectin matrix 3.80E-02 PDCD6IP 1 Maintenance Cellular Function and function function of mitochondria 4.72E-02 SACS 1 Maintenance Cellular Function and growth growth of dendrites 4.72E-02 BDNF 1 Maintenance Small Molecule homeostasis homeostasis of D-glucose 1.02E-03 IRS1, PPARG, PYGL 3 Biochemistry Small Molecule accumulation accumulation of methotrexate 9.63E-03 GGH 1 Biochemistry Small Molecule accumulation accumulation of folic acid 1.92E-02 GGH 1 Biochemistry Small Molecule depletion depletion of spermine 9.63E-03 SAT1 1 Biochemistry Small Molecule detachment detachment of hyaluronic acid 9.63E-03 ADAM10 1 Biochemistry Small Molecule flux flux of spermidine 9.63E-03 SAT1 1 Biochemistry Small Molecule flux flux of spermine 9.63E-03 SAT1 1 Biochemistry Small Molecule formation formation of spermidine 9.63E-03 SAT1 1 Biochemistry Small Molecule generation generation of hydrogen sulfide 9.63E-03 CTH 1 Biochemistry Small Molecule metabolism metabolism of methotrexate 9.63E-03 GGH 1 Biochemistry Small Molecule metabolism metabolism of L-cysteine 2.86E-02 CTH 1 Biochemistry Small Molecule metabolism metabolism of folic acid 2.86E-02 GGH 1 Biochemistry

354

Small Molecule methylation methylation of 2- 9.63E-03 COMT 1 Biochemistry hydroxyestradiol Small Molecule quantity quantity of spermidine 9.63E-03 SAT1 1 Biochemistry Small Molecule quantity quantity of spermine 9.63E-03 SAT1 1 Biochemistry Small Molecule quantity quantity of lysobisphosphatidic 1.92E-02 PDCD6IP 1 Biochemistry acid Small Molecule release release of GABA 9.63E-03 BDNF 1 Biochemistry Small Molecule release release of norepinephrine 4.72E-02 PRKCA 1 Biochemistry Small Molecule storage storage of fat 9.63E-03 PPARG 1 Biochemistry Small Molecule synthesis synthesis of L-cysteine 9.63E-03 CTH 1 Biochemistry Small Molecule synthesis synthesis of malonyl-coenzyme 9.63E-03 FASN 1 Biochemistry A Small Molecule synthesis synthesis of myristic acid 9.63E-03 FASN 1 Biochemistry Small Molecule synthesis synthesis of stearic acid 9.63E-03 FASN 1 Biochemistry Small Molecule synthesis synthesis of palmitic acid 1.92E-02 FASN 1 Biochemistry Small Molecule synthesis synthesis of D-glucose 2.86E-02 PLAU 1 Biochemistry Small Molecule synthesis synthesis of NADPH 2.86E-02 FASN 1 Biochemistry Small Molecule synthesis synthesis of acetyl-coenzyme A 2.86E-02 FASN 1 Biochemistry Small Molecule synthesis synthesis of acylglycerol 2.95E-02 FASN, PLAU 2 Biochemistry Small Molecule synthesis synthesis of nitric oxide 3.69E-02 FASN, IRS1, PLAU 3 Biochemistry

355

Small Molecule synthesis synthesis of melanin 3.80E-02 BMP4 1 Biochemistry Small Molecule oxidation oxidation of fatty acid 1.23E-02 IRS1, IRS2, PPARG 3 Biochemistry Small Molecule oxidation oxidation of NADPH 4.72E-02 FASN 1 Biochemistry Small Molecule beta-oxidation beta-oxidation of fatty acid 2.04E-02 IRS1, IRS2 2 Biochemistry Small Molecule import import of D-glucose 2.21E-02 IRS1, IRS2 2 Biochemistry Small Molecule secretion secretion of progesterone 4.72E-02 BMP4 1 Biochemistry

Appendix 6. 2 The top molecular and cellular functions associated with differentially expressed genes in the GGH-inhibited HCT116 colon cancer cells

No. of Category Function Function Annotation P-value Genes Genes Cell Death apoptosis apoptosis 2.11E-06 AHR, AKAP12, ANKRD1, APP, APPL1, ATAD2, BMP4, CDC42, 61 CDK6, CDKN1A, CRABP2, CYB5A (includes EG:109672), DDIT3, DHRS2, FASN, FGFR3, FHIT, GABPB1, HAUS1, HERPUD1, HIST1H1C, HYOU1, IFNAR2, IGFBP3, IGFBP6, IRS1, MAP4K1, MCM2, MMP7, MST4, NDRG1, NR4A2, PAWR, PDPK1, PHLDA1, PLK2, PPARG, PPIA, PRKCD, PRKRIR, PRMT2, SAT1, SEMA3A, SERPINB5, SOD1, SOX9, SPRY2, SRPX, ST6GAL1, SYK, TCF12, TERF2, TFAP2A, TIA1, TNFRSF6B, TNFSF18, TOPBP1, TPD52, TPD52L1, XPR1, ZNF443 Cell Death apoptosis apoptosis of tumor cell lines 8.21E-05 AKAP12, ANKRD1, APP, ATAD2, BMP4, CDC42, CDK6, 40 CDKN1A, CRABP2, CYB5A (includes EG:109672), DDIT3, FASN, FHIT, IFNAR2, IGFBP3, IGFBP6, MMP7, MST4, NDRG1, NR4A2, PAWR, PDPK1, PLK2, PPARG, PPIA, PRKCD, SAT1, SEMA3A, SERPINB5, SOD1, SOX9, SPRY2, SRPX, ST6GAL1, SYK, TNFRSF6B, TNFSF18, TOPBP1, TPD52, XPR1 Cell Death apoptosis apoptosis of colon cancer cell 2.67E-04 CDC42, CDKN1A, DDIT3, FASN, IGFBP3, MMP7, NDRG1, 13 lines PPARG, PPIA, PRKCD, SOX9, ST6GAL1, TOPBP1

356

Cell Death apoptosis apoptosis of neuroblastoma 6.01E-04 APP, CDC42, CDKN1A, DDIT3, IGFBP3, SOD1, XPR1 7 cell lines Cell Death apoptosis apoptosis of brain cancer cell 1.21E-03 APP, BMP4, IGFBP3, PDPK1, PRKCD, SEMA3A, TNFRSF6B 7 lines Cell Death apoptosis apoptosis of cancer cells 2.07E-03 CDKN1A, DDIT3, FHIT, MCM2, NR4A2, PHLDA1, PRKCD, 8 TFAP2A Cell Death apoptosis apoptosis of embryonic cells 7.73E-03 BMP4, TNFRSF6B 2 Cell Death apoptosis apoptosis of prostate cancer 1.02E-02 AKAP12, FASN, IGFBP3, PAWR, PPIA, PRKCD, SERPINB5, 8 cell lines TPD52 Cell Death apoptosis apoptosis of limb bud cells 1.99E-02 BMP4 1 Cell Death apoptosis apoptosis of hepatoma cell 3.34E-02 ANKRD1, CDKN1A, DDIT3, MMP7, PRKCD 5 lines Cell Death apoptosis apoptosis of carcinoma cell 3.85E-02 DDIT3, FHIT, IGFBP3, IGFBP6, PRKCD, SPRY2 6 lines Cell Death apoptosis apoptosis of melanocytes 3.93E-02 APP 1 Cell Death apoptosis apoptosis of splenocytes 3.93E-02 CDKN1A 1 Cell Death apoptosis apoptosis of stromal fibroblast 3.93E-02 BMP4 1 cells Cell Death cell death cell death 4.03E-06 AHR, AKAP12, ANKRD1, ANXA2, APP, APPL1, ATAD2, ATG12 77 (includes EG:361321), BMP4, CA2, CABLES1, CALB2, CDC42, CDK6, CDKN1A, CRABP2, CYB5A (includes EG:109672), DDIT3, DHRS2, FASN, FGFR3, FHIT, GABPB1, HAUS1, HERPUD1, HIST1H1C, HYOU1, ID2, IFNAR2, IGFBP3, IGFBP6, IRF9, IRS1, KRT19 (human), LAMP1, MAP4K1, MCM2, MGST1, MMP7, MST4, NDRG1, NR4A2, PAWR, PBX1, PDGFA, PDPK1, PHLDA1, PLK2, PPARG, PPIA, PRKACB, PRKCD, PRKRIR, PRMT2, RDX, RRM2B, SAT1, SEMA3A, SERPINB5, SOD1, SOX9, SPRY2, SRPX, ST6GAL1, STC1, SYK, TCF12, TERF2, TFAP2A, TIA1, TNFRSF6B, TNFSF18, TOPBP1, TPD52, TPD52L1, XPR1, ZNF443 Cell Death cell death cell death of tumor cell lines 1.98E-05 AKAP12, ANKRD1, ANXA2, APP, ATAD2, ATG12 (includes 47 EG:361321), BMP4, CABLES1, CDC42, CDK6, CDKN1A, CRABP2, CYB5A (includes EG:109672), DDIT3, FASN, FHIT, IFNAR2, IGFBP3, IGFBP6, LAMP1, MMP7, MST4, NDRG1, NR4A2, PAWR, PDPK1, PLK2, PPARG, PPIA, PRKCD, RDX, RRM2B, SAT1, SEMA3A, SERPINB5, SOD1, SOX9, SPRY2, SRPX, ST6GAL1, SYK, TFAP2A, TNFRSF6B, TNFSF18, TOPBP1, TPD52, XPR1

357

Cell Death cell death cell death of colon cancer cell 3.92E-04 CDC42, CDKN1A, DDIT3, FASN, IGFBP3, MMP7, NDRG1, 14 lines PPARG, PPIA, PRKCD, SOX9, ST6GAL1, TNFRSF6B, TOPBP1 Cell Death cell death cell death of cancer cells 8.68E-04 APP, BMP4, CDKN1A, DDIT3, FHIT, MCM2, NR4A2, PHLDA1, 10 PRKCD, TFAP2A Cell Death cell death cell death of brain cancer cell 1.31E-03 APP, BMP4, IGFBP3, PDPK1, PRKCD, SEMA3A, SOD1, 8 lines TNFRSF6B Cell Death cell death cell death of hippocampal 5.59E-03 APP, HYOU1 2 neurons Cell Death cell death cell death of prostate cancer 6.92E-03 AKAP12, DDIT3, FASN, IGFBP3, PAWR, PPIA, PRKCD, 9 cell lines SERPINB5, TPD52 Cell Death cell death cell death of lung cancer cell 8.30E-03 ATAD2, CDKN1A, DDIT3, FHIT, IGFBP3, IGFBP6, PRKCD, 9 lines RRM2B, SOD1 Cell Death cell death cell death of breast cancer cell 1.23E-02 CDC42, CDKN1A, CRABP2, CYB5A (includes EG:109672), FASN, 12 lines FHIT, IGFBP3, MST4, PLK2, PPARG, SERPINB5, TFAP2A Cell Death cell death cell death of hepatoma cell 1.60E-02 ANKRD1, CDKN1A, DDIT3, MMP7, PRKCD, SOD1 6 lines Cell Death cell death cell death of HCSM cells 1.99E-02 APP 1 Cell Death cell death cell death of carcinoma cell 3.02E-02 DDIT3, FHIT, IGFBP3, IGFBP6, PRKCD, SOD1, SPRY2 7 lines Cell Death cell death cell death of pre- 3.93E-02 SOD1 1 oligodendrocytes Cell Death necrosis necrosis 3.84E-05 AKAP12, ANKRD1, ANXA2, APP, ATAD2, ATG12 (includes 56 EG:361321), BMP4, CABLES1, CDC42, CDK6, CDKN1A, CRABP2, CYB5A (includes EG:109672), DDIT3, FASN, FHIT, HERPUD1, HYOU1, IFNAR2, IGFBP3, IGFBP6, IRS1, LAMP1, MAP4K1, MCM2, MMP7, MST4, NDRG1, NR4A2, PAWR, PDPK1, PHLDA1, PLK2, PPARG, PPIA, PRKCD, PRMT2, RDX, RRM2B, SAT1, SEMA3A, SERPINB5, SOD1, SOX9, SPRY2, SRPX, ST6GAL1, SYK, TCF12, TERF2, TFAP2A, TNFRSF6B, TNFSF18, TOPBP1, TPD52, XPR1 Cell Death survival cell survival 5.46E-03 APP, BMP4, CA2, CALB2, CDK6, CDKN1A, DDIT3, FGFR3, FHIT, 28 HERPUD1, HYOU1, ID2, IGFBP3, IRF9, KRT19 (human), MGST1, NDRG1, PBX1, PDGFA, PLK2, PRKACB, PRKCD, RRM2B, SERPINB5, SOD1, STC1, SYK, TOPBP1 Cell Death cell viability cell viability 5.68E-03 APP, BMP4, CA2, CALB2, CDK6, CDKN1A, DDIT3, FGFR3, FHIT, 27 HERPUD1, HYOU1, ID2, IGFBP3, IRF9, KRT19 (human), MGST1, NDRG1, PBX1, PDGFA, PLK2, PRKACB, PRKCD, RRM2B,

358

SERPINB5, SOD1, STC1, SYK Cell Death cell viability cell viability of epithelial cell 1.11E-02 APP, CALB2, IRF9, MGST1 4 lines Cell Death cell viability cell viability of breast cancer 1.34E-02 CA2, CDKN1A, ID2, IGFBP3, KRT19 (human), PBX1 6 cell lines Cell Death cell viability cell viability of neuroblastoma 1.99E-02 APP 1 cells Cell Death cell viability cell viability of cancer cells 2.31E-02 APP, BMP4, FHIT 3 Cell Death cell viability cell viability of neuroglia 2.65E-02 APP, PDGFA 2 Cell Death cell viability cell viability of neurons 2.73E-02 APP, HERPUD1, IGFBP3 3 Cell Death cell viability cell viability of kidney cell 3.92E-02 APP, HYOU1, IRF9 3 lines Cell Death cell viability cell viability of lung cancer 3.93E-02 FHIT 1 cells Cell Death fragmentation fragmentation of DNA 1.16E-02 APP, CDKN1A, PPIA, PRKCD, SOD1, TPD52L1 6 Cell Death degeneration degeneration of neurons 1.59E-02 APP, SOD1 2 Cell Death killing killing of hippocampal neurons 1.99E-02 APP 1 Cell Death anoikis anoikis of cancer cells 3.93E-02 NR4A2 1 Cell Death cytotoxicity cytotoxicity of carcinoma cell 3.93E-02 PDPK1 1 lines Cell Death cytotoxicity cytotoxicity of lung cancer cell 3.93E-02 PDPK1 1 lines Cell Cycle interphase interphase of hepatoma cell 2.91E-05 AHR, DDIT3, MEIS2, PBX1 4 lines Cell Cycle interphase interphase 3.16E-04 AHR, BMP4, CDC42, CDK6, CDKN1A, DDIT3, FASN, FBXO5, 22 FHIT, GJA1, ID2, KDM5B, MEIS2, PBX1, PLK2, PPARG, RRM2B, SOX9, SPRY2, TCF12, TOPBP1, TPD52L1 Cell Cycle interphase interphase of tumor cell lines 3.88E-03 AHR, CDC42, CDK6, CDKN1A, DDIT3, FASN, FHIT, GJA1, 14 MEIS2, PBX1, RRM2B, SOX9, SPRY2, TOPBP1 Cell Cycle interphase arrest in interphase 1.08E-02 BMP4, CDC42, CDKN1A, DDIT3, FASN, FBXO5, FHIT, KDM5B, 12 PPARG, RRM2B, TCF12, TOPBP1 Cell Cycle interphase interphase of epithelial cells 1.29E-02 CDKN1A, ID2 2 Cell Cycle interphase interphase of breast cell lines 3.06E-02 CDKN1A, KDM5B 2

359

Cell Cycle interphase arrest in interphase of lung 3.92E-02 CDKN1A, FHIT, RRM2B 3 cancer cell lines Cell Cycle G1/S phase transition G1/S phase transition of bone 2.95E-04 CDC42, CDKN1A, GJA1, TOPBP1 4 cancer cell lines Cell Cycle G1/S phase transition G1/S phase 7.70E-04 AHR, CDC42, CDK6, CDKN1A, GJA1, ID2, PLK2, SPRY2, 9 TOPBP1 Cell Cycle G1/S phase transition G1/S phase transition of tumor 1.80E-03 AHR, CDC42, CDKN1A, GJA1, SPRY2, TOPBP1 6 cell lines Cell Cycle G1/S phase transition arrest in G1/S phase transition 1.91E-03 CDC42, CDKN1A, TOPBP1 3 of bone cancer cell lines Cell Cycle G1/S phase transition G1/S phase transition of 1.99E-02 ID2 1 keratinocytes Cell Cycle G1/S phase transition delay in G1/S phase transition 1.99E-02 AHR 1 of hepatoma cell lines Cell Cycle G1/S phase transition arrest in G1/S phase transition 3.93E-02 CDKN1A 1 of cancer cells Cell Cycle G1 phase G1 phase of bone cancer cell 5.94E-04 CDC42, CDK6, CDKN1A, GJA1, TOPBP1 5 lines Cell Cycle G1 phase G1 phase 6.56E-04 AHR, CDC42, CDK6, CDKN1A, DDIT3, FASN, FBXO5, GJA1, ID2, 14 PLK2, PPARG, SPRY2, TCF12, TOPBP1 Cell Cycle G1 phase arrest in G1 phase of colon 1.61E-03 CDC42, CDKN1A, FASN, TOPBP1 4 cancer cell lines Cell Cycle G1 phase G1 phase of hepatoma cell 2.30E-03 AHR, DDIT3 2 lines Cell Cycle G1 phase G1 phase of tumor cell lines 7.45E-03 AHR, CDC42, CDK6, CDKN1A, DDIT3, FASN, GJA1, SPRY2, 9 TOPBP1 Cell Cycle G1 phase arrest in G1 phase 1.57E-02 CDC42, CDKN1A, DDIT3, FASN, FBXO5, PPARG, TCF12, 8 TOPBP1 Cell Cycle G1 phase entry into G1 phase of 1.99E-02 CDKN1A 1 fibroblasts Cell Cycle G1 phase arrest in G1 phase of hepatoma 3.93E-02 DDIT3 1 cell lines Cell Cycle S phase entry into S phase of hepatoma 1.16E-03 MEIS2, PBX1 2 cell lines Cell Cycle S phase re-entry into S phase of 1.99E-02 CDKN1A 1

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fibroblasts Cell Cycle S phase re-entry into S phase 3.48E-02 CDKN1A, PPARG 2 Cell Cycle polyploidy polyploidy of tumor cell lines 1.16E-03 NDRG1, SPRY2 2 Cell Cycle polyploidy polyploidy of bladder cancer 1.99E-02 NDRG1 1 cell lines Cell Cycle polyploidy polyploidy of colon cancer cell 1.99E-02 NDRG1 1 lines Cell Cycle polyploidy polyploidy of mammary cells 1.99E-02 NDRG1 1 Cell Cycle polyploidy polyploidy of sarcoma cell 1.99E-02 SPRY2 1 lines Cell Cycle premature senescence premature senescence of 2.30E-03 BMP4, CDKN1A 2 carcinoma cell lines Cell Cycle premature senescence premature senescence of lung 2.30E-03 BMP4, CDKN1A 2 cancer cell lines Cell Cycle mitosis mitosis of breast cancer cell 2.40E-03 BMP4, CDKN1A, SYK 3 lines Cell Cycle mitosis mitosis of connective tissue 3.78E-03 CDKN1A, FGF9 2 cells Cell Cycle mitosis arrest in mitosis 3.67E-02 CDKN1A, FBXO5, SGOL1 3 Cell Cycle mitosis exit from mitosis of breast 3.93E-02 CDKN1A 1 cancer cell lines Cell Cycle mitosis mitosis of chondrocytes 3.93E-02 FGF9 1 Cell Cycle mitosis mitosis of kidney cells 3.93E-02 CDKN1A 1 Cell Cycle senescence senescence of fibroblasts 2.95E-03 CDKN1A, FASN, SOD1 3 Cell Cycle senescence senescence of cells 3.21E-02 BMP4, CDKN1A, EREG, FASN, SOD1 5 Cell Cycle cell cycle progression cell cycle progression of colon 4.29E-03 CDK6, CDKN1A, PPARG 3 cancer cell lines Cell Cycle cell cycle progression arrest in cell cycle progression 7.73E-03 CDKN1A, PPARG 2 of colon cancer cell lines Cell Cycle cell cycle progression arrest in cell cycle progression 1.29E-02 AHR, CDKN1A, KRT19 (human) 3 of breast cancer cell lines Cell Cycle cell cycle progression cell cycle progression 1.84E-02 AHR, BMP4, CDC42, CDK6, CDKN1A, EREG, FASN, FBXO5, 22 FGF9, HAUS1, IGFBP3, KRT19 (human), MCM2, PAWR, PPARG, PRKCD, SERPINB5, SGOL1, SOD1, SPRY2, SYK, TRIM33

361

Cell Cycle cell cycle progression arrest in cell cycle progression 1.99E-02 BMP4 1 of myeloma cell lines Cell Cycle cell cycle progression arrest in cell cycle progression 1.99E-02 CDKN1A 1 of synovial fibroblasts Cell Cycle cell cycle progression cell cycle progression of colon 1.99E-02 CDKN1A 1 carcinoma cells Cell Cycle cell cycle progression delay in cell cycle progression 1.99E-02 CDKN1A 1 of colon cancer cell lines Cell Cycle cell cycle progression cell cycle progression of tumor 3.06E-02 CDKN1A, FASN 2 cells Cell Cycle cell cycle progression cell cycle progression of tumor 3.07E-02 AHR, BMP4, CDK6, CDKN1A, IGFBP3, KRT19 (human), PPARG, 8 cell lines PRKCD Cell Cycle ploidy ploidy of tumor cell lines 5.93E-03 CDKN1A, NDRG1, SPRY2 3 Cell Cycle ploidy ploidy of cells 2.35E-02 CDKN1A, MST4, NDRG1, SPRY2 4 Cell Cycle G0/G1 phase transition G0/G1 phase transition 7.94E-03 BMP4, CDKN1A, FHIT, SOX9 4 Cell Cycle G0/G1 phase transition G0/G1 phase transition of 1.29E-02 CDKN1A, SOX9 2 breast cancer cell lines Cell Cycle G0/G1 phase transition G0/G1 phase transition of 2.31E-02 CDKN1A, FHIT, SOX9 3 tumor cell lines Cell Cycle G0/G1 phase transition arrest in G0/G1 phase 2.52E-02 BMP4, CDKN1A, FHIT 3 transition Cell Cycle G0/G1 phase transition arrest in G0/G1 phase 3.93E-02 FHIT 1 transition of carcinoma cell lines Cell Cycle G0/G1 phase transition arrest in G0/G1 phase 3.93E-02 CDKN1A 1 transition of colon cancer cell lines Cell Cycle G0 phase arrest in G0 phase of 1.99E-02 CDKN1A 1 fibroblasts Cell Cycle G2 phase delay in G2 phase of colon 1.99E-02 CDKN1A 1 carcinoma cells Cell Cycle G2 phase arrest in G2 phase of colon 2.65E-02 CDKN1A, FASN 2 cancer cell lines Cell Cycle G2 phase arrest in G2 phase of 3.93E-02 CDKN1A 1 mammary epithelial cells

362

Cell Cycle G2/M phase transition initiation of G2/M phase 1.99E-02 CDKN1A 1 transition of brain cancer cell lines Cell Cycle G2/M phase transition arrest in G2/M phase transition 3.93E-02 CDKN1A 1 of bladder cancer cell lines Cell Cycle S/G2 phase transition arrest in S/G2 phase transition 1.99E-02 FHIT 1 of carcinoma cell lines Cell Cycle S/G2 phase transition arrest in S/G2 phase transition 1.99E-02 FHIT 1 of squamous cell carcinoma cell lines Cell Cycle mitogenesis mitogenesis of endometrial 1.99E-02 FGF9 1 stromal cells Cell Cycle mitogenesis mitogenesis of tumor cell lines 3.42E-02 IGFBP3, SPRY2, TRIM33 3 Cell Cycle segregation segregation of chromosomes 3.64E-02 CDC42, NCAPD3, RIOK3, SGOL1 4 Cell Cycle G2/M phase arrest in G2/M phase of breast 3.93E-02 KDM5B 1 cell lines Cell Cycle endoreduplication endoreduplication of tumor 3.93E-02 CDKN1A 1 cell lines Cell Cycle metaphase arrest in metaphase of 3.93E-02 FBXO5 1 embryonic cell lines Cell Cycle metaphase arrest in metaphase of 3.93E-02 FBXO5 1 epithelial cell lines Cell Cycle metaphase arrest in metaphase of kidney 3.93E-02 FBXO5 1 cell lines Cell Cycle polyploidization polyploidization of 3.93E-02 CDKN1A 1 megakaryocytes Cell Cycle prometaphase delay in prometaphase of 3.93E-02 CDC42 1 cervical cancer cell lines Cell Cycle sub-G1 phase arrest in sub-G1 phase of 3.93E-02 CDKN1A 1 mesothelioma cells Cellular Movement migration migration of cells 3.65E-05 ANKS1A, ANXA2, APP, ASAP2, BMP4, CDC42, CKLF, CXADR, 38 EREG, GJA1, HAS3, HEBP1, ID2, IGFBP3, IGFBP6, IRS1, KISS1R, MMP7, MSX2, NDRG1, NOV, PDGFA, PPARG, PPFIA1, PPIA, PRKCD, PRSS1/PRSS3, S100A2, SEMA3A, SERPINB5, SPAG9, SPRY2, ST6GAL1, STC1, SYK, TFAP2A, TNFRSF6B, VIM

363

Cellular Movement migration migration of tumor cell lines 2.17E-04 ANKS1A, ANXA2, APP, CDC42, CKLF, EREG, GJA1, HAS3, 22 IGFBP3, IGFBP6, IRS1, KISS1R, MSX2, NDRG1, NOV, PPFIA1, PRKCD, SEMA3A, SPRY2, ST6GAL1, TFAP2A, VIM Cellular Movement migration migration of breast cancer cell 3.06E-03 ANKS1A, CDC42, GJA1, IGFBP3, NDRG1, PRKCD, SEMA3A, 9 lines TFAP2A, VIM Cellular Movement migration migration of central nervous 3.78E-03 APP, BMP4 2 system cells Cellular Movement migration migration of carcinoma cell 5.79E-03 IRS1, KISS1R, NOV, PRKCD, VIM 5 lines Cellular Movement migration migration of neocortical 1.99E-02 BMP4 1 neurons Cellular Movement migration migration of myofibroblasts 3.93E-02 MMP7 1 Cellular Movement migration migration of 3.93E-02 IGFBP6 1 rhabdomyosarcoma cell lines Cellular Movement cell movement cell movement 1.45E-04 ANKS1A, ANXA2, APP, ASAP2, BMP4, CDC42, CKLF, CXADR, 40 EREG, GJA1, HAS3, HEBP1, ID2, IGFBP3, IGFBP6, IRS1, KISS1R, KRT19 (human), MMP7, MSX2, NDRG1, NOV, PDGFA, PPARG, PPFIA1, PPIA, PRKCD, PRSS1/PRSS3, S100A2, SEMA3A, SERPINB5, SPAG9, SPRY2, ST6GAL1, STC1, SYK, TFAP2A, TNFRSF6B, VIM, ZNF217 Cellular Movement cell movement cell movement of tumor cell 2.16E-04 ANKS1A, ANXA2, APP, CDC42, CKLF, EREG, GJA1, HAS3, 26 lines IGFBP3, IGFBP6, IRS1, KISS1R, KRT19 (human), MSX2, NDRG1, NOV, PPFIA1, PPIA, PRKCD, SEMA3A, SERPINB5, SPRY2, ST6GAL1, SYK, TFAP2A, VIM Cellular Movement cell movement cell movement of breast cancer 2.29E-04 ANKS1A, CDC42, GJA1, IGFBP3, KRT19 (human), NDRG1, 12 cell lines PRKCD, SEMA3A, SERPINB5, SYK, TFAP2A, VIM Cellular Movement cell movement cell movement of peripheral 5.07E-03 APP, PPARG, PPIA 3 blood monocytes Cellular Movement cell movement cell movement of myeloid 2.26E-02 APP, CKLF, CXADR, HEBP1, PPARG, PPIA, SEMA3A, SYK, 9 cells TNFRSF6B Cellular Movement cell movement cell movement of phagocytes 2.66E-02 APP, CKLF, CXADR, HEBP1, PPARG, PPIA, SEMA3A, SYK, 9 TNFRSF6B Cellular Movement cell movement cell movement of neuroglia 3.48E-02 APP, TNFRSF6B 2 Cellular Movement transmigration transmigration of peripheral 1.16E-03 APP, PPARG 2 blood monocytes Cellular Movement chemotaxis chemotaxis of embryonic cell 6.88E-03 APP, CDC42, PPIA 3

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lines Cellular Movement chemotaxis chemotaxis of epithelial cell 6.88E-03 APP, CDC42, PPIA 3 lines Cellular Movement chemotaxis chemotaxis of cells 8.27E-03 ANXA2, APP, BMP4, CDC42, CKLF, CXADR, HEBP1, PPARG, 13 PPIA, PRKCD, SEMA3A, SYK, TNFRSF6B Cellular Movement chemotaxis chemotaxis of myeloid cells 9.08E-03 APP, CKLF, CXADR, HEBP1, PPARG, PPIA, SYK, TNFRSF6B 8 Cellular Movement chemotaxis chemotaxis of kidney cell lines 1.02E-02 APP, CDC42, PPIA 3 Cellular Movement chemotaxis chemotaxis of phagocytes 3.11E-02 APP, CKLF, CXADR, HEBP1, PPIA, SYK, TNFRSF6B 7 Cellular Movement chemotaxis chemotaxis of squamous cell 3.93E-02 SYK 1 carcinoma cell lines Cellular Movement invasion invasion of lung cancer cell 1.96E-02 CDC42, FHIT, PPARG, PRKCD 4 lines Cellular Movement invasion invasion of bronchial epithelial 1.99E-02 CDC42 1 cells Cellular Movement invasion invasion of squamous 1.99E-02 SERPINB5 1 carcinoma cells Cellular Movement invasion invasion of prostate cancer cell 2.50E-02 PRKCD, SERPINB5, SPRY2, VIM 4 lines Cellular Movement invasion invasion of adenocarcinoma 3.93E-02 SERPINB5 1 cells Cellular Movement invasion invasion of gonadal cell lines 3.93E-02 MSX2 1 Cellular Movement infiltration infiltration by microglia 1.99E-02 TNFRSF6B 1 Cellular Movement withdrawal withdrawal of lamellipodia 1.99E-02 SERPINB5 1 Cellular proliferation proliferation of prostate cancer 6.74E-05 CDK6, CDKN1A, CXADR, FASN, HAS3, IGFBP3, LRRC26, MST4, 14 Development cell lines PPARG, PRKCD, SAT1, SERPINB5, SUMO3, TPD52 Cellular proliferation proliferation of tumor cell 1.06E-03 AKR1C3, APP, ATAD2, BMP4, CABLES1, CDC42, CDK6, 42 Development lines CDKN1A, CXADR, EREG, FASN, FES, FGFR3, FHIT, FTL, GCAT, GJA1, HAS3, ID2, IGFBP3, IRS1, KIAA0101, KISS1R, LRRC26, MCM2, MMP7, MST4, MSX2, MTAP, NOV, NR4A2, PDPK1, PPARG, PRKCD, SAT1, SERPINB5, SOD1, SPRY2, SUMO3, SYK, TFAP2A, TPD52 Cellular proliferation proliferation of stromal cells 2.95E-03 BMP4, FGF9, IGFBP3 3 Development Cellular proliferation proliferation of fibroblasts 8.09E-03 BMP4, CDK6, CDKN1A, EREG, PDGFA, SPRY2 6

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Development Cellular proliferation proliferation of keratinocytes 1.39E-02 CDKN1A, EREG, ID2, PRKCD 4 Development Cellular proliferation proliferation of fibroblast cell 1.49E-02 CDKN1A, EREG, PPARG, ZBED1 4 Development lines Cellular proliferation proliferation of chondrocytes 1.59E-02 IGFBP3, STC1 2 Development Cellular proliferation proliferation of lung cancer 1.59E-02 DDIT3, FHIT 2 Development cells Cellular proliferation proliferation of tumor cells 1.67E-02 BMP4, CDKN1A, DDIT3, FHIT, ID2, IGFBP3, NR4A2, PRKCD 8 Development Cellular proliferation proliferation of muscle cells 2.82E-02 BMP4, EREG, IGFBP3, MMP7, PDPK1 5 Development Cellular proliferation proliferation of 3.18E-02 CDKN1A, GCAT, SYK 3 Development lymphoblastoid cell lines Cellular proliferation proliferation of breast cancer 3.85E-02 ATAD2, CDKN1A, GJA1, ID2, IGFBP3, IRS1, MTAP, PPARG, 12 Development cell lines PRKCD, SAT1, SYK, TFAP2A Cellular proliferation proliferation of embryonic 3.93E-02 APP 1 Development stem cells Cellular proliferation proliferation of stromal 3.93E-02 BMP4 1 Development fibroblast cells Cellular differentiation differentiation of trophoblast 3.93E-04 BMP4, NDRG1 2 Development Cellular differentiation differentiation of colon cancer 4.03E-04 CDKN1A, NDRG1, PPARG 3 Development cell lines Cellular differentiation differentiation of chondrocytes 1.14E-03 FGFR3, SOX9, STC1 3 Development Cellular differentiation differentiation of muscle cells 1.39E-03 BMP4, CDKN1A, EREG, IGFBP3, PPARG 5 Development Cellular differentiation differentiation of myoblasts 3.58E-03 BMP4, CDKN1A, IGFBP3 3 Development Cellular differentiation differentiation of tumor cell 4.37E-03 AKR1C3, BMP4, CDC42, CDKN1A, FES, MST4, NDRG1, PPARG, 10 Development lines PRKCD, SACS Cellular differentiation differentiation of cancer cells 1.59E-02 BMP4, CDKN1A 2 Development

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Cellular differentiation arrest in differentiation of 1.99E-02 BMP4 1 Development thymocytes Cellular differentiation differentiation of colony- 1.99E-02 BMP4 1 Development forming erythroid cells Cellular differentiation differentiation of endothelial 1.99E-02 IGFBP3 1 Development progenitor cells Cellular differentiation differentiation of liposarcoma 1.99E-02 PPARG 1 Development Cellular differentiation differentiation of lung cell 1.99E-02 BMP4 1 Development lines Cellular differentiation differentiation of 1.99E-02 BMP4 1 Development myelomonocytic progenitor cells Cellular differentiation differentiation of 1.99E-02 CDKN1A 1 Development neuroblastoma cells Cellular differentiation differentiation of cells 2.59E-02 AHR, AKR1C3, APP, BMP4, CDC42, CDK6, CDKN1A, EREG, 20 Development FES, FGFR3, HIST1H4A (includes others), ID2, IGFBP3, MST4, NDRG1, PPARG, PRKCD, SACS, SOX9, STC1 Cellular differentiation differentiation of connective 2.92E-02 BMP4, CDK6, FGFR3, PPARG, SOX9, STC1 6 Development tissue cells Cellular differentiation differentiation of fibrosarcoma 3.93E-02 PPARG 1 Development cell lines Cellular differentiation differentiation of white 3.93E-02 PPARG 1 Development adipocytes Cellular lifespan lifespan of fibroblast cell lines 2.30E-03 CDK6, CDKN1A 2 Development Cellular senescence senescence of fibroblasts 2.95E-03 CDKN1A, FASN, SOD1 3 Development Cellular development development of myeloma cell 1.99E-02 ID2 1 Development lines Cellular development development of primordial 1.99E-02 BMP4 1 Development germ cells Cellular development endothelial cell development 3.07E-02 BMP4, CDKN1A, FGFR3, HAS3, PPIA, STC1, TFAP2A, 8 Development TNFRSF6B Cellular development development of tumor cell 3.48E-02 HIST1H4A (includes others), ID2 2

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Development lines Cellular development development of astrocytes 3.93E-02 CDK6 1 Development Cellular immortalization immortalization of endothelial 1.99E-02 ZNF217 1 Development cell lines Cellular maturation maturation of erythroblasts 1.99E-02 CDKN1A 1 Development Cellular maturation maturation of dopaminergic 3.93E-02 NR4A2 1 Development neurons Cellular maturation maturation of embryonic stem 3.93E-02 NR4A2 1 Development cell lines Cellular transdifferentiation transdifferentiation of 1.99E-02 SOX9 1 Development epithelial tissue Cellular growth arrest in growth of germ cell 3.93E-02 CDKN1A 1 Development tumor cell lines Cellular growth re-entry into growth of 3.93E-02 FTL 1 Development leukemia cell lines Cellular hematopoiesis hematopoiesis of leukemia cell 3.93E-02 HIST1H4A (includes others) 1 Development lines Cellular Growth and proliferation proliferation of prostate cancer 6.74E-05 CDK6, CDKN1A, CXADR, FASN, HAS3, IGFBP3, LRRC26, MST4, 14 Proliferation cell lines PPARG, PRKCD, SAT1, SERPINB5, SUMO3, TPD52 Cellular Growth and proliferation proliferation of connective 7.15E-04 BMP4, CDK6, CDKN1A, EREG, FGF9, ID2, IGFBP3, PDGFA, 11 Proliferation tissue cells PRKCD, SPRY2, STC1 Cellular Growth and proliferation proliferation of tumor cell 1.06E-03 AKR1C3, APP, ATAD2, BMP4, CABLES1, CDC42, CDK6, 42 Proliferation lines CDKN1A, CXADR, EREG, FASN, FES, FGFR3, FHIT, FTL, GCAT, GJA1, HAS3, ID2, IGFBP3, IRS1, KIAA0101, KISS1R, LRRC26, MCM2, MMP7, MST4, MSX2, MTAP, NOV, NR4A2, PDPK1, PPARG, PRKCD, SAT1, SERPINB5, SOD1, SPRY2, SUMO3, SYK, TFAP2A, TPD52 Cellular Growth and proliferation proliferation of cells 1.85E-03 AKR1C3, ANXA2, APP, APPL1, ATAD2, BMP4, CABLES1, CAV2, 70 Proliferation CD33, CDC42, CDK6, CDKN1A, CKLF, COL6A1, CRABP2, CXADR, DDIT3, EREG, FASN, FES, FGF9, FGFR3, FHIT, FTL, GCAT, GJA1, HAS3, HNRNPU, ID2, IGFBP3, IGFBP6, IRS1, KIAA0101, KISS1R, LEPREL1, LRRC26, MCM2, MMP7, MST4, MSX2, MTAP, NAP1L1, NEU1, NOV, NR4A2, PAWR, PDGFA, PDPK1, PHLDA1, PPARG, PPIA, PRKCD, PRKRIR, RERG, SAT1, SERPINB5, SERPINH1, SOD1, SPRY2, STC1, SUMO3, SYK,

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TACSTD2, TFAP2A, TNFRSF6B, TPD52, TRIM33, TSC22D3, ZBED1, ZNF217 Cellular Growth and proliferation proliferation of stromal cells 2.95E-03 BMP4, FGF9, IGFBP3 3 Proliferation Cellular Growth and proliferation proliferation of fibroblasts 8.09E-03 BMP4, CDK6, CDKN1A, EREG, PDGFA, SPRY2 6 Proliferation Cellular Growth and proliferation proliferation of endometrial 1.02E-02 CABLES1, FGF9 2 Proliferation cells Cellular Growth and proliferation proliferation of keratinocytes 1.39E-02 CDKN1A, EREG, ID2, PRKCD 4 Proliferation Cellular Growth and proliferation proliferation of fibroblast cell 1.49E-02 CDKN1A, EREG, PPARG, ZBED1 4 Proliferation lines Cellular Growth and proliferation proliferation of chondrocytes 1.59E-02 IGFBP3, STC1 2 Proliferation Cellular Growth and proliferation proliferation of lung cancer 1.59E-02 DDIT3, FHIT 2 Proliferation cells Cellular Growth and proliferation proliferation of tumor cells 1.67E-02 BMP4, CDKN1A, DDIT3, FHIT, ID2, IGFBP3, NR4A2, PRKCD 8 Proliferation Cellular Growth and proliferation proliferation of muscle cells 2.82E-02 BMP4, EREG, IGFBP3, MMP7, PDPK1 5 Proliferation Cellular Growth and proliferation proliferation of 3.18E-02 CDKN1A, GCAT, SYK 3 Proliferation lymphoblastoid cell lines Cellular Growth and proliferation proliferation of breast cancer 3.85E-02 ATAD2, CDKN1A, GJA1, ID2, IGFBP3, IRS1, MTAP, PPARG, 12 Proliferation cell lines PRKCD, SAT1, SYK, TFAP2A Cellular Growth and proliferation proliferation of embryonic 3.93E-02 APP 1 Proliferation stem cells Cellular Growth and proliferation proliferation of stromal 3.93E-02 BMP4 1 Proliferation fibroblast cells Cellular Growth and cytostasis cytostasis of smooth muscle 1.16E-03 BMP4, ID2 2 Proliferation cells Cellular Growth and cytostasis cytostasis 5.72E-03 BMP4, CDKN1A, CRABP2, FHIT, ID2, RERG, SERPINB5, 9 Proliferation TRIM33, ZNF217 Cellular Growth and cytostasis cytostasis of tumor cell lines 9.58E-03 BMP4, CDKN1A, CRABP2, FHIT, RERG, SERPINB5, TRIM33 7 Proliferation Cellular Growth and cytostasis cytostasis of breast cancer cell 3.18E-02 CRABP2, RERG, SERPINB5 3

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Proliferation lines Cellular Growth and cytostasis cytostasis of hepatoma cell 3.93E-02 TRIM33 1 Proliferation lines Cellular Growth and colony formation colony formation of fibroblast 5.59E-03 EREG, SPRY2 2 Proliferation cell lines Cellular Growth and colony formation colony formation of lung 2.95E-02 CDKN1A, IGFBP3, SOD1 3 Proliferation cancer cell lines Cellular Growth and formation formation of neural precursor 1.99E-02 APP 1 Proliferation cells Cellular Growth and formation formation of colon cancer cell 3.93E-02 CDKN1A 1 Proliferation lines Cellular Growth and production production of olfactory 1.99E-02 BMP4 1 Proliferation receptor neurons Cellular Growth and stimulation stimulation of prostate cancer 1.99E-02 IGFBP3 1 Proliferation cell lines Cellular Growth and growth arrest in growth of germ cell 3.93E-02 CDKN1A 1 Proliferation tumor cell lines Cellular Growth and growth re-entry into growth of 3.93E-02 FTL 1 Proliferation leukemia cell lines

Appendix 6. 3 The top molecular and cellular functions associated with differentially expressed genes in the GGH- overexpressed MDA-MB-435 breast cancer cells

No. of Category Function Function Annotation P-value Genes Genes Cell-To-Cell Signaling binding binding of prostate cancer cell 6.49E-08 CD44 (includes EG:100330801), HAS2, HAS3, ITGA3, ITGA5, 10 and Interaction lines ITGA6, ITGB1, LGALS3, NRP1 (includes EG:18186), PTPRM Cell-To-Cell Signaling binding binding of skin cell lines 5.45E-04 CYR61, ITGA6, ITGB1 3 and Interaction Cell-To-Cell Signaling binding binding of cells 7.26E-03 A2M, ABCA1, ALCAM, ANXA5, CCL20, CD151, CD44 (includes and Interaction EG:100330801), CD83, CEACAM1 (includes others), CIB1, CYR61, DAG1 (includes EG:114489), HAS2, HAS3, HLA-A, IL8, ITGA3, 39 ITGA5, ITGA6, ITGB1, ITGB5, JAM3, LAMA5, LDLR, LGALS3, LRP8, LRPAP1, LY96 (includes EG:17087), NRP1 (includes

370

EG:18186), NT5E, PLAUR, PROS1, PTPRM, SDC1 (includes EG:20969), SEMA3A, SORT1, SPARC, SPP1 (includes EG:20750), STMN1 Cell-To-Cell Signaling binding binding of keratinocytes 1.89E-02 ITGA3, ITGB1 2 and Interaction Cell-To-Cell Signaling binding binding of breast cancer cell 2.44E-02 CD44 (includes EG:100330801), ITGB1, ITGB5, LGALS3, SPP1 5 and Interaction lines (includes EG:20750) Cell-To-Cell Signaling binding binding of tumor cell lines 2.71E-02 ALCAM, ANXA5, CD44 (includes EG:100330801), CEACAM1 and Interaction (includes others), HAS2, HAS3, HLA-A, ITGA3, ITGA5, ITGA6, 19 ITGB1, ITGB5, JAM3, LAMA5, LGALS3, NRP1 (includes EG:18186), PROS1, PTPRM, SPP1 (includes EG:20750) Cell-To-Cell Signaling binding binding of cancer cells 3.15E-02 CD44 (includes EG:100330801), DAG1 (includes EG:114489), 3 and Interaction PLAUR Cell-To-Cell Signaling binding binding of epithelial cells 3.15E-02 CEACAM1 (includes others), ITGA3, ITGB1 3 and Interaction Cell-To-Cell Signaling binding binding of fibroblast cell lines 3.15E-02 CYR61, ITGA6, ITGB1 3 and Interaction Cell-To-Cell Signaling binding binding of endothelial cell 3.65E-02 CD44 (includes EG:100330801), LGALS3, NT5E, SPARC 4 and Interaction lines Cell-To-Cell Signaling attachment attachment of cells 1.51E-04 CD44 (includes EG:100330801), EGR1, ILK, ITGA3, ITGA5, ITGA6, 12 and Interaction ITGB1, LRPAP1, MGAT5, SPP1 (includes EG:20750), SPRY2, VHL Cell-To-Cell Signaling attachment attachment of tumor cell lines 3.13E-04 CD44 (includes EG:100330801), EGR1, ITGA3, ITGA5, ITGA6, 8 and Interaction ITGB1, MGAT5, SPRY2 Cell-To-Cell Signaling attachment attachment of melanoma cell 2.05E-03 ITGA3, ITGA6, ITGB1 3 and Interaction lines Cell-To-Cell Signaling adhesion adhesion of fibrosarcoma cell 1.27E-03 ITGA5, ITGB1, JAM3, MGAT5 4 and Interaction lines Cell-To-Cell Signaling adhesion adhesion of lymphatic system 4.80E-03 CCL20, ITGA5, ITGB1 3 and Interaction cells Cell-To-Cell Signaling adhesion adhesion of kidney cells 8.55E-03 CADM1, IL8, ITGA3, ITGB1, PLAUR, RRAS, SOD2, TIMP3, VHL, 10 and Interaction ZEB2 Cell-To-Cell Signaling adhesion adhesion of stomach cancer 1.48E-02 CYR61, ITGA3, ITGB1 3 and Interaction cell lines Cell-To-Cell Signaling adhesion adhesion of tumor cell lines 2.97E-02 AFAP1, CD151, CD44 (includes EG:100330801), CD55, CD59 and Interaction (includes EG:25407), CD9, CYP2J2, CYR61, DAB2, HAS3, IL8, 26 ITGA3, ITGA5, ITGA6, ITGB1, JAM3, LGALS3, MGAT5, PARVB,

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PLAUR, PRKCA, RASSF1, SDC1 (includes EG:20969), SNX1, SPP1 (includes EG:20750), VHL Cell-To-Cell Signaling adhesion adhesion of bone marrow cells 3.58E-02 CCL20, ITGA5 2 and Interaction Cell-To-Cell Signaling immune response immune response of 4.80E-03 MCL1, PLA2G6, PROS1 3 and Interaction lymphoma cell lines Cell-To-Cell Signaling formation formation of fibrillar adhesions 6.68E-03 ITGB1, VHL 2 and Interaction Cell-To-Cell Signaling formation formation of fibronectin matrix 3.58E-02 ITGB1, PLAUR 2 and Interaction Cell-To-Cell Signaling response response of embryonic cell 1.29E-02 BIRC2, BIRC3, IFIH1, IRAK2 4 and Interaction lines Cell-To-Cell Signaling response response of epithelial cell lines 1.29E-02 BIRC2, BIRC3, IFIH1, IRAK2 4 and Interaction Cell-To-Cell Signaling response response of kidney cell lines 1.29E-02 BIRC2, BIRC3, IFIH1, IRAK2 4 and Interaction Cell-To-Cell Signaling phagocytosis phagocytosis of lymphoma cell 1.89E-02 PLA2G6, PROS1 2 and Interaction lines Cell-To-Cell Signaling sensitization sensitization of tumor cells 1.89E-02 BCL2, CASP9 (includes EG:100140945) 2 and Interaction Cell-To-Cell Signaling activation activation of leukocyte cell 2.92E-02 HLA-A, LGALS3, SOCS2, VAMP4 4 and Interaction lines Cell-To-Cell Signaling antiviral response antiviral response of 3.58E-02 BIRC2, BIRC3 2 and Interaction embryonic cell lines Cell-To-Cell Signaling antiviral response antiviral response of epithelial 3.58E-02 BIRC2, BIRC3 2 and Interaction cell lines Cell-To-Cell Signaling antiviral response antiviral response of kidney 3.58E-02 BIRC2, BIRC3 2 and Interaction cell lines Cell-To-Cell Signaling stimulation stimulation of T lymphocytes 4.86E-02 CD83, HLA-G, MUC1, TNFSF13B, TYR 5 and Interaction Cellular Movement cell movement cell movement of tumor cell 7.16E-07 A2M, AFAP1, AKAP11, ARFGEF1, ARHGDIB, ARPC1B, CCL20, lines CD151, CD44 (includes EG:100330801), CD9, CDK14, CYP2J2, CYR61, DAB2, EGR1, ELMO1, EPS8, FADD, FOXO3, FSCN1, 85 FYN, GAB2, GPI, GRN, HAS2, HAS3, HTATIP2, HTRA1, ID1, IGFBP6, IGSF8, IL8, ILK, ITGA6, ITGB1, JUN, KLF4, LAMB3, LCP1, LGALS3, LRPAP1, MET, MGAT5, MUC1, MYO10, NCOA3,

372

NES, NOV, NREP, NRP1 (includes EG:18186), PBK, PLA2G6, PLAUR, PODXL, PREX1, PRKCA, PRKCZ, PTPN11, PTPRM, RASSF1, RHOC, RPS6KA5, SDC1 (includes EG:20969), SDCBP, SEMA3A, SEPT9, SHC1 (includes EG:20416), SHC4, SIRPA, SLC2A1, SNAI2, SOD2, SPARC, SPP1 (includes EG:20750), SPRY2, ST3GAL5, STAT3, STMN1, TFAP2A, TFAP2C, TFF2, TGFA, THBS2, VAV3, ZYX Cellular Movement cell movement cell movement 3.62E-06 A2M, ADM, AFAP1, AGGF1, AKAP11, ALCAM, ARFGEF1, ARHGAP24, ARHGDIB, ARPC1B, ASAP1, ASAP2, CCL20, CCND1, CD151, CD44 (includes EG:100330801), CD63, CD9, CDK14, CEACAM1 (includes others), CIB1, CITED2, CLIC4, CYP2J2, CYR61, DAB2, DAG1 (includes EG:114489), EDNRB, EGR1, ELMO1, EPS8, FADD, FOXO3, FSCN1, FYN, GAB2, GPI, GRN, HAS2, HAS3, HLA-G, HTATIP2, HTRA1, ID1, IGFBP6, IGSF8, IL8, ILK, ITGA3, ITGA5, ITGA6, ITGB1, ITGB1BP1, JAM3, JUN, KCNMA1, KLF4, KLHL20, LAMB3, LCP1, LGALS3, LRP8, LRPAP1, MAP2K3, MDK, MET, MGAT5, MGP, MUC1, MYLK, 132 MYO10, NARS, NCOA3, NES, NOV, NREP, NRP1 (includes EG:18186), NTN4, PARP9, PBK, PLA2G6, PLAUR, PODXL, PODXL2, PPAP2A, PPAP2B, PREX1, PRKCA, PRKCZ, PTPN11, PTPRM, RALA, RASSF1, RHOC, ROPN1B, ROPN1L, RPS6KA5, S100A4, SCG2, SDC1 (includes EG:20969), SDCBP, SEMA3A, SEPT9, SERPINA3, SHC1 (includes EG:20416), SHC4, SIRPA, SLC2A1, SNAI2, SOD2, SORT1, SPA17, SPARC, SPATA13, SPP1 (includes EG:20750), SPRY2, SRPX2, ST3GAL5, STAT3, STMN1, TFAP2A, TFAP2C, TFF2, TGFA, THBS2, TIMP3, TUBB2B, VAV3, VCL, VHL, ZNF217, ZYX Cellular Movement cell movement cell movement of gonadal cell 5.01E-04 ASAP1, CD9, CIB1, ITGB1, ITGB1BP1, PLAUR, PODXL2 7 lines Cellular Movement cell movement cell movement of breast cancer 5.81E-04 ARHGDIB, ARPC1B, CD151, CYP2J2, FOXO3, GAB2, GPI, IL8, cell lines ITGA6, ITGB1, JUN, LRPAP1, MET, MGAT5, NRP1 (includes EG:18186), PLAUR, PRKCZ, SDC1 (includes EG:20969), SDCBP, 28 SEMA3A, SEPT9, SNAI2, SPARC, SPP1 (includes EG:20750), STAT3, TFAP2A, TFAP2C, TGFA Cellular Movement cell movement cell movement of brain cancer 7.51E-03 A2M, EGR1, ELMO1, FYN, NREP, NRP1 (includes EG:18186), cell lines PLAUR, PTPRM, SEMA3A, SHC1 (includes EG:20416), SIRPA, 12 VAV3 Cellular Movement cell movement cell movement of fibrosarcoma 1.10E-02 AKAP11, DAB2, GPI, MGAT5, PLAUR, SDC1 (includes EG:20969), 7 cell lines SOD2 Cellular Movement cell movement cell movement of cilia and 1.89E-02 ROPN1L, SPA17 2

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flagella Cellular Movement cell movement cell movement of prostate 1.92E-02 AFAP1, FSCN1, ID1, IGSF8, IL8, LAMB3, LCP1, LGALS3, 11 cancer cell lines NCOA3, NES, PBK Cellular Movement cell movement cell movement of tumor cells 2.03E-02 CD44 (includes EG:100330801), ITGA3, ITGB1, LGALS3, MET, 10 PARP9, PRKCA, SIRPA, SPP1 (includes EG:20750), VHL Cellular Movement cell movement cell movement of germ cells 3.15E-02 PPAP2A, PPAP2B, ROPN1B 3 Cellular Movement cell movement cell movement of ovarian 3.70E-02 CD44 (includes EG:100330801), HAS2, HAS3, HTRA1, PLA2G6, 6 cancer cell lines ST3GAL5 Cellular Movement cell movement cell movement of cancer cells 4.36E-02 CD44 (includes EG:100330801), ITGA3, MET, PARP9, PRKCA, 8 SIRPA, SPP1 (includes EG:20750), VHL Cellular Movement cell movement cell movement of 4.85E-02 CCL20, CD44 (includes EG:100330801), ITGA3, ITGA6, ITGB1, 6 hematopoietic cells SEMA3A Cellular Movement cell movement cell movement of endothelial 4.89E-02 ADM, ALCAM, CYR61, IL8, ITGB1, MET, NTN4, PPAP2B, RHOC 9 cell lines Cellular Movement migration migration of tumor cell lines 1.95E-06 A2M, AFAP1, AKAP11, ARFGEF1, ARHGDIB, CCL20, CD151, CD44 (includes EG:100330801), CYP2J2, CYR61, DAB2, EGR1, ELMO1, FOXO3, FSCN1, FYN, GAB2, GRN, HAS2, HAS3, HTATIP2, ID1, IGFBP6, IGSF8, IL8, ITGA6, ITGB1, JUN, KLF4, LAMB3, LGALS3, MET, MGAT5, MUC1, MYO10, NCOA3, NES, NOV, NREP, NRP1 (includes EG:18186), PLA2G6, PLAUR, 69 PODXL, PREX1, PRKCA, PTPN11, PTPRM, RASSF1, RHOC, RPS6KA5, SDC1 (includes EG:20969), SDCBP, SEMA3A, SHC1 (includes EG:20416), SHC4, SIRPA, SLC2A1, SNAI2, SOD2, SPP1 (includes EG:20750), SPRY2, STAT3, STMN1, TFAP2A, TFAP2C, TGFA, THBS2, VAV3, ZYX Cellular Movement migration migration of cells 1.09E-04 A2M, ADM, AFAP1, AGGF1, AKAP11, ALCAM, ARFGEF1, ARHGAP24, ARHGDIB, ASAP1, ASAP2, CCL20, CD151, CD44 (includes EG:100330801), CD9, CEACAM1 (includes others), CIB1, CITED2, CLIC4, CYP2J2, CYR61, DAB2, DAG1 (includes EG:114489), EDNRB, EGR1, ELMO1, FOXO3, FSCN1, FYN, GAB2, GRN, HAS2, HAS3, HLA-G, HTATIP2, ID1, IGFBP6, IGSF8, IL8, ILK, ITGA3, ITGA5, ITGA6, ITGB1, ITGB1BP1, JAM3, 111 JUN, KCNMA1, KLF4, KLHL20, LAMB3, LGALS3, LRP8, MAP2K3, MDK, MET, MGAT5, MGP, MUC1, MYLK, MYO10, NARS, NCOA3, NES, NOV, NREP, NRP1 (includes EG:18186), NTN4, PARP9, PLA2G6, PLAUR, PODXL, PPAP2A, PPAP2B, PREX1, PRKCA, PRKCZ, PTPN11, PTPRM, RASSF1, RHOC, RPS6KA5, S100A4, SCG2, SDC1 (includes EG:20969), SDCBP, SEMA3A, SERPINA3, SHC1 (includes EG:20416), SHC4, SIRPA,

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SLC2A1, SNAI2, SOD2, SORT1, SPATA13, SPP1 (includes EG:20750), SPRY2, STAT3, STMN1, TFAP2A, TFAP2C, TFF2, TGFA, THBS2, TIMP3, TUBB2B, VAV3, VCL, VHL, ZYX Cellular Movement migration migration of gonadal cell lines 1.10E-03 ASAP1, CIB1, ITGB1, ITGB1BP1, PLAUR 5 Cellular Movement migration migration of thymocytes 1.27E-03 ITGA3, ITGA6, ITGB1, SEMA3A 4 Cellular Movement migration migration of carcinoma cell 6.64E-03 CYR61, HTATIP2, ID1, IL8, ITGB1, MET, NOV, PODXL, RASSF1, 11 lines STMN1, TGFA Cellular Movement migration migration of brain cancer cell 1.27E-02 A2M, EGR1, ELMO1, FYN, NREP, PLAUR, PTPRM, SHC1 10 lines (includes EG:20416), SIRPA, VAV3 Cellular Movement migration migration of germ cells 1.89E-02 PPAP2A, PPAP2B 2 Cellular Movement migration migration of squamous cell 1.99E-02 CD44 (includes EG:100330801), CYP2J2, JUN, MET, ZYX 5 carcinoma cell lines Cellular Movement migration migration of breast cancer cell 2.01E-02 ARHGDIB, CYP2J2, FOXO3, GAB2, IL8, ITGA6, ITGB1, JUN, lines MET, NRP1 (includes EG:18186), PLAUR, SDC1 (includes 20 EG:20969), SDCBP, SEMA3A, SNAI2, SPP1 (includes EG:20750), STAT3, TFAP2A, TFAP2C, TGFA Cellular Movement migration migration of prostate cancer 2.66E-02 AFAP1, FSCN1, ID1, IGSF8, IL8, LAMB3, LGALS3, NCOA3, NES 9 cell lines Cellular Movement migration migration of hematopoietic 2.95E-02 CCL20, ITGA3, ITGA6, ITGB1, SEMA3A 5 progenitor cells Cellular Movement migration migration of fibrosarcoma cell 3.20E-02 AKAP11, DAB2, MGAT5, PLAUR, SDC1 (includes EG:20969), 6 lines SOD2 Cellular Movement migration migration of sarcoma cell lines 3.58E-02 EGR1, SPRY2 2 Cellular Movement invasion invasion of cells 4.19E-06 ADM, ASAP1, BCL2, CCND1, CD151, CD44 (includes EG:100330801), CD9, CDK14, CEACAM1 (includes others), CYP2J2, DAB2, EDNRB, ELMO1, FSCN1, FST, GAB2, GPI, GRN, HAS2, HAS3, HIF1A, HTATIP2, ID1, IER3, IL8, ILK, ITGA3, ITGA5, ITGB1, JAM3, JUN, KCNMA1, KLF4, LCP1, LGALS3, MCAM, MET, MGAT5, MITF, MMP1 (includes EG:300339), MUC1, 71 NCOA3, NES, NOV, NRP1 (includes EG:18186), NUAK1, PARVB, PLA2G6, PLAUR, PODXL, PRKCA, RHOC, S100A6, SDCBP, SEPT9, SLC2A1, SOD2, SPARC, SPP1 (includes EG:20750), SPRY2, ST8SIA1, STAT3, STMN1, TFAP2A, TFAP2C, TFF2, TGFA, THBS2, TIMP3, VHL, ZEB2 Cellular Movement invasion invasion of tumor cell lines 8.35E-06 ADM, ASAP1, BCL2, CD151, CD44 (includes EG:100330801), CD9, CDK14, CYP2J2, DAB2, ELMO1, FSCN1, GAB2, GPI, GRN, HAS2, 62 HAS3, HIF1A, HTATIP2, ID1, IL8, ITGA3, ITGA5, ITGB1, JAM3,

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JUN, KLF4, LCP1, LGALS3, MET, MGAT5, MITF, MMP1 (includes EG:300339), MUC1, NCOA3, NES, NOV, NRP1 (includes EG:18186), NUAK1, PARVB, PLA2G6, PLAUR, PODXL, PRKCA, RHOC, S100A6, SDCBP, SEPT9, SLC2A1, SOD2, SPARC, SPP1 (includes EG:20750), SPRY2, ST8SIA1, STAT3, STMN1, TFAP2A, TFAP2C, TFF2, TGFA, TIMP3, VHL, ZEB2 Cellular Movement invasion invasion of colon cancer cell 8.02E-04 CD44 (includes EG:100330801), FSCN1, HAS2, HAS3, HIF1A, lines KLF4, MET, NRP1 (includes EG:18186), NUAK1, STAT3, TFF2, 12 TGFA Cellular Movement invasion invasion of prostate cancer cell 5.04E-03 FSCN1, IL8, LCP1, LGALS3, MMP1 (includes EG:300339), NCOA3, 11 lines NES, PLAUR, PODXL, SPP1 (includes EG:20750), SPRY2 Cellular Movement invasion invasion of hybrid cells 6.68E-03 CEACAM1 (includes others), SPP1 (includes EG:20750) 2 Cellular Movement invasion invasion of bladder cancer cell 1.29E-02 CD9, DAB2, GRN, SOD2 4 lines Cellular Movement invasion invasion of lung cancer cell 2.38E-02 ASAP1, CD44 (includes EG:100330801), HTATIP2, ID1, ITGB1, 9 lines MET, ST8SIA1, STAT3, STMN1 Cellular Movement invasion invasion of fibrosarcoma cell 2.44E-02 GPI, JAM3, MGAT5, PLAUR, SOD2 5 lines Cellular Movement invasion invasion of breast cancer cell 2.83E-02 ASAP1, CD44 (includes EG:100330801), GAB2, HIF1A, ID1, lines ITGA3, ITGA5, JUN, MET, MUC1, PARVB, PLAUR, PODXL, 18 RHOC, SDCBP, SEPT9, SPARC, SPP1 (includes EG:20750) Cellular Movement invasion invasion of kidney cell lines 3.65E-02 IER3, MET, TFF2, ZEB2 4 Cellular Movement invasion invasion of carcinoma cell 3.72E-02 ASAP1, FSCN1, GRN, HTATIP2, ID1, ITGB1, MET, SPP1 (includes 10 lines EG:20750), STMN1, TIMP3 Cellular Movement invasion invasion of squamous cell 4.25E-02 CD44 (includes EG:100330801), CYP2J2, FSCN1, PLAUR, SPP1 6 carcinoma cell lines (includes EG:20750), TGFA Cellular Movement cytokinesis cytokinesis 5.15E-03 ARL3, CALM1 (includes others), CCNB1, CD2AP, CDKN1A, CETN2, INCENP, NEDD4L, PLK1, RALA, RHOC, SEPT6, SEPT9, 16 STX16, TM4SF1, VAV3 Cellular Movement chemotaxis chemotaxis of breast cancer 1.29E-02 ITGA6, ITGB1, JUN, PRKCZ 4 cell lines Cellular Movement cell rolling cell rolling of eosinophils 1.48E-02 IL8, ITGB1, LGALS3 3 Cellular Movement chemorepulsion chemorepulsion of brain 1.89E-02 NRP1 (includes EG:18186), SEMA3A 2 cancer cell lines Cellular Movement transmigration transmigration of peripheral 3.58E-02 IL8, ITGB1 2 blood leukocytes

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Cellular Movement mobility mobility of cells 4.23E-02 CADM1, ITGA5, TM4SF1 3 Cell Death anoikis anoikis 2.20E-06 BCL2, CASP9 (includes EG:100140945), CEACAM1 (includes others), DAB2, FADD, GRN, HTRA1, ILK, ITGB1, LGALS3, MCL1, 16 NCOA3, NR4A2, PLAUR, PRKCA, TNFRSF10B Cell Death anoikis anoikis of tumor cell lines 1.65E-05 BCL2, CEACAM1 (includes others), DAB2, GRN, HTRA1, ILK, 12 LGALS3, MCL1, NCOA3, NR4A2, PRKCA, TNFRSF10B Cell Death anoikis anoikis of breast cancer cell 3.45E-04 DAB2, ILK, LGALS3, MCL1, NCOA3 5 lines Cell Death cell death cell death of brain cancer cell 4.41E-05 BCL2, BIRC2, BIRC3, CASP1, CD59 (includes EG:25407), CRYAB, lines DHCR24, EPAS1, FOXO3, HIF1A, IL24, JAG1, MCL1, MET, MITF, 22 NRP1 (includes EG:18186), SEMA3A, SIRPA, SNCA, SOD2, STAT3, TNFRSF10B Cell Death cell death cell death of breast cancer cell 1.52E-04 ADM, BAG3, BCL2, BIRC2, BIRC3, BNIP3L, CASP1, CASP9 lines (includes EG:100140945), CCND1, CD44 (includes EG:100330801), CDKN1A, CEACAM1 (includes others), CYCS, CYR61, DAB2, DDIT4, DNAJC15, FADD, FBXO32, FOXO3, HIF1A, HSPB1, IER3, 42 IL24, ILK, JUN, LGALS3, MCL1, MST4, MUC1, NCOA3, PLK1, PLK2, PRKCA, PRKDC, SDC1 (includes EG:20969), SOD2, STMN1, TFAP2A, TFAP2C, TNFRSF10B, UGCG Cell Death cell death cell death of tumor cell lines 2.32E-04 ADM, AKAP12, BAG3, BCHE, BCL2, BCL2A1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BNIP3L, CABLES1, CASP1, CASP9 (includes EG:100140945), CCNB1, CCND1, CD44 (includes EG:100330801), CD55, CD59 (includes EG:25407), CDK5R1, CDKN1A, CDKN2C, CDKN2D, CEACAM1 (includes others), CIB1, CREB3L2, CRYAB, CYCS, CYP2J2, CYR61, DAB2, DCT, DDIT4, DHCR24, DNAJC15, DUT, EGR1, EPAS1, FADD, FAIM3, FBXO32, FOXO3, FST, GLRX, GPC1, GPI, GPR37, GRN, HIF1A, HLA-DRB4, HSPB1, HTATIP2, HTRA1, ID1, IER3, IFI6, IGFBP6, IGFBP7, IL24, IL8, ILK, IRF1 (includes EG:16362), IRF4, ITGB1, ITPK1, JAG1, JUN, KLF4, KLF9, LGALS3, MAOA, MAPKAP1, 138 MAX, MCL1, MDK, MET, MITF, MSRB2, MST4, MT1X, MT2A, MTFP1, MUC1, NAA35, NCOA3, NR4A2, NRP1 (includes EG:18186), NUAK1, PARVA, PBK, PCBP2, PLA2G6, PLAUR, PLK1, PLK2, PPP2R2A, PRAME, PRKAR1A, PRKCA, PRKCZ, PRKDC, PTPN11, RAB32, RASSF1, RHOC, S100A4, S100A6, SAT1, SDC1 (includes EG:20969), SEMA3A, SHC1 (includes EG:20416), SIRPA, SLC11A2, SNCA, SOD2, SPARC, SPP1 (includes EG:20750), SPRY2, SRPX, STAT3, STMN1, TERF1, TFAP2A, TFAP2C, TGFA, TIMP3, TNFAIP3, TNFRSF10B, TNFSF13B, TSG101, TYMP, UBA7, UGCG, VAV3, VHL, YWHAZ,

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ZFYVE16 Cell Death cell death cell death 6.06E-04 ABCC5, ADM, AGGF1, AGTRAP, AKAP12, ALDH1A1, ANTXR1, ANTXR2, APIP, ASAH1, ATP7A, AXIN2, BAG3, BCHE, BCL2, BCL2A1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BLID, BLVRA, BNIP3L, CABLES1, CADM1, CALB2, CALM1 (includes others), CAMK2D, CAMK2N1, CAPN3, CASP1, CASP9 (includes EG:100140945), CCNB1, CCND1, CD44 (includes EG:100330801), CD55, CD59 (includes EG:25407), CD74, CD9, CD96, CDK2, CDK20, CDK5R1, CDKN1A, CDKN2C, CDKN2D, CEACAM1 (includes others), CIB1, CKAP5, CLCF1, CREB3L2, CRYAB, CYCS, CYP2J2, CYR61, DAB2, DAP, DCT, DDIT4, DDR1, DEPTOR, DHCR24, DNAJC15, DPF2, DUSP5, DUT, DYRK3, EDNRB, EGR1, EMP1, EPAS1, ERCC5, FADD, FAIM3, FBXO32, FHL2, FOXO3, FST, GABRG2, GBA, GLRX, GPC1, GPI, GPR37, GRN, HDAC9, HIF1A, HIST1H1C, HLA-DRB4, HLA-G, HRK, HSPB1, HTATIP2, HTRA1, ID1, IER3, IFI6, IFIH1, IGFBP6, IGFBP7, IL24, IL6ST, IL8, ILK, ING3, IRF1 (includes EG:16362), IRF4, ITGA5, ITGA6, ITGB1, ITM2B, ITPK1, JAG1, JUN, KCNMA1, KLF4, KLF9, LAPTM4B, 235 LGALS3, LRPAP1, LUC7L3, MAOA, MAP1S, MAPKAP1, MAPT, MAX, MCAM, MCL1, MDK, MET, MITF, MLLT11, MSRB2, MST4, MT1X, MT2A, MTFP1, MUC1, MYLK, NAA35, NCOA3, NDUFAF4, NFATC1, NFIL3, NR4A2, NRP1 (includes EG:18186), NT5E, NTN4, NUAK1, OPA1, P2RX4, P2RX7, PARVA, PARVB, PBK, PCBP2, PHLDA1, PHLDA2, PLA2G6, PLAUR, PLK1, PLK2, PLSCR1, PPAP2A, PPP2R1B, PPP2R2A, PRAME, PRKAR1A, PRKCA, PRKCZ, PRKDC, PRKRIR, PRMT2, PSMA1, PSMC3, PSME3, PTGR1, PTGS1, PTPN11, PTPN22, PTPRM, RAB32, RAC2, RASSF1, RHOC, RPS6KA2, S100A4, S100A6, SAT1, SCG2, SDC1 (includes EG:20969), SEMA3A, SH3BGRL3, SHC1 (includes EG:20416), SIRPA, SLC11A2, SLC22A1, SLC25A23, SLC2A1, SNAI2, SNCA, SOD2, SORT1, SPARC, SPP1 (includes EG:20750), SPRY2, SRPX, STAT3, STMN1, TERF1, TFAP2A, TFAP2C, TGFA, TGFB1I1, THBS2, TIMP3, TLR1, TNFAIP3, TNFRSF10B, TNFSF13B, TRIB2, TSG101, TXNDC5, TYMP, UBA7, UBR4, UGCG, URI1, VAV3, VHL, WIPF1, YWHAZ, ZFP36, ZFYVE16 Cell Death cell death cell death of neuroblastoma 1.84E-03 ADM, BCHE, BCL2, CASP9 (includes EG:100140945), CDKN1A, cell lines CDKN2D, CREB3L2, CYCS, FADD, GLRX, GPC1, GPR37, ITGB1, 20 MAOA, S100A6, SLC11A2, SNCA, SPP1 (includes EG:20750), TNFRSF10B, UGCG Cell Death cell death cell death of prostate cancer 2.89E-03 AKAP12, BCL2, BIRC2, BIRC3, CASP1, CASP9 (includes cell lines EG:100140945), CDK5R1, EGR1, FADD, FOXO3, FST, HIF1A, ID1, 25 IGFBP7, IL24, ITGB1, MCL1, MET, NCOA3, PLAUR, PRKCA,

378

STAT3, TGFA, TNFRSF10B, TYMP Cell Death cell death cell death of leukemia cell 3.60E-03 BCL2, BCL2A1, BIRC3, CASP9 (includes EG:100140945), CD55, lines CD59 (includes EG:25407), CDKN1A, CEACAM1 (includes others), CYCS, FADD, FAIM3, FOXO3, HLA-DRB4, HSPB1, ITGB1, JUN, 35 KLF4, LGALS3, MAX, MCL1, MET, MSRB2, MUC1, PRKCA, PRKCZ, RHOC, SHC1 (includes EG:20416), SOD2, STMN1, TGFA, TNFAIP3, TNFRSF10B, TNFSF13B, UBA7, UGCG Cell Death cell death cell death of leukocyte cell 4.36E-03 BCL2, CASP9 (includes EG:100140945), FADD, HLA-DRB4, IFIH1, 11 lines IRF4, JUN, LGALS3, MCL1, PRKCZ, SHC1 (includes EG:20416) Cell Death cell death cell death of epithelial cells 4.77E-03 ALDH1A1, BCL2, BCL2A1, BCL6, BIRC2, BIRC3, BNIP3L, CASP1, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CRYAB, CYCS, EMP1, FADD, GPR37, HSPB1, 34 IER3, IL24, IL8, IRF1 (includes EG:16362), ITGB1, MAPT, MCL1, MET, MYLK, P2RX7, PLAUR, PRKDC, PRMT2, RASSF1, SPP1 (includes EG:20750), TGFB1I1, TIMP3, TNFRSF10B Cell Death cell death cell death of lymphoblastoid 5.17E-03 BCL2, CDKN1A, CDKN2C, HLA-DRB4, IRF4, SOD2, TERF1, 8 cell lines VAV3 Cell Death cell death cell death of hematopoietic cell 5.79E-03 BCL2, BIRC3, CASP9 (includes EG:100140945), FADD, HLA- lines DRB4, IFIH1, IRF4, JUN, LGALS3, MCL1, PRKCZ, SHC1 (includes 13 EG:20416), TRIB2 Cell Death cell death cell death of epithelial cell 6.60E-03 BCL2, BCL2A1, BCL6, BIRC2, BIRC3, BNIP3L, CASP1, CASP9 lines (includes EG:100140945), CD44 (includes EG:100330801), CRYAB, CYCS, EMP1, FADD, GPR37, HSPB1, IER3, IL24, IRF1 (includes 29 EG:16362), ITGB1, MAPT, MCL1, MET, P2RX7, PRKDC, PRMT2, RASSF1, SPP1 (includes EG:20750), TGFB1I1, TNFRSF10B Cell Death cell death cell death of central nervous 7.45E-03 BCL2, GBA, GLRX, HDAC9, IL8, LRPAP1, SNCA, TGFA 8 system cells Cell Death cell death cell death of embryonic cell 8.28E-03 BCL2, BCL2A1, BCL6, BIRC2, BIRC3, BNIP3L, CASP1, CASP9 lines (includes EG:100140945), CD44 (includes EG:100330801), CYCS, EMP1, FADD, GPR37, HSPB1, IER3, IL24, IRF1 (includes 28 EG:16362), ITGB1, MAPT, MCL1, P2RX7, PRKDC, PRMT2, RASSF1, SPP1 (includes EG:20750), TGFB1I1, TLR1, TNFRSF10B Cell Death cell death cell death of melanoma cell 9.12E-03 BCL2, BIRC2, CASP9 (includes EG:100140945), CDKN1A, DCT, 13 lines EGR1, IL24, IL8, MCL1, PBK, SAT1, STAT3, TNFRSF10B Cell Death cell death cell death of ovarian cancer 1.19E-02 BCL2, BIRC2, CASP1, CASP9 (includes EG:100140945), CDKN1A, cell lines DAB2, DNAJC15, HTRA1, IL8, PRKDC, SPARC, TGFA, 15 TNFRSF10B, TSG101, UGCG Cell Death cell death cell death of brain cells 1.31E-02 BCL2, GBA, GLRX, HDAC9, LRPAP1, SNCA, TGFA 7

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Cell Death cell death cell death of lymphoma cell 1.36E-02 BCL2, BIRC3, CASP9 (includes EG:100140945), CD55, CD59 lines (includes EG:25407), CDKN1A, DUT, FADD, HLA-DRB4, HSPB1, 19 LGALS3, MCL1, PLA2G6, PRKCZ, SOD2, STAT3, TNFRSF10B, UGCG, YWHAZ Cell Death cell death cell death of muscle 1.43E-02 ADM, APIP, CASP1, CASP9 (includes EG:100140945), CYCS, IL8, 10 MDK, STAT3, TIMP3, TNFRSF10B Cell Death cell death cell death of granule cells 1.48E-02 BCL2, GLRX, HDAC9 3 Cell Death cell death cell death of connective tissue 1.50E-02 ADM, BCL2, BIRC2, BIRC3, BLID, CASP9 (includes cells EG:100140945), CD44 (includes EG:100330801), CDKN1A, CYR61, 18 GPI, ITGA6, ITGB1, LRPAP1, MAPT, MCL1, SNAI2, TIMP3, TNFRSF10B Cell Death cell death cell death of tumor cells 1.67E-02 BCL2, CASP1, CASP9 (includes EG:100140945), CD74, CDK2, CDKN1A, CEACAM1 (includes others), FADD, HIF1A, IL24, IL8, 23 JAG1, MCL1, MUC1, NCOA3, NR4A2, PHLDA1, PLK1, SNAI2, STAT3, TFAP2A, TNFRSF10B, TNFSF13B Cell Death cell death cell death of kidney cells 1.85E-02 BCL2, BCL2A1, BCL6, BIRC2, BIRC3, BLID, BNIP3L, CASP1, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CYCS, EMP1, FADD, GPR37, HSPB1, IER3, IL24, IRF1 (includes 29 EG:16362), ITGB1, MAPT, MCL1, P2RX7, PRKDC, PRMT2, RASSF1, SOD2, SPP1 (includes EG:20750), TGFB1I1, TNFRSF10B Cell Death cell death cell death of endometrial 2.23E-02 ADM, BCL2, CYR61 3 cancer cell lines Cell Death cell death cell death of kidney cell lines 2.28E-02 BCL2, BCL2A1, BCL6, BIRC2, BIRC3, BLID, BNIP3L, CASP1, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CYCS, EMP1, FADD, GPR37, HSPB1, IER3, IL24, IRF1 (includes 28 EG:16362), ITGB1, MAPT, MCL1, P2RX7, PRKDC, PRMT2, RASSF1, SPP1 (includes EG:20750), TGFB1I1, TNFRSF10B Cell Death cell death cell death of fibroblast cell 2.77E-02 BCL2, BLID, CASP9 (includes EG:100140945), CYR61, ITGA6, 8 lines ITGB1, MAPT, TNFRSF10B Cell Death cell death cell death of fibroblasts 2.78E-02 BCL2, BIRC2, BIRC3, CASP9 (includes EG:100140945), CDKN1A, 10 GPI, ITGB1, MCL1, TIMP3, TNFRSF10B Cell Death cell death cell death of muscle cells 2.97E-02 ADM, APIP, CASP1, CASP9 (includes EG:100140945), CYCS, 9 MDK, STAT3, TIMP3, TNFRSF10B Cell Death cell death cell death of glioblastoma cells 3.58E-02 BCL2, IL24 2 Cell Death cell death cell death of 3.65E-02 CDKN1A, JUN, MCL1, TNFRSF10B 4 rhabdomyosarcoma cell lines Cell Death cell death cell death of cervical cancer 3.71E-02 BCL2, BCL2A1, BIRC2, BNIP3L, CASP1, CDKN1A, CDKN2C, 33

380

cell lines CIB1, FADD, FST, IER3, IRF1 (includes EG:16362), ITPK1, JUN, MAPKAP1, MCL1, MUC1, NAA35, NR4A2, PARVA, PCBP2, PLK1, PPP2R2A, PRAME, RAB32, RASSF1, SNCA, SOD2, SPP1 (includes EG:20750), TERF1, TFAP2A, TIMP3, TNFRSF10B Cell Death cell death cell death of cerebral cortex 4.16E-02 BCL2, GBA, LRPAP1, SNCA, TGFA 5 cells Cell Death cell death cell death of liver cells 4.16E-02 BCL2, FADD, IER3, JUN, SOD2 5 Cell Death apoptosis apoptosis 8.55E-05 ADM, AGGF1, AKAP12, ALDH1A1, APIP, ASAH1, BAG3, BCL2, BCL2A1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BLID, BLVRA, BNIP3L, CAPN3, CASP1, CASP9 (includes EG:100140945), CCNB1, CCND1, CD44 (includes EG:100330801), CD55, CD59 (includes EG:25407), CD74, CDK2, CDK5R1, CDKN1A, CDKN2C, CDKN2D, CEACAM1 (includes others), CIB1, CLCF1, CRYAB, CYCS, CYP2J2, CYR61, DAB2, DAP, DCT, DDIT4, DDR1, DEPTOR, DNAJC15, DPF2, DUT, EDNRB, EGR1, EPAS1, ERCC5, FADD, FAIM3, FBXO32, FHL2, FOXO3, FST, GLRX, GRN, HIF1A, HIST1H1C, HLA-G, HRK, HSPB1, HTATIP2, HTRA1, ID1, IER3, IFI6, IFIH1, IGFBP6, IGFBP7, IL24, IL6ST, IL8, ILK, ING3, IRF1 (includes EG:16362), IRF4, ITGA5, ITGA6, ITGB1, ITPK1, JAG1, JUN, KCNMA1, KLF4, KLF9, LGALS3, LRPAP1, LUC7L3, MAOA, MAP1S, MAPKAP1, MAPT, MAX, MCL1, MDK, MET, 180 MITF, MLLT11, MSRB2, MST4, MT2A, MTFP1, MUC1, MYLK, NCOA3, NFATC1, NR4A2, NRP1 (includes EG:18186), NT5E, NTN4, OPA1, P2RX4, P2RX7, PARVA, PARVB, PBK, PCBP2, PHLDA1, PHLDA2, PLA2G6, PLAUR, PLK1, PLK2, PLSCR1, PPP2R2A, PRAME, PRKAR1A, PRKCA, PRKCZ, PRKDC, PRKRIR, PRMT2, PSME3, PTGS1, PTPN11, RAB32, RAC2, RASSF1, RHOC, RPS6KA2, S100A4, S100A6, SAT1, SCG2, SDC1 (includes EG:20969), SEMA3A, SH3BGRL3, SHC1 (includes EG:20416), SIRPA, SNAI2, SNCA, SOD2, SORT1, SPARC, SPP1 (includes EG:20750), SPRY2, SRPX, STAT3, STMN1, TERF1, TFAP2A, TGFA, TIMP3, TLR1, TNFAIP3, TNFRSF10B, TNFSF13B, TRIB2, TSG101, TYMP, UBA7, UBR4, UGCG, URI1, VHL, YWHAZ, ZFYVE16 Cell Death apoptosis apoptosis of breast cancer cell 2.11E-04 ADM, BAG3, BCL2, BIRC2, BIRC3, BNIP3L, CASP1, CASP9 lines (includes EG:100140945), CCND1, CD44 (includes EG:100330801), CDKN1A, CEACAM1 (includes others), CYCS, CYR61, DAB2, 37 DDIT4, FADD, FBXO32, FOXO3, HIF1A, HSPB1, IER3, IL24, ILK, JUN, LGALS3, MCL1, MST4, MUC1, NCOA3, PLK1, PLK2, PRKCA, SDC1 (includes EG:20969), SOD2, TNFRSF10B, UGCG Cell Death apoptosis apoptosis of tumor cell lines 3.24E-04 ADM, AKAP12, BAG3, BCL2, BCL2A1, BCL3, BCL6, BHLHE40, 119

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BIRC2, BIRC3, BNIP3L, CASP1, CASP9 (includes EG:100140945), CCNB1, CCND1, CD44 (includes EG:100330801), CD55, CD59 (includes EG:25407), CDK5R1, CDKN1A, CDKN2C, CDKN2D, CEACAM1 (includes others), CRYAB, CYCS, CYP2J2, CYR61, DAB2, DCT, DDIT4, DNAJC15, DUT, EGR1, EPAS1, FADD, FAIM3, FBXO32, FOXO3, FST, GLRX, GRN, HIF1A, HSPB1, HTATIP2, HTRA1, ID1, IER3, IFI6, IGFBP6, IGFBP7, IL24, IL8, ILK, IRF1 (includes EG:16362), IRF4, ITGB1, ITPK1, JAG1, JUN, KLF4, KLF9, LGALS3, MAOA, MAPKAP1, MAX, MCL1, MET, MITF, MSRB2, MST4, MT2A, MTFP1, MUC1, NCOA3, NR4A2, NRP1 (includes EG:18186), PARVA, PBK, PCBP2, PLA2G6, PLAUR, PLK1, PLK2, PPP2R2A, PRKAR1A, PRKCA, PRKCZ, PRKDC, PTPN11, RAB32, RASSF1, RHOC, S100A4, S100A6, SAT1, SDC1 (includes EG:20969), SEMA3A, SIRPA, SNCA, SOD2, SPARC, SPP1 (includes EG:20750), SPRY2, SRPX, STAT3, STMN1, TERF1, TGFA, TIMP3, TNFAIP3, TNFRSF10B, TNFSF13B, TSG101, TYMP, UBA7, UGCG, VHL, YWHAZ, ZFYVE16 Cell Death apoptosis apoptosis of prostate cancer 1.09E-03 AKAP12, BCL2, BIRC2, BIRC3, CASP9 (includes EG:100140945), cell lines CDK5R1, EGR1, FADD, FOXO3, FST, HIF1A, ID1, IGFBP7, IL24, 24 ITGB1, MCL1, MET, NCOA3, PLAUR, PRKCA, STAT3, TGFA, TNFRSF10B, TYMP Cell Death apoptosis apoptosis of leukemia cell 1.63E-03 BCL2, BCL2A1, BIRC3, CASP9 (includes EG:100140945), CD55, lines CD59 (includes EG:25407), CDKN1A, CEACAM1 (includes others), CYCS, FADD, FAIM3, FOXO3, HSPB1, ITGB1, JUN, KLF4, 33 LGALS3, MAX, MCL1, MET, MSRB2, MUC1, PRKCA, PRKCZ, RHOC, SOD2, STMN1, TGFA, TNFAIP3, TNFRSF10B, TNFSF13B, UBA7, UGCG Cell Death apoptosis apoptosis of tumor cells 4.38E-03 BCL2, CASP1, CASP9 (includes EG:100140945), CDK2, CDKN1A, CEACAM1 (includes others), FADD, HIF1A, IL24, JAG1, MCL1, 21 MUC1, NCOA3, NR4A2, PHLDA1, PLK1, SNAI2, STAT3, TFAP2A, TNFRSF10B, TNFSF13B Cell Death apoptosis apoptosis of epithelial cell 4.73E-03 BCL2, BCL2A1, BCL6, BIRC2, BIRC3, BNIP3L, CASP1, CASP9 lines (includes EG:100140945), CD44 (includes EG:100330801), CRYAB, CYCS, FADD, IER3, IL24, IRF1 (includes EG:16362), ITGB1, 23 MAPT, MCL1, MET, P2RX7, PRMT2, SPP1 (includes EG:20750), TNFRSF10B Cell Death apoptosis apoptosis of embryonic cell 6.05E-03 BCL2, BCL2A1, BCL6, BIRC2, BIRC3, BNIP3L, CASP1, CASP9 lines (includes EG:100140945), CD44 (includes EG:100330801), CYCS, FADD, IER3, IL24, IRF1 (includes EG:16362), ITGB1, MAPT, 22 MCL1, P2RX7, PRMT2, SPP1 (includes EG:20750), TLR1, TNFRSF10B

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Cell Death apoptosis apoptosis of brain cancer cell 6.29E-03 BCL2, CASP1, CRYAB, FOXO3, IL24, JAG1, MCL1, MET, MITF, 14 lines NRP1 (includes EG:18186), SEMA3A, SIRPA, STAT3, TNFRSF10B Cell Death apoptosis apoptosis of splenocytes 6.68E-03 CDKN1A, IL6ST 2 Cell Death apoptosis apoptosis of retinal cells 9.18E-03 BCL2, HSPB1, PLAUR, TIMP3 4 Cell Death apoptosis apoptosis of melanoma cell 1.19E-02 BCL2, BIRC2, CASP9 (includes EG:100140945), CDKN1A, DCT, 12 lines EGR1, IL24, MCL1, PBK, SAT1, STAT3, TNFRSF10B Cell Death apoptosis apoptosis of bladder cancer 1.26E-02 BCL2, CASP9 (includes EG:100140945), CDKN1A, LGALS3, PLK1 5 cell lines Cell Death apoptosis apoptosis of epithelial cells 1.55E-02 ALDH1A1, BCL2, BCL2A1, CRYAB, FADD, IL8, MCL1, MYLK, 11 PLAUR, TIMP3, TNFRSF10B Cell Death apoptosis apoptosis of kidney cell lines 1.85E-02 BCL2, BCL2A1, BCL6, BIRC2, BIRC3, BLID, BNIP3L, CASP1, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CYCS, FADD, IER3, IL24, IRF1 (includes EG:16362), ITGB1, 22 MAPT, MCL1, P2RX7, PRMT2, SPP1 (includes EG:20750), TNFRSF10B Cell Death apoptosis apoptosis of lymphoblastoid 1.96E-02 BCL2, CDKN1A, CDKN2C, IRF4, SOD2, TERF1 6 cell lines Cell Death apoptosis apoptosis of ovarian cancer 1.98E-02 BCL2, BIRC2, CASP1, CASP9 (includes EG:100140945), CDKN1A, 12 cell lines DNAJC15, HTRA1, PRKDC, SPARC, TGFA, TSG101, UGCG Cell Death apoptosis apoptosis of leukocyte cell 2.38E-02 BCL2, CASP9 (includes EG:100140945), FADD, IFIH1, IRF4, 9 lines LGALS3, MCL1, PRKCZ, SHC1 (includes EG:20416) Cell Death apoptosis apoptosis of fibroblast cell 2.45E-02 BCL2, BLID, CASP9 (includes EG:100140945), CYR61, ITGA6, 7 lines ITGB1, TNFRSF10B Cell Death apoptosis apoptosis of lymphoma cell 2.46E-02 BCL2, BIRC3, CASP9 (includes EG:100140945), CD55, CD59 lines (includes EG:25407), CDKN1A, DUT, FADD, HSPB1, MCL1, 16 PLA2G6, PRKCZ, STAT3, TNFRSF10B, UGCG, YWHAZ Cell Death apoptosis apoptosis of hematopoietic cell 2.59E-02 BCL2, BIRC3, CASP9 (includes EG:100140945), FADD, IFIH1, 11 lines IRF4, LGALS3, MCL1, PRKCZ, SHC1 (includes EG:20416), TRIB2 Cell Death apoptosis apoptosis of microvascular 2.95E-02 BCL2, CASP9 (includes EG:100140945), CYCS, FOXO3, 5 endothelial cells TNFRSF10B Cell Death apoptosis apoptosis of fibroblasts 2.97E-02 BCL2, BIRC2, BIRC3, CASP9 (includes EG:100140945), CDKN1A, 9 ITGB1, MCL1, TIMP3, TNFRSF10B Cell Death apoptosis apoptosis of acute 3.58E-02 BCL2, MUC1 2 myeloblastic leukemia cells Cell Death apoptosis apoptosis of microvascular 3.58E-02 CASP9 (includes EG:100140945), CYCS 2

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smooth muscle cells Cell Death apoptosis apoptosis of muscle 3.92E-02 ADM, CASP1, CASP9 (includes EG:100140945), CYCS, IL8, MDK, 8 STAT3, TIMP3 Cell Death apoptosis apoptosis of kidney cancer cell 4.16E-02 BCL2, EPAS1, PRKAR1A, STAT3, VHL 5 lines Cell Death apoptosis apoptosis of synovial cells 4.25E-02 ADM, CD44 (includes EG:100330801), MCL1, SNAI2, TIMP3, 6 TNFRSF10B Cell Death apoptosis apoptosis of cervical cancer 4.36E-02 BCL2, BIRC2, BNIP3L, CASP1, CDKN1A, CDKN2C, FADD, FST, cell lines IER3, IRF1 (includes EG:16362), ITPK1, JUN, MAPKAP1, MCL1, 26 MUC1, NR4A2, PARVA, PCBP2, PLK1, PPP2R2A, RAB32, RASSF1, SNCA, TERF1, TIMP3, TNFRSF10B Cell Death apoptosis apoptosis of cancer cells 4.75E-02 BCL2, CASP1, CASP9 (includes EG:100140945), CDKN1A, FADD, IL24, MCL1, MUC1, NCOA3, NR4A2, PHLDA1, PLK1, TFAP2A, 15 TNFRSF10B, TNFSF13B Cell Death apoptosis apoptosis of carcinoma cell 4.82E-02 BCL2, CASP1, CCND1, CDKN2C, DNAJC15, FOXO3, GRN, lines IGFBP6, JAG1, MCL1, MET, PRKAR1A, PRKCZ, SPRY2, STAT3, 16 TNFRSF10B Cell Death apoptosis apoptosis of vascular 4.89E-02 ADM, AGGF1, BCL2, CASP1, CASP9 (includes EG:100140945), 9 endothelial cells CYCS, FOXO3, IL8, TNFRSF10B Cell Death inhibition inhibition of apoptosis 4.40E-04 ACAA2, ANXA5, BCL2, BCL2A1, BIRC3, CDKN2D, CRYAB, DHCR24, FAIM3, HSPA9, HSPB1, HTATIP2, IFI6, IL6ST, MCL1, 24 MUC1, OPA1, PRKCZ, SNCA, SOCS2, TAX1BP1, TNFAIP3, TXNDC5, YWHAZ Cell Death cell viability cell viability of stomach 6.43E-04 BCL2, CD44 (includes EG:100330801), ITGB1, MET, SPP1 (includes 5 cancer cell lines EG:20750) Cell Death cell viability cell viability of myeloma cell 2.59E-02 AGTRAP, BCL2, CKAP5, MCL1, MET, PLK1, PSMA1, PSMC3, 11 lines SLC25A23, STAT3, TNFSF13B Cell Death cell viability cell viability of B lymphocytes 2.92E-02 BCL2, BCL6, NFIL3, TNFSF13B 4 Cell Death cell viability cell viability of neuroblastoma 4.25E-02 BCL2, GPC1, MDK, S100A6, SLC2A1, SNCA 6 cell lines Cell Death necrosis necrosis 1.01E-03 ADM, AGGF1, AKAP12, ALDH1A1, ANTXR1, ANTXR2, APIP, BAG3, BCHE, BCL2, BCL2A1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BLID, BNIP3L, CABLES1, CASP1, CASP9 (includes EG:100140945), CCNB1, CCND1, CD44 (includes EG:100330801), 169 CD55, CD59 (includes EG:25407), CD74, CDK2, CDK5R1, CDKN1A, CDKN2C, CDKN2D, CEACAM1 (includes others), CIB1, CLCF1, CREB3L2, CRYAB, CYCS, CYP2J2, CYR61, DAB2, DCT,

384

DDIT4, DHCR24, DNAJC15, DUT, EGR1, EMP1, EPAS1, FADD, FAIM3, FBXO32, FOXO3, FST, GABRG2, GBA, GLRX, GPC1, GPI, GPR37, GRN, HDAC9, HIF1A, HLA-DRB4, HLA-G, HRK, HSPB1, HTATIP2, HTRA1, ID1, IER3, IFI6, IFIH1, IGFBP6, IGFBP7, IL24, IL6ST, IL8, ILK, IRF1 (includes EG:16362), IRF4, ITGA6, ITGB1, ITM2B, ITPK1, JAG1, JUN, KLF4, KLF9, LGALS3, LRPAP1, MAOA, MAPKAP1, MAPT, MAX, MCL1, MDK, MET, MITF, MSRB2, MST4, MT1X, MT2A, MTFP1, MUC1, MYLK, NAA35, NCOA3, NFATC1, NFIL3, NR4A2, NRP1 (includes EG:18186), NUAK1, P2RX7, PARVA, PBK, PCBP2, PHLDA1, PLA2G6, PLAUR, PLK1, PLK2, PPP2R2A, PRAME, PRKAR1A, PRKCA, PRKCZ, PRKDC, PRMT2, PTPN11, RAB32, RAC2, RASSF1, RHOC, S100A4, S100A6, SAT1, SDC1 (includes EG:20969), SEMA3A, SHC1 (includes EG:20416), SIRPA, SLC11A2, SNAI2, SNCA, SOD2, SPARC, SPP1 (includes EG:20750), SPRY2, SRPX, STAT3, STMN1, TERF1, TFAP2A, TFAP2C, TGFA, TGFB1I1, TIMP3, TLR1, TNFAIP3, TNFRSF10B, TNFSF13B, TSG101, TYMP, UBA7, UGCG, VAV3, VHL, YWHAZ, ZFYVE16 Cell Death degradation cellular degradation 1.74E-02 GPR37, MAPT, SNCA, TSG101 4 Cell Death cytolysis cytolysis of neuroblastoma cell 1.89E-02 CD55, CD59 (includes EG:25407) 2 lines Cell Death cytolysis cytolysis of tumor cell lines 2.74E-02 BCL2, CD55, CD59 (includes EG:25407), HLA-G, MUC1, 6 TNFRSF10B Cell Death cytolysis cytolysis of prostate cancer 3.58E-02 CD55, MUC1 2 cell lines Cell Death survival survival of chronic 1.89E-02 CD74, IL8 2 lymphocytic leukemia B cells Cell Death survival survival of pre-B lymphocytes 1.89E-02 NFIL3, TNFSF13B 2 Cell Death killing killing of breast cancer cell 3.15E-02 GLRX, SIRPA, TNFRSF10B 3 lines Cell Death loss loss of neurons 3.15E-02 BACE1, MAPT, SNCA 3 Cell Death activation-induced activation-induced cell death 3.52E-02 ADM, CASP9 (includes EG:100140945), CD59 (includes EG:25407), 5 cell death ITGB1, RAC2 Cell Death degeneration degeneration of dopaminergic 3.58E-02 GPR37, SNCA 2 neurons Cell Death degeneration degeneration of neurons 4.23E-02 GPR37, MAPT, SNCA 3

385

Cellular Development proliferation proliferation of prostate cancer 3.36E-04 ADM, AFAP1, ALDH1A1, CCND1, CD44 (includes EG:100330801), cell lines CDKN1A, CEACAM1 (includes others), CTNNBIP1, DAB2, FSCN1, FST, HAS3, HIF1A, IGFBP7, IL24, IL8, ITGB1, LCP1, LGALS3, 32 LZTS1, MET, MST4, NCOA3, NR1H3, RASSF1, RHOC, SAT1, SHC1 (includes EG:20416), STAT3, TGFA, TGFB1I1, URI1 Cellular Development proliferation proliferation of pulmonary 5.45E-04 ITGA5, ITGB1, VHL 3 fibroblasts Cellular Development proliferation proliferation of ovarian cancer 1.46E-03 BCL2, CCND1, CD44 (includes EG:100330801), DAB2, FOXO3, cell lines FST, GRN, HAS2, HAS3, PRKAR1A, SERTAD1, SOD2, SPARC, 16 STAT3, TGFA, TSG101 Cellular Development proliferation proliferation of pancreatic 7.51E-03 ADM, CCND1, CDKN1A, CEACAM1 (includes others), HIF1A, 12 cancer cell lines MET, MTUS1, NCOA3, PRKCA, S100A6, SOD2, STAT3 Cellular Development proliferation proliferation of melanoma 9.18E-03 CD44 (includes EG:100330801), EGR1, MCAM, POU3F2 4 cells Cellular Development proliferation proliferation of epithelial cell 9.31E-03 ABCC5, CASP9 (includes EG:100140945), CCND1, CDK2, lines CDKN1A, CYR61, DAB2, EGR1, ENC1, FSCN1, ITGA5, MET, 17 MST4, PLAUR, QPCT, RASSF1, STAT3 Cellular Development proliferation proliferation of breast cancer 1.17E-02 ASAH1, BCL2, CCND1, CD151, CD44 (includes EG:100330801), cell lines CDC16, CDKN1A, CYR61, DAB2, EEF1A1, GPC1, GRN, HAS2, ID1, IER3, IL24, IRF1 (includes EG:16362), ITGA5, JUN, LGALS3, MET, NCOA3, NDUFAF4, NR1H3, NRP1 (includes EG:18186), 40 PARVB, PBK, PLAUR, PLK1, PRKCA, RALA, SAT1, SEPT9, SOD2, SOX2, SPP1 (includes EG:20750), STAT3, TFAP2A, TFAP2C, TGFA Cellular Development proliferation proliferation of tumor cell 1.32E-02 A2M, ADM, AFAP1, AKR1C3, ALDH1A1, ASAH1, BCL2, BCL6, lines BIRC2, CABLES1, CAMK2N1, CCL20, CCND1, CD151, CD44 (includes EG:100330801), CDC16, CDK14, CDK2, CDK5R1, CDKN1A, CDKN2B, CEACAM1 (includes others), CREG1, CTNNBIP1, CYP2J2, CYR61, DAB2, DUSP5, EED, EEF1A1, EGR1, ENC1, EPS8, ERRFI1, FGFRL1, FNTA, FOXO3, FSCN1, FST, GAB2, GHR, GPC1, GRN, H2AFY, HAS2, HAS3, HIF1A, ID1, IER3, IGFBP7, IL24, IL6ST, IL8, ILK, IRF1 (includes EG:16362), 128 ITGA5, ITGB1, JAG1, JUN, KLF4, LCP1, LDHA, LGALS3, LZTS1, MAX, MDK, MET, MST4, MT1A, MT2A, MTUS1, MUC1, MXI1, NAA35, NCOA3, NDUFAF4, NOV, NR1H3, NR4A2, NRP1 (includes EG:18186), OLIG2, PARVB, PBK, PLA2G6, PLAUR, PLK1, PRAME, PRKAR1A, PRKCA, PTPN11, PTPN22, PTPRR, RALA, RASSF1, RHOC, S100A6, SAT1, SEMA6B, SEPT9, SERTAD1, SHC1 (includes EG:20416), SLC26A2, SLC2A1, SOD2, SOX2, SPARC, SPP1 (includes EG:20750), SPRY2, ST8SIA1,

386

STAT3, STMN1, TERF1, TFAP2A, TFAP2C, TGFA, TGFB1I1, THBS2, TIMP3, TNFRSF10B, TNFSF13B, TRIB2, TSG101, TXNIP, UGCG, URI1, VHL, VMP1, ZFP36 Cellular Development proliferation proliferation of tumor cells 1.43E-02 CASP1, CCND1, CD44 (includes EG:100330801), CDKN1A, CYR61, EGR1, FST, GRN, ID1, ILK, JAG1, LZTS1, MCAM, MUC1, 22 NR4A2, PEG10, PLAUR, PLK1, POU3F2, PRKCA, TNFSF13B, ZMAT3 Cellular Development proliferation proliferation of endothelial 1.53E-02 ADM, AGGF1, ARHGAP24, CD44 (includes EG:100330801), cells CDKN1A, CEACAM1 (includes others), DAB2, ECM1 (includes EG:100332249), EDNRB, FOXO3, HAS3, HIF1A, IL8, ITGB1, 22 MDK, MGP, MYOF, NRP1 (includes EG:18186), PRKCA, PTPRM, SCG2, SEMA6B Cellular Development proliferation proliferation of kidney cancer 1.64E-02 MET, RASSF1, STAT3, TGFA, VHL, VMP1 6 cell lines Cellular Development proliferation proliferation of brain cancer 4.45E-02 A2M, CDKN1A, CDKN2B, CYR61, EGR1, GRN, IL24, IL8, JAG1, 15 cell lines MET, MXI1, PLAUR, PRKCA, SEMA6B, TGFA Cellular Development proliferation proliferation of muscle cells 4.65E-02 APOD, CTNNBIP1, IL8, ITGA5, ITGA6, PLAUR, PRKCZ, PTPN11, SHC1 (includes EG:20416), SPP1 (includes EG:20750), ST8SIA1, 12 TNFAIP3 Cellular Development epithelial- epithelial-mesenchymal 7.99E-04 GPI, HIF1A, ID1, JAG1, NRP1 (includes EG:18186), PLAUR, mesenchymal transition of tumor cell lines STMN1, ZEB2 8 transition Cellular Development epithelial- epithelial-mesenchymal 3.76E-03 CD44 (includes EG:100330801), GPI, HIF1A, ID1, JAG1, NRP1 mesenchymal transition (includes EG:18186), PLAUR, S100A4, SNAI2, STMN1, ZEB2 11 transition Cellular Development epithelial- epithelial-mesenchymal 2.23E-02 GPI, PLAUR, STMN1 mesenchymal transition of breast cancer cell 3 transition lines Cellular Development epithelial- epithelial-mesenchymal 2.23E-02 HIF1A, ID1, NRP1 (includes EG:18186) mesenchymal transition of prostate cancer 3 transition cell lines Cellular Development development endothelial cell development 2.08E-03 ADM, AGGF1, ARHGAP24, CD151, CD44 (includes EG:100330801), CDKN1A, CEACAM1 (includes others), DAB2, ECM1 (includes EG:100332249), EDNRB, FOXO3, HAS3, HIF1A, 27 IL8, ITGB1, KLHL20, LGALS3, MDK, MET, MGP, MYOF, NRP1 (includes EG:18186), PRKCA, PTPRM, SCG2, SEMA6B, TFAP2A Cellular Development development development of tumor cell 2.92E-02 ITGA6, MET, PLAUR, VHL 4 lines

387

Cellular Development development development of vascular 4.47E-02 CD151, KLHL20, LGALS3, TFAP2A 4 endothelial cells Cellular Development differentiation differentiation of tumor cell 2.83E-03 AKR1C3, BCL2, BHLHE40, CCND1, CDK5R1, CDKN1A, CREG1, lines CYR61, FOXO3, FST, HIF1A, IRF1 (includes EG:16362), JAG1, 27 KLF4, MAX, MCL1, MET, MST4, MUC1, PRKAR1A, SNCA, SSBP2, ST8SIA1, STAT3, STIM1, TGFA, VHL Cellular Development differentiation differentiation of cells 3.15E-03 ADM, AKR1C3, BCL2, BCL6, BHLHE40, BIRC2, CCND1, CDK5R1, CDKN1A, CDKN2B, CHD7, CLIC4, CREG1, CTNNBIP1, CYR61, DYRK3, FADD, FOXO3, FSCN1, FST, GAB2, GAS7, GPR55, HAS2, HDAC9, HIF1A, ID1, IL24, IL6ST, IL8, IRF1 (includes EG:16362), ITGA6, ITGB1, JAG1, JUN, KAT2B, KLF4, 71 LRP5, LTBP4, MAPT, MAX, MCL1, MDK, MEF2C, MET, MGP, MLLT11, MST4, MUC1, NCOA3, PPARGC1A, PRAME, PRKAR1A, PRKCA, SNAI2, SNCA, SORT1, SOX2, SPP1 (includes EG:20750), SSBP2, ST8SIA1, STAT3, STIM1, TGFA, TLR1, TNFRSF10B, TNFSF13B, TRIB2, TSG101, TXNIP, VHL Cellular Development differentiation differentiation of endothelial 1.48E-02 ADM, FADD, IL24 3 cell lines Cellular Development differentiation differentiation of leukemia cell 2.79E-02 AKR1C3, CDK5R1, CREG1, FOXO3, HIF1A, KLF4, MAX, MCL1, 14 lines MST4, MUC1, PRKAR1A, ST8SIA1, STAT3, STIM1 Cellular Development differentiation differentiation of keratinocyte 3.15E-02 BHLHE40, CYR61, JAG1 3 cancer cell lines Cellular Development differentiation onset of differentiation of 3.58E-02 CYR61, JAG1 2 keratinocyte cancer cell lines Cellular Development growth arrest in growth of kidney 6.68E-03 STAT3, VHL 2 cancer cell lines Cellular Development growth arrest in growth of tumor cell 1.13E-02 BCL2, CCND1, CDK2, CDKN1A, CDKN2B, DAB2, GAB2, IL6ST, 13 lines IRF1 (includes EG:16362), KLF4, MUC1, STAT3, VHL Cellular Development growth arrest in growth of colon 3.58E-02 CDKN1A, KLF4 2 cancer cell lines Cellular Development growth arrest in growth of leukemia 4.23E-02 BCL2, GAB2, MUC1 3 cell lines Cellular Development tubulation tubulation of endothelial cells 7.02E-03 EDNRB, FOXO3, ID1, IL8, KCNMA1, MGP, PLAUR, PRKCA, 9 STIM1 Cellular Development morphogenesis morphogenesis of breast cell 1.89E-02 CCND1, HIF1A 2 lines Cellular Development outgrowth outgrowth of tumor cell lines 1.89E-02 CCND1, CYR61 2

388

Cellular Development angiogenesis angiogenesis of endothelial 4.23E-02 CD151, KLHL20, MET 3 cells Cellular Growth and proliferation proliferation of prostate cancer 3.36E-04 ADM, AFAP1, ALDH1A1, CCND1, CD44 (includes EG:100330801), Proliferation cell lines CDKN1A, CEACAM1 (includes others), CTNNBIP1, DAB2, FSCN1, FST, HAS3, HIF1A, IGFBP7, IL24, IL8, ITGB1, LCP1, LGALS3, 32 LZTS1, MET, MST4, NCOA3, NR1H3, RASSF1, RHOC, SAT1, SHC1 (includes EG:20416), STAT3, TGFA, TGFB1I1, URI1 Cellular Growth and proliferation proliferation of pulmonary 5.45E-04 ITGA5, ITGB1, VHL 3 Proliferation fibroblasts Cellular Growth and proliferation proliferation of ovarian cancer 1.46E-03 BCL2, CCND1, CD44 (includes EG:100330801), DAB2, FOXO3, Proliferation cell lines FST, GRN, HAS2, HAS3, PRKAR1A, SERTAD1, SOD2, SPARC, 16 STAT3, TGFA, TSG101 Cellular Growth and proliferation proliferation of lung cells 2.67E-03 CCND1, HLA-DPB1, ITGA5, ITGB1, VHL 5 Proliferation Cellular Growth and proliferation proliferation of pancreatic 7.51E-03 ADM, CCND1, CDKN1A, CEACAM1 (includes others), HIF1A, 12 Proliferation cancer cell lines MET, MTUS1, NCOA3, PRKCA, S100A6, SOD2, STAT3 Cellular Growth and proliferation proliferation of melanoma 9.18E-03 CD44 (includes EG:100330801), EGR1, MCAM, POU3F2 4 Proliferation cells Cellular Growth and proliferation proliferation of epithelial cell 9.31E-03 ABCC5, CASP9 (includes EG:100140945), CCND1, CDK2, Proliferation lines CDKN1A, CYR61, DAB2, EGR1, ENC1, FSCN1, ITGA5, MET, 17 MST4, PLAUR, QPCT, RASSF1, STAT3 Cellular Growth and proliferation proliferation of breast cancer 1.17E-02 ASAH1, BCL2, CCND1, CD151, CD44 (includes EG:100330801), Proliferation cell lines CDC16, CDKN1A, CYR61, DAB2, EEF1A1, GPC1, GRN, HAS2, ID1, IER3, IL24, IRF1 (includes EG:16362), ITGA5, JUN, LGALS3, MET, NCOA3, NDUFAF4, NR1H3, NRP1 (includes EG:18186), 40 PARVB, PBK, PLAUR, PLK1, PRKCA, RALA, SAT1, SEPT9, SOD2, SOX2, SPP1 (includes EG:20750), STAT3, TFAP2A, TFAP2C, TGFA Cellular Growth and proliferation proliferation of tumor cell 1.32E-02 A2M, ADM, AFAP1, AKR1C3, ALDH1A1, ASAH1, BCL2, BCL6, Proliferation lines BIRC2, CABLES1, CAMK2N1, CCL20, CCND1, CD151, CD44 (includes EG:100330801), CDC16, CDK14, CDK2, CDK5R1, CDKN1A, CDKN2B, CEACAM1 (includes others), CREG1, CTNNBIP1, CYP2J2, CYR61, DAB2, DUSP5, EED, EEF1A1, EGR1, ENC1, EPS8, ERRFI1, FGFRL1, FNTA, FOXO3, FSCN1, FST, 128 GAB2, GHR, GPC1, GRN, H2AFY, HAS2, HAS3, HIF1A, ID1, IER3, IGFBP7, IL24, IL6ST, IL8, ILK, IRF1 (includes EG:16362), ITGA5, ITGB1, JAG1, JUN, KLF4, LCP1, LDHA, LGALS3, LZTS1, MAX, MDK, MET, MST4, MT1A, MT2A, MTUS1, MUC1, MXI1, NAA35, NCOA3, NDUFAF4, NOV, NR1H3, NR4A2, NRP1

389

(includes EG:18186), OLIG2, PARVB, PBK, PLA2G6, PLAUR, PLK1, PRAME, PRKAR1A, PRKCA, PTPN11, PTPN22, PTPRR, RALA, RASSF1, RHOC, S100A6, SAT1, SEMA6B, SEPT9, SERTAD1, SHC1 (includes EG:20416), SLC26A2, SLC2A1, SOD2, SOX2, SPARC, SPP1 (includes EG:20750), SPRY2, ST8SIA1, STAT3, STMN1, TERF1, TFAP2A, TFAP2C, TGFA, TGFB1I1, THBS2, TIMP3, TNFRSF10B, TNFSF13B, TRIB2, TSG101, TXNIP, UGCG, URI1, VHL, VMP1, ZFP36 Cellular Growth and proliferation proliferation of tumor cells 1.43E-02 CASP1, CCND1, CD44 (includes EG:100330801), CDKN1A, Proliferation CYR61, EGR1, FST, GRN, ID1, ILK, JAG1, LZTS1, MCAM, MUC1, 22 NR4A2, PEG10, PLAUR, PLK1, POU3F2, PRKCA, TNFSF13B, ZMAT3 Cellular Growth and proliferation proliferation of endothelial 1.53E-02 ADM, AGGF1, ARHGAP24, CD44 (includes EG:100330801), Proliferation cells CDKN1A, CEACAM1 (includes others), DAB2, ECM1 (includes EG:100332249), EDNRB, FOXO3, HAS3, HIF1A, IL8, ITGB1, 22 MDK, MGP, MYOF, NRP1 (includes EG:18186), PRKCA, PTPRM, SCG2, SEMA6B Cellular Growth and proliferation proliferation of kidney cancer 1.64E-02 MET, RASSF1, STAT3, TGFA, VHL, VMP1 6 Proliferation cell lines Cellular Growth and proliferation proliferation of cells 2.39E-02 A2M, ABCC5, ADM, AFAP1, AGGF1, AHCY, AKR1C1/AKR1C2, Proliferation AKR1C3, ALDH1A1, APOD, ARHGAP24, ARL3, ASAH1, ATP5G1, AXIN2, BCL2, BCL2A1, BCL3, BCL6, BHLHE40, BIRC2, BST2, CABLES1, CALM1 (includes others), CAMK2N1, CASP1, CASP9 (includes EG:100140945), CCL20, CCND1, CD151, CD44 (includes EG:100330801), CD55, CD63, CD74, CD83, CD9, CDC16, CDK14, CDK2, CDK20, CDK5R1, CDKN1A, CDKN2B, CDKN2C, CDKN2D, CEACAM1 (includes others), CITED2, CLCF1, CREG1, CTDSPL, CTNNBIP1, CYP2J2, CYR61, DAB2, DAP, DDR1, DPAGT1, DUSP5, ECM1 (includes EG:100332249), EDNRB, EED, EEF1A1, EGR1, EIF5A2, ELF4, EMP1, ENC1, EPAS1, EPS8, 233 ERRFI1, ETFB, FGFRL1, FHL2, FLOT1, FNTA, FOXO3, FSCN1, FST, GAB2, GHR, GPC1, GPI, GPNMB, GRN, H2AFY, HAS2, HAS3, HIF1A, HLA-DPB1, HNRNPU, ID1, IER3, IFITM1, IGFBP6, IGFBP7, IL24, IL6ST, IL8, ILK, INSIG1, IRF1 (includes EG:16362), IRF4, ITGA3, ITGA5, ITGA6, ITGB1, JAG1, JUN, KAT2B, KCNMA1, KCNN4, KLF4, LAMA5, LCP1, LDHA, LEPREL1, LGALS3, LRP5, LTBP4, LZTS1, MAGED2, MAPT, MAX, MCAM, MCL1, MDK, MET, MGP, MST4, MT1A, MT2A, MTUS1, MUC1, MXI1, MYOF, NAA35, NCK2, NCOA3, NDUFAF4, NES, NOV, NR1H3, NR4A2, NRP1 (includes EG:18186), NTN4, OLIG2, PARVB, PBK, PDIA5, PEG10, PFDN5, PFN2, PHLDA1, PHLDA2,

390

PLA2G6, PLAUR, PLCE1, PLIN3, PLK1, PLP1 (includes EG:18823), POU3F2, PRAME, PRKAR1A, PRKCA, PRKCZ, PRKRIR, PSMC3, PTGS1, PTPN11, PTPN22, PTPRM, PTPRR, QPCT, RAC2, RALA, RASSF1, RHOC, RPS6KA2, S100A6, SAT1, SCG2, SEMA6B, SEPT9, SERTAD1, SERTAD2, SERTAD3, SF3B3, SHC1 (includes EG:20416), SIRPA, SLC22A1, SLC26A2, SLC2A1, SMARCD3, SNAI2, SOD2, SOX2, SPARC, SPP1 (includes EG:20750), SPRY2, ST8SIA1, STAT3, STMN1, TBC1D8, TERF1, TFAP2A, TFAP2C, TGFA, TGFB1I1, THBS2, TIMP3, TNFAIP3, TNFRSF10B, TNFSF13B, TRIB2, TRIM33, TSG101, TXNIP, TYR, TYRP1, UBE2L6, UBR5, UGCG, URI1, USP4, VAV3, VHL, VMP1, WDR6, ZBED1, ZFP36, ZMAT3, ZNF217, ZYX Cellular Growth and proliferation proliferation of brain cancer 4.45E-02 A2M, CDKN1A, CDKN2B, CYR61, EGR1, GRN, IL24, IL8, JAG1, 15 Proliferation cell lines MET, MXI1, PLAUR, PRKCA, SEMA6B, TGFA Cellular Growth and proliferation proliferation of muscle cells 4.65E-02 APOD, CTNNBIP1, IL8, ITGA5, ITGA6, PLAUR, PRKCZ, PTPN11, Proliferation SHC1 (includes EG:20416), SPP1 (includes EG:20750), ST8SIA1, 12 TNFAIP3 Cellular Growth and colony formation colony formation 1.45E-03 ALDH1A1, BCL2, CABLES1, CADM1, CASP9 (includes Proliferation EG:100140945), CCND1, CDKN1A, CEACAM1 (includes others), CYP2J2, CYR61, DYRK3, EPAS1, FOXO3, GRN, HAS2, IFIH1, IGFBP7, IL6ST, KLF4, LAPTM4B, LGALS3, LUM, LZTS1, MDK, 41 MET, MST4, MT1X, NCOA3, PBK, PHLDA1, PLK1, PRKAR1A, PSMC3, RASSF1, RHOC, RPS6KA5, SERTAD1, SOD2, SPARC, SPRY2, TGFB1I1 Cellular Growth and colony formation colony formation of cells 2.30E-03 ALDH1A1, BCL2, CABLES1, CADM1, CASP9 (includes Proliferation EG:100140945), CCND1, CDKN1A, CEACAM1 (includes others), CYP2J2, CYR61, DYRK3, EPAS1, FOXO3, GRN, HAS2, IFIH1, IGFBP7, IL6ST, KLF4, LAPTM4B, LUM, LZTS1, MDK, MET, 40 MST4, MT1X, NCOA3, PBK, PHLDA1, PLK1, PRKAR1A, PSMC3, RASSF1, RHOC, RPS6KA5, SERTAD1, SOD2, SPARC, SPRY2, TGFB1I1 Cellular Growth and colony formation colony formation of epithelial 4.00E-03 CCND1, LZTS1, MST4, SOD2 4 Proliferation cell lines Cellular Growth and colony formation colony formation of tumor cell 6.60E-03 ALDH1A1, BCL2, CABLES1, CADM1, CASP9 (includes Proliferation lines EG:100140945), CDKN1A, CEACAM1 (includes others), CYP2J2, CYR61, EPAS1, FOXO3, GRN, HAS2, IGFBP7, IL6ST, KLF4, 29 LUM, LZTS1, MDK, MST4, MT1X, NCOA3, PBK, PLK1, PRKAR1A, RASSF1, SERTAD1, SOD2, SPARC Cellular Growth and colony formation colony formation of kidney 9.02E-03 LZTS1, MST4, SOD2 3 Proliferation cell lines

391

Cellular Growth and colony formation colony formation of embryonic 1.48E-02 LZTS1, MST4, SOD2 3 Proliferation cell lines Cellular Growth and formation formation of chondrocytes 6.68E-03 ITGA5, ITGB1 2 Proliferation Cellular Growth and growth arrest in growth of kidney 6.68E-03 STAT3, VHL 2 Proliferation cancer cell lines Cellular Growth and growth arrest in growth of tumor cell 1.13E-02 BCL2, CCND1, CDK2, CDKN1A, CDKN2B, DAB2, GAB2, IL6ST, 13 Proliferation lines IRF1 (includes EG:16362), KLF4, MUC1, STAT3, VHL Cellular Growth and growth arrest in growth of cells 1.31E-02 BCL2, CCND1, CDK2, CDKN1A, CDKN2B, DAB2, EGR1, FOXO3, Proliferation GAB2, IL6ST, IRF1 (includes EG:16362), KLF4, MUC1, NRP1 16 (includes EG:18186), STAT3, VHL Cellular Growth and growth arrest in growth of colon 3.58E-02 CDKN1A, KLF4 2 Proliferation cancer cell lines Cellular Growth and growth arrest in growth of leukemia 4.23E-02 BCL2, GAB2, MUC1 3 Proliferation cell lines Cellular Growth and outgrowth outgrowth of tumor cell lines 1.89E-02 CCND1, CYR61 2 Proliferation Cellular Growth and cytostasis cytostasis 4.86E-02 ABCC5, BCL6, BHLHE40, CCND1, CDKN1A, CDKN2C, Proliferation CDKN2D, CEACAM1 (includes others), CYR61, FOXO3, GPI, ID1, 20 IRF1 (includes EG:16362), JAG1, MXI1, PFDN5, PRKAR1A, PRKCA, TRIM33, ZNF217 Cellular Growth and stimulation stimulation of T lymphocytes 4.86E-02 CD83, HLA-G, MUC1, TNFSF13B, TYR 5 Proliferation

392

Appendix 6. 4 The top molecular and cellular functions associated with differentially expressed genes in the GGH-inhibited MDA-MB-435 breast cancer cells

No. of Category Function Function Annotation P-value Genes Genes Cell Death cell death cell death 1.62E-08 ADM, ADRM1, AGGF1, AKAP12, ALDH1A1, ALKBH8, ANTXR1, 173 BCHE, BCL2A1, BCL2L1, BIRC5, BLVRA, CADM1, CALB2, CAPN3, CASP1, CASP4, CCND1, CCNE1, CD74, CD9, CD96, CDC20 (includes EG:107995), CDK1, CDK2, CDK5, CDKN1A, CDKN1B, CEACAM1 (includes others), CEBPD, CHEK1, CKAP5, CLCF1, CLEC11A, CREM, CTGF, CYR61, DAPK3, DEFB103A/DEFB103B, DEPTOR, DHRS2, DKK1, DPF2, DUSP10, DUSP14, DUSP5, EDNRB, EIF4E, ELAVL1, EMP1, EPAS1, FHL2, FOSB, FOXO3, FXR1, GABPB1, GABRG2, GLIPR1, GSK3B, HBEGF, HDAC4, HEY1, HIF1A, HK1, HLA-DRB4, HMGA2, HOXA5, HRK, HSPA8, ID1, ID2, ID3 (includes EG:15903), ID4, IFI6, IGFBP6, IGFBP7, IL6ST, IL7, IL8, ING3, IRS1, ITGA5, ITPK1, KCNMA1, KLF11, KLF4, KLF9, LAMP2, LMNB1, LPAR1, LUC7L3, MAP1S, MAP3K4, MAPT, MCL1, MCTS1, MET, MGST1, MITF, MLLT11, MSX1, MT1X, MT2A, MUC1, MX1, MYLK, NCAM1, NKX3-1, NME1 (includes EG:18102), NR4A2, NT5E, OBFC2A, P2RX7, PARVA, PCBP2, PCGF2, PDCD10, PDRG1, PHLDA1, PHLDA2, PLA2G6, PLAUR, PMP22, PPP1R15A, PRMT2, PRUNE2, PSIP1, PSMC3, PTGR1, PTN, RAP1B, RASSF1, RHOC, RHOT1, RND3, RPS6KA2, S100A4, SAT1, SFR1, SGK1, SIRPA, SLC25A23, SLC2A1, SMAD3, SMAD6, SNCA, SOD2, SORBS2, SPC25 (includes EG:100144563), SPP1 (includes EG:20750), ST6GAL1, STAT1, STAT3, STAT5A, STC1, STX8, TCF12, TFAP2A, TFRC, TGFB1I1, TGFBR2, TGFBR3, TIMP3, TMED10, TNFAIP3, TOP2A, TOP2B, TRPM2, TXNRD1, UBE2C, UBQLN1, VAV3, ZC3H8 Cell Death cell death cell death of cervical cancer 4.18E-08 ADRM1, BCL2A1, BCL2L1, BIRC5, CASP1, CASP4, CDC20 38 cell lines (includes EG:107995), CDK1, CDKN1A, CDKN1B, CHEK1, EIF4E, ELAVL1, GSK3B, HDAC4, ITPK1, MAP3K4, MCL1, MSX1, MUC1, NME1 (includes EG:18102), NR4A2, OBFC2A, PARVA, PCBP2, PSIP1, RASSF1, SGK1, SNCA, SOD2, SPC25 (includes EG:100144563), SPP1 (includes EG:20750), STAT1, TFAP2A, TIMP3, TOP2A, TOP2B, UBQLN1 Cell Death cell death cell death of tumor cells 1.75E-05 BCL2L1, BIRC5, CASP1, CD74, CDK2, CDKN1A, CDKN1B, 23 CEACAM1 (includes others), CHEK1, GSK3B, HBEGF, HIF1A, HMGA2, IL7, IL8, LPAR1, MCL1, MUC1, NR4A2, PHLDA1,

393

STAT3, TFAP2A, TGFBR2 Cell Death cell death cell death of brain cancer cell 6.52E-05 BCL2L1, BIRC5, CASP1, CDK1, CHEK1, DKK1, EPAS1, FOXO3, 16 lines HIF1A, MCL1, MET, MITF, SIRPA, SNCA, SOD2, STAT3 Cell Death cell death cell death of tumor cell lines 1.40E-04 ADM, ADRM1, AKAP12, BCHE, BCL2A1, BCL2L1, BIRC5, 91 CASP1, CASP4, CCND1, CCNE1, CDC20 (includes EG:107995), CDK1, CDK5, CDKN1A, CDKN1B, CEACAM1 (includes others), CEBPD, CHEK1, CREM, CTGF, CYR61, DAPK3, DKK1, EIF4E, ELAVL1, EPAS1, FOSB, FOXO3, GLIPR1, GSK3B, HBEGF, HDAC4, HIF1A, HLA-DRB4, HOXA5, HSPA8, ID1, ID3 (includes EG:15903), IFI6, IGFBP6, IGFBP7, IL7, IL8, ITPK1, KLF4, KLF9, LAMP2, LMNB1, MAP3K4, MCL1, MET, MITF, MSX1, MT1X, MT2A, MUC1, NCAM1, NME1 (includes EG:18102), NR4A2, OBFC2A, PARVA, PCBP2, PLA2G6, PLAUR, PPP1R15A, PSIP1, RASSF1, RHOC, S100A4, SAT1, SGK1, SIRPA, SNCA, SOD2, SPC25 (includes EG:100144563), SPP1 (includes EG:20750), ST6GAL1, STAT1, STAT3, TFAP2A, TFRC, TGFBR3, TIMP3, TMED10, TNFAIP3, TOP2A, TOP2B, UBE2C, UBQLN1, VAV3 Cell Death cell death cell death of myeloma cell 1.93E-04 BCL2L1, CDK1, CDKN1B, CHEK1, GSK3B, HLA-DRB4, IFI6, 11 lines KLF9, MCL1, NCAM1, TOP2A Cell Death cell death cell death of epithelial cells 5.31E-04 ALDH1A1, BCL2A1, BCL2L1, BIRC5, CASP1, EMP1, HK1, IL8, 26 IRS1, MAPT, MCL1, MET, MYLK, P2RX7, PLAUR, PMP22, PPP1R15A, PRMT2, RASSF1, RND3, SPP1 (includes EG:20750), TCF12, TFRC, TGFB1I1, TIMP3, TRPM2 Cell Death cell death cell death of kidney cells 1.47E-03 BCL2A1, BCL2L1, BIRC5, CASP1, CDKN1B, CTGF, EMP1, 23 GSK3B, HK1, IRS1, MAPT, MCL1, P2RX7, PMP22, PPP1R15A, PRMT2, RASSF1, SOD2, SPP1 (includes EG:20750), TCF12, TFRC, TGFB1I1, TRPM2 Cell Death cell death cell death of prostate cancer 1.79E-03 AKAP12, BCL2L1, BIRC5, CASP1, CDK5, CHEK1, FOXO3, 18 cell lines GLIPR1, GSK3B, HBEGF, HIF1A, ID1, IGFBP7, MCL1, MET, PLAUR, STAT3, TMED10 Cell Death cell death cell death of cancer cells 3.12E-03 BCL2L1, BIRC5, CASP1, CD74, CDKN1A, CDKN1B, CHEK1, 16 GSK3B, IL7, IL8, LPAR1, MCL1, MUC1, NR4A2, PHLDA1, TFAP2A Cell Death cell death cell death of lymphoblastoid 5.74E-03 CCNE1, CDKN1A, CHEK1, HLA-DRB4, SOD2, VAV3 6 cell lines Cell Death cell death cell death of epithelial cell 5.77E-03 BCL2A1, BCL2L1, BIRC5, CASP1, EMP1, HK1, IRS1, MAPT, 20 lines MCL1, MET, P2RX7, PMP22, PPP1R15A, PRMT2, RASSF1, SPP1 (includes EG:20750), TCF12, TFRC, TGFB1I1, TRPM2

394

Cell Death cell death cell death of breast cancer 7.92E-03 ADM, BCL2L1, BIRC5, CASP1, CCND1, CDK1, CDKN1A, 24 cell lines CEACAM1 (includes others), CHEK1, CTGF, CYR61, FOXO3, GSK3B, HIF1A, HOXA5, MCL1, MUC1, NME1 (includes EG:18102), PSIP1, SOD2, STAT1, TFAP2A, TFRC, TGFBR3 Cell Death cell death cell death of embryonic cell 8.95E-03 BCL2A1, BCL2L1, BIRC5, CASP1, EMP1, HK1, IRS1, MAPT, 19 lines MCL1, P2RX7, PMP22, PPP1R15A, PRMT2, RASSF1, SPP1 (includes EG:20750), TCF12, TFRC, TGFB1I1, TRPM2 Cell Death cell death cell death of colon cancer 9.59E-03 BCL2L1, BIRC5, CASP1, CDK1, CDKN1A, CDKN1B, CEACAM1 21 cell lines (includes others), CHEK1, FOXO3, GLIPR1, GSK3B, HOXA5, MCL1, MUC1, PPP1R15A, SGK1, SOD2, SPP1 (includes EG:20750), ST6GAL1, STAT1, UBE2C Cell Death cell death cell death of fibrosarcoma 9.61E-03 BCL2A1, BCL2L1, BIRC5, CDKN1A, SOD2, STAT1, TOP2A 7 cell lines Cell Death cell death cell death of kidney cell lines 1.02E-02 BCL2A1, BCL2L1, BIRC5, CASP1, EMP1, GSK3B, HK1, IRS1, 20 MAPT, MCL1, P2RX7, PMP22, PPP1R15A, PRMT2, RASSF1, SPP1 (includes EG:20750), TCF12, TFRC, TGFB1I1, TRPM2 Cell Death cell death cell death of stomach cancer 1.09E-02 BCL2L1, BIRC5, HIF1A, MET, MT1X 5 cell lines Cell Death cell death cell death of macrophages 2.10E-02 BCL2L1, MCL1, SOD2, STAT3 4 Cell Death cell death cell death of blood cells 2.24E-02 BCL2L1, CASP4, CDK1, CDKN1B, GSK3B, HLA-DRB4, HRK, 20 IL7, IL8, KLF4, MCL1, MCTS1, MX1, P2RX7, PCBP2, SOD2, SPP1 (includes EG:20750), STAT3, STAT5A, ZC3H8 Cell Death cell death cell death of leukemia cell 2.32E-02 BCL2A1, BCL2L1, BIRC5, CDKN1A, CDKN1B, CEACAM1 21 lines (includes others), CEBPD, CREM, FOXO3, GSK3B, HLA-DRB4, HSPA8, IL7, KLF4, MCL1, MET, MUC1, RHOC, SOD2, TNFAIP3, TOP2A Cell Death cell death cell death of immune cells 2.91E-02 BCL2L1, CASP4, CDK1, CDKN1B, GSK3B, HLA-DRB4, IL7, IL8, 19 KLF4, MCL1, MCTS1, MX1, P2RX7, PCBP2, SOD2, SPP1 (includes EG:20750), STAT3, STAT5A, ZC3H8 Cell Death cell death cell death of antigen 3.03E-02 BCL2L1, CDKN1B, MCL1, SOD2, STAT3 5 presenting cells Cell Death apoptosis apoptosis 1.63E-07 ADM, AGGF1, AKAP12, ALDH1A1, BCL2A1, BCL2L1, BIRC5, 129 BLVRA, CAPN3, CASP1, CASP4, CCND1, CCNE1, CD74, CDC20 (includes EG:107995), CDK1, CDK2, CDK5, CDKN1A, CDKN1B, CEACAM1 (includes others), CEBPD, CHEK1, CLCF1, CREM, CTGF, CYR61, DAPK3, DEPTOR, DHRS2, DKK1, DPF2, EDNRB, EIF4E, ELAVL1, EPAS1, FHL2, FOSB, FOXO3, FXR1, GABPB1, GLIPR1, GSK3B, HBEGF, HDAC4, HEY1, HIF1A, HMGA2,

395

HOXA5, HRK, HSPA8, ID1, ID3 (includes EG:15903), IFI6, IGFBP6, IGFBP7, IL6ST, IL7, IL8, ING3, IRS1, ITGA5, ITPK1, KCNMA1, KLF4, KLF9, LMNB1, LPAR1, LUC7L3, MAP1S, MAP3K4, MAPT, MCL1, MCTS1, MET, MITF, MLLT11, MSX1, MT2A, MUC1, MX1, MYLK, NCAM1, NME1 (includes EG:18102), NR4A2, NT5E, P2RX7, PARVA, PCBP2, PDCD10, PHLDA1, PHLDA2, PLA2G6, PLAUR, PPP1R15A, PRMT2, PRUNE2, PTN, RAP1B, RASSF1, RHOC, RHOT1, RND3, RPS6KA2, S100A4, SAT1, SGK1, SIRPA, SMAD3, SMAD6, SNCA, SOD2, SORBS2, SPP1 (includes EG:20750), ST6GAL1, STAT1, STAT3, STAT5A, TCF12, TFAP2A, TFRC, TGFBR2, TGFBR3, TIMP3, TMED10, TNFAIP3, TOP2A, UBQLN1, ZC3H8 Cell Death apoptosis apoptosis of tumor cells 4.18E-06 BCL2L1, BIRC5, CASP1, CDK2, CDKN1A, CDKN1B, CEACAM1 21 (includes others), CHEK1, GSK3B, HBEGF, HIF1A, HMGA2, IL7, LPAR1, MCL1, MUC1, NR4A2, PHLDA1, STAT3, TFAP2A, TGFBR2 Cell Death apoptosis apoptosis of cervical cancer 1.11E-04 BCL2L1, BIRC5, CASP1, CASP4, CDC20 (includes EG:107995), 25 cell lines CDK1, CDKN1A, CDKN1B, CHEK1, ELAVL1, GSK3B, HDAC4, ITPK1, MAP3K4, MCL1, MSX1, MUC1, NME1 (includes EG:18102), NR4A2, PARVA, PCBP2, RASSF1, SGK1, SNCA, TIMP3 Cell Death apoptosis apoptosis of tumor cell lines 3.08E-04 ADM, AKAP12, BCL2A1, BCL2L1, BIRC5, CASP1, CASP4, 78 CCND1, CCNE1, CDC20 (includes EG:107995), CDK1, CDK5, CDKN1A, CDKN1B, CEACAM1 (includes others), CEBPD, CHEK1, CREM, CTGF, CYR61, DAPK3, DKK1, EIF4E, ELAVL1, EPAS1, FOSB, FOXO3, GLIPR1, GSK3B, HBEGF, HDAC4, HIF1A, HOXA5, HSPA8, ID1, ID3 (includes EG:15903), IFI6, IGFBP6, IGFBP7, IL7, IL8, ITPK1, KLF4, KLF9, LMNB1, MAP3K4, MCL1, MET, MITF, MSX1, MT2A, MUC1, NCAM1, NME1 (includes EG:18102), NR4A2, PARVA, PCBP2, PLA2G6, PLAUR, PPP1R15A, RASSF1, RHOC, S100A4, SAT1, SGK1, SIRPA, SNCA, SOD2, SPP1 (includes EG:20750), ST6GAL1, STAT1, STAT3, TFRC, TGFBR3, TIMP3, TMED10, TNFAIP3, TOP2A Cell Death apoptosis apoptosis of mesangial cells 4.57E-04 CDKN1B, CTGF, SOD2 3 Cell Death apoptosis apoptosis of brain cancer cell 6.39E-04 BCL2L1, BIRC5, CASP1, CDK1, CHEK1, DKK1, FOXO3, MCL1, 12 lines MET, MITF, SIRPA, STAT3 Cell Death apoptosis apoptosis of myeloma cell 1.17E-03 BCL2L1, CDKN1B, CHEK1, GSK3B, IFI6, KLF9, MCL1, NCAM1, 9 lines TOP2A Cell Death apoptosis apoptosis of cancer cells 1.52E-03 BCL2L1, BIRC5, CASP1, CDKN1A, CDKN1B, CHEK1, GSK3B, 14 IL7, LPAR1, MCL1, MUC1, NR4A2, PHLDA1, TFAP2A

396

Cell Death apoptosis apoptosis of muscle 2.17E-03 ADM, BCL2L1, CASP1, GSK3B, IL8, PTN, STAT3, TIMP3 8 Cell Death apoptosis apoptosis of splenocytes 2.42E-03 CDKN1A, IL6ST 2 Cell Death apoptosis apoptosis of cardiomyocytes 3.92E-03 ADM, BCL2L1, CASP1, PTN 4 Cell Death apoptosis apoptosis of epithelial cells 4.34E-03 ALDH1A1, BCL2A1, BCL2L1, IL8, MCL1, MYLK, PLAUR, RND3, 9 TIMP3 Cell Death apoptosis apoptosis of breast cancer 5.00E-03 ADM, BCL2L1, BIRC5, CASP1, CCND1, CDK1, CDKN1A, 22 cell lines CEACAM1 (includes others), CHEK1, CTGF, CYR61, FOXO3, GSK3B, HIF1A, HOXA5, MCL1, MUC1, NME1 (includes EG:18102), SOD2, STAT1, TFRC, TGFBR3 Cell Death apoptosis apoptosis of RPE cells 5.52E-03 BCL2L1, PLAUR, TIMP3 3 Cell Death apoptosis apoptosis of muscle cells 6.79E-03 ADM, BCL2L1, CASP1, GSK3B, PTN, STAT3, TIMP3 7 Cell Death apoptosis apoptosis of prostate cancer 7.06E-03 AKAP12, BCL2L1, BIRC5, CDK5, FOXO3, GLIPR1, HBEGF, 15 cell lines HIF1A, ID1, IGFBP7, MCL1, MET, PLAUR, STAT3, TMED10 Cell Death apoptosis apoptosis of fibrosarcoma 1.13E-02 BCL2A1, BCL2L1, BIRC5, CDKN1A, SOD2, STAT1 6 cell lines Cell Death apoptosis apoptosis of phagocytes 1.17E-02 BCL2L1, CDKN1B, GSK3B, IL8, MCL1, P2RX7, SOD2, STAT3 8 Cell Death apoptosis apoptosis of sarcoma cells 1.28E-02 BCL2L1, CASP1, GSK3B, IL7, LPAR1, MUC1 6 Cell Death apoptosis apoptosis of colon cancer 1.44E-02 BCL2L1, BIRC5, CASP1, CDK1, CDKN1A, CEACAM1 (includes 18 cell lines others), CHEK1, FOXO3, GLIPR1, GSK3B, HOXA5, MCL1, MUC1, PPP1R15A, SGK1, SPP1 (includes EG:20750), ST6GAL1, STAT1 Cell Death apoptosis apoptosis of carcinoma cell 2.02E-02 BCL2L1, BIRC5, CASP1, CCND1, CDKN1B, DAPK3, FOXO3, ID3 12 lines (includes EG:15903), IGFBP6, MCL1, MET, STAT3 Cell Death apoptosis apoptosis of epithelial cell 2.40E-02 BCL2A1, BCL2L1, BIRC5, CASP1, IRS1, MAPT, MCL1, MET, 14 lines P2RX7, PPP1R15A, PRMT2, SPP1 (includes EG:20750), TCF12, TFRC Cell Death apoptosis apoptosis of leukemia cell 2.41E-02 BCL2A1, BCL2L1, BIRC5, CDKN1A, CDKN1B, CEACAM1 19 lines (includes others), CEBPD, CREM, FOXO3, GSK3B, HSPA8, IL7, KLF4, MCL1, MET, MUC1, RHOC, SOD2, TNFAIP3 Cell Death apoptosis apoptosis of stomach cancer 2.44E-02 BCL2L1, BIRC5, HIF1A, MET 4 cell lines Cell Death apoptosis apoptosis of 2.88E-02 BCL2L1, CDKN1A, MCL1 3 rhabdomyosarcoma cell lines Cell Death apoptosis apoptosis of leukocytes 2.89E-02 BCL2L1, CASP4, CDKN1B, GSK3B, IL7, IL8, KLF4, MCL1, 14 P2RX7, SOD2, SPP1 (includes EG:20750), STAT3, STAT5A, ZC3H8

397

Cell Death apoptosis apoptosis of leukemia cells 3.03E-02 CASP1, GSK3B, IL7, LPAR1, MUC1 5 Cell Death apoptosis apoptosis of endometrial 3.18E-02 ADM, CYR61 2 cancer cell lines Cell Death apoptosis apoptosis of myeloma cells 3.18E-02 CHEK1, MCL1 2 Cell Death necrosis necrosis 3.56E-06 ADM, ADRM1, AGGF1, AKAP12, ALDH1A1, ANTXR1, BCHE, 121 BCL2A1, BCL2L1, BIRC5, CASP1, CASP4, CCND1, CCNE1, CD74, CDC20 (includes EG:107995), CDK1, CDK2, CDK5, CDKN1A, CDKN1B, CEACAM1 (includes others), CEBPD, CHEK1, CLCF1, CREM, CTGF, CYR61, DAPK3, DKK1, EIF4E, ELAVL1, EMP1, EPAS1, FOSB, FOXO3, GABRG2, GLIPR1, GSK3B, HBEGF, HDAC4, HIF1A, HK1, HLA-DRB4, HMGA2, HOXA5, HRK, HSPA8, ID1, ID3 (includes EG:15903), IFI6, IGFBP6, IGFBP7, IL6ST, IL7, IL8, IRS1, ITPK1, KLF4, KLF9, LAMP2, LMNB1, LPAR1, MAP3K4, MAPT, MCL1, MCTS1, MET, MITF, MSX1, MT1X, MT2A, MUC1, MX1, MYLK, NCAM1, NME1 (includes EG:18102), NR4A2, OBFC2A, P2RX7, PARVA, PCBP2, PHLDA1, PLA2G6, PLAUR, PMP22, PPP1R15A, PRMT2, PSIP1, PTN, RASSF1, RHOC, RND3, S100A4, SAT1, SGK1, SIRPA, SNCA, SOD2, SPC25 (includes EG:100144563), SPP1 (includes EG:20750), ST6GAL1, STAT1, STAT3, STAT5A, TCF12, TFAP2A, TFRC, TGFB1I1, TGFBR2, TGFBR3, TIMP3, TMED10, TNFAIP3, TOP2A, TOP2B, TRPM2, UBE2C, UBQLN1, VAV3, ZC3H8 Cell Death cell viability cell viability of epithelial cell 2.41E-04 ALKBH8, BIRC5, CALB2, CEBPD, MAPT, MGST1, P2RX7, 9 lines PTGR1, TNFAIP3 Cell Death cell viability cell viability of embryonic 2.92E-04 ALKBH8, BIRC5, CEBPD, HBEGF, MAPT, P2RX7, PTGR1, 8 cell lines TNFAIP3 Cell Death cell viability cell viability of leukemia 1.45E-03 CD74, GSK3B, IL7, IL8 4 cells Cell Death cell viability cell viability of kidney cell 2.25E-03 ALKBH8, BIRC5, CEBPD, MAPT, P2RX7, PTGR1, TNFAIP3 7 lines Cell Death cell viability cell viability of breast cancer 3.07E-03 BCL2L1, CCND1, CDK2, CDKN1A, CEBPD, CTGF, HMGA2, 12 cell lines HOXA5, ID2, ID4, SOD2, STAT3 Cell Death cell viability cell viability of brain cancer 4.93E-03 GSK3B, MET, PCGF2, PLAUR, STAT3, STC1 6 cell lines Cell Death cell viability cell viability 8.94E-03 ALKBH8, ANTXR1, BCL2A1, BCL2L1, BIRC5, CADM1, CALB2, 55 CASP1, CCND1, CD74, CDK2, CDKN1A, CDKN1B, CEBPD, CHEK1, CKAP5, CLEC11A, CTGF, DUSP10, DUSP14, DUSP5,

398

ELAVL1, GSK3B, HBEGF, HMGA2, HOXA5, ID2, ID4, IL7, IL8, MAP3K4, MAPT, MCL1, MET, MGST1, MX1, P2RX7, PCGF2, PLAUR, PSMC3, PTGR1, PTN, S100A4, SFR1, SGK1, SLC25A23, SLC2A1, SNCA, SOD2, SPP1 (includes EG:20750), STAT3, STC1, STX8, TGFBR2, TNFAIP3 Cell Death cell viability cell viability of tumor cell 1.10E-02 BCL2A1, BCL2L1, BIRC5, CADM1, CASP1, CCND1, CDK2, 44 lines CDKN1A, CDKN1B, CEBPD, CHEK1, CKAP5, CTGF, DUSP10, DUSP14, DUSP5, ELAVL1, GSK3B, HBEGF, HMGA2, HOXA5, ID2, ID4, IL7, IL8, MAP3K4, MCL1, MET, PCGF2, PLAUR, PSMC3, PTN, S100A4, SFR1, SGK1, SLC25A23, SLC2A1, SNCA, SOD2, SPP1 (includes EG:20750), STAT3, STC1, STX8, TGFBR2 Cell Death cell viability cell viability of cancer cells 1.67E-02 CD74, GSK3B, IL7, IL8, MCL1 5 Cell Death colony survival colony survival of lymphoma 2.42E-03 BCL2L1, CCND1 2 cell lines Cell Death colony survival colony survival of cells 4.56E-03 BCL2L1, CCND1, CDKN1A, CHEK1, PDRG1 5 Cell Death survival cell survival 2.57E-03 ALKBH8, ANTXR1, BCL2A1, BCL2L1, BIRC5, CADM1, CALB2, 60 CASP1, CCND1, CD74, CDK2, CDKN1A, CDKN1B, CEBPD, CHEK1, CKAP5, CLEC11A, CTGF, DEFB103A/DEFB103B, DUSP10, DUSP14, DUSP5, ELAVL1, GSK3B, HBEGF, HMGA2, HOXA5, ID2, ID4, IL6ST, IL7, IL8, ITGA5, MAP3K4, MAPT, MCL1, MET, MGST1, MX1, P2RX7, PCGF2, PDRG1, PLAUR, PSMC3, PTGR1, PTN, S100A4, SFR1, SGK1, SLC25A23, SLC2A1, SNCA, SOD2, SPP1 (includes EG:20750), STAT3, STC1, STX8, TGFBR2, TNFAIP3, TOP2A Cell Death survival survival of chronic 7.02E-03 CD74, IL8 2 lymphocytic leukemia B cells Cell Death survival survival of megakaryocytes 2.19E-02 BCL2L1, IL8 2 Cell Death anoikis anoikis of RPE cells 1.36E-02 BCL2L1, PLAUR 2 Cell Death permeability permeability of endothelial 2.88E-02 IL8, NT5E, RAP1B 3 cell lines Cellular Development proliferation proliferation of prostate 2.76E-08 ADM, AFAP1, ALDH1A1, BCL2L1, BIRC5, CBX7, CCND1, 31 cancer cell lines CDKN1A, CDKN1B, CEACAM1 (includes others), CEBPD, CREM, FSCN1, HAS3, HBEGF, HIF1A, HK1, HMGA2, IGFBP7, IL8, LCP1, MET, NKX3-1, PDCD10, PTN, RASSF1, RHOC, SAT1, SRF, STAT3, TGFB1I1 Cellular Development proliferation proliferation of pancreatic 3.68E-06 ADM, CCND1, CDK5, CDKN1A, CDKN1B, CEACAM1 (includes 14 cancer cell lines others), GSK3B, HBEGF, HIF1A, MET, PTN, SOD2, STAT3,

399

UBE2C Cellular Development proliferation proliferation of tumor cell 1.68E-05 A2M, ADM, AFAP1, AKR1B10, AKR1C3, ALDH1A1, BCL2L1, 97 lines BIRC5, CBX7, CCL20, CCND1, CCNE1, CDK1, CDK2, CDK5, CDKN1A, CDKN1B, CDKN2B, CEACAM1 (includes others), CEBPD, CHEK1, CLEC11A, CREM, CTGF, CYR61, DDX5, DKK1, DUSP5, EED, EEF1A1, EIF4E, ELAVL1, ENC1, ERRFI1, FGFRL1, FOXO3, FSCN1, GAB2, GSK3B, H2AFY, HAS3, HBEGF, HDAC4, HIF1A, HK1, HMGA2, ID1, ID2, IDH2, IGFBP7, IL6ST, IL7, IL8, IRS1, ITGA5, ITGB8, KLF4, LCP1, LDHA, MET, MT1A, MT2A, MUC1, MYL9, NKX3-1, NME1 (includes EG:18102), NOV, NR4A2, PCGF2, PDCD10, PFKFB3, PLA2G6, PLAUR, PTN, RASSF1, RHOC, SAT1, SERTAD1, SGK1, SLC2A1, SMAD3, SOD2, SPP1 (includes EG:20750), SRF, ST8SIA1, STARD13, STAT1, STAT3, STAT5A, SYNM, TFAP2A, TGFB1I1, TGFBR2, TIMP3, TXNIP, UBE2C, UBIAD1 Cellular Development proliferation proliferation of epithelial cell 3.79E-04 BIRC5, CCND1, CDK2, CDKN1A, CYR61, ENC1, FSCN1, ITGA5, 15 lines MET, NME1 (includes EG:18102), PLAUR, PNN, PTN, RASSF1, STAT3 Cellular Development proliferation proliferation of acute 1.10E-03 CASP1, CHEK1, MUC1 3 myeloblastic leukemia cells Cellular Development proliferation proliferation of 1.10E-03 ID1, ID2, ID3 (includes EG:15903) 3 adenocarcinoma cells Cellular Development proliferation proliferation of brain cancer 1.40E-03 A2M, BIRC5, CCNE1, CDKN1A, CDKN1B, CDKN2B, CYR61, IL8, 14 cell lines ITGB8, MET, PCGF2, PLAUR, SMAD3, SYNM Cellular Development proliferation proliferation of vascular 1.91E-03 ELAVL1, FHL1 (includes EG:14199), GSK3B, IL8, PLAUR, 7 smooth muscle cells ST8SIA1, TRIB1 Cellular Development proliferation proliferation of colon cancer 2.30E-03 BCL2L1, BIRC5, CCL20, CCND1, CDK1, CDKN1A, DDX5, 20 cell lines DUSP5, ENC1, HDAC4, ID2, ITGB8, KLF4, MET, MT1A, MUC1, PLA2G6, STAT3, TGFBR2, UBE2C Cellular Development proliferation proliferation of tumor cells 2.96E-03 CASP1, CCND1, CCNE1, CDKN1A, CHEK1, CYR61, HMGA2, 17 ID1, ID2, ID3 (includes EG:15903), MUC1, NKX3-1, NR4A2, PEG10, PLAUR, POU3F2, SMAD3 Cellular Development proliferation proliferation of muscle cells 3.20E-03 ELAVL1, FHL1 (includes EG:14199), GSK3B, HBEGF, IL8, ITGA5, 11 PLAUR, SPP1 (includes EG:20750), ST8SIA1, TNFAIP3, TRIB1 Cellular Development proliferation proliferation of fibroblasts 3.83E-03 CCND1, CD74, CDKN1A, CDKN1B, CDKN2B, IDH2, ITGA5, 11 PGK1, SERTAD1, SOD2, TGFBR2 Cellular Development proliferation proliferation of smooth 4.34E-03 ELAVL1, FHL1 (includes EG:14199), GSK3B, HBEGF, IL8, 10 muscle cells PLAUR, SPP1 (includes EG:20750), ST8SIA1, TNFAIP3, TRIB1

400

Cellular Development proliferation proliferation of leukemia 5.13E-03 CASP1, CHEK1, MUC1, NKX3-1 4 cells Cellular Development proliferation proliferation of leukemia cell 5.52E-03 AKR1C3, ALDH1A1, BCL2L1, BIRC5, CDKN1A, CDKN1B, 15 lines CLEC11A, CREM, FOXO3, GAB2, GSK3B, IL7, MUC1, NKX3-1, TXNIP Cellular Development proliferation proliferation of myeloma cell 6.65E-03 GSK3B, HBEGF, IL6ST, SGK1, SOD2 5 lines Cellular Development proliferation proliferation of acute 7.02E-03 CHEK1, MUC1 2 myeloid leukemia blast cells Cellular Development proliferation proliferation of kidney 7.90E-03 MET, MYL9, RASSF1, STAT3, TGFBR2 5 cancer cell lines Cellular Development proliferation proliferation of carcinoma 8.28E-03 ID1, ID2, ID3 (includes EG:15903), PLAUR 4 cells Cellular Development proliferation proliferation of lung cancer 8.95E-03 BIRC5, CDK2, CDKN1A, CHEK1, CYR61, DKK1, DUSP5, EIF4E, 19 cell lines ERRFI1, H2AFY, HDAC4, HK1, ID1, IDH2, ITGB8, MET, PLAUR, RASSF1, ST8SIA1 Cellular Development proliferation proliferation of cancer cells 9.07E-03 CASP1, CHEK1, CYR61, HMGA2, ID1, ID2, ID3 (includes 14 EG:15903), MUC1, NKX3-1, NR4A2, PEG10, PLAUR, POU3F2, SMAD3 Cellular Development proliferation proliferation of endothelial 1.61E-02 ADM, AGGF1, CDKN1A, CDKN1B, CEACAM1 (includes others), 15 cells DKK1, EDNRB, FOXO3, GBP1, HAS3, HEY1, HIF1A, IL8, PTN, STAT1 Cellular Development proliferation proliferation of blood cells 1.65E-02 A2M, BCL2L1, BIRC5, CCL20, CD74, CDKN1A, CEACAM1 22 (includes others), EPAS1, GSK3B, HOXA5, IL6ST, IL7, ITGA5, KLF4, PLA2G6, PNP, SPP1 (includes EG:20750), SRF, STAT5A, TYR, TYRP1, VAV3 Cellular Development proliferation proliferation of carcinoma 1.82E-02 BIRC5, CDK2, CDK5, CDKN1A, CHEK1, CYR61, DKK1, EIF4E, 19 cell lines FSCN1, H2AFY, HDAC4, HK1, ID1, IDH2, IL6ST, ITGB8, MET, NOV, RASSF1 Cellular Development proliferation proliferation of airway 2.19E-02 CCND1, ITGB8 2 epithelial cells Cellular Development proliferation proliferation of hepatoma 2.19E-02 AKR1B10, BIRC5, CDKN1A, IL8, MET, MT2A, SLC2A1, SPP1 11 cell lines (includes EG:20750), STARD13, STAT3, TGFBR2 Cellular Development proliferation proliferation of cervical 2.53E-02 BIRC5, CDKN1A, CDKN1B, CHEK1, EIF4E, ELAVL1, H2AFY, 13 cancer cell lines HDAC4, NR4A2, PFKFB3, SPP1 (includes EG:20750), STAT3, TFAP2A

401

Cellular Development proliferation proliferation of 2.88E-02 ADM, DKK1, IL8 3 microvascular endothelial cells Cellular Development differentiation differentiation of cells 4.42E-05 ADM, AKR1C3, AZGP1, BCL2L1, CCND1, CDK5, CDKN1A, 54 CDKN1B, CDKN2B, CEBPD, CLEC11A, CTGF, CYR61, DKK1, FOXO3, FSCN1, GAB2, GAS7, HBEGF, HDAC4, HIF1A, HMGA2, HOXA5, ID1, ID2, IL6ST, IL7, IL8, KLF4, LRP5, LTBP4, MAPT, MCL1, MET, MLL5, MLLT11, MUC1, NCAM1, NME1 (includes EG:18102), OGT, PNP, PPARGC1A, SFPQ, SMAD3, SNCA, SPP1 (includes EG:20750), SRF, ST8SIA1, STAT1, STAT3, STAT5A, STC1, TFRC, TXNIP Cellular Development differentiation differentiation of tumor cell 1.42E-04 AKR1C3, AZGP1, CCND1, CDKN1A, CDKN1B, CEBPD, CTGF, 22 lines CYR61, FOXO3, HIF1A, HOXA5, KLF4, MCL1, MET, MUC1, NCAM1, NME1 (includes EG:18102), SFPQ, SNCA, ST8SIA1, STAT1, STAT3 Cellular Development differentiation differentiation of leukemia 3.07E-03 AKR1C3, CEBPD, FOXO3, HIF1A, HOXA5, KLF4, MCL1, MUC1, 12 cell lines SFPQ, ST8SIA1, STAT1, STAT3 Cellular Development differentiation differentiation of myeloid 5.52E-03 BCL2L1, DKK1, IL7 3 progenitor cells Cellular Development differentiation differentiation of epithelial 7.02E-03 FSCN1, ID2 2 cell lines Cellular Development differentiation differentiation of connective 1.25E-02 DKK1, GAB2, GAS7, HMGA2, IL6ST, IL7, IL8, LRP5, 12 tissue cells PPARGC1A, SMAD3, SPP1 (includes EG:20750), STC1 Cellular Development differentiation differentiation of adipocytes 1.46E-02 HMGA2, LRP5, PPARGC1A, SMAD3, SPP1 (includes EG:20750) 5 Cellular Development differentiation differentiation of embryonic 2.15E-02 GAS7, HBEGF, HMGA2, IL6ST, SRF 5 cells Cellular Development differentiation differentiation of cerebral 2.19E-02 BCL2L1, MAPT 2 cortex cells Cellular Development differentiation differentiation of fibroblasts 2.19E-02 DKK1, IL7 2 Cellular Development differentiation differentiation of trophoblast 3.18E-02 HBEGF, SRF 2 cells Cellular Development growth arrest in growth of tumor cell 1.16E-04 CBX7, CCND1, CDK2, CDKN1A, CDKN1B, CDKN2B, GAB2, 13 lines IL6ST, KLF4, MUC1, STAT1, STAT3, TGFBR2 Cellular Development growth arrest in growth of colon 4.57E-04 CDKN1A, KLF4, TGFBR2 3 cancer cell lines Cellular Development growth arrest in growth of germ cell 2.42E-03 CDKN1A, CDKN1B 2

402

tumor cell lines Cellular Development growth arrest in growth of 1.36E-02 CDKN1A, STAT1 2 fibrosarcoma cell lines Cellular Development growth arrest in growth of epithelial 2.19E-02 CCND1, CDK2 2 cell lines Cellular Development growth arrest in growth of brain 3.18E-02 CDKN1A, CDKN2B 2 cancer cell lines Cellular Development tubulation tubulation of endothelial 2.00E-04 EDNRB, FOXO3, GBP1, ID1, ID3 (includes EG:15903), IL8, 9 cells KCNMA1, PLAUR, PTN Cellular Development tubulation tubulation of vascular 2.06E-03 FOXO3, ID1, ID3 (includes EG:15903), IL8, KCNMA1, PTN 6 endothelial cells Cellular Development tubulation tubulation of skin cell lines 2.19E-02 ALCAM, RAP1B 2 Cellular Development maturation maturation of 3.58E-03 BIRC5, CCND1, CDKN1A 3 megakaryocytes Cellular Development maturation maturation of leukemia cell 1.46E-02 CCND1, CDKN1A, FOXO3 3 lines Cellular Development immortalization immortalization 3.92E-03 CBX7, CCND1, ID1, ZNF217 4 Cellular Development immortalization immortalization of 3.18E-02 CCND1, ID1 2 keratinocytes Cellular Development development endothelial cell development 4.23E-03 ADM, AGGF1, CDKN1A, CDKN1B, CEACAM1 (includes others), 18 DKK1, EDNRB, FOXO3, GBP1, HAS3, HEY1, HIF1A, IL8, MET, PTN, STAT1, STC1, TFAP2A Cellular Development development development of stem cells 2.34E-02 CLEC11A, IL6ST, SMAD3 3 Cellular Development development development of bone marrow 2.44E-02 CLEC11A, HOXA5, IL6ST, SMAD3 4 cells Cellular Development epithelial- epithelial-mesenchymal 4.62E-03 GSK3B, HIF1A, HMGA2, ID1, IRS1, PLAUR, S100A4, TGFBR3 8 mesenchymal transition transition Cellular Development morphogenesis morphogenesis of breast cell 7.02E-03 CCND1, HIF1A 2 lines Cellular Development outgrowth outgrowth of tumor cell lines 7.02E-03 CCND1, CYR61 2 Cellular Development transdifferentiation transdifferentiation of 7.02E-03 AZGP1, MUC1 2 pancreatic cancer cell lines Cellular Development hematopoiesis hematopoiesis of bone 1.10E-02 CLEC11A, HOXA5, IL6ST 3 marrow cells

403

Cellular Development hematopoiesis hematopoiesis of 3.18E-02 CLEC11A, IL6ST 2 megakaryocytes Cellular Development hematopoiesis hematopoiesis of stem cells 3.18E-02 CLEC11A, IL6ST 2 Cellular Growth and proliferation proliferation of prostate 2.76E-08 ADM, AFAP1, ALDH1A1, BCL2L1, BIRC5, CBX7, CCND1, 31 Proliferation cancer cell lines CDKN1A, CDKN1B, CEACAM1 (includes others), CEBPD, CREM, FSCN1, HAS3, HBEGF, HIF1A, HK1, HMGA2, IGFBP7, IL8, LCP1, MET, NKX3-1, PDCD10, PTN, RASSF1, RHOC, SAT1, SRF, STAT3, TGFB1I1 Cellular Growth and proliferation proliferation of pancreatic 3.68E-06 ADM, CCND1, CDK5, CDKN1A, CDKN1B, CEACAM1 (includes 14 Proliferation cancer cell lines others), GSK3B, HBEGF, HIF1A, MET, PTN, SOD2, STAT3, UBE2C Cellular Growth and proliferation proliferation of cells 5.94E-06 A2M, ADM, AFAP1, AGGF1, AKR1B10, AKR1C3, ALDH1A1, 170 Proliferation BCL2A1, BCL2L1, BIRC5, CASP1, CAV2, CBX7, CCL20, CCND1, CCNE1, CD74, CD9, CDK1, CDK2, CDK5, CDKN1A, CDKN1B, CDKN2B, CEACAM1 (includes others), CEBPD, CHEK1, CLCF1, CLEC11A, CNOT8, CREM, CTGF, CTSL1, CUL1, CYP20A1, CYR61, DCBLD2, DDX5, DKK1, DUSP5, EDNRB, EED, EEF1A1, EIF4E, ELAVL1, EMP1, ENC1, EPAS1, ERRFI1, FGF13, FGFRL1, FHL1 (includes EG:14199), FHL2, FOXO3, FSCN1, GAB2, GBP1, GPNMB, GSK3B, H2AFY, HAS3, HBEGF, HDAC4, HEY1, HIF1A, HK1, HLA-DPB1, HMGA2, HNRNPU, HOXA5, ID1, ID2, ID3 (includes EG:15903), IDH2, IFITM1, IGFBP6, IGFBP7, IL6ST, IL7, IL8, INSIG1, IRS1, ITGA5, ITGB8, KCNMA1, KLF11, KLF4, LAMA5, LCP1, LDHA, LEPREL1, LRP5, LTBP4, MAPT, MCL1, MCM7, MCTS1, MET, MSX1, MT1A, MT2A, MUC1, MX1, MYL9, NKX3-1, NME1 (includes EG:18102), NOV, NR2F1, NR4A2, PCGF2, PDCD10, PEG10, PFKFB3, PGK1, PHLDA1, PHLDA2, PLA2G6, PLAUR, PLP1 (includes EG:18823), PMP22, PNN, PNP, POU3F2, PPP1R15A, PSMC3, PTN, RASSF1, RHOC, RPS6KA2, SAT1, SCAF11, SERTAD1, SF1, SGK1, SIRPA, SLC2A1, SMAD3, SMARCA2, SMARCD3, SOD2, SPP1 (includes EG:20750), SRF, ST8SIA1, STARD13, STAT1, STAT3, STAT5A, STC1, SUPV3L1, SYNM, TFAP2A, TFRC, TGFB1I1, TGFBR2, TGFBR3, THEM4, TIMP3, TNFAIP3, TOP2A, TRIB1, TXNIP, TYR, TYRP1, UBE2C, UBE2L6, UBIAD1, USP3, VAV3, ZNF217, ZYX Cellular Growth and proliferation proliferation of tumor cell 1.68E-05 A2M, ADM, AFAP1, AKR1B10, AKR1C3, ALDH1A1, BCL2L1, 97 Proliferation lines BIRC5, CBX7, CCL20, CCND1, CCNE1, CDK1, CDK2, CDK5, CDKN1A, CDKN1B, CDKN2B, CEACAM1 (includes others), CEBPD, CHEK1, CLEC11A, CREM, CTGF, CYR61, DDX5, DKK1, DUSP5, EED, EEF1A1, EIF4E, ELAVL1, ENC1, ERRFI1, FGFRL1, FOXO3, FSCN1, GAB2, GSK3B, H2AFY, HAS3, HBEGF, HDAC4,

404

HIF1A, HK1, HMGA2, ID1, ID2, IDH2, IGFBP7, IL6ST, IL7, IL8, IRS1, ITGA5, ITGB8, KLF4, LCP1, LDHA, MET, MT1A, MT2A, MUC1, MYL9, NKX3-1, NME1 (includes EG:18102), NOV, NR4A2, PCGF2, PDCD10, PFKFB3, PLA2G6, PLAUR, PTN, RASSF1, RHOC, SAT1, SERTAD1, SGK1, SLC2A1, SMAD3, SOD2, SPP1 (includes EG:20750), SRF, ST8SIA1, STARD13, STAT1, STAT3, STAT5A, SYNM, TFAP2A, TGFB1I1, TGFBR2, TIMP3, TXNIP, UBE2C, UBIAD1 Cellular Growth and proliferation proliferation of connective 3.57E-04 CCND1, CD74, CDKN1A, CDKN1B, CDKN2B, CTGF, DDX5, 20 Proliferation tissue cells FGF13, ID2, IDH2, IL8, ITGA5, KCNMA1, PGK1, PLAUR, SERTAD1, SMAD3, SOD2, STC1, TGFBR2 Cellular Growth and proliferation proliferation of epithelial cell 3.79E-04 BIRC5, CCND1, CDK2, CDKN1A, CYR61, ENC1, FSCN1, ITGA5, 15 Proliferation lines MET, NME1 (includes EG:18102), PLAUR, PNN, PTN, RASSF1, STAT3 Cellular Growth and proliferation proliferation of acute 1.10E-03 CASP1, CHEK1, MUC1 3 Proliferation myeloblastic leukemia cells Cellular Growth and proliferation proliferation of 1.10E-03 ID1, ID2, ID3 (includes EG:15903) 3 Proliferation adenocarcinoma cells Cellular Growth and proliferation proliferation of brain cancer 1.40E-03 A2M, BIRC5, CCNE1, CDKN1A, CDKN1B, CDKN2B, CYR61, IL8, 14 Proliferation cell lines ITGB8, MET, PCGF2, PLAUR, SMAD3, SYNM Cellular Growth and proliferation proliferation of vascular 1.91E-03 ELAVL1, FHL1 (includes EG:14199), GSK3B, IL8, PLAUR, 7 Proliferation smooth muscle cells ST8SIA1, TRIB1 Cellular Growth and proliferation proliferation of colon cancer 2.30E-03 BCL2L1, BIRC5, CCL20, CCND1, CDK1, CDKN1A, DDX5, 20 Proliferation cell lines DUSP5, ENC1, HDAC4, ID2, ITGB8, KLF4, MET, MT1A, MUC1, PLA2G6, STAT3, TGFBR2, UBE2C Cellular Growth and proliferation proliferation of lung cells 2.91E-03 CCND1, HLA-DPB1, ITGA5, ITGB8 4 Proliferation Cellular Growth and proliferation proliferation of tumor cells 2.96E-03 CASP1, CCND1, CCNE1, CDKN1A, CHEK1, CYR61, HMGA2, 17 Proliferation ID1, ID2, ID3 (includes EG:15903), MUC1, NKX3-1, NR4A2, PEG10, PLAUR, POU3F2, SMAD3 Cellular Growth and proliferation proliferation of muscle cells 3.20E-03 ELAVL1, FHL1 (includes EG:14199), GSK3B, HBEGF, IL8, ITGA5, 11 Proliferation PLAUR, SPP1 (includes EG:20750), ST8SIA1, TNFAIP3, TRIB1 Cellular Growth and proliferation proliferation of fibroblasts 3.83E-03 CCND1, CD74, CDKN1A, CDKN1B, CDKN2B, IDH2, ITGA5, 11 Proliferation PGK1, SERTAD1, SOD2, TGFBR2 Cellular Growth and proliferation proliferation of smooth 4.34E-03 ELAVL1, FHL1 (includes EG:14199), GSK3B, HBEGF, IL8, 10 Proliferation muscle cells PLAUR, SPP1 (includes EG:20750), ST8SIA1, TNFAIP3, TRIB1 Cellular Growth and proliferation proliferation of leukemia 5.13E-03 CASP1, CHEK1, MUC1, NKX3-1 4

405

Proliferation cells Cellular Growth and proliferation proliferation of leukemia cell 5.52E-03 AKR1C3, ALDH1A1, BCL2L1, BIRC5, CDKN1A, CDKN1B, 15 Proliferation lines CLEC11A, CREM, FOXO3, GAB2, GSK3B, IL7, MUC1, NKX3-1, TXNIP Cellular Growth and proliferation proliferation of myeloma cell 6.65E-03 GSK3B, HBEGF, IL6ST, SGK1, SOD2 5 Proliferation lines Cellular Growth and proliferation proliferation of acute 7.02E-03 CHEK1, MUC1 2 Proliferation myeloid leukemia blast cells Cellular Growth and proliferation proliferation of kidney 7.90E-03 MET, MYL9, RASSF1, STAT3, TGFBR2 5 Proliferation cancer cell lines Cellular Growth and proliferation proliferation of carcinoma 8.28E-03 ID1, ID2, ID3 (includes EG:15903), PLAUR 4 Proliferation cells Cellular Growth and proliferation proliferation of lung cancer 8.95E-03 BIRC5, CDK2, CDKN1A, CHEK1, CYR61, DKK1, DUSP5, EIF4E, 19 Proliferation cell lines ERRFI1, H2AFY, HDAC4, HK1, ID1, IDH2, ITGB8, MET, PLAUR, RASSF1, ST8SIA1 Cellular Growth and proliferation proliferation of cancer cells 9.07E-03 CASP1, CHEK1, CYR61, HMGA2, ID1, ID2, ID3 (includes 14 Proliferation EG:15903), MUC1, NKX3-1, NR4A2, PEG10, PLAUR, POU3F2, SMAD3 Cellular Growth and proliferation proliferation of epithelial 1.50E-02 BCL2A1, CCND1, CDKN1A, CDKN2B, DDX5, FHL2, HBEGF, 12 Proliferation cells ID1, ID2, ITGB8, NME1 (includes EG:18102), SMAD3 Cellular Growth and proliferation proliferation of endothelial 1.61E-02 ADM, AGGF1, CDKN1A, CDKN1B, CEACAM1 (includes others), 15 Proliferation cells DKK1, EDNRB, FOXO3, GBP1, HAS3, HEY1, HIF1A, IL8, PTN, STAT1 Cellular Growth and proliferation proliferation of blood cells 1.65E-02 A2M, BCL2L1, BIRC5, CCL20, CD74, CDKN1A, CEACAM1 22 Proliferation (includes others), EPAS1, GSK3B, HOXA5, IL6ST, IL7, ITGA5, KLF4, PLA2G6, PNP, SPP1 (includes EG:20750), SRF, STAT5A, TYR, TYRP1, VAV3 Cellular Growth and proliferation proliferation of carcinoma 1.82E-02 BIRC5, CDK2, CDK5, CDKN1A, CHEK1, CYR61, DKK1, EIF4E, 19 Proliferation cell lines FSCN1, H2AFY, HDAC4, HK1, ID1, IDH2, IL6ST, ITGB8, MET, NOV, RASSF1 Cellular Growth and proliferation proliferation of airway 2.19E-02 CCND1, ITGB8 2 Proliferation epithelial cells Cellular Growth and proliferation proliferation of hepatoma 2.19E-02 AKR1B10, BIRC5, CDKN1A, IL8, MET, MT2A, SLC2A1, SPP1 11 Proliferation cell lines (includes EG:20750), STARD13, STAT3, TGFBR2 Cellular Growth and proliferation proliferation of cervical 2.53E-02 BIRC5, CDKN1A, CDKN1B, CHEK1, EIF4E, ELAVL1, H2AFY, 13 Proliferation cancer cell lines HDAC4, NR4A2, PFKFB3, SPP1 (includes EG:20750), STAT3,

406

TFAP2A Cellular Growth and proliferation proliferation of lymphatic 2.68E-02 BCL2L1, BIRC5, CCL20, DKK1, EDNRB, PLP1 (includes 6 Proliferation system cells EG:18823) Cellular Growth and proliferation proliferation of 2.88E-02 ADM, DKK1, IL8 3 Proliferation microvascular endothelial cells Cellular Growth and growth arrest in growth of tumor cell 1.16E-04 CBX7, CCND1, CDK2, CDKN1A, CDKN1B, CDKN2B, GAB2, 13 Proliferation lines IL6ST, KLF4, MUC1, STAT1, STAT3, TGFBR2 Cellular Growth and growth arrest in growth of cells 2.29E-04 CBX7, CCND1, CCNE1, CDK2, CDKN1A, CDKN1B, CDKN2B, 15 Proliferation FOXO3, GAB2, IL6ST, KLF4, MUC1, STAT1, STAT3, TGFBR2 Cellular Growth and growth arrest in growth of colon 4.57E-04 CDKN1A, KLF4, TGFBR2 3 Proliferation cancer cell lines Cellular Growth and growth arrest in growth of germ cell 2.42E-03 CDKN1A, CDKN1B 2 Proliferation tumor cell lines Cellular Growth and growth arrest in growth of 1.36E-02 CDKN1A, STAT1 2 Proliferation fibrosarcoma cell lines Cellular Growth and growth arrest in growth of epithelial 2.19E-02 CCND1, CDK2 2 Proliferation cell lines Cellular Growth and growth arrest in growth of brain 3.18E-02 CDKN1A, CDKN2B 2 Proliferation cancer cell lines Cellular Growth and colony formation colony formation of cells 2.82E-04 ALDH1A1, BCL2L1, BIRC5, CADM1, CCND1, CDKN1A, 30 Proliferation CDKN1B, CEACAM1 (includes others), CHEK1, CLEC11A, CYR61, EPAS1, FOXO3, HMGA2, IGFBP7, IL6ST, IL7, KLF4, MET, MT1X, PCGF2, PHLDA1, PPP1R15A, PSMC3, PTN, RASSF1, RHOC, SERTAD1, SOD2, TGFB1I1 Cellular Growth and colony formation colony formation of tumor 1.18E-02 ALDH1A1, BCL2L1, CADM1, CDKN1A, CDKN1B, CEACAM1 19 Proliferation cell lines (includes others), CYR61, EPAS1, FOXO3, HMGA2, IGFBP7, IL6ST, KLF4, MT1X, PCGF2, PTN, RASSF1, SERTAD1, SOD2 Cellular Growth and colony formation colony formation of lung 2.42E-02 CADM1, CDKN1A, CYR61, RASSF1, SOD2 5 Proliferation cancer cell lines Cellular Growth and cytostasis cytostasis 1.28E-03 BIRC5, CCND1, CDKN1A, CDKN1B, CEACAM1 (includes others), 18 Proliferation CYR61, FOXO3, HDAC4, ID1, ID2, NME1 (includes EG:18102), NR2F1, PPP1R15A, SMAD3, SMARCA2, STAT1, TOP2A, ZNF217 Cellular Growth and cytostasis cytostasis of tumor cell lines 2.11E-03 BIRC5, CCND1, CDKN1A, CDKN1B, CEACAM1 (includes others), 14 Proliferation CYR61, FOXO3, NME1 (includes EG:18102), NR2F1, PPP1R15A, SMAD3, SMARCA2, STAT1, TOP2A

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Cellular Growth and cytostasis cytostasis of smooth muscle 7.02E-03 ID1, ID2 2 Proliferation cells Cellular Growth and cytostasis cytostasis of colon cancer 1.25E-02 BIRC5, CDKN1B, FOXO3, PPP1R15A 4 Proliferation cell lines Cellular Growth and cytostasis cytostasis of breast cancer 2.72E-02 CCND1, PPP1R15A, SMAD3, SMARCA2, TOP2A 5 Proliferation cell lines Cellular Growth and outgrowth outgrowth of tumor cell lines 7.02E-03 CCND1, CYR61 2 Proliferation Cellular Growth and contact growth contact growth inhibition of 1.10E-02 BIRC5, FOXO3, PPP1R15A 3 Proliferation inhibition colon cancer cell lines Cellular Growth and contact growth contact growth inhibition of 1.61E-02 BIRC5, CDKN1A, FOXO3, NME1 (includes EG:18102), PPP1R15A, 7 Proliferation inhibition tumor cell lines SMAD3, TOP2A Cellular Growth and contact growth contact growth inhibition of 2.19E-02 NME1 (includes EG:18102), PPP1R15A 2 Proliferation inhibition cervical cancer cell lines Cellular Growth and formation formation of syncytia 2.19E-02 CD9, EIF4E 2 Proliferation Cellular Growth and formation formation of cells 2.70E-02 CD9, CDKN1A, EIF4E, IL7, IL8, ITGA5, ORC6 (includes 9 Proliferation EG:23594), SMARCA2, STAT5A Cellular Movement cell movement cell movement 5.57E-07 A2M, ADM, AFAP1, AGGF1, ALCAM, ALPP/ALPPL2, ARHGDIB, 91 ARPC1B, CCL20, CCND1, CD9, CDK1, CDK5, CDKN1B, CEACAM1 (includes others), CLEC11A, CTGF, CTSL1, CYR61, DAG1 (includes EG:114489), DCBLD2, DEFB103A/DEFB103B, DKK1, EDNRB, ELMO1, FERMT2, FHL1 (includes EG:14199), FOXO3, FSCN1, GAB2, GSK3B, HAS3, HBEGF, HEY1, ID1, ID2, ID3 (includes EG:15903), IGFBP6, IL7, IL8, IRS1, ITGA5, ITGB8, KCNMA1, KLF4, LCP1, LPAR1, LPP, MAP2K3, MAPK8IP3, MET, MMP3, MUC1, MX1, MYLK, NME1 (includes EG:18102), NOV, NREP, PARP9, PLA2G6, PLAUR, PMP22, PPAP2B, PTN, PTPRF, RAP1B, RASSF1, RHOC, S100A4, SERPINA3, SIRPA, SLC2A1, SMAD3, SOD2, SP100, SPP1 (includes EG:20750), ST3GAL5, ST6GAL1, STARD13, STAT3, STC1, SYNM, TFAP2A, TFF2, TGFBR2, TIMP3, TRIB1, TUBB2B, VAV3, ZNF217, ZYX Cellular Movement cell movement cell movement of tumor cell 4.13E-05 A2M, AFAP1, ARHGDIB, ARPC1B, CCL20, CD9, CDK5, 53 lines CDKN1B, CTGF, CTSL1, CYR61, DCBLD2, DEFB103A/DEFB103B, ELMO1, FERMT2, FOXO3, FSCN1, GAB2, GSK3B, HAS3, ID1, IGFBP6, IL8, IRS1, KLF4, LCP1, LPAR1, MAPK8IP3, MET, MMP3, MUC1, MX1, NME1 (includes EG:18102), NOV, NREP, PLA2G6, PLAUR, PTN, RASSF1, RHOC,

408

SIRPA, SLC2A1, SOD2, SPP1 (includes EG:20750), ST3GAL5, ST6GAL1, STARD13, STAT3, SYNM, TFAP2A, TFF2, VAV3, ZYX Cellular Movement cell movement cell movement of vascular 4.20E-03 ADM, DKK1, ID1, ID3 (includes EG:15903), IL8, TGFBR2 6 endothelial cells Cellular Movement cell movement cell movement of endothelial 5.30E-03 ADM, AGGF1, CD9, CDKN1B, CEACAM1 (includes others), 18 cells CYR61, DKK1, FOXO3, HAS3, HEY1, ID1, ID3 (includes EG:15903), IL8, MAP2K3, PTN, SP100, STC1, TGFBR2 Cellular Movement cell movement cell movement of endothelial 7.94E-03 ADM, ALCAM, CYR61, IL8, MET, PPAP2B, RAP1B, RHOC 8 cell lines Cellular Movement cell movement cell movement of fibroblasts 8.28E-03 CCND1, HBEGF, LPP, TGFBR2 4 Cellular Movement cell movement cell movement of 1.50E-02 ADM, DKK1, IL8, TGFBR2 4 microvascular endothelial cells Cellular Movement cell movement cell movement of prostate 1.66E-02 AFAP1, FSCN1, ID1, IL8, IRS1, LCP1, MX1, PTN 8 cancer cell lines Cellular Movement cell movement cell movement of connective 2.15E-02 CCND1, HBEGF, KCNMA1, LPP, TGFBR2 5 tissue cells Cellular Movement cell movement cell movement of epithelial 2.21E-02 HBEGF, PLAUR, PMP22, PTPRF, SMAD3, TGFBR2 6 cells Cellular Movement migration migration of cells 2.19E-06 A2M, ADM, AFAP1, AGGF1, ALCAM, ALPP/ALPPL2, ARHGDIB, 80 CCL20, CD9, CDK1, CDK5, CDKN1B, CEACAM1 (includes others), CLEC11A, CTGF, CTSL1, CYR61, DAG1 (includes EG:114489), DCBLD2, DEFB103A/DEFB103B, DKK1, EDNRB, ELMO1, FHL1 (includes EG:14199), FOXO3, FSCN1, GAB2, HAS3, HBEGF, HEY1, ID1, ID2, ID3 (includes EG:15903), IGFBP6, IL7, IL8, IRS1, ITGA5, ITGB8, KCNMA1, KLF4, LPAR1, LPP, MAP2K3, MAPK8IP3, MET, MMP3, MUC1, MYLK, NME1 (includes EG:18102), NOV, NREP, PARP9, PLA2G6, PLAUR, PMP22, PPAP2B, PTN, PTPRF, RAP1B, RASSF1, RHOC, S100A4, SERPINA3, SIRPA, SLC2A1, SOD2, SP100, SPP1 (includes EG:20750), ST6GAL1, STAT3, STC1, SYNM, TFAP2A, TFF2, TGFBR2, TIMP3, TUBB2B, VAV3, ZYX Cellular Movement migration migration of tumor cell lines 1.82E-04 A2M, AFAP1, ARHGDIB, CCL20, CDK5, CDKN1B, CTSL1, 42 CYR61, DCBLD2, ELMO1, FOXO3, FSCN1, GAB2, HAS3, ID1, IGFBP6, IL8, IRS1, KLF4, LPAR1, MAPK8IP3, MET, MMP3, MUC1, NME1 (includes EG:18102), NOV, NREP, PLA2G6, PLAUR, PTN, RASSF1, RHOC, SIRPA, SLC2A1, SOD2, SPP1 (includes EG:20750), ST6GAL1, STAT3, SYNM, TFAP2A, VAV3,

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ZYX Cellular Movement migration migration of brain cancer cell 3.05E-04 A2M, ELMO1, LPAR1, MAPK8IP3, NREP, PLAUR, PTN, SIRPA, 10 lines SYNM, VAV3 Cellular Movement migration migration of endothelial cells 4.55E-03 ADM, AGGF1, CD9, CDKN1B, CYR61, DKK1, FOXO3, HAS3, 17 HEY1, ID1, ID3 (includes EG:15903), IL8, MAP2K3, PTN, SP100, STC1, TGFBR2 Cellular Movement migration migration of vascular 6.79E-03 ADM, AGGF1, CDKN1B, DKK1, FOXO3, ID1, ID3 (includes 10 endothelial cells EG:15903), PTN, SP100, TGFBR2 Cellular Movement migration migration of carcinoma cell 7.17E-03 CYR61, ID1, IL8, IRS1, MET, NOV, PTN, RASSF1 8 lines Cellular Movement migration migration of eosinophils 1.25E-02 ALPP/ALPPL2, IL8, PLAUR, SIRPA 4 Cellular Movement migration migration of dermal 1.36E-02 HBEGF, TGFBR2 2 fibroblasts Cellular Movement migration migration of fibroblasts 1.87E-02 HBEGF, LPP, TGFBR2 3 Cellular Movement migration migration of connective 2.10E-02 HBEGF, KCNMA1, LPP, TGFBR2 4 tissue cells Cellular Movement invasion invasion of cells 4.26E-06 ADM, AZGP1, CCND1, CD9, CDK5, CDKN1B, CEACAM1 49 (includes others), CTSL1, DKK1, EDNRB, ELMO1, FSCN1, FXYD5, GAB2, GBP1, HAS3, HBEGF, HIF1A, ID1, ID2, IL8, ITGA5, KCNMA1, KLF4, LCP1, LPAR1, MARCKS, MET, MITF, MMP1 (includes EG:300339), MMP3, MUC1, MX1, MYH10, NME1 (includes EG:18102), NOV, PLA2G6, PLAUR, RHOC, RND3, SLC2A1, SOD2, SP100, SPP1 (includes EG:20750), ST8SIA1, STAT3, TFAP2A, TFF2, TIMP3 Cellular Movement invasion invasion of tumor cell lines 4.16E-05 ADM, AZGP1, CD9, CDK5, CDKN1B, DKK1, ELMO1, FSCN1, 41 FXYD5, GAB2, HAS3, HBEGF, HIF1A, ID1, ID2, IL8, ITGA5, KLF4, LCP1, LPAR1, MARCKS, MET, MITF, MMP1 (includes EG:300339), MUC1, MX1, NME1 (includes EG:18102), NOV, PLA2G6, PLAUR, RHOC, RND3, SLC2A1, SOD2, SP100, SPP1 (includes EG:20750), ST8SIA1, STAT3, TFAP2A, TFF2, TIMP3 Cellular Movement invasion invasion of hybrid cells 2.42E-03 CEACAM1 (includes others), SPP1 (includes EG:20750) 2 Cellular Movement invasion invasion of colon cancer cell 3.64E-03 FSCN1, HAS3, HIF1A, KLF4, MET, NME1 (includes EG:18102), 8 lines STAT3, TFF2 Cellular Movement invasion invasion of breast cancer cell 1.65E-02 CDKN1B, FXYD5, GAB2, HIF1A, ID1, ID2, ITGA5, MET, MUC1, 13 lines PLAUR, RHOC, SP100, SPP1 (includes EG:20750) Cellular Movement invasion invasion of squamous cell 1.90E-02 FSCN1, HBEGF, NME1 (includes EG:18102), PLAUR, SPP1 5

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carcinoma cell lines (includes EG:20750) Cellular Movement invasion invasion of prostate cancer 1.93E-02 FSCN1, IL8, LCP1, MMP1 (includes EG:300339), MX1, PLAUR, 7 cell lines SPP1 (includes EG:20750) Cellular Movement transmigration transmigration of vascular 2.42E-03 ID1, ID3 (includes EG:15903) 2 endothelial cells Cellular Movement transmigration transmigration of eosinophils 7.02E-03 ALPP/ALPPL2, SIRPA 2 Cellular Movement movement movement of vascular 2.92E-03 ADM, AGGF1, CDKN1B, DKK1, FOXO3, ID1, ID3 (includes 11 endothelial cells EG:15903), IL8, PTN, SP100, TGFBR2 Cell Morphology morphology morphology of cells 1.40E-05 ADM, CCND1, CD9, CDKN1A, DAPK3, EDNRB, FERMT2, HAS3, 27 IGFBP7, IL6ST, KLF4, MAP2K3, MET, MITF, MUC1, PEG10, PHLDA1, PITPNM1, PLAUR, PMP22, RASSF1, SIRPA, SOD2, STARD13, TGFBR2, THEM4, TIMP3 Cell Morphology morphology morphology of melanoma 7.02E-03 MITF, SOD2 2 cell lines Cell Morphology morphology morphology of kidney cell 1.50E-02 MAP2K3, PEG10, PLAUR, PMP22 4 lines Cell Morphology morphology morphology of fibroblast cell 2.19E-02 CCND1, PEG10 2 lines Cell Morphology morphology morphology of tumor cell 2.51E-02 ADM, CCND1, CD9, CDKN1A, HAS3, IL6ST, KLF4, MITF, 12 lines RASSF1, SOD2, STARD13, TIMP3 Cell Morphology tubulation tubulation of cells 4.83E-05 ALCAM, CD9, EDNRB, FOXO3, GBP1, ID1, ID3 (includes 12 EG:15903), IL8, KCNMA1, PLAUR, PTN, RAP1B Cell Morphology tubulation tubulation of endothelial 2.00E-04 EDNRB, FOXO3, GBP1, ID1, ID3 (includes EG:15903), IL8, 9 cells KCNMA1, PLAUR, PTN Cell Morphology tubulation tubulation of vascular 2.06E-03 FOXO3, ID1, ID3 (includes EG:15903), IL8, KCNMA1, PTN 6 endothelial cells Cell Morphology tubulation tubulation of skin cell lines 2.19E-02 ALCAM, RAP1B 2 Cell Morphology cell spreading cell spreading of brain cancer 9.62E-04 EDNRB, MARCKS, SIRPA, SYNM 4 cell lines Cell Morphology cell spreading cell spreading 6.09E-03 ANTXR1, EDNRB, IL8, ITGA5, LPP, MAPT, MARCKS, PARVA, 13 PMP22, RAP1B, SIRPA, SYNM, ZYX Cell Morphology cell spreading cell spreading of fibroblasts 2.19E-02 IL8, LPP 2 Cell Morphology morphogenesis morphogenesis of breast cell 7.02E-03 CCND1, HIF1A 2 lines Cell Morphology size size of cells 1.17E-02 CCND1, CDKN1B, DEPTOR, GAB2, KCNMA1, PPARGC1A, 8

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SYNM, UCN2 Cell Morphology shape shape of cells 1.36E-02 IL8, MAPT 2 Cell Morphology shape change shape change of tumor cell 1.59E-02 AKAP12, CCNE1, EDNRB, IL8, ITGA5, MARCKS, MET, RAP1B, 10 lines SIRPA, SYNM Cell Morphology length length of microtubules 2.19E-02 CKAP5, MAPT 2 Cell Morphology permeability permeability of skin cell 2.19E-02 IL8, RAP1B 2 lines Cell Morphology permeability permeability of endothelial 2.88E-02 IL8, NT5E, RAP1B 3 cell lines Cell Morphology formation formation of pseudopodia 2.88E-02 CDC42EP2, CDC42EP4, CDC42EP5 3

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Appendix 7 The list of differentially expressed genes associated with the top networks in response to GGH modulation

Appendix 7. 1 The top networks matched by the genes differentially expressed in the GGH-overexpressed HCT116 colon cancer cells

Focus No. Top Functions Score Genes in Network Genes 1 Cellular Movement, 40 24 ↓ADAM10, ↓AKAP12*, Akt, ↓BDNF, ↓BMP4*, CD3, ↓CRABP2, ↑FGFBP1 (includes Dermatological Diseases and EG:14181), ↑GDF15, hCG, IgG, ↑IRS1, ↓IRS2, Jnk, ↑KLK6, ↑LCN2, NFkB (complex), P38 Conditions, Carbohydrate MAPK,p85 (pik3r), ↓PDE4B*, PDGF BB, ↓PDLIM1, ↑PHLDA1*, PI3K (complex), ↑PLAU, Metabolism ↓PLK2, ↓PRKACB*,↓PRKCA, ↓RND3, ↓SACS, ↓SEMA3A, ↓SLC2A3, ↑TACSTD2, TCR, ↑UPP1 2 Cell Cycle, Cancer, 20 15 ADCK3, AGTR1, BRAF, CEP55, CHMP4C, CHRNA3, ↑COMT, Cyclin A, DHFR, E2F4, ESR1, Hematological Disease ↓FBXO5, HEY1, ↑HIST1H1C, ↓HNRNPU, IL1A, MCM6, ↓NAP1L1*, NFKB1, NFKBIZ, ↓NR2F2, NR3C1, ↓PDCD6IP, ↓PDE4B*, ↓PLK2, POLD1, ↓PPP1R14C, RFC4, ↓RPL35A, ↓SERBP1, ↓SPAG9*, TCEB1, ↓TOPBP1, TP53 (includes EG:22059), ↓ZNF91 3 DNA Replication, 17 13 ACTB, ↓BAMBI, BRCA2, ↓EPS8, ↑FASN, ↑HMGCS1, HNRNPC, ING1, MCM6, ↓MSH6, Recombination, and Repair, ↓NAP1L1*, NONO, ↓PHLDB2*, ↓PMS1, POLD1, POLE2, POLE3, ↓POLE4, POLE, PRMT1, Cancer, Renal and Urological ↓PVRL3*, ↓RFC1, ↓ RFC4, Rnr, ↓RPS23, ↓SAP30 (includes EG:60406), SMARCA4, Disease SMARCC1, SMC3, THRB, TOP1, TP53 (includes EG:22059), UBE3A, ↓UBQLN1, YWHAG 4 Cellular Growth and 15 12 ↑ABCG1, ABCG2, ↓ANXA2, ↓CAV2, CCND1, CDK5, ↓CDK6, CDKN2C, CSF2, ERK1/2, Proliferation, Cancer, Cell estrogen receptor, F11R, FBXO7, FLT1, ↓HSPH1, ICAM3, IGFBP5, ↓MNS1, OCLN, ↓PDGFC, Death ↑PPARG*, ↓PPIA, PRODH, ↑PTGES, ↑SAT1, SERPINF1, SGK1, SMAD4, Smad, ↓SNTB1, TGFBR1, ↓TIMP2 (includes EG:21858), ↓TRIM33, ↑TSC22D1, TSPYL2 5 Cell-To-Cell Signaling and 15 12 ↓ADAM19, AGT, ↓AKAP12*, ↓C12orf29, ↑CA2*, CD82, ↑CDH1, CDKN2B, CEBPA, CSF3R, Interaction, Hematological DRAP1, ENG, ↑FES, HIF1A, ↑HIST1H2BJ/HIST1H2BK, ↓HLA-DRA, IFNG (includes System Development and EG:15978), ITGAM (includes EG:16409), ITGAX, NCF2, ↑NFIB, ↑PBX1, ↑PLA2G16, ↑PTGES, Function, Immune Cell RFX5, SELP, Smad, SPINT2, TCF7L2, ↓TGFB2, TGFB1 (includes EG:21803), TGFBR2, TGM2, Trafficking TJP1 (includes EG:21872)

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers; ↑ upregulated; ↓ downregulated.

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Appendix 7. 2 The top networks matched by the genes differentially expressed in the GGH-inhibited HCT116 colon cancer cells

Focus No. Top Functions Score Genes in Network Genes 1 Hereditary Disorder, 37 27 ↑ABLIM1*, ↓AKAP12, ↑CRABP2, ↓CYP24A1, ↓DDIT3, ↑EREG, FSH, hCG, Hdac, Neurological Disease, ↓HERPUD1, Histone h3, IgG, ↑KDM5B, ↑LAMP1, Lh, MAP2K1/2, ↑MRPS6, ↑ MSMO1, Cardiovascular System ↓PDGFA, ↑PHLDA1*, ↓PRKACB*, ↑REEP1, ↑SACS, ↓SEMA3A, ↑SLC20A1, ↓SLC2A3, Development and Function ↑SMYD3, ↓SPAG9*, ↑ST6GAL1, ↓STC1, ↓TACSTD2, TCR, ↓UPP1, ↓ZBED1, ↓ZNF217 2 Cell Death, Cellular 35 26 26s Proteasome, ↓AHR, ↓ANXA2, ↑APOBEC3F, ↑APOBEC3G, ↑APP, ↓BMP4*, caspase, Movement, Hematological ↓CDK6, ↑CDKN1A, Cyclin A, Cyclin D, Cyclin E, E2f, ERK1/2, ↓FBXO5, ↓FGF9, ↑FHIT, System Development and ↑HIST1H2BJ/HIST1H2BK, ↓HIST1H4A (includes others)*, ↓IGFBP3, ↓IGFBP6, Igm, Function ↓KIAA0101, ↑MAP4K1, ↑MMP7*, ↑NEU1, P38 MAPK, ↓PAWR, ↓PPARG*, ↑PPIA, ↑SULF2, ↑SYK, ↓TFAP2A, ↓TOPBP1 3 Cancer, Cell Cycle, 29 23 Akt, CD3, ↓CDC42, ↑FGFR3, ↓GJA1, ↑GRB14, ↓HAS3, Hsp70, Hsp90, ↓HYOU1*, ↑IFI27, Connective Tissue IFN Beta, ↑IFNAR2, Interferon alpha, ↑IRF9, ↓IRS1, Jnk, LDL, Mapk, ↑NDRG1, NFkB (complex), Development and Function ↓NR4A2, PDGF BB, ↓PDPK1, PI3K (complex), ↑PRKCD, ↑PSMB9, ↓RIOK3, ↑RRAS2, ↓SLC16A6, ↑SPRY2, ↑TAP1, ↑TERF2, ↑TNFSF18, ↑TSC22D3* 4 Cell Cycle, Infectious Disease, 25 21 AGTR1, ↑AKR1A1, ↓ALYREF, ↓ANKRD1, AP3D1, ↓ATG12 (includes EG:361321), BCLAF1, Cellular Response to ↓DCLK1, ↑FASN, HIPK2, ↑HIST1H1C, Histone h4, HNRNPA2B1, ↑ID2*, IRF3, LBR (includes Therapeutics EG:368360), MAVS, ↓MEIS2, MYH10, ↑NAP1L1*, Notch, NPAT, ↓PBX1, ↑PPIA, ↑PRSS1/PRSS3, ↑RPL35A, ↑SAP18, ↑SERBP1, ↑SRI*, ↑SRPX, ↑TMEM173, TP53 (includes EG:22059), ↑VPS41 (includes EG:218035), ↓WSB1, ZNF143 5 Cellular Assembly and 22 19 ↑ADARB1, ↑AKR1C3, ↓BMP4*, ↑C12orf29, ↑CA2*, ↓CDK6, ↓CPLX1, ↑EPDR1, ERK1/2, Organization, Carbohydrate FGFR1, ↓FTL, ↓GJA1, ↑GRIN1, HAS1, HOXA13, ↓HOXB6, IL13, ↓MSX2, MUC5AC/MUC5B, Metabolism, Drug Metabolism ↑OTUD6B, PADI4, PGR (includes EG:18667), ↑PHLDA1*, SBDS, SMPD1, SNCA, SNCG, SP1, ↑SPRY2, TGM2, ↓TIA1*, ↑TMC6, UGCG, ↑UGDH, VHL

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers; ↑ upregulated; ↓ downregulated.

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Appendix 7. 3 The top networks matched by the genes differentially expressed in the GGH-overexpressed MDA-MB-435 breast cancer cells

Focus No. Top Functions Score Genes in Network Genes 1 Infectious Disease, 37 35 ↑ALDH3A2*, ↑ARMCX1*, ↓BAIAP2L1, ↑C7orf58*, ↑CAPRIN1, ↑CCND1, ↑CDKN2C, Dermatological Diseases and ↓CPNE3, ↓DNAJB6*, ↓EEF1A1, ↓ EIF5A2*, ↑GPC6, ↑H2AFJ, ↑HIST2H2BE (includes Conditions, Tissue others), ↓IDH3A, ↑ IFIH1, ↑LDLR, ↓LRPAP1, ↓MAPKAP1, ↓MNS1, ↑MORC4, ↑MYBL1, Morphology ↑NT5E, ↓PCBP2*, ↑PDLIM3*, ↓PTBP3*, ↓RBMS3, ↑RGS2 (includes EG:19735), ↑RUFY3, ↑SF3B3, ↓TMSB15A, ↓TYMP, ↓UBE2N, ↑ZBED5, ↓ZFP106* 2 Cellular Development, 30 32 ↑CALM1 (includes others), ↑CAMK2D, ↓CCDC50, ↓CD63, ↑CD74*, ↑CDC42EP1, Developmental Disorder, ↓FASTKD1, ↓FUCA1, ↑HLA-DMB, HLA-DR, ↑HLA-F, IFN Beta, ↑IL24*, ↑INSIG1*, Hereditary Disorder ↑KLHL2, ↑LUC7L3, NFkB (complex), ↓NGFRAP1*, ↑PDLIM1, ↑RFTN1, ↑RFX5, ↑RIOK3, ↓RIPK4, ↑RUSC2, ↑SYTL4, ↑TAP1, ↓TAX1BP1, ↓TBC1D4, ↓TFAM, ↑TJP2, ↓TNFAIP3, ↓TRAF3IP2, ↑TRIM8, ↑UBA7, ↑VMP1 3 Molecular Transport, Cell 30 32 ↑A2M, Ap2 alpha, ↑ARHGAP22, ↓ASAH1, ↑ATP1B1*, ↓ATP1B3*, ↑BAG3, ↓BCL2, ↓CTSH, Death, Tumor Morphology ↓CTSL2, ↓CYP27A1, ↑DYNLT3*, ↓ELOVL2, ↑EMP1, ↓FYCO1, hCG, ↑IFITM1, IgG, ↑ITGA10, ↑KRT15, ↓LGALS3, ↓M6PR, ↓MAP1LC3B, ↑MUC1*, ↓NDRG2*, ↑NES, ↓PLS3*, ↓RGS20, ↑SLC20A1, ↑TM4SF1, ↑TMEM158, ↑TNFRSF10B, ↓TNFSF13B*, ↓TSPAN7*, ↓UPP1 4 Cellular Movement, Cell-To- 26 30 ↓ANXA5, ↓ATP1A1*, ↑CD9, ↑CD151*, ↑CDC42EP5, Collagen(s), ↑CYR61, ↓DAB2*, Cell Signaling and Interaction, ↓EDNRB, ERK1/2, ↓FHL2*, Focal adhesion kinase, ↑GAS1 (includes EG:14451)*, ↓GRN, Tissue Development ↓HSPB1, ↓IGSF8, ↑IL6ST*, ↑ILK, ↑ITGA3, ↑ITGA5, ↑ITGA6, ↑ITGB1*, Laminin, ↑LMO4*, ↑MGAT5, ↓MPRIP, ↑PARVA*, ↓PARVB, Pkg, ↑PLAUR*, ↓PPP2R2A, ↑PTPN11, ↓SDCBP, ↑SHC1 (includes EG:20416), ↑TFF2 5 Cell Death, Dermatological 26 30 ↑AP1M1, ↑ARHGDIB, ↑BIRC2, ↑BIRC3*, ↓CANX, CD3, CK1, ↑COPB1, ↓CST1, ↓CYP2J2, Diseases and Conditions, ↑DNAJC15, ↑GPR37, ↑HLA-A, ↑HLA-B, ↑HLA-G, IKK (complex), Immunoglobulin, Integrin, Immunological Disease ↑JUN, ↓NFATC1, ↓P2RX7, ↓PEG10, ↓PHB2, ↑PHLDA2, ↑PODXL, ↑PRKCA, ↑RAC2, ↓RHOBTB3, ↑RNF128, ↑SLC38A2, ↑STIM1, ↓TFAP2A*, ↓TYR, ↑UBE2L6, ↓VAV3*

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers; ↑ upregulated; ↓ downregulated.

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Appendix 7. 4 The top networks matched by the genes differentially expressed in the GGH-inhibited MDA-MB-435 breast cancer cells

Focus No. Top Functions Score Genes in Network Genes 1 Developmental Disorder, 34 31 ↓ACSL3, ↑ARPC1B, ↑CALB2, ↓CAP2, ↑ CASP1*, ↑CASP4, ↓CAV2, ↑CD68, ↓CDC42EP4, Renal and Urological Disease, ↓COL13A1*, ↓CREBZF, ↑EMP1, estrogen receptor, ↑FGF13, ↑FJX1, FSH, ↑GPRC5A, hCG, Protein Synthesis ↑HMGA2, Lh, ↑MAMLD1, ↑MT1X, ↑MT2A, ↓NFIB, ↓NR0B1, ↓PTPRF, ↑RQCD1, ↑SGK1*, ↑SLC20A1, ↑ STC1, ↓TCF12, ↑TGFBR2*, ↓TGFBR3, ↑TMEM158, ↑UPP1 2 Cellular Movement, Cellular 32 30 ↑ADM, ↑AKAP12*, AMPK, ↓AZGP1, ↓CDC42EP5, Ctbp, ↑CYR61, ↑DDX5, ↑DKK1, Growth and Proliferation, Cell ↑EPAS1, ERK1/2, ↑FHL1 (includes EG:14199), ↓HDAC4, ↑HEY1, ↑HIF1A*, ↓HLA-DRB3 Death (includes others), ↑IL6ST*,↑ ITGA5, ↓KCNMA1, ↑KIAA1199, ↑LDHA*, ↓LRP5, ↓NR4A2*, ↓P2RX7, ↓PARVA*, ↑PFKFB3, ↑PLAUR*, ↑PTN, ↑RAP1B, ↑SLC2A1, ↑SNCA*, SRC (family), ↑TFF2, ↑TRPM2, Vegf 3 Tumor Morphology, Cell 32 30 ↓ADD3*, ↓BBX, ↑C11orf82, ↓C12orf76, ↑CCND1, ↑CDKN2B, ↓CEACAM1 (includes Cycle, Connective Tissue others)*, ↓CEBPD, Creb, ↓CRIP2, ↑DNAJB6*, ↑ENC1, ↑GPC6, ↓H2AFJ, ↑HBE1, Histone h3, Development and Function Histone h4, ↓HNRPDL, ↑ID2*, ↑LDLR, ↑METTL1*, ↑MT1G, ↑NME1 (includes EG:18102), Notch, ↑NT5E, ↓OGT*, ↑PCBP2*, ↑PDLIM5, ↓PHGDH, Pkc(s), ↑RBM39, ↑SERTAD1, ↑SPC25 (includes EG:100144563), ↓SUGP2, ↓ZFP106* 4 Cellular Movement, 30 29 ↓AMPH, ↑BCL2A1, ↑CCL20, CD3, ↓CD74*, ↓CLEC11A, ↑DEFB103A/DEFB103B, ↑EDNRB, Hematological System ↑EEF1A1, F Actin, ↓FUCA1, ↓HLA-DMB, ↓HLA-DRA*, IFN Beta, ↑INSIG1*, ↑ITGB8, Development and Function, ↓LUC7L3, ↓LYST*, ↑MAP2K3, ↓MAP3K3, MHC Class II (complex), ↑MLKL, NFkB (complex), Immune Cell Trafficking ↑PDRG1, ↑PPAP2B, ↑RFTN1, ↓RFX5, ↑SMAD6, ↑TFAM, ↑TFRC, Tgf beta, ↑TIMP3, ↑TNFAIP3, ↓TRAF3IP2, ↑TXNRD1* 5 Cell Death, Cellular Assembly 25 26 ↑A2M, Ap1, ↓CAPRIN1, ↑CTGF, ↑DUSP5, ↑DUSP10*, ↑FHL2*, ↑FOSB, ↑HBEGF, and Organization, Cellular ↓IGFBP6, IL1, ↑KLHL21, LDL, ↓LMCD1, MAP2K1/2, Mapk, ↑MGST1, ↑MMP3, ↑MMP1 Compromise (includes EG:300339), ↑MYL9*, ↑NOV, P38 MAPK, PDGF BB, ↓ PHLDA1*, ↑PPP1R15A, Rb, ↑RND3, ↓S100A4*, ↓SLC2A3, ↓SMARCA2, SMOOTH MUSCLE ACTIN, ↑SRF, ↓TOP2A, ↓TOP2B, ↑TRIB1

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers; ↑ upregulated; ↓ downregulated.

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Appendix 8 Genes with altered promoter methylation and expression in response to GGH modulation

Appendix 8. 1 Genes with altered promoter methylation and expression in the GGH-overexpressed HCT116 colon cancer cells

DNA Gene Probe ID Methylation Expression Entrez CpG Gene Symbol Description β-Value Chromosome Fold Change DNA Gene ID Island Gene Expression Difference (vs. Control) Methylation (vs. Control)

Hypermethylated and Downregulated SUSD2 sushi domain containing 2 0.26 -2.27 cg23349242 ILMN_1693270 56241 22 No MNS1 meiosis-specific nuclear structural 1 0.45 -1.70 cg14797887 ILMN_2157240 55329 15 Yes ZNF91 zinc finger protein 91 0.28 -1.70 cg21029904 ILMN_1802053 7644 19 No HLA-DRA major histocompatibility complex, class II, 0.33 -1.37 cg19248557 ILMN_2157441 3122 6 No DR alpha

Hypomethylated and Upregulated FGFBP1 fibroblast growth factor binding protein 1 -0.28 4.08 cg13929970 ILMN_1785404 9982 4 No

Appendix 8. 2 Genes with altered promoter methylation and expression in the GGH-inhibited HCT116 colon cancer cells

DNA Gene Probe ID Methylation Expression Entrez CpG Gene Symbol Description β-Value Chromosome Fold Change DNA Gene Gene ID Island Difference (vs. Control) Methylation Expression (vs. Control)

Hypermethylated and Downregulated

IGFBP3 insulin-like growth factor binding protein 3 0.29 -1.84 cg15898840 ILMN_1746085 3486 7 Yes IGFBP3 insulin-like growth factor binding protein 3 0.21 -1.84 cg04796162 ILMN_1746085 3486 7 Yes SUSD2 sushi domain containing 2 0.23 -1.49 cg03599338 ILMN_1693270 56241 22 No

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ALDH1A3 aldehyde dehydrogenase 1 family, member 0.47 -1.34 cg19224278 ILMN_2139970 220 15 No A3

Hypomethylated and Upregulated SNTB1 syntrophin, beta 1 (dystrophin-associated -0.34 2.75 cg14992108 ILMN_1793410 6641 8 No protein A1, 59kDa, basic component 1) FAT1 FAT tumor suppressor homolog 1 -0.23 2.04 cg03098643 ILMN_3247578 2195 4 Yes (Drosophila) HIST1H2BK histone cluster 1, H2bk -0.34 1.87 cg02704907 ILMN_1796179 85236 6 No FAT1 FAT tumor suppressor homolog 1 -0.23 1.76 cg03098643 ILMN_1754795 2195 4 Yes (Drosophila) CRABP2 cellular retinoic acid binding protein 2 -0.33 1.72 cg27493997 ILMN_1690170 1382 1 Yes OSR1 odd-skipped related 1 (Drosophila) -0.54 1.69 cg02742971 ILMN_2197128 130497 2 Yes OSR1 odd-skipped related 1 (Drosophila) (OSR1) -0.26 1.69 cg06509239 ILMN_2197128 130497 2 No PRSS3 protease, serine, 3 (mesotrypsin) (PRSS3) -0.25 1.60 cg18540325 ILMN_1685699 5646 9 No SERPINB1 serpin peptidase inhibitor, clade B -0.37 1.55 cg06148264 ILMN_1679133 1992 6 No (ovalbumin), member 1 GTF3C1 general transcription factor IIIC, polypeptide -0.21 1.52 cg19571071 ILMN_1789839 2975 16 No 1, alpha 220kDa

AKR1C3 aldo-keto reductase family 1, member C3 (3- -0.30 1.47 cg19118077 ILMN_1713124 8644 10 No alpha hydroxysteroid dehydrogenase, type II) CYB5A cytochrome b5 type A (microsomal) -0.25 1.47 cg27662877 ILMN_1714167 1528 18 Yes MMP7 matrix metallopeptidase 7 (matrilysin, -0.49 1.44 cg25511807 ILMN_2192072 4316 11 No uterine) VAMP1 vesicle-associated membrane protein 1 -0.21 1.33 cg25750404 ILMN_1737611 6843 12 Yes (synaptobrevin 1) SULF2 sulfatase 2 -0.33 1.32 cg11979959 ILMN_1667460 55959 20 Yes

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Appendix 8. 3 Genes with altered promoter methylation and expression in the GGH-overexpressed MDA-MB-435 breast cancer cells

DNA Gene Probe ID Methylation Expression Entrez CpG Gene Symbol Description β Chromosome -Value Fold Change DNA Gene Gene ID Island Difference (vs. Control) Methylation Expression (vs. Control)

Hypermethylated and Downregulated TYR tyrosinase (oculocutaneous albinism IA) 0.26 -31.95 cg03417466 ILMN_1788774 7299 11 No TSPAN7 tetraspanin 7 0.34 -21.45 cg20450764 ILMN_2120695 7102 X No TSPAN7 tetraspanin 7 0.34 -9.29 cg20450764 ILMN_1809291 7102 X No APOD apolipoprotein D 0.23 -5.41 cg05624196 ILMN_1780170 347 3 No CTSL2 cathepsin L2 0.23 -3.78 cg13234643 ILMN_1748352 1515 9 Yes PLSCR1 phospholipid scramblase 1 0.28 -3.63 cg20586531 ILMN_1745242 5359 3 No GPR143 G protein-coupled receptor 143 0.55 -3.44 cg11325578 ILMN_1756261 4935 X Yes MSRB2 methionine sulfoxide reductase B2 0.46 -3.20 cg17431739 ILMN_1657977 22921 10 No KCNAB1 potassium voltage-gated channel, shaker- 0.36 -2.95 cg23873703 ILMN_1744968 7881 3 No related subfamily, beta member 1 EDNRB endothelin receptor type B 0.25 -2.61 cg24745738 ILMN_1751904 1910 13 No CMTM8 CKLF-like MARVEL transmembrane 0.25 -2.35 cg10553028 ILMN_1710124 152189 3 Yes domain containing 8 EPAS1 endothelial PAS domain protein 1 0.21 -2.11 cg17518825 ILMN_1704753 2034 2 No FUCA1 fucosidase, alpha-L- 1, tissue 0.27 -2.06 cg24792360 ILMN_1752728 2517 1 No RAB38 RAB38, member RAS oncogene family 0.21 -2.00 cg00027674 ILMN_2134974 23682 11 No SOX10 SRY (sex determining region Y)-box 10 0.38 -1.75 cg06614002 ILMN_1653750 6663 22 No C8orf38 chromosome 8 open reading frame 38 0.22 -1.71 cg21197871 ILMN_1727618 137682 8 No FECH ferrochelatase (protoporphyria) 0.27 -1.64 cg05243804 ILMN_1774091 2235 18 No C6orf218 chromosome 6 open reading frame 218 0.21 -1.59 cg22572779 ILMN_1665824 221718 6 No

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GREB1 growth regulation by estrogen in breast 0.26 -1.56 cg10612997 ILMN_1721170 9687 2 No cancer 1 ITPKB inositol 1, 4, 5-trisphosphate 3-kinase B 0.52 -1.47 cg01259619 ILMN_1700432 3707 1 Yes POP1 processing of precursor 1, ribonuclease 0.45 -1.47 cg26804057 ILMN_1768273 10940 8 No P/MRP subunit (S. cerevisiae) VAV3 vav 3 guanine nucleotide exchange factor 0.34 -1.45 cg19918758 ILMN_2399463 10451 1 No ITGB1BP1 integrin beta 1 binding protein 1 0.24 -1.43 cg07974891 ILMN_1690099 9270 2 No ZNF274 zinc finger protein 274 0.28 -1.39 cg00647741 ILMN_1688629 10782 19 No AGL amylo-1, 6-glucosidase, 4-alpha- 0.27 -1.38 cg14453641 ILMN_2371825 178 1 No glucanotransferase VAV3 vav 3 guanine nucleotide exchange factor 0.34 -1.38 cg19918758 ILMN_1657679 10451 1 No ABCC5 ATP-binding cassette, sub-family C 0.21 -1.34 cg16378421 ILMN_1706531 10057 3 No (CFTR/MRP), member 5 GAS2 growth arrest-specific 2 0.32 -1.32 cg03547797 ILMN_1804569 2620 11 No

Hypomethylated and Upregulated S100A4 S100 calcium binding protein A4 -0.24 13.13 cg26894575 ILMN_1684306 6275 1 No S100A4 S100 calcium binding protein A4 -0.24 10.51 cg26894575 ILMN_1688780 6275 1 No FGFRL1 fibroblast growth factor receptor-like 1 -0.21 6.83 cg00940891 ILMN_1795865 53834 4 Yes PARVA parvin, alpha -0.29 5.04 cg26320696 ILMN_3307892 55742 11 Yes PARVA parvin, alpha -0.25 5.04 cg01781266 ILMN_3307892 55742 11 Yes NNMT nicotinamide N-methyltransferase -0.37 4.80 cg14209518 ILMN_1715508 4837 11 No HTATIP2 HIV-1 Tat interactive protein 2, 30kDa -0.35 4.75 cg15576195 ILMN_1664303 10553 11 Yes CRYAB crystallin, alpha B -0.46 4.48 cg13084335 ILMN_1729216 1410 11 No CPVL carboxypeptidase, vitellogenic-like -0.46 4.11 cg24448259 ILMN_2400759 54504 7 No KLRC2 killer cell lectin-like receptor subfamily C, -0.34 4.09 cg22432908 ILMN_2059357 3822 12 No member 2 COL8A1 collagen, type VIII, alpha 1 -0.33 3.93 cg02441647 ILMN_1685433 1295 3 No RAC2 ras-related C3 botulinum toxin substrate 2 -0.28 3.93 cg18265887 ILMN_1709795 5880 22 No (rho family, small GTP binding protein Rac2)

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PTPN22 protein tyrosine phosphatase, non-receptor -0.47 3.79 cg14385738 ILMN_2246328 26191 1 No type 22 (lymphoid) PTPN22 protein tyrosine phosphatase, non-receptor -0.32 3.79 cg00916635 ILMN_2246328 26191 1 No type 22 (lymphoid)

CDC42EP5 CDC42 effector protein (Rho GTPase -0.58 3.76 cg09227563 ILMN_1774982 148170 19 No binding) 5 IGFBP7 insulin-like growth factor binding protein 7 -0.22 3.66 cg00884221 ILMN_2062468 3490 4 Yes FKBP11 FK506 binding protein 11, 19 kDa -0.66 3.63 cg21908259 ILMN_1787345 51303 12 Yes FKBP11 FK506 binding protein 11, 19 kDa -0.24 3.63 cg15147435 ILMN_1787345 51303 12 Yes A2M alpha-2-macroglobulin -0.29 3.57 cg12058490 ILMN_1745607 2 12 No CPVL carboxypeptidase, vitellogenic-like -0.46 3.46 cg24448259 ILMN_1682928 54504 7 No COL8A1 collagen, type VIII, alpha 1 -0.33 3.39 cg02441647 ILMN_2402392 1295 3 No TMEM42 transmembrane protein 42 -0.44 3.19 cg23844090 ILMN_1760245 131616 3 Yes TMEM42 transmembrane protein 42 -0.44 3.19 cg04682845 ILMN_1760245 131616 3 No TMEM47 transmembrane protein 47 -0.21 3.03 cg14346048 ILMN_2129234 83604 X Yes KDELR3 KDEL (Lys-Asp-Glu-Leu) endoplasmic -0.56 2.99 cg23583739 ILMN_1722820 11015 22 Yes reticulum protein retention receptor 3

CXorf26 chromosome X open reading frame 26 -0.24 2.97 cg25591670 ILMN_1768176 51260 X No KDELR3 KDEL (Lys-Asp-Glu-Leu) endoplasmic -0.56 2.91 cg23583739 ILMN_1713901 11015 22 Yes reticulum protein retention receptor 3 NES nestin -0.31 2.87 cg12356261 ILMN_1738147 10763 1 Yes CHN1 chimerin (chimaerin) 1 -0.24 2.79 cg11695601 ILMN_1678493 1123 2 No CHN1 chimerin (chimaerin) 1 -0.22 2.79 cg18440474 ILMN_1678493 1123 2 Yes MT1A metallothionein 1A -0.31 2.74 cg09137696 ILMN_1691156 4489 16 Yes HLA-DPA1 major histocompatibility complex, class II, -0.45 2.73 cg13906813 ILMN_1772218 3113 6 No DP alpha 1 PARVA parvin, alpha -0.29 2.72 cg26320696 ILMN_1756408 55742 11 Yes PARVA parvin, alpha -0.25 2.72 cg01781266 ILMN_1756408 55742 11 Yes CTDSPL CTD (carboxy-terminal domain, RNA -0.48 2.71 cg27201297 ILMN_2392189 10217 3 Yes polymerase II, polypeptide A) small phosphatase-like

421

FGFRL1 fibroblast growth factor receptor-like 1 -0.21 2.58 cg00940891 ILMN_2348367 53834 4 Yes PTPN22 protein tyrosine phosphatase, non-receptor -0.47 2.57 cg14385738 ILMN_1695640 26191 1 No type 22 (lymphoid)

PTPN22 protein tyrosine phosphatase, non-receptor -0.32 2.57 cg00916635 ILMN_1695640 26191 1 No type 22 (lymphoid)

CYBRD1 cytochrome b reductase 1 -0.37 2.44 cg10731149 ILMN_2087692 79901 2 No CYBRD1 cytochrome b reductase 1 -0.37 2.27 cg10731149 ILMN_1712305 79901 2 No KDELR3 KDEL (Lys-Asp-Glu-Leu) endoplasmic -0.56 2.25 cg23583739 ILMN_1798952 11015 22 Yes reticulum protein retention receptor 3

DACT3 dapper, antagonist of beta-catenin, homolog -0.37 2.24 cg06454084 ILMN_1733851 147906 19 No 3 (Xenopus laevis) CD55 CD55 molecule, decay accelerating factor -0.37 2.22 cg06792598 ILMN_1800540 1604 1 Yes for complement (Cromer blood group) MDK midkine (neurite growth-promoting factor 2) -0.25 2.20 cg18925548 ILMN_2349393 4192 11 No BST2 bone marrow stromal cell antigen 2 -0.24 2.19 cg01254505 ILMN_1723480 684 19 No SH3BGRL3 SH3 domain binding glutamic acid-rich -0.47 2.16 cg01414934 ILMN_1737163 83442 1 No protein like 3 SOX2 SRY (sex determining region Y)-box 2 -0.33 2.05 cg01340005 ILMN_2177156 6657 3 No ACSS2 acyl-CoA synthetase short-chain family -0.34 2.05 cg13368786 ILMN_2336595 55902 20 No member 2 ZCCHC24 zinc finger, CCHC domain containing 24 -0.41 2.04 cg17470697 ILMN_1754660 219654 10 No ACSS2 acyl-CoA synthetase short-chain family -0.34 1.99 cg13368786 ILMN_1714197 55902 20 No member 2 WDR6 WD repeat domain 6 -0.44 1.98 cg13415434 ILMN_1669484 11180 3 Yes TNFRSF10B tumor necrosis factor receptor superfamily, -0.41 1.98 cg02008544 ILMN_1699265 8795 8 Yes member 10b

STARD8 StAR-related lipid transfer (START) domain -0.49 1.89 cg13370916 ILMN_1799600 9754 X No containing 8 STARD8 StAR-related lipid transfer (START) domain -0.29 1.89 cg20832009 ILMN_1799600 9754 X No containing 8

RCN1 reticulocalbin 1, EF-hand calcium binding -0.27 1.88 cg12311132 ILMN_1800276 5954 11 No domain

422

MAOA monoamine oxidase A -0.54 1.88 cg15014034 ILMN_1663640 4128 X Yes MGP matrix Gla protein -0.31 1.86 cg13302154 ILMN_2071809 4256 12 No MGP matrix Gla protein -0.31 1.84 cg13302154 ILMN_1651958 4256 12 No ARHGAP22 Rho GTPase activating protein 22 -0.34 1.84 cg25290938 ILMN_1676361 58504 10 Yes ARHGAP22 Rho GTPase activating protein 22 -0.22 1.84 cg20506783 ILMN_1676361 58504 10 No ZNF35 zinc finger protein 35 -0.67 1.82 cg11612291 ILMN_1692100 7584 3 Yes RRAS related RAS viral (r-ras) oncogene homolog -0.36 1.81 cg17383958 ILMN_1780825 6237 19 Yes WDR33 WD repeat domain 33 -0.26 1.77 cg22854546 ILMN_2383913 55339 2 No EPS8 epidermal growth factor receptor pathway -0.42 1.75 cg09761310 ILMN_1651699 2059 12 Yes substrate 8 HIST1H2BK histone cluster 1, H2bk -0.36 1.75 cg02704907 ILMN_1796179 85236 6 No WDR33 WD repeat domain 33 -0.26 1.74 cg22854546 ILMN_1670172 55339 2 No ITIH5L inter-alpha (globulin) inhibitor H5-like -0.40 1.74 cg15521097 ILMN_1709177 347365 X No P2RX6 purinergic receptor P2X, ligand-gated ion -0.34 1.69 cg05442902 ILMN_3243924 9127 22 No channel, 6 SHC4 SHC (Src homology 2 domain containing) -0.21 1.67 cg16711185 ILMN_1807050 399694 15 No family, member 4 LEPREL1 leprecan-like 1 -0.20 1.66 cg20270599 ILMN_1657373 55214 3 No UBB ubiquitin B -0.20 1.66 cg03954587 ILMN_1762436 7314 17 Yes SLC26A4 solute carrier family 26, member 4 -0.30 1.65 cg04473302 ILMN_1652465 5172 7 Yes ARMCX1 armadillo repeat containing, X-linked 1 -0.42 1.65 cg18731813 ILMN_2180677 51309 X No HDAC9 histone deacetylase 9 -0.29 1.60 cg08285151 ILMN_1781173 9734 7 No PLAUR plasminogen activator, urokinase receptor -0.61 1.58 cg06540636 ILMN_1691508 5329 19 No PLAUR plasminogen activator, urokinase receptor -0.61 1.57 cg06540636 ILMN_2374340 5329 19 No H1F0 H1 histone family, member 0 -0.26 1.55 cg07141002 ILMN_1757467 3005 22 Yes TIPARP TCDD-inducible poly(ADP-ribose) -0.34 1.54 cg24262469 ILMN_1765578 25976 3 No polymerase STX2 syntaxin 2 -0.26 1.53 cg08169325 ILMN_1747775 2054 12 No SLC4A7 solute carrier family 4, sodium bicarbonate -0.20 1.52 cg06798189 ILMN_2200917 9497 3 No cotransporter, member 7

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PTPN22 protein tyrosine phosphatase, non-receptor -0.47 1.52 cg14385738 ILMN_1715885 26191 1 No type 22 (lymphoid) PTPN22 protein tyrosine phosphatase, non-receptor -0.32 1.52 cg00916635 ILMN_1715885 26191 1 No type 22 (lymphoid)

STK19 serine/threonine kinase 19 -0.45 1.50 cg24870273 ILMN_1659782 8859 6 No PGAP2 FGF receptor activating protein 1 -0.53 1.49 cg02940164 ILMN_1755405 27315 11 Yes ARMCX1 armadillo repeat containing, X-linked 1 -0.42 1.48 cg18731813 ILMN_1788192 51309 X No VHL von Hippel-Lindau tumor suppressor -0.21 1.46 cg20916523 ILMN_1738579 7428 3 No SERPINB1 serpin peptidase inhibitor, clade B -0.39 1.46 cg06148264 ILMN_1679133 1992 6 No (ovalbumin), member 1 SERPINB1 serpin peptidase inhibitor, clade B -0.27 1.46 cg27056119 ILMN_1679133 1992 6 Yes (ovalbumin), member 1 STX2 syntaxin 2 -0.26 1.45 cg08169325 ILMN_2344216 2054 12 No C6orf150 chromosome 6 open reading frame 150 -0.39 1.45 cg09527362 ILMN_1706645 115004 6 No C6orf150 chromosome 6 open reading frame 150 -0.25 1.45 cg00463577 ILMN_1706645 115004 6 Yes UBB ubiquitin B -0.20 1.44 cg03954587 ILMN_2191428 7314 17 Yes SOX2 SRY (sex determining region Y)-box 2 -0.33 1.43 cg01340005 ILMN_1770635 6657 3 No SLC22A4 solute carrier family 22 (organic -0.30 1.41 cg27372468 ILMN_2050911 6583 5 Yes cation/ergothioneine transporter), member 4 WDR33 WD repeat domain 33 -0.26 1.41 cg22854546 ILMN_1716086 55339 2 No DAK dihydroxyacetone kinase 2 homolog (S. -0.22 1.37 cg25406518 ILMN_1678619 26007 11 No cerevisiae) GRIA3 glutamate receptor, ionotrophic, AMPA 3 -0.35 1.36 cg23424962 ILMN_1782043 2892 X No HAS3 hyaluronan synthase 3 -0.46 1.36 cg12265604 ILMN_1794501 3038 16 No PLXNA3 plexin A3 -0.57 1.34 cg07428182 ILMN_1719972 55558 X Yes BRSK1 BR serine/threonine kinase 1 -0.28 1.34 cg25443975 ILMN_2185845 84446 19 No PEX7 peroxisomal biogenesis factor 7 -0.23 1.33 cg03807235 ILMN_1729650 5191 6 Yes HOXC8 homeobox C8 -0.28 1.33 cg05022306 ILMN_1718285 3224 12 Yes COL8A1 collagen, type VIII, alpha 1 -0.33 1.32 cg02441647 ILMN_2246563 1295 3 No

424

ARL3 ADP-ribosylation factor-like 3 -0.35 1.32 cg18151487 ILMN_1780444 403 10 No LONRF3 LON peptidase N-terminal domain and ring -0.52 1.31 cg06944050 ILMN_1720452 79836 X Yes finger 3 GTF2A1 general transcription factor IIA, 1, -0.27 1.31 cg17498523 ILMN_1725247 2957 14 Yes 19/37kDa ELF4 E74-like factor 4 (ets domain transcription -0.28 1.30 cg06428055 ILMN_1652082 2000 X Yes factor)

Appendix 8. 4 Genes with altered promoter methylation and expression in the GGH-inhibited MDA-MB-435 breast cancer cell

DNA Gene Probe ID Methylation Expression Gene Entrez CpG Description β-Value Chromosome Symbol Fold Change DNA Gene Gene ID Island Difference (vs. Control) Methylation Expression (vs. Control)

Hypermethylated and Downregulated CTHRC1 collagen triple helix repeat containing 1 0.55 -4.44 cg19188612 ILMN_1725090 115908 8 No CTHRC1 collagen triple helix repeat containing 1 0.55 -3.20 cg19188612 ILMN_2117508 115908 8 No CDC42EP5 CDC42 effector protein (Rho GTPase 0.24 -2.53 cg09227563 ILMN_1774982 148170 19 No binding) 5 LMCD1 LIM and cysteine-rich domains 1 0.40 -2.40 cg08935301 ILMN_1754969 29995 3 No AMPH amphiphysin 0.35 -2.17 cg09966445 ILMN_1685834 273 7 No HLA-DMA major histocompatibility complex, class II, 0.21 -1.98 cg24421410 ILMN_1695311 3108 6 No DM alpha PCOLCE procollagen C-endopeptidase enhancer 0.27 -1.69 cg26777475 ILMN_1707070 5118 7 No PCOLCE procollagen C-endopeptidase enhancer 0.23 -1.69 cg02797569 ILMN_1707070 5118 7 No IL11RA interleukin 11 receptor, alpha 0.21 -1.63 cg21504624 ILMN_1664912 3590 9 No SORBS2 sorbin and SH3 domain containing 2 0.28 -1.60 cg20544605 ILMN_1716407 8470 4 No SORBS2 sorbin and SH3 domain containing 2 0.28 -1.53 cg20544605 ILMN_2407879 8470 4 No GYG2 glycogenin 2 0.24 -1.50 cg05113908 ILMN_2319424 8908 X Yes AUTS2 autism susceptibility candidate 2 0.31 -1.50 cg15753757 ILMN_1749081 26053 7 Yes

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RABGAP1 RAB GTPase activating protein 1 0.22 -1.40 cg15209169 ILMN_2061950 23637 9 No MYO1B myosin IB 0.22 -1.37 cg15096140 ILMN_2093027 4430 2 No GAS7 growth arrest-specific 7 0.38 -1.36 cg13991233 ILMN_1745994 8522 17 No ALCAM activated leukocyte cell adhesion molecule 0.21 -1.35 cg16338347 ILMN_1670870 214 3 No PARVA parvin, alpha 0.73 -1.33 cg01781266 ILMN_3307892 55742 11 Yes PC pyruvate carboxylase 0.22 -1.33 cg14897096 ILMN_1671489 5091 11 No PAM peptidylglycine alpha-amidating 0.28 -1.31 cg20131596 ILMN_2313901 5066 5 No monooxygenase

Hypomethylated and Upregulated CXorf26 chromosome X open reading frame 26 -0.40 10.42 cg12901064 ILMN_1768176 51260 X No CXorf26 chromosome X open reading frame 26 -0.34 10.42 cg25591670 ILMN_1768176 51260 X No BCHE butyrylcholinesterase -0.22 2.77 cg21995126 ILMN_2176592 590 3 No CDKN1A cyclin-dependent kinase inhibitor 1A (p21, -0.29 2.57 cg03714916 ILMN_1784602 1026 6 No Cip1) DYNLT3 dynein, light chain, Tctex-type 3 -0.49 2.34 cg20119635 ILMN_1681890 6990 X No BCHE butyrylcholinesterase -0.22 2.31 cg21995126 ILMN_1685641 590 3 No APCDD1L adenomatosis polyposis coli down-regulated -0.22 1.93 cg23418591 ILMN_1689431 164284 20 No 1-like EDNRB endothelin receptor type B -0.85 1.89 cg24745738 ILMN_1751904 1910 13 No SLC22A18AS solute carrier family 22 (organic cation -0.25 1.86 cg08999895 ILMN_1691048 5003 11 No transporter), member 18 antisense MRGPRX4 MAS-related GPR, member X4 -0.35 1.83 cg16446783 ILMN_1722227 117196 11 No LEPREL1 leprecan-like 1 -0.21 1.81 cg20270599 ILMN_1657373 55214 3 No RENBP renin binding protein -0.52 1.73 cg04287191 ILMN_1780057 5973 X No FRMD4A FERM domain containing 4A -0.24 1.71 cg25464840 ILMN_1678961 55691 10 No FHOD3 formin homology 2 domain containing 3 -0.42 1.71 cg08568512 ILMN_1761322 80206 18 Yes FHL2 four and a half LIM domains 2 -0.26 1.70 cg10635061 ILMN_2355831 2274 2 No AGPAT9 1-acylglycerol-3-phosphate O- -0.24 1.70 cg26071978 ILMN_1794875 84803 4 No acyltransferase 9

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RHEB Ras homolog enriched in brain -0.22 1.66 cg03998173 ILMN_1657949 6009 7 Yes CCND1 cyclin D1 -0.23 1.65 cg07426960 ILMN_1688480 595 11 No FHL2 four and a half LIM domains 2 -0.26 1.64 cg10635061 ILMN_1668411 2274 2 No AKR1B10 aldo-keto reductase family 1, member B10 -0.27 1.62 cg11693019 ILMN_1672148 57016 7 No (aldose reductase) C1GALT1 core 1 synthase, glycoprotein-N- -0.32 1.58 cg13109289 ILMN_1662578 56913 7 No acetylgalactosamine 3-beta- galactosyltransferase, 1 DYNLT3 dynein, light chain, Tctex-type 3 -0.49 1.57 cg20119635 ILMN_2162564 6990 X No PTN pleiotrophin -0.45 1.53 cg11521965 ILMN_1813753 5764 7 No PTN pleiotrophin -0.23 1.53 cg00653387 ILMN_1813753 5764 7 No PHLDA2 pleckstrin homology-like domain, family -0.22 1.52 cg26799802 ILMN_1671557 7262 11 Yes A, member 2 UBIAD1 UbiA prenyltransferase domain containing -0.31 1.45 cg11858029 ILMN_1651872 29914 1 No 1 ZCCHC6 zinc finger, CCHC domain containing 6 -0.22 1.42 cg05065037 ILMN_1779677 79670 9 No TYRP1 tyrosinase-related protein 1 -0.38 1.41 cg25989745 ILMN_2054652 7306 9 No SIRPB1 signal-regulatory protein beta 1 -0.32 1.41 cg25799433 ILMN_1733997 10326 20 No RASSF1 Ras association (RalGDS/AF-6) domain -0.22 1.41 cg00777121 ILMN_1734205 11186 3 Yes family 1 CLDN14 claudin 14 -0.21 1.41 cg15310162 ILMN_1661194 23562 21 No CLDN14 claudin 14 -0.20 1.41 cg11099899 ILMN_1661194 23562 21 No AEBP1 AE binding protein 1 -0.35 1.38 cg06852744 ILMN_1736178 165 7 No PMP22 peripheral myelin protein 22 -0.74 1.37 cg08343834 ILMN_1785646 5376 17 No PLAUR plasminogen activator, urokinase receptor -0.29 1.37 cg06540636 ILMN_2374340 5329 19 No AFAP1 actin filament associated protein 1 -0.39 1.33 cg15957394 ILMN_1701998 60312 4 Yes

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Appendix 9 The list of differentially methylated and expressed genes associated with the top networks in response to GGH modulation

Appendix 9. 1 The top networks matched by the genes with altered expression and promoter methylation in the GGH- overexpressed HCT116 colon cancer cells

Focus No. Top Functions Score Genes in Network Genes Hypermethylated and Downregulated 1 Cell Morphology, Connective Tissue Development 3 1 E2F4, ZNF91 and Function, Digestive System Development and Function 2 Cell Cycle, Cell-To-Cell Signaling and Interaction, 3 1 CCND1, CDK4, MAPK1, MNS1 Connective Tissue Development and Function 3 Genetic Disorder, Immunological Disease, Gene 2 1 CD82, CIITA, CREB1, DRAP1, EBI3, HLA-DRA, HLA-DRB1, IFNG Expression (includes EG:15978), IL27, IL1B, POU2F1, RAD21, RFX5, RFXANK, RFXAP, SIN3A, SMARCA4, SMC3, XBP1 Hypomethylated and Upregulated 1 Cell Cycle, DNA Replication, Recombination, and 3 1 FBXW7, FGFBP1, IgG, MAPK14, Mek, Pkc(s), TP53 (includes Repair, Cellular Compromise EG:22059)

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base).

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Appendix 9. 2 The top networks matched by the genes with altered expression and promoter methylation in the GGH-inhibited HCT116 colon cancer cells

Focus No. Top Functions Score Genes in Network Genes Hypermethylated and Downregulated 1 Lipid Metabolism, Molecular Transport, Small 3 1 ALDH1A3, IL1A, PGR (includes EG:18667), TNF Molecule Biochemistry 2 Gene Expression, Organismal Development, 2 1 Ap1, BAZ1A, BMP7, CASP7, caspase, EPAS1, FANCC, GH1, Hdac, Cellular Growth and Proliferation HN, Igf, IGFBP3*, IGFBP4, Igfbp, LEPR, LYVE1, MECP2, MMP19, N- cor, NKX3-1, NR4A1, PLG, PPARA, RXRA, SLPI, SMAD2, SUZ12, TGFBR1, THBS2, TOPBP1, TP63, USF1, USF2, VDR, ZNF217 Hypomethylated and Upregulated 1 Cancer, Reproductive System Disease, Cellular 9 5 AKT1, CASP1, CCL27, CD44 (includes EG:100330801), CEBPA, Development CHI3L1, CSF2, CTNNB1, E2F1, ERBB2, ERK1/2, ESR1, FN1, FOS, IGFBP5, IL1, IL5, IL1B, JUN, KRAS, LGALS3, MMP3, MMP7, MMP8, MTOR, PLAU, PRSS1/PRSS3, RB1, SERPINB1, SMAD1/5, SNTB1, SULF2, TGFB1 (includes EG:21803), TGFBR2, TNF 2 Embryonic Development, Organismal 3 1 OSR1*, WNK1 Development, Tissue Development 3 Developmental Disorder, Endocrine System 2 1 AKR1C3, PGR (includes EG:18667), SBDS Disorders, Gastrointestinal Disease 4 Cancer, Embryonic Development, Tissue 2 1 GTF3C1, MTOR, RPTOR Development 5 Reproductive System Development and Function, 2 1 CRABP2, ESR1, IgG, RARA Drug Metabolism, Lipid Metabolism

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Appendix 9. 3 The top networks matched by the genes with altered expression and promoter methylation in the GGH- overexpressed MDA-MB-435 breast cancer cells Focus No. Top Functions Score Genes in Network Genes Hypermethylated and Downregulated 1 Cell Morphology, Reproductive System 28 13 ABCC5, ABCF2, ALDOC, APOD, BMP4, CORO1A, CTSL2, EDNRB, EIF5A, EPAS1, Disease, Cardiovascular System Development ESR1, FAM13A, FUCA1, GREB1, HIF1A, HLA-DRB3 (includes others), ITGB1, ITGB1BP1, and Function KDM4B, KIAA1199, LGALS3, MAP2K1, MAPK1, NFkB (complex), PLSCR1, RAB38, RLN1/RLN2, STC2, TAF9B, TCR, TMEM45A, TMPRSS6, TSPAN7*, TYR, VAV3* 2 Carbohydrate Metabolism, Cell Death, Cellular 2 1 MSRB2, PARP1 Assembly and Organization 3 Cardiovascular System Development and 2 1 AGTR1, KCNAB1 Function, Organ Morphology, Organismal Injury and Abnormalities 4 Cell Death, Cellular Function and Maintenance, 2 1 TRIM28, ZNF274 Reproductive System Development and Function 5 RNA Damage and Repair, Developmental 2 1 POP1 (includes EG:10940), RMRP Disorder, Genetic Disorder Hypomethylated and Upregulated 1 Cellular Movement, Cardiovascular System 23 14 Ap1, BCL2, BST2, CBL, CD55, CHI3L1, CSK, CYCS, ELF4, EPS8, HAS3, HDAC9, HDC, Development and Function, Cancer IGFBP7, IL8, IL1B, KLRC2, LILRA4, MAL, MAPK1, MMP13, MYEF2, NES, NNMT, P38 MAPK, PI3K (complex), PTPN22*, RAC2, S100A3, S100A4*, SERPINB1*, SPP1 (includes EG:20750), SRC, TIMP1, TNF 2 Cellular Development, Cell Cycle, Cancer 21 13 A2M, Akt, ARMCX1*, BMP2, CCND1, CDCA7L, COL2A1 (includes EG:1280), CRYAB, CTNNB1, DLX5, EN1, EZH2, GADD45B, HIC1, HIST1H2BJ/HIST1H2BK, MAOA, Mapk, MDK, MEIS1, MT1A, PLAC1, PTEN, RB1, RCN1 (includes EG:19672), RIN2, RRAS, SATB1, SLC22A4, SMAD3, SOX2*, SP1, SPP1 (includes EG:20750), TMEM47, TWIST2, WDR6 3 Cellular Movement, Skeletal and Muscular 19 12 A2M, Ap1, ARHGAP22*, ATF6, BMP2, CASP9 (includes EG:100140945), CDC42EP5, System Development and Function, Cell Death COL8A1*, Collagen type IV, ERK1/2,FGFR1, GADD45B, GRIA3, hCG, HTATIP2, ILK,ITGB5, KDELR3*, LIMS1, LRP1 (includes EG:16971), MMP2, MMP14, MYC, NCOA5, PARVA*, PLAUR*, SHC4, SMAD3, SPP1 (includes EG:20750), SPRY2, TCR, TNFRSF10B, UXT, VHL, ZNF217 Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Appendix 9. 4 The top networks matched by the genes with altered expression and promoter methylation in the GGH-inhibited MDA-MB-435 breast cancer cells

Focus No. Top Functions Score Genes in Network Genes Hypermethylated and Downregulated 1 Cellular Development, Cell-To-Cell Signaling 17 8 AKT1, ALCAM, AMPH, AR, ASAH1, AUTS2, C19orf29, CCL5, CCL26, CD48, and Interaction, Hematological System CDC42EP5, CLEC11A, CXCL16, DHCR24, EBI3, ELAVL1, ERAP2, ERK1/2, Development and Function G0S2, GAS7, HLA-DMA, IFNG (includes EG:15978), IL13, IL27, IL36A, ILK, Interferon alpha, LIMS1, MYO1B, NFkB (complex), P2RY6, PARVA, RFTN1, TPMT, U1 snRNP 2 Cellular Compromise, Cellular Development, 3 1 CELF1, RABGAP1 Connective Tissue Development and Function 3 Gene Expression, Cardiovascular Disease, 3 1 LMCD1, SRF Cellular Growth and Proliferation 4 Gene Expression, Cardiovascular Disease, 3 1 ELAVL1, PCOLCE* Cellular Growth and Proliferation Hypomethylated and Upregulated 1 Cellular Growth and Proliferation, Tumor 20 10 AGPAT9, AHSA1, ALDH3A1, ANXA5, BCL2, CCND1, CDKN1A, CHRNA3, Morphology, Cell Death CINP, Cyclin A, DHCR7, DNAJB4, DTL, DYNLT3*, EDNRB, EIF4EBP1, ERK1/2, Histone H1, IgG, Jnk, KRT10, MAP2K2, MTOR, NFkB (complex), PCBP1, PHLDA2, PLAUR, PTN*, RASSF1, RB1CC1, RHEB, TNFSF13, TRIM29, TSPYL2, USP11 2 Cellular Movement, Cell Cycle, Cell-To-Cell 11 6 AFAP1, BAX, CCL2, CDKN1B, CSF2, EGFR, ESR1, ETS1, EZH2, FHL2*, Signaling and Interaction FHOD3, FN1, hCG, ITGA3, ITGA4, ITGA5, ITGA6, ITGB1, JUN, LGALS1, Mapk, Mek, MMP1 (includes EG:300339), MYC, NRG1 (includes EG:112400), PI3K (complex), PLAU, PMP22, PRKCA, RB1, SLC22A18AS, TGFA, TSG101, TYRP1, VEGFA 3 Cell-To-Cell Signaling and Interaction, 2 1 SIRPB1, TYROBP Molecular Transport, Small Molecule Biochemistry

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Appendix 10 The list of genes associated with the GGH-specific gene expression analysis

Appendix 10. 1 The list of genes with altered expression associated with the function of GGH in the GGH-modulated HCT116 colon cancer cells

Fold Change (vs. Control) Gene Description Accession Probe_ID Symbol GGH GGH Overexpression Inhibition

Downregulated in GGH Overexpression and Upregulated in GGH Inhibition

POLE4 -6.53 1.59 polymerase (DNA-directed), epsilon 4 NM_019896.2 ILMN_1660063 (p12 subunit) PVRL3 -3.86 1.41 poliovirus receptor-related 3 NM_015480.1 ILMN_2188521 PVRL3 -3.25 1.59 poliovirus receptor-related 3 NM_015480.1 ILMN_1727633 TNFRSF6B -2.61 2.49 tumor necrosis factor receptor NM_032945.2 ILMN_2331231 superfamily, member 6b, decoy TNFRSF6B -1.79 2.16 tumor necrosis factor receptor NM_032945.2 ILMN_2331232 superfamily, member 6b, decoy PDE4B -1.77 3.24 phosphodiesterase 4B, cAMP-specific NM_002600.3 ILMN_2340259 (phosphodiesterase E4 dunce homolog, Drosophila) TRIM33 -1.69 1.33 tripartite motif-containing 33 NM_015906.3 ILMN_1682316 PYGL -1.68 1.37 phosphorylase, glycogen, liver NM_002863.3 ILMN_1696187 ALG6 -1.59 1.66 asparagine-linked glycosylation 6 NM_013339.2 ILMN_1771411 homolog (S. cerevisiae, alpha-1,3- glucosyltransferase) MTAP -1.59 1.36 methylthioadenosine phosphorylase NM_002451.3 ILMN_1753639 MCOLN2 -1.54 1.51 mucolipin 2 NM_153259.2 ILMN_1660462 SACS -1.52 1.52 spastic ataxia of Charlevoix-Saguenay NM_014363.3 ILMN_2131523 (sacsin) MRPS31 -1.50 1.61 mitochondrial ribosomal protein S31 NM_005830.2 ILMN_1654552 TNFRSF6B -1.49 2.18 tumor necrosis factor receptor NM_003823.2 ILMN_1661825 superfamily, member 6b, decoy ABHD10 -1.48 1.77 abhydrolase domain containing 10 NM_018394.1 ILMN_1770031 RPS23 -1.45 1.56 ribosomal protein S23 NM_001025.4 ILMN_1772459 PLK2 -1.44 1.45 polo-like kinase 2 (Drosophila) NM_006622.2 ILMN_1717706 SERBP1 -1.43 1.52 SERPINE1 mRNA binding protein 1 NM_001018069.1 ILMN_1773968 CRABP2 -1.43 1.72 cellular retinoic acid binding protein 2 NM_001878.2 ILMN_1690170 HNRNPU -1.38 1.38 heterogeneous nuclear NM_031844.2 ILMN_1743677 ribonucleoprotein U (scaffold attachment factor A) PDE4B -1.38 1.39 phosphodiesterase 4B, cAMP-specific NM_002600.3 ILMN_1782922 (phosphodiesterase E4 dunce homolog, Drosophila) TMEM154 -1.36 1.52 transmembrane protein 154 NM_152680.1 ILMN_2088124 TMEM30A -1.35 1.52 transmembrane protein 30A NM_018247.2 ILMN_1735680 RPL35A -1.34 1.46 ribosomal protein L35a NM_000996.2 ILMN_1756360 PPP1R14C -1.33 1.44 protein phosphatase 1, regulatory NM_030949.2 ILMN_1664855 (inhibitor) subunit 14C SNTB1 -1.33 2.75 syntrophin, beta 1 (dystrophin- NM_021021.2 ILMN_1793410

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associated protein A1, 59kDa, basic component 1) GRSF1 -1.33 1.41 G-rich RNA sequence binding factor NM_001098477.1 ILMN_1806601 1 C12orf29 -1.32 1.48 chromosome 12 open reading frame NM_001009894.2 ILMN_1792671 29 NAP1L1 -1.32 1.38 nucleosome assembly protein 1-like 1 NM_139207.1 ILMN_1699208 PPIA -1.31 1.43 peptidylprolyl isomerase A (cyclophilin NM_021130.3 ILMN_1704529 A) ANKS1A -1.31 1.47 ankyrin repeat and sterile alpha motif NM_015245.2 ILMN_1813669 domain containing 1A CCDC5 -1.30 1.47 coiled-coil domain containing 5 (spindle NM_138443.2 ILMN_1745946 associated)

Upregulated in GGH Overexpression and Downregulated in GGH Inhibition ANXA10 1.62 -6.85 annexin A10 NM_007193.3 ILMN_1699421 TACSTD2 1.37 -2.38 tumor-associated calcium signal NM_002353.1 ILMN_1739001 transducer 2 TMEM200A 1.67 -1.89 transmembrane protein 200A NM_052913.2 ILMN_1725387 UPP1 1.90 -1.79 uridine phosphorylase 1 NM_003364.2 ILMN_1798256 PBX1 1.68 -1.73 pre-B-cell leukemia homeobox 1 NM_002585.1 ILMN_1784678 PHF19 1.37 -1.62 PHD finger protein 19 NM_001009936.1 ILMN_1756676 RERG 2.97 -1.49 RAS-like, estrogen-regulated, growth NM_032918.1 ILMN_1746359 inhibitor IRS1 1.38 -1.47 insulin receptor substrate 1 NM_005544.1 ILMN_1759232 PPARG 1.37 -1.39 peroxisome proliferator-activated NM_015869.4 ILMN_1800225 receptor gamma RRP7A 1.32 -1.36 ribosomal RNA processing 7 homolog NM_015703.3 ILMN_1688178 A (S. cerevisiae) FES 1.31 -1.35 feline sarcoma oncogene NM_002005.2 ILMN_1693650

Appendix 10. 2 The list of genes with altered expression associated with the function of GGH in the GGH-modulated MDA-MB-435 breast cancer cells

Fold Change (vs. Control) Gene Description Accession Probe_ID Symbol GGH GGH Overexpression Inhibition

Downregulated in GGH Overexpression and Upregulated in GGH Inhibition SPP1 -4.51 5.88 secreted phosphoprotein 1 NM_001040058.1 ILMN_2374449 SPP1 -4.13 4.90 secreted phosphoprotein 1 NM_000582.2 ILMN_1651354 PRSS7 -1.94 4.66 protease, serine, 7 (enterokinase) NM_002772.1 ILMN_1695969 PRSS7 -1.51 4.41 protease, serine, 7 (enterokinase) NM_002772.1 ILMN_2220845 TYR -31.95 4.07 tyrosinase (oculocutaneous albinism NM_000372.4 ILMN_1788774 IA) CYB5R2 -1.34 3.05 cytochrome b5 reductase 2 NM_016229.3 ILMN_1739576 BCHE -4.82 2.31 butyrylcholinesterase NM_000055.2 ILMN_1685641 BCHE -4.50 2.77 butyrylcholinesterase NM_000055.1 ILMN_2176592 ADM -2.63 2.31 adrenomedullin NM_001124.1 ILMN_1708934 TMEM166 -3.05 2.07 transmembrane protein 166 NM_032181.1 ILMN_1702973

433

ORC5L -1.35 2.07 origin recognition complex, subunit 5- NM_002553.2 ILMN_1705093 like (yeast) PNLIPRP3 -1.34 2.05 pancreatic lipase-related protein 3 NM_001011709.1 ILMN_1678655 DYNC1I1 -5.83 2.03 dynein, cytoplasmic 1, intermediate NM_004411.3 ILMN_1690397 chain 1 TM4SF19 -2.66 1.94 transmembrane 4 L six family NM_138461.2 ILMN_2413644 member 19 TM4SF19 -1.71 1.44 PREDICTED: transmembrane 4 L six XM_001134247.1 ILMN_1808325 family member 19, transcript variant 2 DGKI -1.48 1.91 diacylglycerol kinase, iota NM_004717.2 ILMN_1718266 EDNRB -2.61 1.89 endothelin receptor type B NM_000115.1 ILMN_1751904 SNCA -11.04 1.89 synuclein, alpha (non A4 component NM_007308.1 ILMN_1701933 of amyloid precursor) SNCA -5.90 1.49 synuclein, alpha (non A4 component NM_000345.2 ILMN_1766165 of amyloid precursor) FAM176A -2.61 1.82 family with sequence similarity 176, NM_001135032.1 ILMN_3194638 member A FAM176A -2.28 1.82 family with sequence similarity 176, NM_001135032.1 ILMN_3271098 member A FAM13B -1.32 1.82 family with sequence similarity 13, NM_001101800.1 ILMN_3258795 member B CGGBP1 -1.37 1.82 CGG triplet repeat binding protein 1 NM_003663.3 ILMN_2387090 CAPN3 -5.07 1.78 calpain 3, (p94) NM_173087.1 ILMN_2332691 CAPN3 -4.74 1.68 calpain 3, (p94) NM_024344.1 ILMN_1687971 DNAJB6 -2.46 1.77 DnaJ (Hsp40) homolog, subfamily B, NM_058246.3 ILMN_1793770 member 6 RENBP -1.94 1.73 renin binding protein NM_002910.4 ILMN_1780057 TMEM199 -1.43 1.71 transmembrane protein 199 NM_152464.1 ILMN_1748481 FHL2 -1.38 1.70 four and a half LIM domains 2 NM_201557.2 ILMN_1668411 MET -1.59 1.70 met proto-oncogene (hepatocyte NM_000245.2 ILMN_1715175 growth factor receptor) ST3GAL5 -1.43 1.69 ST3 beta-galactoside alpha-2,3- NM_001042437.1 ILMN_1713496 sialyltransferase 5 PCLO -2.85 1.66 piccolo (presynaptic cytomatrix NM_033026.5 ILMN_3230160 protein) PCTP -1.70 1.64 phosphatidylcholine transfer protein NM_021213.1 ILMN_1802257 UPP1 -2.28 1.64 uridine phosphorylase 1 NM_003364.2 ILMN_1798256 FMN2 -1.38 1.61 formin 2 NM_020066.3 ILMN_1764795 IFI6 -1.77 1.58 interferon, alpha-inducible protein 6 NM_022872.2 ILMN_2347798 CLEC2D -1.56 1.55 C-type lectin domain family 2, NM_013269.3 ILMN_1711702 member D SPAG1 -1.55 1.55 sperm associated antigen 1 NM_003114.3 ILMN_2367681 IMPAD1 -1.68 1.50 inositol monophosphatase domain NM_017813.2 ILMN_1696311 containing 1 ZNF697 -1.36 1.50 zinc finger protein 697 NM_001080470.1 ILMN_3249501 EEF1A1 -1.63 1.50 eukaryotic translation elongation NM_001402.5 ILMN_3251737 factor 1 alpha 1 CKMT1B -2.78 1.49 creatine kinase, mitochondrial 1B NM_020990.3 ILMN_1763491 ING3 -1.45 1.48 inhibitor of growth family, member 3 NM_019071.2 ILMN_2237746 NOV -5.12 1.48 nephroblastoma overexpressed gene NM_002514.2 ILMN_1787186 HERC6 -1.88 1.47 hect domain and RLD 6 NM_017912.3 ILMN_1654639

434

POU3F2 -1.65 1.47 POU class 3 homeobox 2 NM_005604.2 ILMN_1725772 OVOS2 -1.61 1.47 ovostatin 2 NM_001080502.1 ILMN_2255001 ALDH1A1 -9.45 1.46 aldehyde dehydrogenase 1 family, NM_000689.3 ILMN_2096372 member A1 MIR1978 -3.55 1.46 microRNA 1978 NR_031742.1 ILMN_3310491 ORC6L -1.41 1.46 origin recognition complex, subunit 6 NM_014321.2 ILMN_1731070 like (yeast) SLC37A3 -1.33 1.45 solute carrier family 37 (glycerol-3- NM_032295.2 ILMN_2307598 phosphate transporter), member 3 ARPC1B -1.31 1.45 actin related protein 2/3 complex, NM_005720.2 ILMN_2085760 subunit 1B, 41kDa ST7 -1.57 1.44 suppression of tumorigenicity 7 NM_021908.2 ILMN_1746137 TNFAIP3 -1.36 1.44 tumor necrosis factor, alpha-induced NM_006290.2 ILMN_1702691 protein 3 CAPZA2 -1.30 1.43 capping protein (actin filament) NM_006136.2 ILMN_1768870 muscle Z-line, alpha 2 RBM28 -1.51 1.41 RNA binding motif protein 28 NM_018077.1 ILMN_1793033 TYRP1 -1.38 1.41 tyrosinase-related protein 1 NM_000550.1 ILMN_2054652 BTBD7 -1.42 1.41 BTB (POZ) domain containing 7 NM_018167.3 ILMN_1757298 TUBB4 -4.85 1.41 tubulin, beta 4 NM_006087.2 ILMN_1682459 CDK2 -1.76 1.40 cyclin-dependent kinase 2 NM_001798.2 ILMN_1665559 LHFPL2 -1.69 1.40 lipoma HMGIC fusion partner-like 2 NM_005779.1 ILMN_1747744 ANTXR1 -1.34 1.39 anthrax toxin receptor 1 NM_032208.1 ILMN_1780894 PCBP2 -1.43 1.39 poly(rC) binding protein 2 NM_005016.5 ILMN_3251155 IGSF5 -1.36 1.39 immunoglobulin superfamily, NM_001080444.1 ILMN_2344221 member 5 TFAM -1.38 1.39 transcription factor A, mitochondrial NM_003201.1 ILMN_1715661 C2orf7 -1.31 1.38 chromosome 2 open reading frame 7 NM_032319.1 ILMN_2229940 CKMT1A -2.81 1.37 creatine kinase, mitochondrial 1A NM_001015001.1 ILMN_1732066 PPARGC1A -3.30 1.37 peroxisome proliferator-activated NM_013261.3 ILMN_1750062 receptor gamma, coactivator 1 alpha NCRNA00152 -1.31 1.37 non-protein coding RNA 152 NR_024204.1 ILMN_3220934 EPAS1 -2.11 1.36 endothelial PAS domain protein 1 NM_001430.3 ILMN_1704753 FAM107B -1.67 1.35 family with sequence similarity 107, NM_031453.2 ILMN_1758672 member B XYLB -1.44 1.35 xylulokinase homolog (H. influenzae) NM_005108.2 ILMN_1794349 HRK -1.58 1.34 harakiri, BCL2 interacting protein NM_003806.1 ILMN_2193706 (contains only BH3 domain) ITPK1 -2.51 1.34 inositol 1,3,4-triphosphate 5/6 kinase NM_014216.3 ILMN_1715674 MEX3C -1.36 1.34 mex-3 homolog C (C. elegans) NM_016626.3 ILMN_1658182 FAM84B -1.50 1.33 family with sequence similarity 84, NM_174911.3 ILMN_1670807 member B VEPH1 -2.89 1.33 ventricular zone expressed PH NM_024621.1 ILMN_1684336 domain homolog 1 (zebrafish) LACTB2 -1.45 1.33 lactamase, beta 2 NM_016027.1 ILMN_1660635 CPN1 -1.59 1.32 carboxypeptidase N, polypeptide 1 NM_001308.2 ILMN_1808674 LCP1 -1.57 1.32 lymphocyte cytosolic protein 1 (L- NM_002298.2 ILMN_1662932 plastin) RRS1 -1.32 1.31 RRS1 ribosome biogenesis regulator NM_015169.3 ILMN_1767658 homolog (S. cerevisiae) RNF175 -2.43 1.31 ring finger protein 175 NM_173662.2 ILMN_1741281 OR7E156P -1.50 1.31 olfactory receptor, family 7, NR_002171.1 ILMN_2090351

435

subfamily E, member 156 pseudogene M6PR -1.50 1.31 mannose-6-phosphate receptor (cation NM_002355.2 ILMN_2084353 dependent) SIRPA -1.41 1.31 signal-regulatory protein alpha NM_080792.2 ILMN_2372974 HIATL1 -1.36 1.30 hippocampus abundant transcript-like NM_032558.2 ILMN_1737964 1 AADACL1 -1.33 1.30 arylacetamide deacetylase-like 1 NM_020792.3 ILMN_2061446

Upregulated in GGH Overexpression and Downregulated in GGH Inhibition CTHRC1 1.77 -4.44 collagen triple helix repeat containing NM_138455.2 ILMN_1725090 1 NNMT 4.80 -3.24 nicotinamide N-methyltransferase NM_006169.2 ILMN_1715508 CTHRC1 1.76 -3.20 collagen triple helix repeat containing NM_138455.2 ILMN_2117508 1 HLA-DOA 1.44 -3.09 major histocompatibility complex, NM_002119.3 ILMN_1659075 class II, DO alpha FSCN1 3.31 -3.03 fascin homolog 1, actin-bundling NM_003088.2 ILMN_1808707 protein (Strongylocentrotus purpuratus) CDC42EP5 3.76 -2.53 CDC42 effector protein NM_145057.2 ILMN_1774982 (Rho GTPase binding) 5 CHN1 2.79 -2.30 chimerin (chimaerin) 1 NM_001025201.1 ILMN_1678493 SLC2A3 1.94 -2.24 solute carrier family 2 (facilitated NM_006931.1 ILMN_1775708 glucose transporter), member 3 HLA-DRB6 2.91 -2.20 major histocompatibility complex, NR_001298.1 ILMN_2066066 class II, DR beta 6 (pseudogene) HLA-DQA1 6.26 -2.19 PREDICTED: major XM_936128.2 ILMN_1808405 histocompatibility complex, class II, DQ alpha 1, transcript variant 10 PHF21A 1.75 -2.11 PHD finger protein 21A NM_016621.2 ILMN_1699496 C21orf34 1.39 -2.11 chromosome 21 open reading frame NM_001005734.1 ILMN_1690703 34 S100A4 13.13 -2.10 S100 calcium binding protein A4 NM_019554.2 ILMN_1684306 AHNAK 5.17 -2.09 AHNAK nucleoprotein NM_024060.2 ILMN_1752159 HLA-DPB1 2.37 -2.05 major histocompatibility complex, NM_002121.4 ILMN_1749070 class II, DP beta 1 COL8A1 3.93 -2.04 collagen, type VIII, alpha 1 NM_020351.2 ILMN_1685433 HLA-DMB 2.74 -2.01 major histocompatibility complex, NM_002118.3 ILMN_1761733 class II, DM beta HLA-DPA1 2.73 -1.99 major histocompatibility complex, NM_033554.2 ILMN_1772218 class II, DP alpha 1 HLA-DMA 1.80 -1.98 major histocompatibility complex, NM_006120.2 ILMN_1695311 class II, DM alpha IFITM1 1.43 -1.96 interferon induced transmembrane NM_003641.3 ILMN_1801246 protein 1 (9-27) PHLDA1 1.90 -1.95 pleckstrin homology-like domain, NM_007350.3 ILMN_3251550 family A, member 1 CROP 1.37 -1.94 cisplatin resistance-associated NM_016424.3 ILMN_1728180 overexpressed protein PRR4 2.61 -1.86 proline rich 4 (lacrimal) NM_001098538.1 ILMN_1753665 TMEM173 1.39 -1.85 transmembrane protein 173 NM_198282.1 ILMN_2145116 HEY2 2.08 -1.85 hairy/enhancer-of-split related with NM_012259.1 ILMN_1682034 YRPW motif 2

436

HLA-DRA 2.45 -1.84 major histocompatibility complex, NM_019111.3 ILMN_1689655 class II, DR alpha AHNAK2 1.35 -1.84 AHNAK nucleoprotein 2 NM_138420.2 ILMN_3243156 HLA-DRB3 2.07 -1.83 major histocompatibility complex, NM_022555.3 ILMN_1717261 class II, DR beta 3 ZNF627 1.30 -1.82 zinc finger protein 627 NM_145295.2 ILMN_2197519 HLA-DRB4 3.04 -1.81 major histocompatibility complex, NM_021983.4 ILMN_2159694 class II, DR beta 4 GABRG2 1.47 -1.79 gamma-aminobutyric acid (GABA) A NM_198904.1 ILMN_1800270 receptor, gamma 2 CYBRD1 2.27 -1.79 cytochrome b reductase 1 NM_024843.2 ILMN_1712305 S100A4 10.51 -1.78 S100 calcium binding protein A4 NM_019554.2 ILMN_1688780 BBS2 1.78 -1.78 Bardet-Biedl syndrome 2 NM_031885.2 ILMN_2230035 CYBRD1 2.44 -1.76 cytochrome b reductase 1 NM_024843.2 ILMN_2087692 MUC1 1.90 -1.75 mucin 1, cell surface associated NM_001044391.1 ILMN_1756992 H2AFY 1.43 -1.73 H2A histone family, member Y NM_138609.2 ILMN_1674034 COL8A1 3.39 -1.73 collagen, type VIII, alpha 1 NM_020351.2 ILMN_2402392 CTPS2 1.40 -1.70 CTP synthase II NM_175859.1 ILMN_2392352 KLF9 2.09 -1.69 Kruppel-like factor 9 NM_001206.2 ILMN_1778523 COL9A1 2.33 -1.68 collagen, type IX, alpha 1 NM_001851.3 ILMN_1814701 CD74 2.88 -1.65 CD74 molecule, major NM_004355.2 ILMN_2379644 histocompatibility complex, class II invariant chain IAH1 2.16 -1.64 isoamyl acetate-hydrolyzing esterase NM_001039613.1 ILMN_2217329 1 homolog (S. cerevisiae) PRMT2 1.43 -1.64 protein arginine methyltransferase 2 NM_206962.1 ILMN_2259119 CA5B 3.48 -1.64 carbonic anhydrase VB, NM_007220.3 ILMN_1672807 mitochondrial FOXO3 1.41 -1.64 forkhead box O3 NM_201559.2 ILMN_1681703 ZNF451 1.42 -1.63 zinc finger protein 451 NM_001031623.2 ILMN_1706734 NPAL3 1.75 -1.63 NIPA-like domain containing 3 NM_020448.3 ILMN_1684210 CMIP 1.52 -1.63 c-Maf-inducing protein NM_030629.1 ILMN_1738075 NRCAM 1.39 -1.62 neuronal cell adhesion molecule NM_005010.3 ILMN_2411236 C14orf139 1.48 -1.61 PREDICTED: chromosome 14 open XR_017875.1 ILMN_1684357 reading frame 139 FAM134B 3.25 -1.61 family with sequence similarity 134, NM_001034850.1 ILMN_2387952 member B CRYL1 1.44 -1.60 crystallin, lambda 1 NM_015974.2 ILMN_1714397 CD74 2.59 -1.59 CD74 molecule, major NM_001025159.1 ILMN_1736567 histocompatibility complex, class II invariant chain C14orf147 1.93 -1.59 chromosome 14 open reading frame NM_138288.3 ILMN_1699676 147 ARHGAP15 2.02 -1.58 Rho GTPase activating protein 15 NM_018460.2 ILMN_2208413 HLA-B 1.93 -1.57 major histocompatibility complex, NM_005514.5 ILMN_1778401 class I, B NDE1 1.32 -1.57 nudE nuclear distribution gene E NM_017668.1 ILMN_1739805 homolog 1 (A. nidulans) SH3PXD2A 1.59 -1.57 SH3 and PX domains 2A NM_014631.2 ILMN_1743103 SNX1 1.59 -1.56 sorting nexin 1 NM_003099.3 ILMN_2365484 ZCCHC24 2.04 -1.56 zinc finger, CCHC domain containing NM_153367.2 ILMN_1754660 24

437

AHNAK 2.99 -1.55 AHNAK nucleoprotein NM_001620.1 ILMN_1792495 C1R 1.72 -1.54 complement component 1, r NM_001733.4 ILMN_1677198 subcomponent DMRT2 1.76 -1.54 doublesex and mab-3 related NM_006557.4 ILMN_1751785 transcription factor 2 IGFBP7 3.66 -1.54 insulin-like growth factor binding NM_001553.1 ILMN_2062468 protein 7 DOCK11 2.13 -1.53 dedicator of cytokinesis 11 NM_144658.3 ILMN_1765860 C5orf13 1.96 -1.53 open reading frame 13 NM_004772.1 ILMN_1680738 DDX50 1.32 -1.52 DEAD (Asp-Glu-Ala-Asp) box NM_024045.1 ILMN_1712320 polypeptide 50 UBE2L6 1.65 -1.52 ubiquitin-conjugating enzyme E2L 6 NM_004223.3 ILMN_1703108 EML1 2.01 -1.52 echinoderm microtubule associated NM_004434.2 ILMN_1729455 protein like 1 PTGR1 2.15 -1.52 prostaglandin reductase 1 NM_012212.2 ILMN_2225537 SNX1 1.52 -1.52 sorting nexin 1 NM_003099.3 ILMN_2365479 CAPRIN1 1.46 -1.51 cell cycle associated protein 1 NM_203364.2 ILMN_1782897 DAG1 1.38 -1.50 dystroglycan 1 (dystrophin-associated NM_004393.2 ILMN_1658425 glycoprotein 1) FKBP11 3.63 -1.49 FK506 binding protein 11, 19 kDa NM_016594.1 ILMN_1787345 HLA-DRA 2.45 -1.49 major histocompatibility complex, NM_019111.3 ILMN_2157441 class II, DR alpha FZD2 2.31 -1.49 frizzled homolog 2 (Drosophila) NM_001466.2 ILMN_1653711 BBS2 1.86 -1.49 Bardet-Biedl syndrome 2 NM_031885.2 ILMN_1767612 ZHX3 1.49 -1.48 zinc fingers and homeoboxes 3 NM_015035.3 ILMN_1774387 CD96 2.73 -1.48 CD96 molecule NM_005816.4 ILMN_2415786 NMT2 1.56 -1.48 N-myristoyltransferase 2 NM_004808.1 ILMN_1656378 OPN3 1.62 -1.48 opsin 3 NM_014322.2 ILMN_1716988 TMEM216 1.51 -1.48 transmembrane protein 216 NM_016499.3 ILMN_1732577 ZNF521 1.55 -1.47 zinc finger protein 521 NM_015461.1 ILMN_2225548 HNRNPU 1.39 -1.47 heterogeneous nuclear NM_004501.3 ILMN_2370135 ribonucleoprotein U (scaffold attachment factor A) H2AFJ 1.34 -1.47 H2A histone family, member J NM_177925.2 ILMN_1708728 SERPINA3 5.74 -1.46 serpin peptidase inhibitor, clade A NM_001085.4 ILMN_1788874 (alpha-1 antiproteinase, antitrypsin), member 3 CEACAM1 1.53 -1.46 carcinoembryonic antigen-related cell NM_001024912.1 ILMN_1716815 adhesion molecule 1 (biliary glycoprotein) LTBP4 1.76 -1.45 latent transforming growth factor beta NM_001042545.1 ILMN_1665219 binding protein 4 OSBPL5 1.49 -1.45 oxysterol binding protein-like 5 NM_145638.1 ILMN_2307032 ZNF217 1.94 -1.45 zinc finger protein 217 NM_006526.2 ILMN_1755303 CD74 2.42 -1.45 CD74 molecule, major NM_001025159.1 ILMN_1761464 histocompatibility complex, class II invariant chain C10orf33 1.41 -1.44 chromosome 10 open reading frame NM_032709.1 ILMN_1684497 33 PTGR1 1.74 -1.44 prostaglandin reductase 1 NM_012212.2 ILMN_1704531 SNORA18 1.48 -1.43 small nucleolar RNA, H/ACA box 18 NR_002959.1 ILMN_3244348 TUBB2B 2.16 -1.43 tubulin, beta 2B NM_178012.3 ILMN_1680874

438

YPEL3 1.67 -1.43 yippee-like 3 (Drosophila) NM_031477.4 ILMN_1791147 TM2D3 1.63 -1.43 TM2 domain containing 3 NM_078474.2 ILMN_1761120 TMEM42 3.19 -1.42 transmembrane protein 42 NM_144638.1 ILMN_1760245 C5orf15 1.40 -1.42 chromosome 5 open reading frame 15 NM_020199.1 ILMN_1695917 TMEM136 2.35 -1.41 transmembrane protein 136 NM_174926.1 ILMN_1815346 TIPARP 1.54 -1.40 TCDD-inducible poly(ADP-ribose) NM_015508.3 ILMN_1765578 polymerase SMARCD3 1.62 -1.40 SWI/SNF related, matrix associated, NM_003078.3 ILMN_2309180 actin dependent regulator of chromatin, subfamily d, member 3 NINJ1 1.76 -1.40 ninjurin 1 NM_004148.3 ILMN_1815086 IFITM2 1.62 -1.39 interferon induced transmembrane NM_006435.2 ILMN_1673352 protein 2 (1-8D) . RTN3 1.34 -1.39 reticulon 3 NM_201430.1 ILMN_2363065 LOC400464 1.47 -1.39 similar to FLJ43276 protein NM_001013670.1 ILMN_2320480 CD96 2.98 -1.39 CD96 molecule NM_198196.2 ILMN_1711573 C5orf53 1.44 -1.39 chromosome 5 open reading frame 53 NM_001007189.1 ILMN_1685854 AGGF1 1.35 -1.38 angiogenic factor with G patch and NM_018046.3 ILMN_2064917 FHA domains 1 LRP5 1.85 -1.38 low density lipoprotein receptor- NM_002335.1 ILMN_1702775 related protein 5 RFX5 1.33 -1.38 regulatory factor X, 5 (influences NM_001025603.1 ILMN_1741200 HLA class II expression) COL13A1 4.16 -1.37 collagen, type XIII, alpha 1 NM_080815.2 ILMN_2311052 ITIH5L 1.74 -1.36 inter-alpha (globulin) inhibitor H5- NM_198510.1 ILMN_1709177 like AHNAK 5.17 -1.36 AHNAK nucleoprotein NM_001620.1 ILMN_1714567 KLRC3 2.01 -1.36 killer cell lectin-like receptor NM_002261.2 ILMN_2386790 subfamily C, member 3 NFE2L3 1.42 -1.36 nuclear factor (erythroid-derived 2)- NM_004289.5 ILMN_2049766 like 3 YPEL5 1.97 -1.36 yippee-like 5 (Drosophila) NM_016061.1 ILMN_1711069 GAS7 1.98 -1.36 growth arrest-specific 7 NM_201433.1 ILMN_1745994 TRIM9 1.55 -1.35 tripartite motif-containing 9 NM_015163.4 ILMN_1763433 CADM1 3.96 -1.35 cell adhesion molecule 1 NM_014333.3 ILMN_1680132 KIAA0586 1.53 -1.35 KIAA0586 NM_014749.2 ILMN_1651684 ALCAM 1.51 -1.35 activated leukocyte cell adhesion NM_001627.2 ILMN_1670870 molecule ZSWIM4 1.54 -1.34 zinc finger, SWIM-type containing 4 NM_023072.1 ILMN_2150654 CSTF3 1.83 -1.34 cleavage stimulation factor, 3' pre- NM_001033505.1 ILMN_1762002 RNA, subunit 3, 77kDa SYTL2 2.13 -1.34 synaptotagmin-like 2 NM_206928.1 ILMN_1682929 MUC1 1.47 -1.34 mucin 1, cell surface associated NM_001044390.1 ILMN_2371911 SLC6A16 1.69 -1.34 solute carrier family 6, member 16 NM_014037.2 ILMN_1723287 TXNIP 2.36 -1.34 thioredoxin interacting protein NM_006472.2 ILMN_1697448 IGFBP6 1.36 -1.34 insulin-like growth factor binding NM_002178.2 ILMN_1669362 protein 6 PARVA 5.04 -1.33 parvin, alpha NM_018222.3 ILMN_3307892 PC 2.14 -1.33 pyruvate carboxylase NM_000920.2 ILMN_1671489 CSTF3 1.97 -1.33 cleavage stimulation factor, 3' pre- NM_001033505.1 ILMN_2397880 RNA, subunit 3, 77kDa CRIP2 1.53 -1.33 cysteine-rich protein 2 NM_001312.2 ILMN_1694432

439

SAT1 1.51 -1.33 spermidine/spermine N1- NM_002970.1 ILMN_1753342 acetyltransferase 1 TCEAL8 1.55 -1.33 transcription elongation factor A NM_001006684.1 ILMN_2402272 (SII)-like 8 STAT3 1.74 -1.33 signal transducer and activator of NM_213662.1 ILMN_2401978 transcription 3 (acute-phase response factor) TMEM47 3.03 -1.33 transmembrane protein 47 NM_031442.2 ILMN_2129234 C2orf34 1.35 -1.32 chromosome 2 open reading frame 34 NM_024766.2 ILMN_1738099 C5orf21 1.56 -1.32 chromosome 5 open reading frame 21 NM_032042.3 ILMN_1654542 CREBZF 1.67 -1.32 CREB/ATF bZIP transcription factor NM_001039618.1 ILMN_1784847 AGTPBP1 1.31 -1.32 ATP/GTP binding protein 1 NM_015239.1 ILMN_1718071 NR4A2 3.22 -1.32 nuclear receptor subfamily 4, group NM_006186.2 ILMN_2339955 A, member 2 CDC42EP4 3.36 -1.32 CDC42 effector protein (Rho GTPase NM_012121.4 ILMN_1745223 binding) 4 DPF2 1.41 -1.32 D4, zinc and double PHD fingers NM_006268.3 ILMN_1734317 family 2 KCNMA1 2.25 -1.31 potassium large conductance calcium- NM_002247.2 ILMN_2297765 activated channel, subfamily M, alpha member 1 COL13A1 5.76 -1.31 collagen, type XIII, alpha 1 NM_080805.2 ILMN_2370624 CKAP5 1.36 -1.30 cytoskeleton associated protein 5 NM_001008938.1 ILMN_1748770 CD9 1.30 -1.30 CD9 molecule NM_001769.2 ILMN_1695423

440

Appendix 11 The top 50 genes with most differentially altered expression in response to FPGS modulation

Appendix 11. 1 The top 50 genes with most differentially downregulated compared with controls in the FPGS-overexpressed HCT116 colon cancer cells

Gene Fold Description Accession Probe ID Symbol Change CCNA2 -4.84 cyclin A2 NM_001237.2 ILMN_1786125 DLGAP5 -4.06 discs, large (Drosophila) homolog-associated protein 5 NM_014750.3 ILMN_1749829 PPIL5 -3.78 peptidylprolyl isomerase (cyclophilin)-like 5 NM_203467.1 ILMN_1715616 KPNA2 -3.66 karyopherin alpha 2 (RAG cohort 1, importin alpha 1) NM_002266.2 ILMN_1721868 HMMR -3.63 hyaluronan-mediated motility receptor (RHAMM) NM_012485.1 ILMN_1781942 AURKA -3.62 aurora kinase A NM_198434.1 ILMN_2357438 CBX1 -3.60 chromobox homolog 1 (HP1 beta homolog Drosophila ) NM_006807.3 ILMN_1770244 RRM1 -3.59 ribonucleotide reductase M1 polypeptide NM_001033.2 ILMN_1771593 DLGAP5 -3.56 discs, large (Drosophila) homolog-associated protein 5 NM_014750.3 ILMN_3239771 AURKA -3.55 aurora kinase A NM_198436.1 ILMN_1680955 CDC20 -3.54 cell division cycle 20 homolog (S. cerevisiae) NM_001255.2 ILMN_1663390 MAD2L1 -3.53 MAD2 mitotic arrest deficient-like 1 (yeast) NM_002358.2 ILMN_2112460 HMMR -3.44 hyaluronan-mediated motility receptor (RHAMM) NM_012484.1 ILMN_2409220 GINS2 -3.43 GINS complex subunit 2 (Psf2 homolog) NM_016095.1 ILMN_1809590 BUB1 -3.41 BUB1 budding uninhibited by benzimidazoles 1 homolog NM_004336.2 ILMN_2202948 (yeast) THOC4 -3.38 THO complex 4 NM_005782.2 ILMN_2364062 POLA2 -3.31 polymerase (DNA directed), alpha 2 (70kD subunit) NM_002689.2 ILMN_1696713 RPS7 -3.26 ribosomal protein S7 NM_001011.3 ILMN_2070072 C6orf173 -3.24 chromosome 6 open reading frame 173 NM_001012507.1 ILMN_1763907 FAM83D -3.21 family with sequence similarity 83, member D NM_030919.2 ILMN_1781943 KIAA0101 -3.19 KIAA0101 NM_014736.4 ILMN_2285996 MND1 -3.17 meiotic nuclear divisions 1 homolog (S. cerevisiae) NM_032117.2 ILMN_1671906 ANLN -3.16 anillin, actin binding protein NM_018685.2 ILMN_1739645 CDC2 -3.13 cell division cycle 2, G1 to S and G2 to M NM_001786.2 ILMN_1710428 TTK -3.13 TTK protein kinase NM_003318.3 ILMN_1788166 PTTG3P -3.08 pituitary tumor-transforming 3 (pseudogene) NR_002734.1 ILMN_2049021 CDC2 -3.07 cell division cycle 2, G1 to S and G2 to M NM_001786.2 ILMN_1747911 CCNB1 -3.06 cyclin B1 NM_031966.2 ILMN_1712803 KIF20A -3.05 kinesin family member 20A NM_005733.1 ILMN_1695658 PPIH -3.04 peptidylprolyl isomerase H (cyclophilin H) NM_006347.3 ILMN_1801913 CCDC34 -3.04 coiled-coil domain containing 34 NM_030771.1 ILMN_2288784 C11orf82 -3.03 open reading frame 82. NM_145018.2 ILMN_1790100 CCDC34 -3.02 coiled-coil domain containing 34 NM_030771.1 ILMN_1657547 VRK1 -3.01 vaccinia related kinase 1 NM_003384.2 ILMN_1805828 KIF11 -3.00 kinesin family member 11 NM_004523.2 ILMN_2143155 TYMS -2.99 thymidylate synthetase NM_001071.1 ILMN_1806040 CENPM -2.98 centromere protein M NM_001002876.1 ILMN_2368718 CEP55 -2.93 centrosomal protein 55kDa NM_018131.3 ILMN_1747016 NDC80 -2.91 NDC80 homolog, kinetochore complex component (S. NM_006101.1 ILMN_1664511 cerevisiae)

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ZWINT -2.88 ZW10 interactor NM_001005413.1 ILMN_2362549 BIRC5 -2.87 baculoviral IAP repeat-containing 5 NM_001168.2 ILMN_2349459 OIP5 -2.87 Opa interacting protein 5 NM_007280.1 ILMN_2196984 SPC25 -2.86 SPC25, NDC80 kinetochore complex component, homolog NM_020675.3 ILMN_1814281 (S. cerevisiae) MCM4 -2.85 minichromosome maintenance complex component 4 NM_005914.2 ILMN_1737205 DKK1 -2.85 dickkopf homolog 1 (Xenopus laevis) NM_012242.2 ILMN_1773337 CACYBP -2.85 calcyclin binding protein NM_014412.2 ILMN_1726574 SKP2 -2.84 S-phase kinase-associated protein 2 (p45) NM_005983.2 ILMN_1791002 ZWINT -2.83 ZW10 interactor NM_001005413.1 ILMN_2362545 SKP2 -2.83 S-phase kinase-associated protein 2 (p45) NM_032637.2 ILMN_1665538 BUB1B -2.83 BUB1 budding uninhibited by benzimidazoles 1 homolog beta NM_001211.4 ILMN_1797307 (yeast)

Appendix 11. 2 The top 50 genes with most differentially upregulated compared with controls in the FPGS-overexpressed HCT116 colon cancer cells

Gene Fold Description Accession Probe ID Symbol Change GDF15 15.30 growth differentiation factor 15 NM_004864.1 ILMN_2188862 TNFRSF6B 11.17 tumor necrosis factor receptor superfamily, member 6b, NM_032945.2 ILMN_2331231 decoy DDIT4 9.12 DNA-damage-inducible transcript 4 NM_019058.2 ILMN_1661599 TNFRSF6B 8.81 tumor necrosis factor receptor superfamily, member 6b, NM_032945.2 ILMN_2331232 decoy DDIT3 7.49 DNA-damage-inducible transcript 3 NM_004083.4 ILMN_1676984 TNFRSF6B 7.09 tumor necrosis factor receptor superfamily, member 6b, NM_003823.2 ILMN_1661825 decoy TRIB3 7.00 tribbles homolog 3 (Drosophila) NM_021158.3 ILMN_1787815 PCK2 6.04 phosphoenolpyruvate carboxykinase 2 (mitochondrial) NM_004563.2 ILMN_1671791 RBCK1 5.87 RanBP-type and C3HC4-type zinc finger containing 1 NM_031229.2 ILMN_1751330 PRIC285 5.13 peroxisomal proliferator-activated receptor A interacting NM_033405.2 ILMN_1787509 complex 285 NDRG1 4.95 N-myc downstream regulated gene 1 NM_006096.2 ILMN_1809931 ASNS 4.89 asparagine synthetase NM_133436.1 ILMN_2398107 RPS29 4.73 ribosomal protein S29 NM_001030001.1 ILMN_2298818 ASNS 4.60 asparagine synthetase NM_133436.1 ILMN_1796417 LOC399959 4.50 hypothetical LOC399959 NR_024430.1 ILMN_3244176 CTHRC1 4.45 collagen triple helix repeat containing 1 NM_138455.2 ILMN_1725090 TSC22D3 4.43 TSC22 domain family, member 3 NM_198057.2 ILMN_1748124 MAP1LC3B 4.35 microtubule-associated protein 1 light chain 3 beta NM_022818.3 ILMN_1703244 MXD4 4.17 MAX dimerization protein 4 NM_006454.2 ILMN_1756541 C4orf34 4.13 chromosome 4 open reading frame 34 NM_174921.1 ILMN_1713892 FGF19 4.05 fibroblast growth factor 19 NM_005117.2 ILMN_1760909 ISG20 4.04 interferon stimulated exonuclease gene 20kDa NM_002201.4 ILMN_1659913 ABCC3 4.03 ATP-binding cassette, sub-family C (CFTR/MRP), member NM_003786.2 ILMN_1677814 3 PYGB 4.01 phosphorylase, glycogen; brain NM_002862.3 ILMN_1778360 IRF9 3.94 interferon regulatory factor 9 NM_006084.4 ILMN_1745471

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ANGPTL4 3.82 angiopoietin-like 4 NM_139314.1 ILMN_1707727 CEBPB 3.81 CCAAT/enhancer binding protein (C/EBP), beta NM_005194.2 ILMN_1693014 PLAU 3.80 plasminogen activator, urokinase NM_002658.2 ILMN_1656057 ATF3 3.79 activating transcription factor 3 NM_001040619.1 ILMN_2374865 YPEL5 3.77 yippee-like 5 (Drosophila) NM_016061.1 ILMN_1711069 ITGA3 3.73 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA- NM_002204.1 ILMN_1685397 3 receptor) C4orf34 3.72 chromosome 4 open reading frame 34 NM_174921.1 ILMN_2224907 SLC16A5 3.71 solute carrier family 16, member 5 (monocarboxylic acid NM_004695.2 ILMN_1755649 transporter 6) VGF 3.68 VGF nerve growth factor inducible NM_003378.2 ILMN_1757497 KLF4 3.67 Kruppel-like factor 4 (gut) NM_004235.3 ILMN_2137789 LEMD1 3.61 LEM domain containing 1 NM_001001552.3 ILMN_1785444 PSAT1 3.60 phosphoserine aminotransferase 1 NM_021154.3 ILMN_1692938 TNFRSF10B 3.59 tumor necrosis factor receptor superfamily, member 10b NM_003842.3 ILMN_1699265 RBCK1 3.52 RanBP-type and C3HC4-type zinc finger containing 1 NM_006462.3 ILMN_2406313 STC2 3.52 stanniocalcin 2 NM_003714.2 ILMN_1691884 H1F0 3.52 H1 histone family, member 0 NM_005318.2 ILMN_1757467 OPTN 3.51 optineurin NM_001008213.1 ILMN_2381899 HSPA5 3.50 heat shock 70kDa protein 5 (glucose-regulated protein, NM_005347.2 ILMN_1773865 78kDa) TRNP1 3.47 TMF1-regulated nuclear protein 1 NM_001013642.2 ILMN_1695946 FAM84B 3.47 family with sequence similarity 84, member B NM_174911.3 ILMN_1670807 SLC22A18 3.36 solute carrier family 22 (organic cation transporter), NM_002555.3 ILMN_2382505 member 18 ASS1 3.36 argininosuccinate synthetase 1 NM_054012.3 ILMN_2395451 CREB3L2 3.34 cAMP responsive element binding protein 3-like 2 NM_194071.2 ILMN_1751097 BTG1 3.32 B-cell translocation gene 1, anti-proliferative NM_001731.1 ILMN_1775743 GADD45A 3.29 growth arrest and DNA-damage-inducible, alpha NM_001924.2 ILMN_2052208

Appendix 11. 3 The top 50 genes with most differentially downregulated compared with controls in the FPGS-inhibited HCT116 colon cancer cells

Gene Fold Description Accession Probe ID Symbol Change BMP4 -2.57 bone morphogenetic protein 4 NM_130851.1 ILMN_1740900 DNTTIP2 -2.11 deoxynucleotidyltransferase, terminal, interacting protein 2 NM_014597.3 ILMN_1708345 PKIB -2.01 protein kinase (cAMP-dependent, catalytic) inhibitor beta NM_032471.4 ILMN_2337263 NR2F2 -2.00 nuclear receptor subfamily 2, group F, member 2 NM_021005.2 ILMN_2094360 PER2 -1.97 period homolog 2 (Drosophila) NM_022817.2 ILMN_1738095 RERG -1.73 RAS-like, estrogen-regulated, growth inhibitor NM_032918.1 ILMN_1746359 ROCK2 -1.73 Rho-associated, coiled-coil containing protein kinase 2 NM_004850.3 ILMN_1659099 AXIN2 -1.72 axin 2 (conductin, axil) NM_004655.2 ILMN_1724480 PVRL3 -1.71 poliovirus receptor-related 3 NM_015480.1 ILMN_2188521 DUSP6 -1.71 dual specificity phosphatase 6 NM_022652.2 ILMN_2396020 STXBP6 -1.70 syntaxin binding protein 6 (amisyn) NM_014178.6 ILMN_2172969 PVRL3 -1.64 poliovirus receptor-related 3 NM_015480.1 ILMN_1727633 ENC1 -1.64 ectodermal-neural cortex (with BTB-like domain) NM_003633.1 ILMN_1779147

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STXBP6 -1.62 syntaxin binding protein 6 (amisyn) NM_014178.6 ILMN_1750912 FIP1L1 -1.62 FIP1 like 1 (S. cerevisiae) NM_030917.2 ILMN_1768743 EVI1 -1.62 ecotropic viral integration site 1 NM_005241.1 ILMN_1803367 NFIB -1.61 nuclear factor I/B NM_005596.2 ILMN_1778991 BMP4 -1.58 bone morphogenetic protein 4 NM_001202.2 ILMN_1709734 CNTNAP2 -1.57 contactin associated protein-like 2 NM_014141.4 ILMN_1690223 ZDHHC6 -1.56 zinc finger, DHHC-type containing 6 NM_022494.1 ILMN_2046003 TTC37 -1.56 tetratricopeptide repeat domain 37 NM_014639.2 ILMN_1795911 HTR7 -1.54 5-hydroxytryptamine (serotonin) receptor 7 (adenylate NM_000872.3 ILMN_1744217 cyclase-coupled) WDR33 -1.54 WD repeat domain 33 NM_001006622.1 ILMN_1670172 RAD23B -1.53 RAD23 homolog B (S. cerevisiae) NM_002874.3 ILMN_1722662 GAS6 -1.53 growth arrest-specific 6 NM_000820.1 ILMN_1779558 SEC23B -1.53 Sec23 homolog B (S. cerevisiae) NM_032985.4 ILMN_1657483 PPAP2B -1.52 phosphatidic acid phosphatase type 2B NM_003713.3 ILMN_2388800 EPM2AIP1 -1.51 EPM2A (laforin) interacting protein 1 NM_014805.2 ILMN_1682658 C11orf82 -1.50 chromosome 11 open reading frame 82 NM_145018.2 ILMN_1790100 DUSP6 -1.50 dual specificity phosphatase 6 NM_001946.2 ILMN_1677466 SLC2A3 -1.49 solute carrier family 2 (facilitated glucose transporter), NM_006931.1 ILMN_1775708 member 3 C8orf38 -1.49 chromosome 8 open reading frame 38 NM_152416.2 ILMN_1727618 ZC3H14 -1.49 zinc finger CCCH-type containing 14 NM_024824.3 ILMN_1714805 LGALS3 -1.48 lectin, galactoside-binding, soluble, 3 (galectin 3) NM_002306.1 ILMN_1803788 FAM83D -1.48 family with sequence similarity 83, member D NM_030919.2 ILMN_1781943 SFRS3 -1.47 splicing factor, arginine/serine-rich 3 NM_003017.3 ILMN_1723212 MCM10 -1.47 minichromosome maintenance complex component 10 NM_018518.3 ILMN_2413898 TANK -1.46 TRAF family member-associated NFKB activator NM_004180.2 ILMN_2292387 PKIA -1.46 protein kinase (cAMP-dependent, catalytic) inhibitor alpha NM_181839.1 ILMN_2337974 SEMA3A -1.46 sema domain, immunoglobulin domain (Ig), short basic NM_006080.2 ILMN_1765641 domain, secreted, (semaphorin) 3A PALLD -1.45 palladin, cytoskeletal associated protein NM_016081.3 ILMN_1698732 NOL8 -1.45 nucleolar protein 8 NM_017948.4 ILMN_2062370 C10orf6 -1.45 chromosome 10 open reading frame 6 NM_018121.2 ILMN_1710207 LAMB1 -1.44 laminin, beta 1 NM_002291.1 ILMN_2214790 JARID2 -1.44 jumonji, AT rich interactive domain 2 NM_004973.2 ILMN_1764177 BUB3 -1.44 BUB3 budding uninhibited by benzimidazoles 3 homolog NM_004725.2 ILMN_1778764 (yeast) ZNF721 -1.44 zinc finger protein 721 NM_133474.2 ILMN_1805271 LOC400986 -1.44 PREDICTED: protein immuno-reactive with anti-PTH XM_001126815.1 ILMN_1811117 polyclonal antibodies TMEM97 -1.44 transmembrane protein 97 NM_014573.2 ILMN_1710962 TANK -1.44 TRAF family member-associated NFKB activator NM_004180.2 ILMN_1715069

444

Appendix 11. 4 The top 50 genes with most differentially upregulated compared with controls in the FPGS-inhibited HCT116 colon cancer cells

Gene Fold Description Accession Probe ID Symbol Change GDF15 3.47 growth differentiation factor 15 NM_004864.1 ILMN_2188862 DDIT4 3.44 DNA-damage-inducible transcript 4 NM_019058.2 ILMN_1661599 CDKN1A 2.94 cyclin-dependent kinase inhibitor 1A (p21, Cip1) NM_000389.2 ILMN_1784602 LCN2 2.72 lipocalin 2 NM_005564.3 ILMN_1692223 CYP24A1 2.58 cytochrome P450, family 24, subfamily A, polypeptide 1 NM_000782.3 ILMN_1685663 ANXA10 2.36 annexin A10 NM_007193.3 ILMN_1699421 IFI27 2.32 interferon, alpha-inducible protein 27 NM_005532.3 ILMN_2058782 IRF9 2.12 interferon regulatory factor 9 NM_006084.4 ILMN_1745471 PHLDB2 2.11 pleckstrin homology-like domain, family B, member 2 NM_145753.1 ILMN_1719792 IFIT1 2.11 interferon-induced protein with tetratricopeptide repeats 1 NM_001548.3 ILMN_1707695 VGF 2.11 VGF nerve growth factor inducible NM_003378.2 ILMN_1757497 UCA1 2.07 urothelial cancer associated 1 (non-protein coding) NR_015379.2 ILMN_3239254 TRIOBP 2.07 TRIO and F-actin binding protein NM_138632.2 ILMN_1753413 SLC1A3 2.06 solute carrier family 1 (glial high affinity glutamate NM_004172.3 ILMN_1738552 transporter), member 3 RPS29 2.06 ribosomal protein S29 NM_001030001.1 ILMN_2298818 TRNP1 2.02 TMF1-regulated nuclear protein 1 NM_001013642.2 ILMN_1695946 RGS2 2.00 regulator of G-protein signalling 2, 24kDa NM_002923.1 ILMN_2197365 PHLDB2 1.98 pleckstrin homology-like domain, family B, member 2 NM_145753.1 ILMN_2179778 IL8 1.91 interleukin 8 NM_000584.2 ILMN_2184373 PCK2 1.89 phosphoenolpyruvate carboxykinase 2 (mitochondrial) NM_004563.2 ILMN_1671791 BST2 1.88 bone marrow stromal cell antigen 2 NM_004335.2 ILMN_1723480 ASNS 1.88 asparagine synthetase NM_133436.1 ILMN_1796417 ALDH1A3 1.85 aldehyde dehydrogenase 1 family, member A3 NM_000693.2 ILMN_1807439 S100A14 1.85 S100 calcium binding protein A14 NM_020672.1 ILMN_1783287 HERC5 1.83 hect domain and RLD 5 NM_016323.2 ILMN_1729749 ISG15 1.82 ISG15 ubiquitin-like modifier NM_005101.1 ILMN_2054019 ALDH1A3 1.80 aldehyde dehydrogenase 1 family, member A3 NM_000693.1 ILMN_2139970 ANKRA2 1.80 ankyrin repeat, family A (RFXANK-like), 2 NM_023039.2 ILMN_2185563 CA2 1.80 carbonic anhydrase II NM_000067.1 ILMN_1662795 IFIT2 1.79 interferon-induced protein with tetratricopeptide repeats 2 NM_001547.4 ILMN_1739428 CA2 1.79 carbonic anhydrase II NM_000067.1 ILMN_2199439 ID1 1.78 inhibitor of DNA binding 1, dominant negative helix- NM_181353.1 ILMN_1664861 loop-helix protein HYAL1 1.77 hyaluronoglucosaminidase 1 NM_153282.1 ILMN_1739813 RPS27L 1.75 ribosomal protein S27-like NM_015920.3 ILMN_1712678 MXD4 1.74 MAX dimerization protein 4 NM_006454.2 ILMN_1756541 ID3 1.71 inhibitor of DNA binding 3, dominant negative helix- NM_002167.2 ILMN_1732296 loop-helix protein TP53INP1 1.70 tumor protein p53 inducible nuclear protein 1 NM_033285.2 ILMN_2214197 GPR110 1.70 G protein-coupled receptor 110 NM_153840.2 ILMN_2241124 KLF9 1.70 Kruppel-like factor 9 NM_001206.2 ILMN_1778523 ATF4 1.68 activating transcription factor 4 (tax-responsive enhancer NM_182810.1 ILMN_2358457 element B67) ACTA2 1.67 actin, alpha 2, smooth muscle, aorta NM_001613.1 ILMN_1671703 PRKACB 1.66 protein kinase, cAMP-dependent, catalytic, beta NM_002731.2 ILMN_1771523

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TP53I3 1.66 tumor protein p53 inducible protein 3 NM_147184.1 ILMN_2358919 PSAT1 1.66 phosphoserine aminotransferase 1 NM_021154.3 ILMN_1692938 LGALS1 1.65 lectin, galactoside-binding, soluble, 1 NM_002305.3 ILMN_1723978 DDB2 1.65 damage-specific DNA binding protein 2, 48kDa NM_000107.1 ILMN_1660817 AARS 1.65 alanyl-tRNA synthetase NM_001605.2 ILMN_1662364 ATF3 1.64 activating transcription factor 3 NM_001040619.1 ILMN_2374865 CRABP2 1.64 cellular retinoic acid binding protein 2 NM_001878.2 ILMN_1690170 IGFBP6 1.64 insulin-like growth factor binding protein 6 NM_002178.2 ILMN_1669362

Appendix 11. 5 The top 50 genes with most differentially downregulated compared with controls in the FPGS-overexpressed MDA-MB-435 breast cancer cells

Gene Fold Description Accession Probe ID Symbol Change TSPAN7 -22.33 tetraspanin 7 NM_004615.2 ILMN_2120695 TYR -20.19 tyrosinase (oculocutaneous albinism IA) NM_000372.4 ILMN_1788774 APOD -11.66 apolipoprotein D NM_001647.2 ILMN_1780170 TRIM48 -11.38 tripartite motif-containing 48 NM_024114.2 ILMN_1762021 BCHE -11.02 butyrylcholinesterase NM_000055.1 ILMN_2176592 IGSF11 -10.38 immunoglobulin superfamily, member 11 NM_001015887.1 ILMN_1753502 DCT -9.55 dopachrome tautomerase (dopachrome delta- NM_001922.2 ILMN_1701783 isomerase, tyrosine-related protein 2) CHCHD6 -9.43 coiled-coil-helix-coiled-coil-helix domain containing NM_032343.1 ILMN_1785161 6 ALDH1A1 -9.30 aldehyde dehydrogenase 1 family, member A1 NM_000689.3 ILMN_2096372 BCHE -8.69 butyrylcholinesterase NM_000055.2 ILMN_1685641 TSPAN7 -7.59 tetraspanin 7 NM_004615.2 ILMN_1809291 GPM6B -5.99 glycoprotein M6B NM_001001995.1 ILMN_1704665 DYNC1I1 -5.14 dynein, cytoplasmic 1, intermediate chain 1 NM_004411.3 ILMN_1690397 MYO10 -5.12 myosin X NM_012334.1 ILMN_2232712 GPM6B -4.82 glycoprotein M6B NM_001001995.1 ILMN_1735438 TUBB4 -4.74 tubulin, beta 4 NM_006087.2 ILMN_1682459 ALDH1A1 -4.69 aldehyde dehydrogenase 1 family, member A1 NM_000689.3 ILMN_1709348 CAPN3 -4.55 calpain 3, (p94) NM_173087.1 ILMN_2332691 CAPN3 -4.52 calpain 3, (p94) NM_024344.1 ILMN_1687971 PIR -4.51 pirin (iron-binding nuclear protein) NM_001018109.1 ILMN_1761247 C4orf18 -4.49 chromosome 4 open reading frame 18 NM_016613.5 ILMN_1761941 PIR -4.46 pirin (iron-binding nuclear protein) NM_001018109.1 ILMN_2383383 FAM65B -4.26 family with sequence similarity 65, member B NM_015864.2 ILMN_1726597 GYG2 -4.24 glycogenin 2 NM_003918.2 ILMN_2319424 PPARGC1A -4.23 peroxisome proliferator-activated receptor gamma, NM_013261.3 ILMN_1750062 coactivator 1 alpha SLC24A5 -4.01 solute carrier family 24, member 5 NM_205850.2 ILMN_1786045 STK32A -3.78 serine/threonine kinase 32A NM_145001.2 ILMN_1756612 MITF -3.76 microphthalmia-associated transcription factor NM_198158.1 ILMN_2304186 LOC729384 -3.61 tripartite motif protein 49-like NM_001105522.1 ILMN_3242226 GHR -3.58 growth hormone receptor NM_000163.2 ILMN_1775814 DNAJB6 -3.56 DnaJ (Hsp40) homolog, subfamily B, member 6 NM_058246.3 ILMN_1793770

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MSRB2 -3.52 methionine sulfoxide reductase B2 NM_012228.2 ILMN_1657977 C18orf51 -3.48 chromosome 18 open reading frame 51 NM_001044369.1 ILMN_1670718 C18orf51 -3.47 chromosome 18 open reading frame 51 NM_001044369.1 ILMN_2173500 AIF1L -3.41 allograft inflammatory factor 1-like NM_031426.2 ILMN_3246401 PKNOX2 -3.22 PBX/knotted 1 homeobox 2 NM_022062.2 ILMN_1807689 SLC5A4 -3.13 solute carrier family 5 (low affinity glucose NM_014227.1 ILMN_1678650 cotransporter), member 4 NOV -3.13 nephroblastoma overexpressed gene NM_002514.2 ILMN_1787186 TBC1D7 -3.08 TBC1 domain family, member 7 NM_016495.2 ILMN_1661622 CPN1 -2.99 carboxypeptidase N, polypeptide 1 NM_001308.2 ILMN_1808674 HIBCH -2.97 3-hydroxyisobutyryl-Coenzyme A hydrolase NM_198047.1 ILMN_1656977 NBL1 -2.94 neuroblastoma, suppression of tumorigenicity 1 NM_005380.4 ILMN_2405009 KCNAB1 -2.93 potassium voltage-gated channel, shaker-related NM_003471.2 ILMN_1744968 subfamily, beta member 1 QPCT -2.91 glutaminyl-peptide cyclotransferase NM_012413.3 ILMN_1741727 FAM81A -2.86 family with sequence similarity 81, member A NM_152450.2 ILMN_1699623 GYG2 -2.84 glycogenin 2 NM_003918.1 ILMN_1684017 TXNDC5 -2.83 thioredoxin domain containing 5 NM_022085.3 ILMN_2403965 SPRYD5 -2.83 SPRY domain containing 5 NM_032681.1 ILMN_1753648 ST6GALNAC3 -2.81 ST6 (alpha-N-acetyl-neuraminyl-2, 3-beta-galactosyl- NM_152996.1 ILMN_2127379 1, 3)-N-acetylgalactosaminide alpha-2, 6- sialyltransferase 3 VEPH1 -2.80 ventricular zone expressed PH domain homolog 1 NM_024621.1 ILMN_1684336 (zebrafish)

Appendix 11. 6 The top 50 genes with most differentially upregulated compared with controls in the FPGS-overexpressed MDA-MB-435 breast cancer cells

Gene Fold Description Accession Probe ID Symbol Change IL24 12.91 interleukin 24 NM_006850.2 ILMN_1774685 HLA-DQA1 9.13 PREDICTED: major histocompatibility complex, class XM_936128.2 ILMN_1808405 II, DQ alpha 1, transcript variant 10 C21orf34 8.79 chromosome 21 open reading frame 34 NM_001005734.1 ILMN_1690703 C20orf100 7.51 chromosome 20 open reading frame 100 NM_032883.1 ILMN_2082209 CNN3 7.16 calponin 3, acidic NM_001839.2 ILMN_1782439 NNMT 6.82 nicotinamide N-methyltransferase NM_006169.2 ILMN_1715508 SPRR2D 6.26 small proline-rich protein 2D NM_006945.3 ILMN_2191967 CDC42EP5 5.99 CDC42 effector protein (Rho GTPase binding) 5 NM_145057.2 ILMN_1774982 AHNAK 5.96 AHNAK nucleoprotein NM_024060.2 ILMN_1752159 COL13A1 5.71 collagen, type XIII, alpha 1 NM_080805.2 ILMN_2370624 MT1E 5.62 metallothionein 1E NM_175617.3 ILMN_2173611 CYR61 5.62 cysteine-rich, angiogenic inducer, 61 NM_001554.3 ILMN_2188264 SYTL2 5.58 synaptotagmin-like 2 NM_206928.1 ILMN_1682929 LAIR2 5.50 leukocyte-associated immunoglobulin-like receptor 2 NM_002288.3 ILMN_1807491 BIRC3 5.23 baculoviral IAP repeat-containing 3 NM_001165.3 ILMN_1776181 KLRC2 5.21 killer cell lectin-like receptor subfamily C, member 2 NM_002260.3 ILMN_2059357 LAIR2 5.18 leukocyte-associated immunoglobulin-like receptor 2 NM_021270.2 ILMN_2323933 IGFBP7 5.18 insulin-like growth factor binding protein 7 NM_001553.1 ILMN_2062468

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FST 5.10 follistatin NM_013409.1 ILMN_1700081 MT1X 5.00 metallothionein 1X NM_005952.2 ILMN_1775170 CYBRD1 4.90 cytochrome b reductase 1 NM_024843.2 ILMN_1712305 CYBRD1 4.90 cytochrome b reductase 1 NM_024843.2 ILMN_2087692 PLOD2 4.85 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 NM_000935.2 ILMN_1771599 IL24 4.81 interleukin 24 NM_181339.1 ILMN_2407799 SYTL2 4.69 synaptotagmin-like 2 NM_206929.1 ILMN_2336609 HTATIP2 4.64 HIV-1 Tat interactive protein 2, 30kDa NM_006410.3 ILMN_1664303 FAM133A 4.61 family with sequence similarity 133, member A NM_173698.1 ILMN_1781742 OLFM1 4.58 olfactomedin 1 NM_014279.4 ILMN_1742025 IL8 4.57 interleukin 8 NM_000584.2 ILMN_2184373 COL13A1 4.53 collagen, type XIII, alpha 1 NM_080815.2 ILMN_2311052 FAM129A 4.34 family with sequence similarity 129, member A NM_052966.2 ILMN_1810725 SPRR2F 4.27 small proline-rich protein 2F NM_001014450.1 ILMN_1674367 LOC399959 4.24 hypothetical LOC399959 NR_024430.1 ILMN_3244176 NR4A2 4.23 nuclear receptor subfamily 4, group A, member 2 NM_006186.2 ILMN_2339955 CTHRC1 4.19 collagen triple helix repeat containing 1 NM_138455.2 ILMN_1725090 NR4A2 4.15 nuclear receptor subfamily 4, group A, member 2 NM_006186.2 ILMN_1782305 HLA-DOA 4.12 major histocompatibility complex, class II, DO alpha NM_002119.3 ILMN_1659075 CATSPER1 4.12 cation channel, sperm associated 1 NM_053054.2 ILMN_1789394 ATP9A 4.11 ATPase, class II, type 9A. NM_006045.1 ILMN_2089073 M160 4.10 scavenger receptor cysteine-rich type 1 protein M160 NM_174941.3 ILMN_1802780 PARVA 4.08 parvin, alpha NM_018222.3 ILMN_3307892 PHLDB2 4.07 pleckstrin homology-like domain, family B, member 2 NM_145753.1 ILMN_2179778 COL8A1 4.06 collagen, type VIII, alpha 1 NM_020351.2 ILMN_1685433 HLA-DRB4 4.04 major histocompatibility complex, class II, DR beta 4 NM_021983.4 ILMN_2159694 C1orf24 4.03 chromosome 1 open reading frame 24 NM_052966.1 ILMN_1667966 RAC2 4.02 ras-related C3 botulinum toxin substrate 2 (rho family, NM_002872.3 ILMN_1709795 small GTP binding protein Rac2) EGR1 3.99 early growth response 1 NM_001964.2 ILMN_1762899 CTHRC1 3.97 collagen triple helix repeat containing 1 NM_138455.2 ILMN_2117508 APCDD1L 3.94 adenomatosis polyposis coli down-regulated 1-like NM_153360.1 ILMN_1689431 CD74 3.91 CD74 molecule, major histocompatibility complex, NM_001025159.1 ILMN_1736567 class II invariant chain

Appendix 11. 7 The top 50 genes with most differentially downregulated compared with controls in the FPGS-inhibited MDA-MB-435 breast cancer cells

Gene Fold Description Accession Probe ID Symbol Change HLA-DQA1 -5.75 PREDICTED: major histocompatibility complex, class II, XM_936128.2 ILMN_1808405 DQ alpha 1, transcript variant 10 THBS2 -5.25 thrombospondin 2 NM_003247.2 ILMN_1678842 AIF1L -4.57 allograft inflammatory factor 1-like NM_031426.2 ILMN_3246401 C21orf34 -4.48 chromosome 21 open reading frame 34 NM_001005734.1 ILMN_1690703 HLA-DOA -3.95 major histocompatibility complex, class II, DO alpha NM_002119.3 ILMN_1659075 C1S -3.26 complement component 1, s subcomponent NM_001734.2 ILMN_1781626

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HLA-DMB -3.17 major histocompatibility complex, class II, DM beta NM_002118.3 ILMN_1761733 CTHRC1 -2.88 collagen triple helix repeat containing 1 NM_138455.2 ILMN_1725090 HLA-DPA1 -2.87 major histocompatibility complex, class II, DP alpha 1 NM_033554.2 ILMN_1772218 HLA-DRB4 -2.82 major histocompatibility complex, class II, DR beta 4 NM_021983.4 ILMN_2159694 HLA-DPB1 -2.76 major histocompatibility complex, class II, DP beta 1 NM_002121.4 ILMN_1749070 AIF1L -2.67 allograft inflammatory factor 1-like NM_031426.2 ILMN_1770725 CHN1 -2.66 chimerin (chimaerin) 1 NM_001025201.1 ILMN_1678493 FAM69B -2.66 PREDICTED: family with sequence similarity 69, member B XM_001130258.1 ILMN_1757440 HLA-DRB6 -2.57 major histocompatibility complex, class II, DR beta 6 NR_001298.1 ILMN_2066066 (pseudogene) CAP2 -2.55 CAP, adenylate cyclase-associated protein, 2 (yeast) NM_006366.2 ILMN_1691237 CTSL2 -2.48 cathepsin L2 NM_001333.2 ILMN_1748352 JAM3 -2.47 junctional adhesion molecule 3 NM_032801.3 ILMN_1769575 AKR1C3 -2.44 aldo-keto reductase family 1, member C3 (3-alpha NM_003739.4 ILMN_1713124 hydroxysteroid dehydrogenase, type II) ARHGDIB -2.37 Rho GDP dissociation inhibitor (GDI) beta NM_001175.4 ILMN_1678143 OPLAH -2.31 5-oxoprolinase (ATP-hydrolysing) NM_017570.2 ILMN_1711030 CTHRC1 -2.30 collagen triple helix repeat containing 1 NM_138455.2 ILMN_2117508 HLA-DRA -2.22 major histocompatibility complex, class II, DR alpha NM_019111.3 ILMN_1689655 C7orf52 -2.20 chromosome 7 open reading frame 52 NM_198571.1 ILMN_2198859 FSCN1 -2.19 fascin homolog 1, actin-bundling protein (Strongylocentrotus NM_003088.2 ILMN_1808707 purpuratus) IFITM1 -2.08 interferon induced transmembrane protein 1 (9-27) NM_003641.3 ILMN_1801246 HTRA1 -2.08 HtrA serine peptidase 1 NM_002775.3 ILMN_1676563 HLA-DRB3 -2.07 major histocompatibility complex, class II, DR beta 3 NM_022555.3 ILMN_1717261 RBPMS2 -2.07 RNA binding protein with multiple splicing 2 NM_194272.1 ILMN_1808238 CYTSB -2.06 cytospin B NM_001033555.1 ILMN_3255061 C1R -2.03 complement component 1, r subcomponent NM_001733.4 ILMN_1677198 LRP5 -2.02 low density lipoprotein receptor-related protein 5 NM_002335.1 ILMN_1702775 ZNF562 -2.00 zinc finger protein 562 NM_017656.2 ILMN_1672940 HIST1H2BD -1.99 histone cluster 1, H2bd NM_138720.1 ILMN_1651496 GAS1 -1.97 growth arrest-specific 1 NM_002048.1 ILMN_1772910 HLA-DMA -1.96 major histocompatibility complex, class II, DM alpha NM_006120.2 ILMN_1695311 HLA-B -1.96 major histocompatibility complex, class I, B NM_005514.5 ILMN_1778401 GAS1 -1.93 growth arrest-specific 1 NM_002048.1 ILMN_2062701 TNFAIP6 -1.90 tumor necrosis factor, alpha-induced protein 6 NM_007115.2 ILMN_1785732 FAHD1 -1.89 fumarylacetoacetate hydrolase domain containing 1 NM_001018104.1 ILMN_1701457 AKAP12 -1.89 A kinase (PRKA) anchor protein (gravin) 12 NM_005100.2 ILMN_2308950 RAB3IL1 -1.89 RAB3A interacting protein (rabin3)-like 1 NM_013401.2 ILMN_1741632 HLA-DRA -1.89 major histocompatibility complex, class II, DR alpha NM_019111.3 ILMN_2157441 SCARF2 -1.88 scavenger receptor class F, member 2 NM_153334.3 ILMN_1655405 CATSPER1 -1.87 cation channel, sperm associated 1 NM_053054.2 ILMN_1789394 LOC613037 -1.85 nuclear pore complex interacting protein pseudogene NR_002555.2 ILMN_2070052 AHNAK -1.84 AHNAK nucleoprotein NM_024060.2 ILMN_1752159 NRCAM -1.84 neuronal cell adhesion molecule NM_005010.3 ILMN_2411236 PRPF4 -1.83 PRP4 pre-mRNA processing factor 4 homolog (yeast) NM_004697.3 ILMN_1697440 NBL1 -1.83 neuroblastoma, suppression of tumorigenicity 1 NM_182744.1 ILMN_1789599

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Appendix 11. 8 The top 50 genes with most differentially upregulated compared with controls in the FPGS-inhibited MDA-MB-435 breast cancer cells

Gene Fold Description Accession Probe ID Symbol Change BCHE 7.02 butyrylcholinesterase NM_000055.1 ILMN_2176592 HAPLN1 6.12 hyaluronan and proteoglycan link protein 1 NM_001884.2 ILMN_1678812 HAPLN1 6.06 hyaluronan and proteoglycan link protein 1 NM_001884.2 ILMN_2210519 BCHE 5.66 butyrylcholinesterase NM_000055.2 ILMN_1685641 CXorf26 5.01 chromosome X open reading frame 26 NM_016500.3 ILMN_1768176 SLFN11 4.08 schlafen family member 11 NM_152270.2 ILMN_1752520 S100A4 4.04 S100 calcium binding protein A4 NM_019554.2 ILMN_1684306 S100A4 3.83 S100 calcium binding protein A4 NM_019554.2 ILMN_1688780 TRIM48 3.67 tripartite motif-containing 48 NM_024114.2 ILMN_1762021 IL1RAPL1 3.41 interleukin 1 receptor accessory protein-like 1 NM_014271.2 ILMN_2160428 RAB38 3.27 RAB38, member RAS oncogene family NM_022337.1 ILMN_2134974 RENBP 3.13 renin binding protein NM_002910.4 ILMN_1780057 ANKS1A 2.83 ankyrin repeat and sterile alpha motif domain containing 1A NM_015245.2 ILMN_1813669 SLFN11 2.72 schlafen family member 11 NM_152270.2 ILMN_2162860 SLITRK4 2.60 SLIT and NTRK-like family, member 4 NM_173078.2 ILMN_2199768 CPVL 2.54 carboxypeptidase, vitellogenic-like NM_031311.3 ILMN_2400759 FCRLA 2.50 Fc receptor-like A NM_032738.3 ILMN_1691071 TMEM45A 2.46 transmembrane protein 45A NM_018004.1 ILMN_2148913 CSRP2 2.45 cysteine and glycine-rich protein 2 NM_001321.1 ILMN_1660806 KDELR3 2.43 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein NM_016657.1 ILMN_1713901 retention receptor 3 CPVL 2.27 carboxypeptidase, vitellogenic-like NM_019029.2 ILMN_1682928 TMEM45A 2.22 transmembrane protein 45A NM_018004.1 ILMN_1770922 IFI6 2.21 interferon, alpha-inducible protein 6 NM_022872.2 ILMN_2347798 ST3GAL5 2.20 ST3 beta-galactoside alpha-2, 3-sialyltransferase 5 NM_001042437.1 ILMN_1713496 CNN3 2.19 calponin 3, acidic NM_001839.2 ILMN_1782439 KDELR3 2.18 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein NM_006855.2 ILMN_1722820 retention receptor 3 MT1X 2.09 metallothionein 1X NM_005952.2 ILMN_1775170 KDELR3 2.06 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein NM_016657.1 ILMN_1798952 retention receptor 3 MT1G 2.05 metallothionein 1G NM_005950.1 ILMN_1715401 ATP6V0E2 2.05 ATPase, H+ transporting V0 subunit e2 NM_145230.2 ILMN_1785095 AKR1B1 2.04 aldo-keto reductase family 1, member B1 (aldose reductase) NM_001628.2 ILMN_1701731 DYNC1I1 1.99 dynein, cytoplasmic 1, intermediate chain 1 NM_004411.3 ILMN_1690397 PLSCR1 1.97 phospholipid scramblase 1 NM_021105.1 ILMN_1745242 MIR1974 1.97 microRNA 1974 NR_031738.1 ILMN_3308961 C20orf191 1.96 chromosome 20 open reading frame 191 NM_001039379.1 ILMN_2044027 IFI27L2 1.95 interferon, alpha-inducible protein 27-like 2 NM_032036.2 ILMN_1740319 PPARGC1A 1.95 peroxisome proliferator-activated receptor gamma, coactivator 1 NM_013261.3 ILMN_1750062 alpha MTE 1.94 metallothionein E NM_175621.2 ILMN_2136089 S100A3 1.94 S100 calcium binding protein A3 NM_002960.1 ILMN_1712545 PNLIPRP3 1.92 pancreatic lipase-related protein 3 NM_001011709.1 ILMN_1678655 CEACAM1 1.92 carcinoembryonic antigen-related cell adhesion molecule 1 NM_001024912.1 ILMN_1716815 (biliary glycoprotein)

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CYB5R2 1.91 cytochrome b5 reductase 2 NM_016229.3 ILMN_1739576 ATP1B1 1.91 ATPase, Na+/K+ transporting, beta 1 polypeptide NM_001001787.1 ILMN_2407824 MT1E 1.90 metallothionein 1E NM_175617.3 ILMN_2173611 IFI6 1.90 interferon, alpha-inducible protein 6 NM_022873.2 ILMN_1687384 ITPK1 1.90 inositol 1, 3, 4-triphosphate 5/6 kinase NM_014216.3 ILMN_1715674 GPC6 1.90 glypican 6 NM_005708.2 ILMN_1805216 C7orf23 1.87 chromosome 7 open reading frame 23 NM_024315.2 ILMN_1751143 HBG1 1.87 hemoglobin, gamma A NM_000559.2 ILMN_1796678 IFI27L2 1.87 interferon, alpha-inducible protein 27-like 2 NM_032036.2 ILMN_3238560

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Appendix 12 The list of differentially expressed genes associated with the top functions in response to FPGS modulation

Appendix 12. 1 The top molecular and cellular functions associated with differentially expressed genes in the FPGS- overexpressed HCT116 colon cancer cells No. of Category Function Function Annotation P-value Genes Genes Cell Cycle cell cycle cell cycle progression 1.27E-15 ABTB1, ACLY, ANAPC11, ANAPC4, ANLN, ASNS, ATF3, 206 progression AURKA, AURKB, AXL, BCL6, BEX2, BIRC5, BLM, BMP4, BMPR2, BOP1, BORA, BRCA1, BRCA2, BUB1 (includes EG:100307076), BUB1B, C15orf63, CAMK2N1, CAT, CAV1, CCNA2, CCNB1, CCNB1IP1, CCNE1, CD24, CDC123, CDC20 (includes EG:107995), CDC23, CDC25A, CDC25C, CDC42, CDC7 (includes EG:12545), CDCA5, CDCA8, CDH1, CDK1, CDK2, CDK6, CDKN1A, CDKN2B, CDKN3, CEBPB (includes EG:1051), CENPA, CENPE, CENPF, CEP192, CHAF1A, CHEK1, CHEK2, CKAP2, CKAP5, CLTC, CSE1L, CSNK2A1, CUL1, DDX17, DEK, DLGAP5, DNMT1, DTL, E2F2, EHF, EIF4G2, EZH2, FANCA, FASN, FBXO5, FOS, FOXC1, FOXM1, FOXO3, FOXO4, FYN, GADD45A, GMNN, GPS1, GRN, HAUS1, HAUS6, HAUS7, HAUS8, HBEGF, HCFC1, HDAC4, HJURP, HMG20B, HMGB1, HRAS, HSPA1A/HSPA1B, ID1, ID3 (includes EG:15903), IER3, IGFBP3, IL8, INCENP, ING2, IRF1 (includes EG:16362), IRF7, ITGB4, KAT2B, KIF11, KIF15, KIF22, KIF2A, KIF2C, KIF4A, KIFC1, KLF6, KNTC1, LEP, LGALS3, LIF, LIG1, LRP5, LSM1, MAD2L1, MAPK1, MAPK3, MAPRE3, MCM2, MELK, METAP2, MLF1, MMS22L, MSLN, NAE1, NDC80, NEDD1, NEK2, NME1 (includes EG:18102), NOLC1, NUDT1, NUF2, NUSAP1, ORC6 (includes EG:23594), PA2G4, PARP1, PAWR, PCNA, PES1, PHB, PKMYT1, PLK1, PNN, POLD1, POLD4, PPP1R15A, PRKCA, PRMT5, PRPF4, PSMB5, PSRC1, PTTG1, RAD21, RAF1, RAN, RCC1, RHOD, RPA1, RPS6KA2, RRM1, RUNX1, S100A4, SASS6, SCRIB, SGOL1, SHC1 (includes EG:20416), SKA1, SKA3, SKP2 (includes EG:27401), SMC3, SPC25 (includes EG:100144563), SREBF1 (includes EG:176574), SSBP2, STAT1, STAT3, STIL, SUGT1, TCP1, TFDP1, TGFA, TOP2A, TP53INP1, TPX2, TSC22D1, TSG101, TTK, TXN (includes EG:116484), UBE2C, UPF1, UQCRFS1, USP16, VEGFA, VHL, WEE1, XBP1 (includes EG:140614), ZAK,

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ZC3HC1, ZW10, ZWINT Cell Cycle cell cycle cell cycle progression of 6.53E-05 AURKA, BIRC5, C15orf63, CCNA2, CDC25C, CDKN1A, 14 progression cervical cancer cell lines CSE1L, CSNK2A1, IER3, MMS22L, PLK1, PRPF4, RPA1, TCP1 Cell Cycle cell cycle cell cycle progression of tumor 1.79E-04 AURKA, BCL6, BIRC5, BMP4, BRCA1, C15orf63, CAMK2N1, 53 progression cell lines CAV1, CCNA2, CCNE1, CDC25C, CDK2, CDK6, CDKN1A, CEBPB (includes EG:1051), CKAP2, CSE1L, CSNK2A1, EHF, EZH2, FANCA, FOXM1, FOXO3, HBEGF, HRAS, IER3, IGFBP3, KIF2A, LGALS3, LIF, LIG1, LSM1, MELK, MLF1, MMS22L, MSLN, PCNA, PHB, PLK1, POLD1, POLD4, PRPF4, RAF1, RPA1, RRM1, S100A4, SSBP2, STAT3, TCP1, TSG101, VHL, WEE1, XBP1 (includes EG:140614) Cell Cycle cell cycle arrest in cell cycle progression 6.00E-04 AURKA, BMP4, BRCA1, C15orf63, CAV1, CCNA2, CCNE1, 56 progression CDC123, CDC25A, CDK2, CDK6, CDKN1A, CDKN2B, CDKN3, CEBPB (includes EG:1051), CKAP2, CUL1, DDX17, E2F2, EHF, EIF4G2, EZH2, FASN, FOXM1, FOXO3, FOXO4, GADD45A, HRAS, ID1, ID3 (includes EG:15903), IL8, ING2, IRF7, KAT2B, LGALS3, LIF, MAD2L1, MELK, MLF1, PA2G4, PLK1, POLD1, PPP1R15A, PRPF4, RAF1, RRM1, S100A4, SKP2 (includes EG:27401), SSBP2, STAT1, TCP1, TP53INP1, TSG101, WEE1, XBP1 (includes EG:140614), ZAK Cell Cycle cell cycle delay in cell cycle progression 3.45E-03 AURKA, BIRC5, CDC7 (includes EG:12545), CDKN1A, LIG1, 8 progression PCNA, POLD4, TFDP1 Cell Cycle cell cycle cell cycle progression of 5.75E-03 MSLN, S100A4, STAT3 3 progression pancreatic cancer cell lines Cell Cycle cell cycle delay in cell cycle progression 1.79E-02 AURKA, BIRC5, CDKN1A, LIG1, PCNA, POLD4 6 progression of tumor cell lines Cell Cycle cell cycle arrest in cell cycle progression 1.99E-02 DDX17, PLK1, TP53INP1 3 progression of embryonic cell lines Cell Cycle cell cycle cell cycle progression of 1.99E-02 CCNE1, E2F2, SKP2 (includes EG:27401) 3 progression hepatocytes Cell Cycle segregation segregation of chromosomes 1.27E-15 AURKB, BRCA1, BUB1 (includes EG:100307076), CCNA2, 40 CCNB1, CCNB2, CDC42, CDK5RAP2, CENPF, CENPW, CHMP2A, DSN1 (includes EG:100002916), ECT2, ESPL1, HDAC4, HJURP, INCENP, KIF2C, NCAPD2, NCAPD3, NCAPG, NCAPG2, NCAPH, NDC80, NDEL1, NEK2, NUF2, NUSAP1, RCC1, RIOK3, SGOL1, SKA1, SKA3, SMC2, SMC4, SPC25 (includes EG:100144563), SRPK1, TOP2A, ZW10, ZWINT

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Cell Cycle segregation segregation of sister chromatids 2.62E-04 CCNA2, ESPL1, NCAPD2, NDC80, NUSAP1, SMC4, ZW10, 8 ZWINT Cell Cycle mitosis mitosis 3.88E-15 ANAPC11, ANAPC4, ANLN, AURKA, AURKB, AXL, BIRC5, 105 BMP4, BORA, BRCA1, BRCA2, BUB1 (includes EG:100307076), BUB1B, CCNB1, CCNB1IP1, CCNE1, CDC123, CDC20 (includes EG:107995), CDC23, CDC25A, CDC25C, CDC7 (includes EG:12545), CDCA5, CDCA8, CDK1, CDKN1A, CENPA, CENPE, CENPF, CEP192, CHEK1, CKAP2, CKAP5, CLTC, CSE1L, DLGAP5, FBXO5, FOXC1, FOXM1, FYN, GMNN, GRN, HAUS1, HAUS6, HAUS7, HAUS8, HBEGF, HDAC4, HMG20B, HRAS, IGFBP3, INCENP, KIF11, KIF15, KIF22, KIF2A, KIF2C, KIF4A, KIFC1, KNTC1, LEP, LRP5, MAD2L1, MAPK1, MAPK3, NAE1, NDC80, NEDD1, NEK2, NOLC1, NUF2, NUSAP1, ORC6 (includes EG:23594), PKMYT1, PLK1, PRKCA, PRMT5, PRPF4, PTTG1, RAD21, RAN, RCC1, SASS6, SCRIB, SGOL1, SHC1 (includes EG:20416), SKA1, SKA3, SMC3, SPC25 (includes EG:100144563), STIL, SUGT1, TCP1, TGFA, TOP2A, TPX2, TTK, TXN (includes EG:116484), UBE2C, USP16, VEGFA, WEE1, ZC3HC1, ZW10, ZWINT Cell Cycle mitosis mitosis of tumor cell lines 7.45E-09 AURKA, BIRC5, BMP4, BRCA1, BRCA2, CCNB1, CCNE1, 37 CDC20 (includes EG:107995), CDC25A, CDC25C, CDC7 (includes EG:12545), CDCA8, CDK1, CDKN1A, CENPE, CHEK1, CSE1L, DLGAP5, FBXO5, FOXM1, GRN, HDAC4, HMG20B, MAD2L1, NAE1, PKMYT1, PLK1, PRPF4, PTTG1, RAD21, SPC25 (includes EG:100144563), STIL, TCP1, TOP2A, TTK, WEE1, ZC3HC1 Cell Cycle mitosis delay in mitosis 6.25E-08 BRCA1, CCNB1, CDC20 (includes EG:107995), CDC25C, 13 CDK1, CENPE, DLGAP5, FOXM1, MAD2L1, PLK1, PTTG1, RAD21, TOP2A Cell Cycle mitosis delay in mitosis of tumor cell 6.83E-08 BRCA1, CCNB1, CDC20 (includes EG:107995), CDC25C, 12 lines CDK1, CENPE, DLGAP5, MAD2L1, PLK1, PTTG1, RAD21, TOP2A Cell Cycle mitosis mitosis of cervical cancer cell 8.04E-08 AURKA, BIRC5, BRCA2, CCNB1, CDC20 (includes 26 lines EG:107995), CDC25A, CDC25C, CDCA8, CENPE, CSE1L, DLGAP5, FBXO5, HDAC4, HMG20B, MAD2L1, PLK1, PRPF4, PTTG1, RAD21, SPC25 (includes EG:100144563), STIL, TCP1, TOP2A, TTK, WEE1, ZC3HC1 Cell Cycle mitosis delay in mitosis of cervical 3.11E-07 CCNB1, CDC20 (includes EG:107995), CDC25C, CENPE, 10 cancer cell lines DLGAP5, MAD2L1, PLK1, PTTG1, RAD21, TOP2A

454

Cell Cycle mitosis arrest in mitosis 7.51E-07 AURKB, BIRC5, BUB1 (includes EG:100307076), BUB1B, 20 CCNE1, CDKN1A, CENPE, CHEK1, CKAP2, CSE1L, FBXO5, HDAC4, KNTC1, MAD2L1, PLK1, SGOL1, TCP1, TTK, ZW10, ZWINT Cell Cycle mitosis arrest in mitosis of tumor cell 5.49E-04 BIRC5, CCNE1, CDKN1A, CENPE, CHEK1, CSE1L, FBXO5, 12 lines HDAC4, MAD2L1, PLK1, TCP1, TTK Cell Cycle mitosis exit from mitosis 1.13E-03 ANLN, BIRC5, CCNE1, CDC23, CDKN1A, MAD2L1, NDC80, 8 ZW10 Cell Cycle mitosis entry into mitosis 1.92E-03 AURKA, CCNB1IP1, CDC25A, CDC25C, CDKN1A, CHEK1, 9 NAE1, PKMYT1, ZC3HC1 Cell Cycle mitosis mitosis of colon cancer cell 3.68E-03 BIRC5, CDC25A, CDKN1A, CHEK1, MAD2L1, NAE1, PLK1 7 lines Cell Cycle mitosis arrest in mitosis of colon cancer 4.41E-03 BIRC5, CDKN1A, MAD2L1, PLK1 4 cell lines Cell Cycle mitosis arrest in mitosis of cervical 5.50E-03 BIRC5, CENPE, CSE1L, FBXO5, HDAC4, PLK1, TCP1, TTK 8 cancer cell lines Cell Cycle mitosis entry into mitosis of tumor cell 6.23E-03 AURKA, CDC25A, CDC25C, CHEK1, NAE1, PKMYT1, 7 lines ZC3HC1 Cell Cycle mitosis entry into mitosis of colon 1.99E-02 CDC25A, CHEK1, NAE1 3 cancer cell lines Cell Cycle interphase interphase 4.63E-09 ACIN1, ACVR1, ANAPC10, ANAPC4, ATF5, AURKA, 139 BABAM1, BCL3, BIRC5, BLM, BMP4, BRCA1, BRCC3, BRSK1, CAMK2N1, CASP3, CCNA2, CCNB1, CCNE1, CCNE2, CDC14B, CDC23, CDC25A, CDC25C, CDC42, CDC7 (includes EG:12545), CDCA5, CDH1, CDK1, CDK2, CDK2AP1, CDK5RAP3, CDK6, CDKN1A, CDKN2B, CDKN3, CEBPD, CENPF, CHEK1, CHEK2, COPS5, CREG1, CRLF3, CSE1L, CSNK2A1, DDIT3, DPP4, DTL, E2F5, ELAC2, EZH2, FAM175A, FAM188A, FANCD2, FASN, FBXO5, FOXM1, FOXN3, FOXO3, FOXO4, GADD45A, GDF15, GMNN, GPN3, GSPT1, HBEGF, HES1 (includes EG:15205), HOXA10, HRAS, ID1, ID2, ID3 (includes EG:15903), IL15 (includes EG:16168), ILKAP, ING2, ITGA5, ITGAV, ITGB1, KDM5B, KLF4, KLF6, KPNA2, KRT7, LAS1L, LGALS1, LGALS3, MAD2L1, MCM10 (includes EG:307126), MCM7, MCMBP, MELK, MMS22L, MSH2, MSLN, MTBP, MXD4, MYB, MYBL1, NAE1, NASP, NCOA3, PBX1, PLAU, PLAUR, PLK1, PNPT1, POLA1, POLD1, POLD4, PRNP, PTGES3, PTOV1, RAF1, RBBP8, RBCK1, RCC1, RGCC, RPA1, RXRA, SHC1 (includes

455

EG:20416), SKP2 (includes EG:27401), SLBP, SOX9, SPHK2, STAT1, TCP1, TFDP1, TFRC, TGFA, TIMELESS, TIMP2 (includes EG:21858), TIPIN, TOPBP1, TUSC2, TYMS, VHL, WEE1, XPC, YWHAQ Cell Cycle interphase interphase of tumor cell lines 2.49E-05 ACIN1, ATF5, BRCA1, CAMK2N1, CASP3, CCNA2, CCNB1, 85 CCNE1, CDC25A, CDC25C, CDC42, CDC7 (includes EG:12545), CDH1, CDK1, CDK2, CDK5RAP3, CDK6, CDKN1A, CDKN2B, CEBPD, CHEK1, CHEK2, COPS5, CREG1, CSE1L, DDIT3, DPP4, E2F5, EZH2, FAM188A, FASN, FOXM1, FOXO3, GADD45A, GDF15, GMNN, GSPT1, HES1 (includes EG:15205), HOXA10, HRAS, ILKAP, ITGA5, ITGAV, ITGB1, KLF4, LAS1L, LGALS1, LGALS3, MAD2L1, MCM10 (includes EG:307126), MSH2, MSLN, MTBP, MXD4, MYB, NAE1, NASP, NCOA3, PBX1, PLAU, PLAUR, PLK1, POLA1, POLD4, PRNP, PTGES3, RAF1, RBBP8, RBCK1, RPA1, RXRA, SHC1 (includes EG:20416), SKP2 (includes EG:27401), SOX9, SPHK2, STAT1, TCP1, TGFA, TOPBP1, TUSC2, TYMS, VHL, WEE1, XPC, YWHAQ Cell Cycle interphase interphase of colon cancer cell 4.28E-05 BRCA1, CAMK2N1, CDC42, CDH1, CDKN1A, CHEK1, 23 lines CHEK2, FASN, FOXO3, GADD45A, GMNN, GSPT1, ITGA5, KLF4, LAS1L, LGALS1, MXD4, NAE1, TGFA, TOPBP1, TYMS, WEE1, YWHAQ Cell Cycle interphase arrest in interphase 1.68E-04 ACIN1, ATF5, AURKA, BIRC5, BLM, BMP4, BRCA1, 75 CAMK2N1, CASP3, CCNA2, CCNB1, CCNE1, CDC25A, CDC25C, CDC42, CDC7 (includes EG:12545), CDK1, CDK2, CDK5RAP3, CDKN1A, CDKN2B, CEBPD, CHEK1, CHEK2, CSE1L, CSNK2A1, DDIT3, DPP4, E2F5, EZH2, FAM188A, FASN, FBXO5, FOXM1, FOXO3, GADD45A, GDF15, GPN3, GSPT1, HES1 (includes EG:15205), HOXA10, HRAS, ING2, ITGA5, ITGAV, ITGB1, KDM5B, KLF4, KLF6, LAS1L, LGALS1, LGALS3, MAD2L1, MMS22L, MSH2, MTBP, PLAU, PLAUR, PLK1, PNPT1, PTGES3, RAF1, RBCK1, RPA1, RXRA, SKP2 (includes EG:27401), SPHK2, TCP1, TFRC, TIMP2 (includes EG:21858), TOPBP1, TUSC2, TYMS, WEE1, XPC Cell Cycle interphase interphase of fibroblasts 3.61E-04 CCNB1, CCNE2, CDC25C, CDC7 (includes EG:12545), 10 CDKN1A, CHEK1, CHEK2, GADD45A, ID3 (includes EG:15903), SKP2 (includes EG:27401) Cell Cycle interphase arrest in interphase of colon 5.28E-04 BRCA1, CAMK2N1, CDC42, CDKN1A, CHEK1, CHEK2, 15 cancer cell lines FASN, GADD45A, GSPT1, ITGA5, KLF4, LAS1L, LGALS1, TOPBP1, TYMS

456

Cell Cycle interphase arrest in interphase of tumor 5.58E-04 ACIN1, ATF5, BRCA1, CAMK2N1, CASP3, CCNA2, CCNB1, 59 cell lines CCNE1, CDC25A, CDC25C, CDC42, CDC7 (includes EG:12545), CDK1, CDK2, CDK5RAP3, CDKN1A, CDKN2B, CEBPD, CHEK1, CHEK2, CSE1L, DDIT3, DPP4, EZH2, FAM188A, FASN, FOXM1, FOXO3, GADD45A, GDF15, GSPT1, HES1 (includes EG:15205), HOXA10, HRAS, ITGA5, ITGAV, ITGB1, KLF4, LAS1L, LGALS1, LGALS3, MAD2L1, MSH2, MTBP, PLAU, PLAUR, PLK1, PTGES3, RAF1, RBCK1, RPA1, RXRA, SPHK2, TCP1, TOPBP1, TUSC2, TYMS, WEE1, XPC Cell Cycle interphase delay in initiation of interphase 1.50E-03 CCNE1, CDKN1A, CHEK2, CREG1, ELAC2, FANCD2, IL15 11 (includes EG:16168), NASP, NCOA3, POLA1, STAT1 Cell Cycle interphase arrest in interphase of breast 1.17E-02 BRCA1, CDC25A, CDC25C, CDK1, CDKN1A, CHEK1, 15 cancer cell lines GDF15, HOXA10, LGALS1, LGALS3, RBCK1, RXRA, SPHK2, TYMS, WEE1 Cell Cycle interphase interphase of breast cancer cell 1.30E-02 BRCA1, CCNE1, CDC25A, CDC25C, CDH1, CDK1, CDKN1A, 21 lines CHEK1, GDF15, HES1 (includes EG:15205), HOXA10, LGALS1, LGALS3, MSH2, RBBP8, RBCK1, RXRA, SOX9, SPHK2, TYMS, WEE1 Cell Cycle checkpoint control checkpoint control 1.44E-07 BUB1 (includes EG:100307076), BUB1B, CCNE2, CDC20 24 (includes EG:107995), CDC25A, CDC25C, CDC7 (includes EG:12545), CDK10, CDK2, CHEK1, CHEK2, FANCD2, FANCG, KNTC1, MAD2L1, MCM7, NDC80, RBBP8, TIPIN, XPC, ZAK, ZW10, ZWILCH, ZWINT Cell Cycle checkpoint control checkpoint control of 3.21E-02 CHEK1, CHEK2 2 lymphoma cell lines Cell Cycle checkpoint control checkpoint control of mitotic 3.21E-02 CDC20 (includes EG:107995), NDC80 2 spindle Cell Cycle M phase M phase 2.29E-07 ANAPC10, ANAPC4, ANLN, AURKA, BIRC5, BRCA2, 51 BUB1B, CCDC99, CCNB1, CCNE1, CDC20 (includes EG:107995), CDC23, CDC42, CDCA8, CDK1, CDKN1A, CENPE, CENPF, CENPV, CEP55, DIAPH3, DLGAP5, ECT2, FBXO5, INCENP, KIF14, KIF20A, KIF20B, KIF23, KIF4A, KIFC1, MAD2L1, MOB1A, MPHOSPH9, MYH14, NCAPD2, NME1 (includes EG:18102), NUF2, NUSAP1, PLK1, PPP1CC, PRC1 (includes EG:233406), PTTG1, RACGAP1, RHOC, RPS6KA2, SEPT4, SKA1, STX16, TOP2A, ZW10 Cell Cycle M phase delay in initiation of M phase 3.32E-04 BIRC5, BUB1B, CCNB1, CDC42, ECT2, MAD2L1, MYH14, 9 PLK1, TOP2A Cell Cycle M phase M phase of tumor cell lines 1.19E-03 CCDC99, CCNE1, CDC42, CEP55, DIAPH3, ECT2, FBXO5, 24

457

KIF14, KIF20A, KIF20B, KIF23, KIF4A, KIFC1, MAD2L1, MOB1A, MYH14, NCAPD2, NUF2, PLK1, PPP1CC, PTTG1, STX16, TOP2A, ZW10 Cell Cycle M phase delay in M phase of tumor cell 3.58E-03 CDC42, ECT2, MAD2L1, MYH14, PLK1, TOP2A 6 lines Cell Cycle M phase M phase of epithelial cell lines 5.75E-03 FBXO5, NME1 (includes EG:18102), RPS6KA2 3 Cell Cycle M phase M phase of cervical cancer cell 1.11E-02 CCDC99, CDC42, CEP55, DIAPH3, ECT2, KIF14, KIF20A, 17 lines KIF23, KIF4A, KIFC1, MAD2L1, NCAPD2, NUF2, PLK1, STX16, TOP2A, ZW10 Cell Cycle M phase delay in M phase of cervical 1.22E-02 CDC42, ECT2, MAD2L1, PLK1, TOP2A 5 cancer cell lines Cell Cycle M phase arrest in M phase 1.47E-02 CCDC99, CCNE1, CDC20 (includes EG:107995), ECT2, FBXO5, 10 MAD2L1, NUF2, PLK1, RPS6KA2, ZW10 Cell Cycle M phase M phase of lung cancer cell 1.99E-02 MYH14, PPP1CC, PTTG1 3 lines Cell Cycle recombination DNA recombination 3.49E-06 BLM, BRCA1, BRCA2, CDK2, DMC1, ERCC1, EXO1 (includes 21 EG:26909), HJURP, KPNA2, MMS22L, MSH2, MSH6, PRKDC, RAD21, RAD51C, RAD54B, RAD54L, RMI1 (includes EG:306734), SERTAD1, SETMAR, TNPO3 Cell Cycle formation formation of mitotic spindle 3.75E-06 BIRC5, CEP192, CKAP2, CKAP5, HAUS1, HAUS6, HAUS7, 20 HAUS8, KIF11, KIF2A, KIF2C, KIF4A, KIFC1, NEDD1, NEK2, NUF2, PLK1, RCC1, SASS6, TPX2 Cell Cycle formation formation of centriole 6.23E-03 CENPJ, CEP135, CUL1, MAD2L1, NEDD1, PLK4, SASS6 7 Cell Cycle G2/M phase G2/M phase 1.13E-05 ANAPC10, ANAPC4, ATF5, AURKA, BABAM1, BIRC5, BLM, 43 BRCA1, BRCC3, BRSK1, CCNA2, CCNB1, CDC14B, CDC25A, CDC25C, CDK1, CDK2, CDKN1A, CDKN2B, CHEK1, CHEK2, CSE1L, DPP4, DTL, ELAC2, FAM175A, FOXM1, GADD45A, GMNN, KDM5B, LGALS1, MAD2L1, MELK, MSH2, MYB, PLAU, PLK1, POLD4, RBBP8, RBCK1, RPA1, SPHK2, WEE1 Cell Cycle G2/M phase G2/M phase of tumor cell lines 1.72E-03 BRCA1, CDC25A, CDC25C, CDKN1A, CHEK1, CHEK2, 17 CSE1L, FOXM1, GMNN, MSH2, MYB, POLD4, RBBP8, RBCK1, RPA1, SPHK2, WEE1 Cell Cycle G2/M phase G2/M phase of breast cancer 5.50E-03 BRCA1, CDC25C, CHEK1, MSH2, RBBP8, RBCK1, SPHK2, 8 cell lines WEE1 Cell Cycle G2/M phase arrest in G2/M phase of breast 6.68E-03 BRCA1, CDC25C, CHEK1, RBCK1, SPHK2, WEE1 6 cancer cell lines

458

Cell Cycle G2/M phase arrest in G2/M phase 1.51E-02 AURKA, BRCA1, CDC25C, CDKN1A, CHEK1, CHEK2, 13 CSE1L, FOXM1, KDM5B, RBCK1, RPA1, SPHK2, WEE1 Cell Cycle G2/M phase arrest in G2/M phase of tumor 1.94E-02 BRCA1, CDC25C, CDKN1A, CHEK1, CHEK2, CSE1L, 11 cell lines FOXM1, RBCK1, RPA1, SPHK2, WEE1 Cell Cycle G2/M phase G2/M phase of colon cancer 2.28E-02 CHEK1, CHEK2, GMNN, WEE1 4 cell lines Cell Cycle G2/M phase arrest in G2/M phase of colon 3.21E-02 CHEK1, CHEK2 2 cancer cell lines Cell Cycle G2 phase G2 phase 2.59E-05 ANAPC10, ANAPC4, ATF5, AURKA, BABAM1, BIRC5, BLM, 52 BRCA1, BRCC3, BRSK1, CCNA2, CCNB1, CDC14B, CDC25A, CDC25C, CDK1, CDK2, CDKN1A, CDKN2B, CENPF, CHEK1, CHEK2, CSE1L, CSNK2A1, DPP4, DTL, E2F5, ELAC2, FAM175A, FASN, FOXM1, FOXN3, GADD45A, GMNN, KDM5B, KPNA2, LGALS1, MAD2L1, MELK, MMS22L, MSH2, MYB, PLAU, PLK1, POLD4, RBBP8, RBCK1, RPA1, SPHK2, TCP1, WEE1, XPC Cell Cycle G2 phase G2 phase of cervical cancer cell 6.04E-04 CCNA2, CCNB1, CDC25A, CDC25C, CHEK1, CSE1L, 11 lines GADD45A, PLK1, RPA1, TCP1, XPC Cell Cycle G2 phase arrest in G2 phase of cervical 1.92E-03 CCNA2, CCNB1, CHEK1, CSE1L, GADD45A, PLK1, RPA1, 9 cancer cell lines TCP1, XPC Cell Cycle G2 phase G2 phase of tumor cell lines 3.14E-03 ATF5, BRCA1, CCNA2, CCNB1, CDC25A, CDC25C, CDK1, 29 CDKN1A, CHEK1, CHEK2, CSE1L, DPP4, FASN, FOXM1, GADD45A, GMNN, MAD2L1, MSH2, MYB, PLAU, PLK1, POLD4, RBBP8, RBCK1, RPA1, SPHK2, TCP1, WEE1, XPC Cell Cycle G2 phase arrest in G2 phase of fibroblasts 3.58E-03 CCNB1, CDC25C, CDKN1A, CHEK1, CHEK2, GADD45A 6 Cell Cycle G2 phase arrest in G2 phase 5.20E-03 ATF5, AURKA, BLM, BRCA1, CCNA2, CCNB1, CDC25C, 31 CDK1, CDKN1A, CHEK1, CHEK2, CSE1L, CSNK2A1, DPP4, E2F5, FASN, FOXM1, GADD45A, KDM5B, LGALS1, MAD2L1, MMS22L, MSH2, PLAU, PLK1, RBCK1, RPA1, SPHK2, TCP1, WEE1, XPC Cell Cycle G2 phase arrest in G2 phase of tumor cell 9.28E-03 ATF5, BRCA1, CCNA2, CCNB1, CDC25C, CDK1, CDKN1A, 24 lines CHEK1, CHEK2, CSE1L, DPP4, FASN, FOXM1, GADD45A, MAD2L1, MSH2, PLAU, PLK1, RBCK1, RPA1, SPHK2, TCP1, WEE1, XPC Cell Cycle G2 phase G2 phase of colon cancer cell 1.22E-02 BRCA1, CDKN1A, CHEK1, CHEK2, FASN, GADD45A, 8 lines GMNN, WEE1 Cell Cycle G2 phase arrest in G2 phase of colon 1.79E-02 BRCA1, CDKN1A, CHEK1, CHEK2, FASN, GADD45A 6

459

cancer cell lines Cell Cycle G2 phase G2 phase of breast cancer cell 1.86E-02 BRCA1, CDC25C, CDK1, CHEK1, MSH2, RBBP8, RBCK1, 9 lines SPHK2, WEE1 Cell Cycle G2 phase arrest in G2 phase of breast 2.15E-02 BRCA1, CDC25C, CDK1, CHEK1, RBCK1, SPHK2, WEE1 7 cancer cell lines Cell Cycle G2 phase arrest in G2 phase of mammary 3.21E-02 CDKN1A, CHEK1 2 epithelial cells Cell Cycle prometaphase prometaphase 4.19E-05 BIRC5, BUB1B, CCDC99, CCNE1, CDC42, ECT2, KIFC1, 10 MAD2L1, NUF2, ZW10 Cell Cycle prometaphase delay in initiation of 1.84E-04 BIRC5, BUB1B, CDC42, ECT2, MAD2L1 5 prometaphase Cell Cycle prometaphase prometaphase of tumor cell 3.58E-03 CCDC99, CCNE1, CDC42, ECT2, NUF2, ZW10 6 lines Cell Cycle prometaphase prometaphase of cervical 6.38E-03 CCDC99, CDC42, ECT2, NUF2, ZW10 5 cancer cell lines Cell Cycle prometaphase arrest in prometaphase 1.22E-02 CCDC99, CCNE1, MAD2L1, NUF2, ZW10 5 Cell Cycle prometaphase arrest in prometaphase of tumor 2.28E-02 CCDC99, CCNE1, NUF2, ZW10 4 cell lines Cell Cycle cycling cycling of centrosome 6.20E-05 BRCA2, CDK1, CDK2, CENPJ, CEP135, CEP192, CUL1, 15 MAD2L1, NEDD1, NEK2, PLK1, PLK4, SASS6, TTK, TUBE1 Cell Cycle S phase S phase 1.17E-04 BRCA1, CAMK2N1, CCNA2, CCNE1, CDC25A, CDC25C, 49 CDC7 (includes EG:12545), CDH1, CDK1, CDK2AP1, CDKN1A, CHEK1, CHEK2, CRLF3, E2F5, FANCD2, FBXO5, FOXM1, FOXO3, HBEGF, ID1, ID3 (includes EG:15903), ITGB1, LGALS1, MCM10 (includes EG:307126), MCM7, MCMBP, MSLN, MXD4, MYBL1, NAE1, NCOA3, PBX1, PNPT1, POLA1, POLD1, PTOV1, RAF1, RCC1, RGCC, RPA1, SHC1 (includes EG:20416), SKP2 (includes EG:27401), SLBP, SOX9, TGFA, TIMELESS, TIPIN, TYMS Cell Cycle S phase S phase of tumor cell lines 5.69E-03 CAMK2N1, CCNA2, CCNE1, CDC25A, CDC25C, CDC7 27 (includes EG:12545), CDH1, CDK1, CDKN1A, CHEK1, E2F5, FOXM1, FOXO3, ITGB1, MCM10 (includes EG:307126), MSLN, MXD4, NAE1, PBX1, POLA1, RAF1, RPA1, SHC1 (includes EG:20416), SKP2 (includes EG:27401), SOX9, TGFA, TYMS Cell Cycle S phase S phase of fibroblasts 1.22E-02 CDC7 (includes EG:12545), CDKN1A, CHEK2, ID3 (includes 5 EG:15903), SKP2 (includes EG:27401)

460

Cell Cycle S phase S phase of colon cancer cell 1.49E-02 CAMK2N1, CDH1, CDKN1A, FOXO3, MXD4, NAE1, TGFA 7 lines Cell Cycle polyploidization polyploidization of cells 4.26E-04 AURKB, BIRC5, BUB1B, CDKN1A, STMN1, TOP2A, TPX2 7 Cell Cycle polyploidization polyploidization of tumor cell 1.14E-02 AURKB, BUB1B, STMN1, TOP2A 4 lines Cell Cycle G2/M phase G2/M phase transition 4.36E-04 ANAPC10, ANAPC4, ATF5, BIRC5, BLM, CCNA2, CCNB1, 22 transition CDC25C, CDK1, CDK2, CDKN1A, CDKN2B, CHEK2, DPP4, ELAC2, FOXM1, GADD45A, LGALS1, MAD2L1, MELK, PLAU, PLK1 Cell Cycle G2/M phase arrest in G2/M phase transition 1.99E-02 CCNB1, CDC25C, GADD45A 3 transition of fibroblasts Cell Cycle G2/M phase arrest in G2/M phase transition 2.95E-02 ATF5, BLM, CCNA2, CCNB1, CDC25C, CDK1, CDKN1A, 12 transition DPP4, GADD45A, LGALS1, MAD2L1, PLAU Cell Cycle ploidy ploidy 7.34E-04 AURKA, AURKB, BIRC5, BUB1B, CDK8, CDKN1A, CEBPD, 22 CENPH, CKAP2, CLK1, EIF2AK3, HRAS, JAK1 (includes EG:16451), MAD2L1, NDRG1, NEK2, NME1 (includes EG:18102), PLK1, STMN1, TACC3, TOP2A, TPX2 Cell Cycle ploidy ploidy of colon cancer cell lines 1.03E-03 BIRC5, CENPH, CKAP2, MAD2L1 4 Cell Cycle ploidy ploidy of cells 5.83E-03 AURKA, AURKB, BIRC5, CDK8, CDKN1A, CEBPD, CENPH, 18 CKAP2, CLK1, EIF2AK3, HRAS, JAK1 (includes EG:16451), MAD2L1, NDRG1, NEK2, NME1 (includes EG:18102), PLK1, TACC3 Cell Cycle ploidy ploidy of tumor cell lines 1.22E-02 BIRC5, CDKN1A, CEBPD, CENPH, CKAP2, MAD2L1, 8 NDRG1, TACC3 Cell Cycle mitotic exit mitotic exit 9.42E-04 BIRC5, CDC20 (includes EG:107995), MAD2L1, UBE2C, WEE1 5 Cell Cycle senescence senescence of cells 1.35E-03 ACLY, BLM, BMP4, BRCA1, BUB1 (includes EG:100307076), 28 CAT, CAV1, CDKN1A, CDKN2B, CENPA, CHEK2, CSNK2A1, DEK, EHF, FASN, HJURP, HRAS, HSPA1A/HSPA1B, ID1, NUDT1, PARP1, PSMB5, RAF1, RUNX1, SREBF1 (includes EG:176574), TSC22D1, UQCRFS1, VEGFA Cell Cycle senescence senescence of liver cell lines 5.75E-03 ACLY, FASN, SREBF1 (includes EG:176574) 3 Cell Cycle senescence senescence of tumor cell lines 2.45E-02 BMP4, CDKN1A, CDKN2B, CHEK2, DEK, EHF, HJURP, 11 HSPA1A/HSPA1B, ID1, PARP1, RAF1 Cell Cycle spindle checkpoint spindle checkpoint of cells 1.92E-03 AXIN2, BIRC5, BUB1 (includes EG:100307076), CDK5RAP2, 9 DLGAP5, ERCC6L, PLK1, PRPF4, TTK

461

Cell Cycle spindle checkpoint spindle checkpoint of tumor 6.38E-03 AXIN2, DLGAP5, ERCC6L, PLK1, PRPF4 5 cell lines Cell Cycle G1 phase G1 phase 1.99E-03 ACIN1, ACVR1, BCL3, BIRC5, BLM, BRCA1, CASP3, 65 CCNE1, CCNE2, CDC23, CDC25A, CDC25C, CDC42, CDCA5, CDK2, CDK5RAP3, CDK6, CDKN1A, CDKN2B, CDKN3, CEBPD, CHEK2, COPS5, CREG1, DDIT3, EZH2, FAM188A, FASN, FBXO5, FOXM1, FOXO4, GADD45A, GDF15, GPN3, GSPT1, HES1 (includes EG:15205), HOXA10, HRAS, ID2, ILKAP, ING2, ITGA5, ITGAV, ITGB1, KLF4, KLF6, LAS1L, LGALS1, LGALS3, MTBP, NASP, NCOA3, PNPT1, PRNP, PTGES3, RAF1, RCC1, SKP2 (includes EG:27401), TFDP1, TFRC, TIMP2 (includes EG:21858), TOPBP1, TUSC2, TYMS, YWHAQ Cell Cycle G1 phase arrest in G1 phase of colon 3.28E-03 BRCA1, CDC42, CDKN1A, FASN, GSPT1, ITGA5, KLF4, 11 cancer cell lines LAS1L, LGALS1, TOPBP1, TYMS Cell Cycle G1 phase G1 phase of colon cancer cell 3.78E-03 BRCA1, CDC42, CDKN1A, FASN, GSPT1, ITGA5, KLF4, 12 lines LAS1L, LGALS1, TOPBP1, TYMS, YWHAQ Cell Cycle G1 phase arrest in G1 phase 3.94E-03 ACIN1, BIRC5, BLM, BRCA1, CASP3, CCNE1, CDC25A, 43 CDC42, CDK2, CDK5RAP3, CDKN1A, CDKN2B, CHEK2, DDIT3, EZH2, FAM188A, FASN, FBXO5, FOXM1, GDF15, GPN3, GSPT1, HES1 (includes EG:15205), HOXA10, ING2, ITGA5, ITGAV, ITGB1, KLF4, KLF6, LAS1L, LGALS1, LGALS3, MTBP, PNPT1, PTGES3, RAF1, SKP2 (includes EG:27401), TFRC, TIMP2 (includes EG:21858), TOPBP1, TUSC2, TYMS Cell Cycle G1 phase arrest in G1 phase of tumor cell 1.77E-02 ACIN1, BRCA1, CASP3, CCNE1, CDC25A, CDC42, CDK2, 32 lines CDK5RAP3, CDKN1A, CDKN2B, DDIT3, EZH2, FAM188A, FASN, FOXM1, GDF15, GSPT1, HES1 (includes EG:15205), HOXA10, ITGA5, ITGAV, ITGB1, KLF4, LAS1L, LGALS1, LGALS3, MTBP, PTGES3, RAF1, TOPBP1, TUSC2, TYMS Cell Cycle G1 phase G1 phase of tumor cell lines 1.91E-02 ACIN1, BRCA1, CASP3, CCNE1, CDC25A, CDC25C, CDC42, 42 CDK2, CDK5RAP3, CDK6, CDKN1A, CDKN2B, COPS5, CREG1, DDIT3, EZH2, FAM188A, FASN, FOXM1, GDF15, GSPT1, HES1 (includes EG:15205), HOXA10, ILKAP, ITGA5, ITGAV, ITGB1, KLF4, LAS1L, LGALS1, LGALS3, MTBP, NASP, NCOA3, PRNP, PTGES3, RAF1, SKP2 (includes EG:27401), TOPBP1, TUSC2, TYMS, YWHAQ Cell Cycle cytokinesis cytokinesis 2.66E-03 ANLN, AURKA, BIRC5, BRCA2, CCNB1, CDC20 (includes 29 EG:107995), CDKN1A, CENPV, CEP55, DIAPH3, ECT2, INCENP, KIF14, KIF20A, KIF20B, KIF23, KIF4A, KIFC1,

462

MYH14, NME1 (includes EG:18102), NUSAP1, PLK1, PPP1CC, PRC1 (includes EG:233406), RACGAP1, RHOC, SEPT4, STX16, TOP2A Cell Cycle homologous homologous recombination of 3.45E-03 BLM, BRCA1, BRCA2, CDK2, DMC1, HJURP, MMS22L, 8 recombination DNA RAD51C Cell Cycle homologous homologous recombination 1.10E-02 BLM, BRCA1, BRCA2, CDK2, DMC1, HJURP, MMS22L, 10 recombination RAD21, RAD51C, RAD54B Cell Cycle morphology morphology of chromosomes 5.75E-03 NCAPG, NCAPG2, SMC2 3 Cell Cycle sister chromatid sister chromatid exchange of 5.75E-03 BLM, RMI1 (includes EG:306734), SERTAD1 3 exchange tumor cell lines Cell Cycle metaphase metaphase 6.68E-03 CDC20 (includes EG:107995), CDCA8, CENPE, FBXO5, PTTG1, 6 RPS6KA2 Cell Cycle metaphase arrest in metaphase of 3.21E-02 FBXO5, RPS6KA2 2 embryonic cell lines Cell Cycle metaphase arrest in metaphase of epithelial 3.21E-02 FBXO5, RPS6KA2 2 cell lines Cell Cycle metaphase arrest in metaphase of kidney 3.21E-02 FBXO5, RPS6KA2 2 cell lines Cell Cycle organization organization of chromosomes 9.89E-03 BRCA1, CDCA8, CENPH, CENPV, EZH2, KIF4A, SUV39H1 7 Cell Cycle anaphase anaphase 1.14E-02 ANAPC10, ANAPC4, MAD2L1, NCAPD2, SKA1, TOP2A 6 Cell Cycle premature premature senescence of tumor 1.22E-02 BMP4, CDKN1A, DEK, EHF, ID1 5 senescence cell lines Cell Cycle replication replication of centriole 1.79E-02 CENPJ, CEP135, CUL1, NEDD1, PLK4, SASS6 6 Cell Cycle S phase checkpoint S phase checkpoint control 2.09E-02 CDC25A, CHEK1, FANCD2, MCM7, TIPIN 5 control Cell Cycle contact growth contact growth inhibition of 2.09E-02 BIRC5, CD24, CDH1, FOXO3, PPP1R15A 5 inhibition colon cancer cell lines Cell Cycle G1/S phase transition G1/S phase 2.65E-02 ACVR1, BIRC5, BLM, CCNE1, CCNE2, CDC25A, CDC42, 31 CDCA5, CDK6, CDKN1A, CDKN2B, CDKN3, CHEK2, COPS5, CREG1, FOXM1, GADD45A, GSPT1, HES1 (includes EG:15205), HRAS, ID2, ITGAV, ITGB1, KLF4, NASP, NCOA3, PRNP, RCC1, TFDP1, TOPBP1, YWHAQ Cell Cycle G0 phase G0 phase 2.82E-02 BMP4, CCNE1, CDKN1A, CEBPD, CHEK1, FOXO3, HRAS, 13 IL15 (includes EG:16168), PLAUR, RXRA, SOX9, STAT1, VHL Cell Cycle DNA replication DNA replication checkpoint of 3.21E-02 CENPE, CHEK1 2

463

checkpoint cells Cellular Assembly and segregation segregation of chromosomes 1.27E-15 AURKB, BRCA1, BUB1 (includes EG:100307076), CCNA2, 40 Organization CCNB1, CCNB2, CDC42, CDK5RAP2, CENPF, CENPW, CHMP2A, DSN1 (includes EG:100002916), ECT2, ESPL1, HDAC4, HJURP, INCENP, KIF2C, NCAPD2, NCAPD3, NCAPG, NCAPG2, NCAPH, NDC80, NDEL1, NEK2, NUF2, NUSAP1, RCC1, RIOK3, SGOL1, SKA1, SKA3, SMC2, SMC4, SPC25 (includes EG:100144563), SRPK1, TOP2A, ZW10, ZWINT Cellular Assembly and segregation segregation of sister chromatids 2.62E-04 CCNA2, ESPL1, NCAPD2, NDC80, NUSAP1, SMC4, ZW10, 8 Organization ZWINT Cellular Assembly and alignment alignment of chromosomes 2.30E-11 AURKA, BIRC5, CCNA2, CENPE, DLGAP5, KIF14, KIF18A, 17 Organization KIF22, KIF2C, KIFC1, NCAPD2, NCAPD3, NCAPG, NCAPG2, SGOL1, SMC4, TTK Cellular Assembly and alignment alignment of sister chromatids 1.99E-02 NDC80, RAD21, TOP2A 3 Organization Cellular Assembly and chromosomal chromosomal congression of 1.06E-06 CENPE, KIF14, KIF18A, KIF22, KIF2C, KIFC1, NDC80, 8 Organization congression chromosomes SGOL2 Cellular Assembly and chromosomal chromosomal congression of 1.84E-04 CDC23, CDCA5, CENPE, CENPF, SEH1L 5 Organization congression metaphase plate Cellular Assembly and organization organization of nucleus 1.23E-06 BORA, BRCA1, CDCA8, CENPH, CENPV, CHAF1A, CKAP5, 22 Organization EZH2, KIF11, KIF2A, KIF4A, NDC80, PLK1S1, PSRC1, RAN, RCC1, SEH1L, SPC25 (includes EG:100144563), STMN1, SUV39H1, TPX2, TTK Cellular Assembly and organization organization of organelle 3.21E-05 A4GALT, ABCA1, ARHGEF2, ATL2, ATL3, BCS1L, BLZF1, 59 Organization BORA, BRCA1, CAV1, CAV2, CDCA8, CDK5RAP2, CENPH, CENPV, CHAF1A, CKAP5, CST3, DNAJB6, EPS15, EZH2, FANCG, GAA, HAUS1, HAUS6, HAUS7, HAUS8, HOOK2, ITGB4, KIF11, KIF2A, KIF4A, NAGPA, NDC80, OPTN, PEX6 (includes EG:117265), PLK1, PLK1S1, PRC1 (includes EG:233406), PRDX3, PRNP, PSRC1, RAB6A, RAN, RCC1, RHOF, RRBP1, SEC23IP, SEH1L, SERP1, SPC25 (includes EG:100144563), STMN1, SURF4, SUV39H1, TMED2, TMED9, TPX2, TTK, UXT Cellular Assembly and organization organization of mitotic spindle 3.62E-05 BORA, CKAP5, KIF11, KIF2A, NDC80, PLK1S1, PSRC1, 13 Organization RAN, RCC1, SPC25 (includes EG:100144563), STMN1, TPX2, TTK Cellular Assembly and organization organization of Golgi apparatus 4.71E-03 ATL2, ATL3, BLZF1, OPTN, RAB6A, SEC23IP, SURF4, 9

464

Organization TMED2, TMED9 Cellular Assembly and organization organization of chromosomes 9.89E-03 BRCA1, CDCA8, CENPH, CENPV, EZH2, KIF4A, SUV39H1 7 Organization Cellular Assembly and organization organization of centrosome 1.72E-02 CDK5RAP2, CKAP5, HAUS1, HAUS6, HAUS7, HAUS8, 8 Organization PLK1, UXT Cellular Assembly and formation formation of spindle fibers 2.15E-06 BIRC5, CEP192, CKAP2, CKAP5, HAUS1, HAUS6, HAUS7, 21 Organization HAUS8, KIF11, KIF2A, KIF2C, KIF4A, KIFC1, NDRG1, NEDD1, NEK2, NUF2, PLK1, RCC1, SASS6, TPX2 Cellular Assembly and formation formation of mitotic spindle 3.75E-06 BIRC5, CEP192, CKAP2, CKAP5, HAUS1, HAUS6, HAUS7, 20 Organization HAUS8, KIF11, KIF2A, KIF2C, KIF4A, KIFC1, NEDD1, NEK2, NUF2, PLK1, RCC1, SASS6, TPX2 Cellular Assembly and formation formation of cellular inclusion 2.26E-03 ATXN1, BSCL2, DNAJA1, DNAJB2, HSF1 (includes EG:15499), 11 Organization bodies HSPA1A/HSPA1B, HSPA4, MAPT, PSMC4, SACS, SQSTM1 Cellular Assembly and formation formation of nucleus 3.45E-03 CDK1, CDKN1A, ESPL1, LBR (includes EG:368360), LMNB1, 8 Organization NUP107, SERTAD1, TMEM48 Cellular Assembly and formation formation of centriole 6.23E-03 CENPJ, CEP135, CUL1, MAD2L1, NEDD1, PLK4, SASS6 7 Organization Cellular Assembly and formation formation of nuclear foci 6.68E-03 BLM, BRCA1, CHEK1, GMNN, MMS22L, TERF2 6 Organization Cellular Assembly and formation formation of chromosome 8.00E-03 BRCA1, CENPA, CENPV, CHAF1A, EZH2, HIRIP3, LEPREL4, 10 Organization components MTA2, SUV39H1, TERF2 Cellular Assembly and formation formation of chromatin 2.99E-02 BRCA1, CENPV, CHAF1A, EZH2, HIRIP3, MTA2, SUV39H1 7 Organization Cellular Assembly and attachment attachment of spindle fibers 3.98E-05 AURKB, BUB1B, BUB3 (includes EG:12237), CASC5, NDC80, 7 Organization NUF2, SGOL1 Cellular Assembly and attachment attachment of chromosomes 3.21E-02 AURKB, BUB1B 2 Organization Cellular Assembly and binding binding of chromosome 1.72E-03 CCNE1, CDC7 (includes EG:12545), DNMT1, GMNN, HMGN2, 10 Organization components LBR (includes EG:368360), PRMT1, RPA1, SUV39H1, ZW10 Cellular Assembly and binding binding of chromatin 2.04E-03 CCNE1, CDC7 (includes EG:12545), DNMT1, GMNN, HMGN2, 8 Organization LBR (includes EG:368360), RPA1, SUV39H1 Cellular Assembly and binding binding of chromosomes 3.21E-02 KIF22, RAN 2 Organization Cellular Assembly and quantity quantity of centrosome 3.95E-03 AURKA, CDKN1A, CHMP1B, CHMP2A, CHMP4C, KIF23, 10 Organization PLK1, SKP2 (includes EG:27401), TACC3, TPP2

465

Cellular Assembly and quantity quantity of intercellular 5.75E-03 CDH1, GDF15, TJP1 (includes EG:21872) 3 Organization junctions Cellular Assembly and quantity quantity of chromosome 2.28E-02 BRCA2, MAD2L1, MMS22L, SGOL1 4 Organization components Cellular Assembly and dissociation dissociation of centrosome 5.75E-03 CKAP2, NEK2, PLK1 3 Organization Cellular Assembly and structure structure of kinetochores 5.75E-03 NCAPD2, NCAPG2, SMC2 3 Organization Cellular Assembly and missegregation missegregation of 1.22E-02 AURKB, CCNE1, CENPA, ERCC6L, KIF4A 5 Organization chromosomes Cellular Assembly and cohesion cohesion of sister chromatids 1.49E-02 CDCA5, CHEK1, ESCO2, NAA50, PDS5B, PLK1, SMC3 7 Organization Cellular Assembly and nucleation nucleation of microtubules 1.79E-02 BIRC5, CENPJ, HAUS8, MAPT, NDEL1, NEDD1 6 Organization Cellular Assembly and replication replication of centriole 1.79E-02 CENPJ, CEP135, CUL1, NEDD1, PLK4, SASS6 6 Organization Cellular Assembly and density density of microtubules 1.99E-02 CKAP5, KIF2C, STMN1 3 Organization Cellular Assembly and elongation elongation of mitotic spindle 1.99E-02 KIF23, PRC1 (includes EG:233406), SPC25 (includes 3 Organization EG:100144563) Cellular Assembly and permeability permeability of mitochondria 1.99E-02 ALOX5, CAV1, HK1 3 Organization Cellular Assembly and separation separation of sister chromatids 1.99E-02 FEN1, PTTG1, SGOL1 3 Organization Cellular Assembly and amplification amplification of centrosome 2.15E-02 AURKA, BRCA1, CCNE1, CDC25C, KLF4, NEK2, PLK1 7 Organization Cellular Assembly and retraction retraction of neurites 2.28E-02 GNA12, LIF, PITPNM1, ROCK1 4 Organization Cellular Assembly and assembly assembly of olfactory cilia 3.21E-02 BBS1, BBS10 2 Organization Cellular Assembly and budding budding of vesicles 3.21E-02 VPS37B, VPS37C 2 Organization DNA Replication, segregation segregation of chromosomes 1.27E-15 AURKB, BRCA1, BUB1 (includes EG:100307076), CCNA2, 40 Recombination, and CCNB1, CCNB2, CDC42, CDK5RAP2, CENPF, CENPW, Repair CHMP2A, DSN1 (includes EG:100002916), ECT2, ESPL1,

466

HDAC4, HJURP, INCENP, KIF2C, NCAPD2, NCAPD3, NCAPG, NCAPG2, NCAPH, NDC80, NDEL1, NEK2, NUF2, NUSAP1, RCC1, RIOK3, SGOL1, SKA1, SKA3, SMC2, SMC4, SPC25 (includes EG:100144563), SRPK1, TOP2A, ZW10, ZWINT DNA Replication, segregation segregation of sister chromatids 2.62E-04 CCNA2, ESPL1, NCAPD2, NDC80, NUSAP1, SMC4, ZW10, 8 Recombination, and ZWINT Repair DNA Replication, alignment alignment of chromosomes 2.30E-11 AURKA, BIRC5, CCNA2, CENPE, DLGAP5, KIF14, KIF18A, 17 Recombination, and KIF22, KIF2C, KIFC1, NCAPD2, NCAPD3, NCAPG, NCAPG2, Repair SGOL1, SMC4, TTK DNA Replication, alignment alignment of sister chromatids 1.99E-02 NDC80, RAD21, TOP2A 3 Recombination, and Repair DNA Replication, metabolism metabolism of DNA 1.44E-08 ACIN1, ADM, AIFM1, ALOX5, BCL2L1, BIK, BIRC5, BNIP3, 82 Recombination, and BRCA1, BSG (includes EG:12215), CASP3, CCNA2, CCNE1, Repair CDC25A, CDC7 (includes EG:12545), CDK2, CDK2AP1, CDKN1A, CHEK1, CUL1, CUL4B, DHX9, DNASE1L1, DNASE2, DSCC1, DUT, ESCO2, FOXM1, GMNN, GRN, HRAS, HUWE1, IL8, ISG20, JUN, KEAP1, KIAA0101, KPNA2, KRT7, LEP, LIG1, MCM2, NAE1, NAP1L1, NOL8, NT5E, ORC1 (includes EG:18392), ORC5 (includes EG:26429), ORC6 (includes EG:23594), PARP1, PCNA, PDGFC, PLK1, POLA1, POLD1, POLD4, POLE2, POLE3, POLG2, PTMS, RAN, RECQL4, RFC1, RFC3, RPA1, S100A4, SET, SMC3, SOD2, SSBP1, STAT1, TFAM, THOC1, TIAM1, TIMELESS, TIPIN, TMPO, TOPBP1, TYMP, UBQLN1, UPF1, XRCC6 DNA Replication, repair repair of DNA 2.31E-08 ALKBH1, ALKBH2, APTX, BABAM1, BCL2L1, BLM, 65 Recombination, and BRCA1, BRCA2, BRCC3, C9orf80, CDCA5, CDK2, CDKN1A, Repair CHEK1, CHEK2, DDX1, DEK, ERCC1, ERCC8, EXO1 (includes EG:26909), EYA3, FAM175A, FBXO6, FEN1, GADD45A, HMGB1, HMGB2, HUWE1, KPNA2, LIG1, MSH2, MSH6, NAE1, NR4A2, OBFC2A, OBFC2B, PARP1, PARP2, PARP3, PCNA, POLA1, POLD1, PRKDC, PRMT6, PRPF19, RAD21, RAD23B, RAD51C, RAD54B, RAD54L, RBBP8, RECQL4, RNF8, RPA1, RRM1, SETMAR, SOD2, TERF2, THOC1, TRIM28, UPF1, USP1, XPC, XRCC2, XRCC6 DNA Replication, replication DNA replication 6.87E-08 BRCA1, CCNA2, CCNE1, CDC25A, CDC7 (includes EG:12545), 53 Recombination, and CDK2, CDK2AP1, CDKN1A, CHEK1, CUL1, CUL4B, DSCC1, Repair DUT, ESCO2, FOXM1, GMNN, HRAS, HUWE1, IL8,

467

KIAA0101, KRT7, LEP, MCM2, NAE1, NAP1L1, NOL8, ORC1 (includes EG:18392), ORC5 (includes EG:26429), ORC6 (includes EG:23594), PCNA, PDGFC, PLK1, POLA1, POLD1, POLD4, POLE2, POLE3, POLG2, PTMS, RECQL4, RFC1, RFC3, RPA1, SET, SMC3, SSBP1, TFAM, TIMELESS, TIPIN, TMPO, TOPBP1, TYMP, UPF1 DNA Replication, replication initiation of replication of DNA 1.07E-03 CCNE1, CDC7 (includes EG:12545), CDK2AP1, CHEK1, MCM2, 10 Recombination, and ORC1 (includes EG:18392), ORC5 (includes EG:26429), POLA1, Repair TIMELESS, TIPIN DNA Replication, replication replication of centriole 1.79E-02 CENPJ, CEP135, CUL1, NEDD1, PLK4, SASS6 6 Recombination, and Repair DNA Replication, checkpoint control checkpoint control 1.44E-07 BUB1 (includes EG:100307076), BUB1B, CCNE2, CDC20 24 Recombination, and (includes EG:107995), CDC25A, CDC25C, CDC7 (includes Repair EG:12545), CDK10, CDK2, CHEK1, CHEK2, FANCD2, FANCG, KNTC1, MAD2L1, MCM7, NDC80, RBBP8, TIPIN, XPC, ZAK, ZW10, ZWILCH, ZWINT DNA Replication, checkpoint control checkpoint control of 3.21E-02 CHEK1, CHEK2 2 Recombination, and lymphoma cell lines Repair DNA Replication, checkpoint control checkpoint control of mitotic 3.21E-02 CDC20 (includes EG:107995), NDC80 2 Recombination, and spindle Repair DNA Replication, chromosomal chromosomal congression of 1.06E-06 CENPE, KIF14, KIF18A, KIF22, KIF2C, KIFC1, NDC80, 8 Recombination, and congression chromosomes SGOL2 Repair DNA Replication, double-stranded double-stranded DNA break 1.13E-06 APTX, BABAM1, BLM, BRCA1, BRCA2, BRCC3, CDCA5, 27 Recombination, and DNA break repair repair CHEK2, DDX1, EYA3, FAM175A, FEN1, KPNA2, NAE1, Repair NR4A2, OBFC2A, PARP1, PCNA, PRKDC, PRPF19, RAD21, RBBP8, RNF8, RPA1, SETMAR, TERF2, XRCC6 DNA Replication, double-stranded double-stranded DNA break 7.36E-04 BLM, BRCA1, BRCA2, KPNA2, NAE1, NR4A2, PARP1, 13 Recombination, and DNA break repair repair of cells PCNA, PRKDC, RBBP8, RPA1, TERF2, XRCC6 Repair DNA Replication, formation formation of spindle fibers 2.15E-06 BIRC5, CEP192, CKAP2, CKAP5, HAUS1, HAUS6, HAUS7, 21 Recombination, and HAUS8, KIF11, KIF2A, KIF2C, KIF4A, KIFC1, NDRG1, Repair NEDD1, NEK2, NUF2, PLK1, RCC1, SASS6, TPX2 DNA Replication, formation formation of mitotic spindle 3.75E-06 BIRC5, CEP192, CKAP2, CKAP5, HAUS1, HAUS6, HAUS7, 20 Recombination, and HAUS8, KIF11, KIF2A, KIF2C, KIF4A, KIFC1, NEDD1,

468

Repair NEK2, NUF2, PLK1, RCC1, SASS6, TPX2 DNA Replication, formation formation of centriole 6.23E-03 CENPJ, CEP135, CUL1, MAD2L1, NEDD1, PLK4, SASS6 7 Recombination, and Repair DNA Replication, formation formation of nuclear foci 6.68E-03 BLM, BRCA1, CHEK1, GMNN, MMS22L, TERF2 6 Recombination, and Repair DNA Replication, formation formation of chromosome 8.00E-03 BRCA1, CENPA, CENPV, CHAF1A, EZH2, HIRIP3, LEPREL4, 10 Recombination, and components MTA2, SUV39H1, TERF2 Repair DNA Replication, formation formation of chromatin 2.99E-02 BRCA1, CENPV, CHAF1A, EZH2, HIRIP3, MTA2, SUV39H1 7 Recombination, and Repair DNA Replication, condensation condensation of chromosomes 2.71E-06 ACIN1, CDCA5, KIF4A, MAD2L1, NCAPD2, NCAPD3, 12 Recombination, and NCAPG, NCAPH, NUSAP1, SMC2, SMC4, TOP2A Repair DNA Replication, recombination DNA recombination 3.49E-06 BLM, BRCA1, BRCA2, CDK2, DMC1, ERCC1, EXO1 (includes 21 Recombination, and EG:26909), HJURP, KPNA2, MMS22L, MSH2, MSH6, PRKDC, Repair RAD21, RAD51C, RAD54B, RAD54L, RMI1 (includes EG:306734), SERTAD1, SETMAR, TNPO3 DNA Replication, recombination recombination 3.62E-05 BLM, BRCA1, BRCA2, CDK2, DMC1, ERCC1, EXO1 (includes 22 Recombination, and EG:26909), HJURP, HMGB1, KPNA2, MMS22L, MSH2, Repair MSH6, PRKDC, RAD21, RAD51C, RAD54B, RAD54L, RMI1 (includes EG:306734), SERTAD1, SETMAR, TNPO3 DNA Replication, organization organization of mitotic spindle 3.62E-05 BORA, CKAP5, KIF11, KIF2A, NDC80, PLK1S1, PSRC1, 13 Recombination, and RAN, RCC1, SPC25 (includes EG:100144563), STMN1, TPX2, Repair TTK DNA Replication, organization organization of chromosomes 9.89E-03 BRCA1, CDCA8, CENPH, CENPV, EZH2, KIF4A, SUV39H1 7 Recombination, and Repair DNA Replication, spindle checkpoint spindle checkpoint of cells 1.92E-03 AXIN2, BIRC5, BUB1 (includes EG:100307076), CDK5RAP2, 9 Recombination, and DLGAP5, ERCC6L, PLK1, PRPF4, TTK Repair DNA Replication, spindle checkpoint spindle checkpoint of tumor 6.38E-03 AXIN2, DLGAP5, ERCC6L, PLK1, PRPF4 5 Recombination, and cell lines Repair DNA Replication, catabolism catabolism of ATP 2.32E-03 ABCA2, ABCD3, ACLY, ATP5D, ATP5F1, BLM, HSP90AA1, 18 Recombination, and KIF20B, MSH2, MSH6, PEX6 (includes EG:117265), PSMC1,

469

Repair PSMC2, PSMC4, PSMC5, PSMC6, RFC3, RHOBTB3 DNA Replication, cleavage cleavage of DNA 3.07E-03 FEN1, FTH1 (includes EG:14319), MBD4, N4BP2, NME1 9 Recombination, and (includes EG:18102), NME2, PCNA, RAF1, TOP2A Repair DNA Replication, homologous homologous recombination of 3.45E-03 BLM, BRCA1, BRCA2, CDK2, DMC1, HJURP, MMS22L, 8 Recombination, and recombination DNA RAD51C Repair DNA Replication, homologous homologous recombination 1.10E-02 BLM, BRCA1, BRCA2, CDK2, DMC1, HJURP, MMS22L, 10 Recombination, and recombination RAD21, RAD51C, RAD54B Repair DNA Replication, quantity quantity of centrosome 3.95E-03 AURKA, CDKN1A, CHMP1B, CHMP2A, CHMP4C, KIF23, 10 Recombination, and PLK1, SKP2 (includes EG:27401), TACC3, TPP2 Repair DNA Replication, damage DNA damage 4.13E-03 APTX, BIRC5, BRCA1, CASP3, CAT, CENPE, DCLRE1A, 23 Recombination, and ERCC1, GMNN, LIG1, MCM10 (includes EG:307126), NQO1, Repair PARP1, PBK, PLK1, PRKDC, RPA1, RRM2, RUNX1, SMOX, SOD2, SQSTM1, TOP2A DNA Replication, excision repair excision repair 4.69E-03 APTX, BRCA2, CDKN1A, ERCC1, ERCC8, EXO1 (includes 16 Recombination, and EG:26909), FEN1, HMGB1, HMGB2, HUWE1, PCNA, PRMT6, Repair RAD23B, RPA1, THOC1, XPC DNA Replication, morphology morphology of chromosomes 5.75E-03 NCAPG, NCAPG2, SMC2 3 Recombination, and Repair DNA Replication, sister chromatid sister chromatid exchange of 5.75E-03 BLM, RMI1 (includes EG:306734), SERTAD1 3 Recombination, and exchange tumor cell lines Repair DNA Replication, synthesis synthesis of DNA 9.56E-03 ADRM1, BIRC5, BMP4, BRCA2, CAV1, CCNA2, CDC25A, 54 Recombination, and CDC25C, CDCA4, CDK2, CDK2AP1, CDK6, CDKN1A, Repair CDKN2B, CHEK1, CHEK2, DNMT1, FEN1, FOS, HBEGF, HRAS, HSPA8, IGFBP3, IL8, ITGB1, KLF2, LEP, LGALS1, LGALS3, LIF, LIG1, PCNA, PIK3CB, PLK1, POLA1, POLD1, POLG2, PPM1G, PRNP, RAF1, RECQL, RECQL4, RFC2, RFC3, RPA1, SDC4, SKP2 (includes EG:27401), SLC29A1, TGFA, TIMP2 (includes EG:21858), TIPIN, TPMT, VEGFA, YY1 DNA Replication, synthesis initiation of synthesis of DNA 1.99E-02 CCNA2, CDK2, CDK6 3 Recombination, and Repair

470

DNA Replication, breakage breakage of DNA 9.91E-03 BIRC5, BRCA1, CENPE, DCLRE1A, ERCC1, LIG1, PARP1, 9 Recombination, and SOD2, TOP2A Repair DNA Replication, re-replication re-replication of DNA 1.14E-02 CCNA2, CUL1, CUL4B, GMNN, HUWE1, NAE1 6 Recombination, and Repair DNA Replication, nicking nicking of DNA 1.14E-02 FTH1 (includes EG:14319), MBD4, N4BP2, TOP2A 4 Recombination, and Repair DNA Replication, elongation elongation of mitotic spindle 1.99E-02 KIF23, PRC1 (includes EG:233406), SPC25 (includes 3 Recombination, and EG:100144563) Repair DNA Replication, incorporation incorporation of DNA 1.99E-02 CDKN1A, RPA1, TPMT 3 Recombination, and Repair DNA Replication, S phase checkpoint S phase checkpoint control 2.09E-02 CDC25A, CHEK1, FANCD2, MCM7, TIPIN 5 Recombination, and control Repair DNA Replication, amplification amplification of centrosome 2.15E-02 AURKA, BRCA1, CCNE1, CDC25C, KLF4, NEK2, PLK1 7 Recombination, and Repair DNA Replication, DNA replication DNA replication checkpoint of 3.21E-02 CENPE, CHEK1 2 Recombination, and checkpoint cells Repair DNA Replication, chromosomal chromosomal aberration 3.21E-02 POLD4, SMC3 2 Recombination, and aberration Repair RNA Post- processing processing of RNA 6.86E-13 AARS, ADARB1, ARL6IP4, BARD1, CELF1, CLK1, CLP1 87 Transcriptional (includes EG:10978), CPSF3, CPSF6, CSTF1, CSTF2, CSTF3, Modification DDX17, DDX20, DDX39A, DDX47, EFTUD2, ESRP1, EXOSC3, EXOSC7, FARS2, GEMIN2, GEMIN4, GEMIN6, HCFC1, HEATR1, HNRNPA1, HNRNPA2B1, HNRNPD, HNRNPH1, HNRNPH3, HNRNPM, HNRNPR, HNRNPU, INTS8, IVNS1ABP, LAS1L, LSM1, LSM3 (includes EG:27258), MBNL1, MBNL2, NOL8, NOLC1, NOP56, NOP58, NUDT21, PABPC4, PES1, PHF5A, PNN, PNPT1, PPAN, PPIG, PRPF19, PRPF3, PRPF31, PRPF4, PTBP1, RBM3, RPS15, RPS24, RPS7, RSRC1, SARS, SF3A3, SF3B2, SF3B3, SF3B4, SNRNP40, SNRPA, SNRPB2, SNRPD1, SNRPF, SRPK1, SRSF1, SRSF4,

471

SYNCRIP, TBP, TCERG1, THOC1, TRA2A, TSEN2, U2AF2, USP39, UTP20, WDR12, WDR75 RNA Post- processing processing of mRNA 2.66E-07 BARD1, CPSF3, CPSF6, CSTF1, CSTF2, CSTF3, DDX39A, 41 Transcriptional EFTUD2, GEMIN2, GEMIN6, HCFC1, HNRNPA1, Modification HNRNPA2B1, HNRNPH3, HNRNPM, HNRNPR, LSM3 (includes EG:27258), NUDT21, PHF5A, PNN, PNPT1, PRPF19, PRPF3, PRPF31, PTBP1, RSRC1, SF3A3, SF3B2, SF3B3, SF3B4, SNRPA, SNRPB2, SNRPD1, SRPK1, SRSF1, SRSF4, TBP, TCERG1, TRA2A, U2AF2, USP39 RNA Post- processing processing of rRNA 3.26E-05 DDX47, EXOSC3, EXOSC7, GEMIN4, HEATR1, LAS1L, 17 Transcriptional NOL8, NOLC1, NOP56, NOP58, PES1, RPS15, RPS24, RPS7, Modification UTP20, WDR12, WDR75 RNA Post- processing processing of tRNA 1.99E-02 AARS, FARS2, SARS 3 Transcriptional Modification RNA Post- splicing splicing of RNA 1.60E-05 ARL6IP4, CELF1, CLK1, CLP1 (includes EG:10978), DDX39A, 36 Transcriptional DDX47, EFTUD2, ESRP1, GEMIN2, GEMIN6, HCFC1, Modification HNRNPH1, HNRNPH3, HNRNPM, IVNS1ABP, LSM1, MBNL1, MBNL2, PHF5A, PNN, PPAN, PPIG, PRPF19, PRPF3, PRPF31, PTBP1, RSRC1, SNRPD1, SNRPF, SRSF1, SRSF4, TCERG1, TRA2A, TSEN2, U2AF2, USP39 RNA Post- splicing splicing of mRNA 7.18E-03 DDX39A, GEMIN6, HCFC1, HNRNPH3, HNRNPM, PHF5A, 18 Transcriptional PNN, PRPF19, PRPF31, PTBP1, RSRC1, SNRPD1, SRSF1, Modification SRSF4, TCERG1, TRA2A, U2AF2, USP39 RNA Post- polyadenylation polyadenylation of mRNA 1.13E-03 BARD1, CPSF3, CSTF1, CSTF2, CSTF3, PNPT1, SNRPA, TBP 8 Transcriptional Modification Cell Death cell death cell death of tumor cell lines 3.29E-12 A4GALT, ABCE1, ADAM17, ADM, ADRM1, AHSA1, AIFM1, 321 AKAP12, ALOX5, ANXA2, APOPT1, ASNS, ATAD2, ATF3, ATF4, ATG12 (includes EG:361321), ATG5 (includes EG:11793), ATP2A2, AURKA, B2M, BACH2, BAG3, BARD1, BCL2L1, BCL3, BCL6, BCLAF1, BEX2, BIK, BIRC3, BIRC5, BLM, BMP4, BNIP3, BNIP3L, BRCA1, BRCA2, BTG1, BUB1B, C15orf63, C9orf80, CAPNS1, CASP3, CASP6, CAT, CAV1, CCNB1, CCNE1, CCT2, CD24, CD55, CD99, CDC20 (includes EG:107995), CDC25A, CDC25C, CDC42, CDC7 (includes EG:12545), CDCA2, CDCP1, CDH1, CDK1, CDK6, CDK8, CDKN1A, CEBPB (includes EG:1051), CEBPD, CHEK1, CHEK2, CKAP2, COPS5, CRABP2, CRBN, CREB1, CREB3L2, CSE1L, CSF2RA, CSNK2A1, CTCF (includes EG:10664), CTSB,

472

CYB5A (includes EG:109672), CYFIP2, DCK, DDIT3, DDIT4, DEK, DEPDC1, DHCR24, DIRAS3, DKK1, DNAJB1, DNM1L, DPP4, DSG2, DUT, EGR1, EHF, EIF2AK3, EIF4G2, EPAS1, ERN1, FAF1 (includes EG:11124), FASN, FBXO32, FLNB, FOS, FOSB, FOXO3, FTH1 (includes EG:14319), FUBP1, GADD45A, GAL, GDF15, GNAS, GPM6A, GPR37, GRN, GSN, HAUS8, HBEGF, HDAC4, HES1 (includes EG:15205), HMMR, HNRNPA1, HNRNPC, HOXA5, HRAS, HSF1 (includes EG:15499), HSP90AB1, HSPA1A/HSPA1B, HSPA4, HSPA5, HSPA8, HSPB11, HSPD1, HUWE1, ID1, ID3 (includes EG:15903), IER3, IGF2R, IGFBP3, IGFBP6, IL15 (includes EG:16168), IL18 (includes EG:16173), IL32, IL8, ILK, ING2, IRF1 (includes EG:16362), ITGAV, ITGB1, ITGB3BP, ITGB4, JAK1 (includes EG:16451), JUN, JUND, JUP, KIF14, KLF2, KLF4, KLF5, KLF6, KLF9, LAMP2, LCN2, LEP, LETM1, LGALS1, LGALS3, LGALS3BP, LIG1, LMNB1, LMO2, LSMD1, LTBR, MAD2L1, MAP2K4, MAP3K7 (includes EG:172842), MAPK1, MAPK3, MAPKAP1, MBP, MCM10 (includes EG:307126), MDC1, MELK, MMS22L, MOAP1, MSH2, MSRB2, MST1R, MSX1, MT2A, MTA2, MYB, MZF1, NCL, NCOA3, NDRG1, NEK2, NFKB2, NFKBIA, NFKBIB, NFKBIZ, NME1 (includes EG:18102), NOC2L, NOS3, NQO1, NR4A2, NRAS, NRP1 (includes EG:18186), NUF2, OBFC2A, PARP1, PAWR, PBK, PCNA, PDCD4, PELP1, PHB, PIK3CB, PINK1, PKMYT1, PLAU, PLAUR, PLK1, PMAIP1, PPP1R13L, PPP1R15A, PPP2CA, PRAME, PRDM1, PRDX1, PRKCA, PRKDC, PRNP, PRPF19, PTTG1, RAC3, RAD21, RAD23B, RAD51C, RAF1, RBM17, REPS2, RHOC, RPA1, RRM1, RUNX1, S100A4, SAT1, SDC1 (includes EG:20969), SEMA3B, SHC1 (includes EG:20416), SIVA1, SKP2 (includes EG:27401), SLC25A6, SLC29A1, SLC3A2, SLPI, SMOX, SND1, SOD2, SOX9, SPC25 (includes EG:100144563), SPHK2, SQSTM1, SRPK1, SRPX, SRSF1, STAT1, STAT3, STMN1, STOML2, SYVN1, TACC3, TBK1, TCP1, TFAP2C, TFRC, TGFA, TGM2, THOC1, TIAM1, TMBIM6, TMED10, TNFRSF10B, TNFRSF18, TNFRSF21, TNFRSF6B, TNFSF14, TOP2A, TOPBP1, TPD52, TRIAP1, TRIM28, TSG101, TTF1, TTK, TUBA1A, TXN (includes EG:116484), TXNDC17, TYMP, TYMS, UBE2C, UBQLN1, UBTF, UNC5B, UQCRFS1, UXT, VEGFA, VHL, VPS28 (includes EG:300052), WEE1, WNT16, XBP1 (includes EG:140614), XPC, XPO1, XPR1, YARS, YBX1, YY1, ZNF148 Cell Death cell death cell death 3.86E-09 A4GALT, ABCA3, ABCC3, ABCC5, ABCE1, ACIN1, ACVR1, 527 ADAM17, ADM, ADRM1, AHSA1, AIFM1, AKAP12, ALDH2,

473

ALOX5, ANGPTL4, ANLN, ANTXR1, ANXA1, ANXA2, APOPT1, ASNS, ATAD2, ATF3, ATF4, ATF6, ATG12 (includes EG:361321), ATG3 (includes EG:171415), ATG5 (includes EG:11793), ATP2A2, AURKA, AURKAIP1, AURKB, AXIN2, AXL, AZI2, B2M, BACH2, BAG3, BAG4, BARD1, BCL2L1, BCL3, BCL6, BCLAF1, BEX2, BIK, BIRC3, BIRC5, BLM, BMP4, BNIP3, BNIP3L, BRCA1, BRCA2, BRCC3, BSG (includes EG:12215), BTG1, BUB1B, C15orf63, C1QBP, C9orf80, CALB2, CAMK2N1, CAPNS1, CAPRIN2, CARS, CASP3, CASP6, CAT, CAV1, CCNA2, CCNB1, CCNE1, CCT2, CD24, CD55, CD99, CDC20 (includes EG:107995), CDC25A, CDC25C, CDC42, CDC45, CDC7 (includes EG:12545), CDCA2, CDCP1, CDH1, CDK1, CDK2, CDK2AP1, CDK5RAP3, CDK6, CDK8, CDKN1A, CEBPB (includes EG:1051), CEBPD, CHEK1, CHEK2, CKAP2, CKAP5, CLDN3, COMT, COPS5, CR2, CRABP2, CRBN, CREB1, CREB3L2, CRLF1, CSE1L, CSF2RA, CSNK2A1, CST3, CTCF (includes EG:10664), CTNNBL1, CTSB, CUL4B, CXCL1, CYB5A (includes EG:109672), CYBA, CYFIP2, DCK, DDIT3, DDIT4, DDN, DDR1, DDX17, DDX47, DEK, DEPDC1, DEPTOR, DHCR24, DHRS2, DIRAS3, DKK1, DLST, DNAJB1, DNAJB2, DNM1L, DPP4, DSG2, DUSP19, DUSP5, DUSP6, DUT, EFNA1, EFNB2, EGR1, EHF, EIF2AK3, EIF3C/EIF3CL, EIF4A3, EIF4G2, EIF5A, ELF3, EPAS1, ERN1, ESPL1, F2R, F2RL1, FAF1 (includes EG:11124), FANCA, FANCD2, FANCG, FASN, FBXO32, FCAR, FGF19, FGFR3, FHL2, FLNB, FOS, FOSB, FOXM1, FOXO3, FOXO4, FTH1 (includes EG:14319), FUBP1, FXR1, GABPB1, GADD45A, GAL, GCLC, GDF15, GMNN, GNAS, GPM6A, GPR37, GRN, GSN, H6PD, HAUS1, HAUS8, HBEGF, HDAC4, HERPUD1, HES1 (includes EG:15205), HEY1, HIST1H1C, HK1, HLA-G, HMGB1, HMMR, HNRNPA1, HNRNPC, HOXA5, HRAS, HSF1 (includes EG:15499), HSP90AA1, HSP90AB1, HSPA1A/HSPA1B, HSPA4, HSPA5, HSPA8, HSPB11, HSPD1, HUWE1, HYOU1, ICAM3, ID1, ID2, ID3 (includes EG:15903), IER3, IFIH1, IGF2R, IGFBP3, IGFBP6, IL15 (includes EG:16168), IL17RD, IL18 (includes EG:16173), IL27RA, IL32, IL8, ILK, ING2, INPP1, INPP5J, IRF1 (includes EG:16362), IRF9, ISG15, ITGA2, ITGA5, ITGAV, ITGB1, ITGB3BP, ITGB4, ITM2B, ITPR3, JAK1 (includes EG:16451), JUN, JUND, JUP, KEAP1, KIAA1967, KIF11, KIF14, KIF18A, KIFC1, KIFC2, KLF11, KLF2, KLF4, KLF5, KLF6, KLF9, LAMP2, LCN2, LEP, LEPR, LETM1, LGALS1, LGALS3, LGALS3BP, LGMN, LIF, LIG1, LILRB1,

474

LMNB1, LMO2, LSMD1, LTBR, MAD2L1, MALT1, MAOB, MAP2K4, MAP3K7 (includes EG:172842), MAP4K1, MAPK1, MAPK3, MAPKAP1, MAPT, MBP, MC1R, MCM10 (includes EG:307126), MCM2, MDC1, MELK, METAP2, MGST1, MMS22L, MOAP1, MSH2, MSRB2, MST1R, MSX1, MT2A, MTA2, MVP, MYB, MZF1, NAE1, NCL, NCOA3, NCSTN, NDC80, NDEL1, NDRG1, NDUFAB1, NDUFS3, NEK2, NFIL3, NFKB2, NFKBIA, NFKBIB, NFKBIZ, NME1 (includes EG:18102), NME2, NOC2L, NOS3, NQO1, NR4A2, NRAS, NRP1 (includes EG:18186), NT5E, NUF2, NUP62, OBFC2A, ODC1, P2RX4, PARK7, PARP1, PAWR, PBK, PBX1, PCNA, PDCD4, PELP1, PHB, PHLDA1, PIK3CB, PINK1, PKMYT1, PLAU, PLAUR, PLK1, PMAIP1, PMP22, PNPT1, POR, PPAP2A, PPM1G, PPM1M, PPP1CC, PPP1R13L, PPP1R15A, PPP1R8, PPP2CA, PPP2R1B, PRAME, PRDM1, PRDX1, PRDX3, PRDX5, PRDX6, PRELID1, PRKCA, PRKDC, PRKRIR, PRNP, PRPF19, PSMA1, PSMA3, PSMA4, PSMA6, PSMC3, PSMC4, PSMC5, PSME3, PTGS1, PTP4A2, PTTG1, RAC2, RAC3, RAD21, RAD23B, RAD51C, RAF1, RASD2, RBM17, REPS2, RHOC, RIF1 (includes EG:295602), RNF130, RPA1, RPS6KA2, RPS6KA3, RRM1, RRM2, RUNX1, S100A4, SAT1, SCRIB, SDC1 (includes EG:20969), SEMA3B, SEPT4, SET, SETMAR, SFR1, SHC1 (includes EG:20416), SHFM1, SIVA1, SKP2 (includes EG:27401), SLC25A23, SLC25A6, SLC29A1, SLC3A2, SLC9A1, SLPI, SMAD6, SMOX, SNCG, SND1, SNRPA1, SOD2, SOX9, SPC25 (includes EG:100144563), SPHK2, SQSTM1, SRPK1, SRPX, SRSF1, SRXN1, STAT1, STAT2, STAT3, STK3, STMN1, STOML2, STX8, SYVN1, TACC3, TAF1B, TAF9, TBC1D9, TBK1, TCP1, TERF2, TFAP2C, TFDP1, TFRC, TGFA, TGFB1I1, TGFBR2, TGM2, THOC1, TIAL1, TIAM1, TIMP1, TIMP2 (includes EG:21858), TMBIM6, TMED10, TNFAIP1, TNFRSF10B, TNFRSF12A, TNFRSF18, TNFRSF21, TNFRSF6B, TNFSF14, TNFSF15, TOP2A, TOPBP1, TP53INP1, TPD52, TPMT, TPP2, TRIAP1, TRIB3, TRIM24, TRIM28, TSG101, TSPO, TTF1, TTK, TUBA1A, TXN (includes EG:116484), TXNDC17, TYMP, TYMS, UBA3 (includes EG:117553), UBE2C, UBQLN1, UBR4, UBTF, ULBP1, UNC5B, UNG, UQCRFS1, USP7, UTP11L, UXT, VEGFA, VHL, VPS28 (includes EG:300052), WEE1, WNT10B, WNT16, XBP1 (includes EG:140614), XPC, XPO1, XPR1, YARS, YBX1, YWHAQ, YY1, ZAK, ZFP36, ZNF148 Cell Death cell death cell death of breast cancer cell 1.35E-06 ADM, ATG5 (includes EG:11793), B2M, BAG3, BARD1, 84 BCL2L1, BEX2, BIK, BIRC3, BIRC5, BNIP3, BNIP3L,

475

lines BRCA1, CASP3, CASP6, CAV1, CDC42, CDH1, CDK1, CDKN1A, CHEK1, CRABP2, CSE1L, CTCF (includes EG:10664), CYB5A (includes EG:109672), DDIT4, DIRAS3, FASN, FBXO32, FOXO3, FTH1 (includes EG:14319), GADD45A, HOXA5, HSPA1A/HSPA1B, HSPA5, HSPD1, IER3, IGFBP3, ILK, ITGB3BP, ITGB4, JUN, KLF5, LGALS3, MAD2L1, MAPK1, MAPK3, MOAP1, MSH2, NCOA3, NFKBIA, NME1 (includes EG:18102), NOS3, NQO1, PARP1, PDCD4, PELP1, PHB, PIK3CB, PINK1, PLK1, PMAIP1, PRKCA, PRKDC, PRNP, PTTG1, SDC1 (includes EG:20969), SIVA1, SLC25A6, SLC29A1, SND1, SOD2, SPHK2, SRPK1, STAT1, STMN1, TBK1, TFAP2C, TFRC, THOC1, TNFRSF10B, TYMS, VEGFA, XBP1 (includes EG:140614) Cell Death cell death cell death of prostate cancer 1.55E-04 ABCE1, AIFM1, AKAP12, ALOX5, BCL2L1, BIRC3, BIRC5, 50 cell lines BRCA1, CASP3, CAT, CAV1, CHEK1, CREB1, DDIT3, DIRAS3, EGR1, EHF, FASN, FOS, FOXO3, GADD45A, GDF15, HBEGF, HSPA1A/HSPA1B, HSPD1, ID1, IGFBP3, ITGB1, LGALS1, MAP3K7 (includes EG:172842), NCOA3, NFKB2, PARP1, PAWR, PLAU, PLAUR, PMAIP1, PRKCA, RAC3, RAF1, SPHK2, STAT3, TGFA, TMBIM6, TMED10, TNFRSF10B, TPD52, TXN (includes EG:116484), TYMP, YY1 Cell Death cell death cell death of cervical cancer 2.08E-04 A4GALT, ADRM1, AIFM1, APOPT1, ATG5 (includes 77 cell lines EG:11793), ATP2A2, BCL2L1, BIRC5, BNIP3L, BUB1B, C15orf63, C9orf80, CASP3, CAT, CDC20 (includes EG:107995), CDC7 (includes EG:12545), CDCA2, CDK1, CDK8, CDKN1A, CHEK1, CSNK2A1, CTSB, DDIT3, EIF4G2, FLNB, FOS, GADD45A, HAUS8, HDAC4, HSF1 (includes EG:15499), HSPA1A/HSPA1B, HSPB11, IER3, IL18 (includes EG:16173), IL32, IRF1 (includes EG:16362), JAK1 (includes EG:16451), JUN, KIF14, LETM1, LSMD1, MAD2L1, MAPK1, MAPKAP1, MCM10 (includes EG:307126), MMS22L, MSX1, NFKBIZ, NME1 (includes EG:18102), NR4A2, NUF2, OBFC2A, PARP1, PAWR, PINK1, PLK1, PRAME, PRPF19, RAD51C, RAF1, RBM17, RPA1, SOD2, SPC25 (includes EG:100144563), SQSTM1, STAT1, TACC3, TBK1, TCP1, TNFRSF10B, TNFRSF21, TOP2A, TTK, TXN (includes EG:116484), TXNDC17, UBQLN1 Cell Death cell death cell death of lymphoma cell 2.79E-04 A4GALT, ADAM17, ANXA2, BACH2, BCL2L1, BIK, BIRC3, 41 lines BIRC5, CASP3, CAT, CD24, CD55, CD99, CDKN1A, CHEK2, DUT, EIF2AK3, ERN1, FTH1 (includes EG:14319), FUBP1, HNRNPA1, HNRNPC, IL15 (includes EG:16168), JAK1 (includes EG:16451), LGALS1, LGALS3, LMNB1, MAPK1, NCL,

476

NFKB2, NFKBIA, PMAIP1, RAD23B, RUNX1, SOD2, STAT3, TGM2, TIAM1, TNFRSF10B, TNFRSF6B, XBP1 (includes EG:140614) Cell Death cell death cell death of vascular 1.19E-03 ADM, ALDH2, ATF3, AXL, BCL2L1, CASP3, CAV1, DDIT3, 22 endothelial cells F2RL1, FOXO3, HSPA5, HSPD1, IL18 (includes EG:16173), IL32, IL8, KLF5, MAPK1, MAPK3, PMAIP1, TNFRSF10B, TNFSF15, VEGFA Cell Death cell death cell death of colon cancer cell 2.23E-03 ADAM17, AHSA1, ATF3, BAG3, BCL2L1, BIK, BIRC5, 62 lines BNIP3, CASP3, CASP6, CCNB1, CDC42, CDC7 (includes EG:12545), CDK1, CDKN1A, CHEK1, CHEK2, CKAP2, CSE1L, CYFIP2, DDIT3, DSG2, FASN, FOXO3, GADD45A, GDF15, GSN, HOXA5, HSF1 (includes EG:15499), HSPD1, IGF2R, IGFBP3, ITGAV, ITGB1, LEP, LGALS3, LSMD1, LTBR, MOAP1, MST1R, MYB, NDRG1, NOC2L, PARP1, PPP1R15A, RAF1, SOD2, SOX9, SPHK2, SQSTM1, SRPK1, STAT1, TGM2, TNFRSF10B, TNFRSF6B, TNFSF14, TOPBP1, TRIAP1, UBE2C, UXT, VPS28 (includes EG:300052), WEE1 Cell Death cell death cell death of ovarian cancer cell 2.66E-03 AIFM1, BARD1, BCL2L1, BIRC5, BRCA2, CASP3, CDC7 29 lines (includes EG:12545), CDKN1A, DDIT3, DIRAS3, HRAS, IL8, KLF2, KLF6, MAD2L1, MAPK1, NFKBIA, PMAIP1, PRKDC, RBM17, SLC3A2, SLPI, TBK1, TGFA, TNFRSF10B, TSG101, WEE1, XPC, YARS Cell Death cell death cell death of lung cancer cell 3.60E-03 ADAM17, AIFM1, ATAD2, ATG5 (includes EG:11793), 46 lines BCL2L1, BIRC3, BIRC5, BRCA2, CASP3, CDCP1, CDKN1A, CEBPB (includes EG:1051), CHEK1, CREB1, DDIT3, FOXO3, GADD45A, HSF1 (includes EG:15499), ID3 (includes EG:15903), IGFBP3, IGFBP6, KLF6, LCN2, LGALS3, MAP3K7 (includes EG:172842), MDC1, MTA2, NQO1, PARP1, PLK1, PPP2CA, RAF1, REPS2, RRM1, SEMA3B, SKP2 (includes EG:27401), SRSF1, STAT3, TBK1, TGM2, TNFRSF10B, TRIAP1, TTF1, UQCRFS1, XPC, YBX1 Cell Death cell death cell death of fibrosarcoma cell 3.72E-03 BCL2L1, BIRC3, BIRC5, CDKN1A, FTH1 (includes EG:14319), 17 lines ING2, NFKB2, NFKBIZ, PMAIP1, SOD2, STAT1, TBK1, TMBIM6, TNFRSF10B, TOP2A, TRIM28, YBX1 Cell Death cell death cell death of neuroblastoma cell 3.73E-03 ADM, AIFM1, ATG5 (includes EG:11793), BCL2L1, BIRC5, 34 lines CASP3, CAT, CDC42, CDKN1A, CREB3L2, DDIT3, DNAJB1, DPP4, GAL, GNAS, GPR37, HSF1 (includes EG:15499), HSPA1A/HSPA1B, IGFBP3, ITGB1, LEP, MBP, NFKBIB, PARP1, PINK1, PRNP, SQSTM1, SYVN1, TGM2, TNFRSF10B, TUBA1A, TXN (includes EG:116484), VEGFA,

477

XPR1 Cell Death cell death cell death of carcinoma cell 8.41E-03 ADAM17, AIFM1, ATG5 (includes EG:11793), AURKA, 39 lines BCL2L1, BIRC5, CASP3, CDCP1, CHEK1, COPS5, CREB1, DDIT3, FOXO3, GRN, HSF1 (includes EG:15499), HSPA5, ID3 (includes EG:15903), IGFBP3, IGFBP6, KLF2, LCN2, MAP3K7 (includes EG:172842), MDC1, NEK2, NQO1, PARP1, RAF1, REPS2, RRM1, SEMA3B, SKP2 (includes EG:27401), SRSF1, STAT3, TBK1, TGM2, TNFRSF10B, TTF1, UQCRFS1, YBX1 Cell Death cell death cell death of epithelial cell lines 2.56E-02 ATG5 (includes EG:11793), BAG4, BCL2L1, BCL6, BIK, 51 BIRC3, BIRC5, BNIP3, BNIP3L, CASP3, CAT, CDC42, CDH1, CUL4B, DDN, DDX17, DLST, ERN1, FGF19, GPR37, HK1, HSPA5, HSPD1, IER3, IGFBP3, IL17RD, IRF1 (includes EG:16362), ITGAV, ITGB1, MAP2K4, MAPT, MOAP1, NDEL1, NDUFAB1, PMAIP1, PMP22, PPP1R15A, PRDX3, PRKDC, PRNP, RAD21, SRXN1, SYVN1, TFRC, TGFB1I1, TNFRSF10B, TP53INP1, TRIB3, UNC5B, UXT, YWHAQ Cell Death necrosis necrosis 4.89E-11 A4GALT, ABCE1, ADAM17, ADM, ADRM1, AHSA1, AIFM1, 395 AKAP12, ALDH2, ALOX5, ANTXR1, ANXA1, ANXA2, APOPT1, ASNS, ATAD2, ATF3, ATF4, ATF6, ATG12 (includes EG:361321), ATG5 (includes EG:11793), ATP2A2, AURKA, AXL, B2M, BACH2, BAG3, BAG4, BARD1, BCL2L1, BCL3, BCL6, BCLAF1, BEX2, BIK, BIRC3, BIRC5, BLM, BMP4, BNIP3, BNIP3L, BRCA1, BRCA2, BTG1, BUB1B, C15orf63, C9orf80, CAPNS1, CASP3, CASP6, CAT, CAV1, CCNB1, CCNE1, CCT2, CD24, CD55, CD99, CDC20 (includes EG:107995), CDC25A, CDC25C, CDC42, CDC45, CDC7 (includes EG:12545), CDCA2, CDCP1, CDH1, CDK1, CDK2, CDK6, CDK8, CDKN1A, CEBPB (includes EG:1051), CEBPD, CHEK1, CHEK2, CKAP2, COMT, COPS5, CR2, CRABP2, CRBN, CREB1, CREB3L2, CRLF1, CSE1L, CSF2RA, CSNK2A1, CST3, CTCF (includes EG:10664), CTSB, CUL4B, CXCL1, CYB5A (includes EG:109672), CYBA, CYFIP2, DCK, DDIT3, DDIT4, DDN, DDX17, DEK, DEPDC1, DHCR24, DIRAS3, DKK1, DLST, DNAJB1, DNAJB2, DNM1L, DPP4, DSG2, DUT, EFNA1, EFNB2, EGR1, EHF, EIF2AK3, EIF4G2, EPAS1, ERN1, F2R, F2RL1, FAF1 (includes EG:11124), FANCA, FANCD2, FASN, FBXO32, FCAR, FGF19, FLNB, FOS, FOSB, FOXO3, FOXO4, FTH1 (includes EG:14319), FUBP1, GADD45A, GAL, GDF15, GNAS, GPM6A, GPR37, GRN, GSN, HAUS8, HBEGF, HDAC4, HERPUD1, HES1 (includes EG:15205), HK1, HLA-G, HMGB1, HMMR, HNRNPA1, HNRNPC, HOXA5, HRAS, HSF1 (includes

478

EG:15499), HSP90AA1, HSP90AB1, HSPA1A/HSPA1B, HSPA4, HSPA5, HSPA8, HSPB11, HSPD1, HUWE1, HYOU1, ID1, ID3 (includes EG:15903), IER3, IFIH1, IGF2R, IGFBP3, IGFBP6, IL15 (includes EG:16168), IL17RD, IL18 (includes EG:16173), IL27RA, IL32, IL8, ILK, ING2, IRF1 (includes EG:16362), ITGA2, ITGAV, ITGB1, ITGB3BP, ITGB4, ITM2B, ITPR3, JAK1 (includes EG:16451), JUN, JUND, JUP, KIF14, KLF2, KLF4, KLF5, KLF6, KLF9, LAMP2, LCN2, LEP, LEPR, LETM1, LGALS1, LGALS3, LGALS3BP, LGMN, LIF, LIG1, LILRB1, LMNB1, LMO2, LSMD1, LTBR, MAD2L1, MAP2K4, MAP3K7 (includes EG:172842), MAP4K1, MAPK1, MAPK3, MAPKAP1, MAPT, MBP, MC1R, MCM10 (includes EG:307126), MCM2, MDC1, MELK, MMS22L, MOAP1, MSH2, MSRB2, MST1R, MSX1, MT2A, MTA2, MVP, MYB, MZF1, NAE1, NCL, NCOA3, NDEL1, NDRG1, NDUFAB1, NEK2, NFIL3, NFKB2, NFKBIA, NFKBIB, NFKBIZ, NME1 (includes EG:18102), NOC2L, NOS3, NQO1, NR4A2, NRAS, NRP1 (includes EG:18186), NUF2, OBFC2A, PARK7, PARP1, PAWR, PBK, PCNA, PDCD4, PELP1, PHB, PHLDA1, PIK3CB, PINK1, PKMYT1, PLAU, PLAUR, PLK1, PMAIP1, PMP22, PNPT1, PPP1R13L, PPP1R15A, PPP2CA, PRAME, PRDM1, PRDX1, PRDX3, PRELID1, PRKCA, PRKDC, PRNP, PRPF19, PTTG1, RAC2, RAC3, RAD21, RAD23B, RAD51C, RAF1, RBM17, REPS2, RHOC, RPA1, RRM1, RUNX1, S100A4, SAT1, SDC1 (includes EG:20969), SEMA3B, SET, SHC1 (includes EG:20416), SIVA1, SKP2 (includes EG:27401), SLC25A6, SLC29A1, SLC3A2, SLPI, SMOX, SND1, SOD2, SOX9, SPC25 (includes EG:100144563), SPHK2, SQSTM1, SRPK1, SRPX, SRSF1, SRXN1, STAT1, STAT3, STMN1, STOML2, SYVN1, TACC3, TAF1B, TBK1, TCP1, TERF2, TFAP2C, TFDP1, TFRC, TGFA, TGFB1I1, TGFBR2, TGM2, THOC1, TIAM1, TIMP1, TMBIM6, TMED10, TNFRSF10B, TNFRSF18, TNFRSF21, TNFRSF6B, TNFSF14, TNFSF15, TOP2A, TOPBP1, TP53INP1, TPD52, TRIAP1, TRIB3, TRIM28, TSG101, TTF1, TTK, TUBA1A, TXN (includes EG:116484), TXNDC17, TYMP, TYMS, UBA3 (includes EG:117553), UBE2C, UBQLN1, UBTF, UNC5B, UNG, UQCRFS1, UXT, VEGFA, VHL, VPS28 (includes EG:300052), WEE1, WNT16, XBP1 (includes EG:140614), XPC, XPO1, XPR1, YARS, YBX1, YWHAQ, YY1, ZNF148 Cell Death apoptosis apoptosis of tumor cell lines 5.03E-10 A4GALT, ABCE1, ADAM17, ADM, AHSA1, AIFM1, AKAP12, 270 ALOX5, APOPT1, ASNS, ATAD2, ATF3, ATG5 (includes EG:11793), AURKA, B2M, BACH2, BAG3, BARD1, BCL2L1, BCL3, BCL6, BCLAF1, BEX2, BIK, BIRC3, BIRC5, BLM,

479

BMP4, BNIP3, BNIP3L, BRCA1, BRCA2, C15orf63, CAPNS1, CASP3, CASP6, CAT, CAV1, CCNB1, CCNE1, CCT2, CD24, CD55, CD99, CDC20 (includes EG:107995), CDC25A, CDC42, CDC7 (includes EG:12545), CDCA2, CDCP1, CDH1, CDK1, CDK6, CDK8, CDKN1A, CEBPB (includes EG:1051), CEBPD, CHEK1, CHEK2, CKAP2, COPS5, CRABP2, CREB1, CSE1L, CSF2RA, CSNK2A1, CTCF (includes EG:10664), CTSB, CYB5A (includes EG:109672), CYFIP2, DDIT3, DDIT4, DEPDC1, DIRAS3, DKK1, DNAJB1, DNM1L, DSG2, DUT, EGR1, EHF, EIF2AK3, EIF4G2, EPAS1, ERN1, FAF1 (includes EG:11124), FASN, FBXO32, FLNB, FOS, FOSB, FOXO3, FTH1 (includes EG:14319), FUBP1, GADD45A, GAL, GDF15, GRN, GSN, HBEGF, HDAC4, HES1 (includes EG:15205), HMMR, HNRNPA1, HNRNPC, HOXA5, HRAS, HSF1 (includes EG:15499), HSP90AB1, HSPA1A/HSPA1B, HSPA4, HSPA5, HSPA8, HSPD1, HUWE1, ID1, ID3 (includes EG:15903), IER3, IGF2R, IGFBP3, IGFBP6, IL15 (includes EG:16168), IL18 (includes EG:16173), IL32, IL8, ILK, ING2, IRF1 (includes EG:16362), ITGAV, ITGB1, ITGB3BP, ITGB4, JAK1 (includes EG:16451), JUN, JUND, JUP, KIF14, KLF2, KLF4, KLF5, KLF6, KLF9, LCN2, LEP, LGALS1, LGALS3, LGALS3BP, LIG1, LMNB1, LMO2, MAP2K4, MAP3K7 (includes EG:172842), MAPK1, MAPK3, MAPKAP1, MCM10 (includes EG:307126), MDC1, MELK, MOAP1, MSRB2, MST1R, MSX1, MT2A, MTA2, MYB, MZF1, NCL, NCOA3, NDRG1, NFKB2, NFKBIA, NFKBIB, NFKBIZ, NME1 (includes EG:18102), NOC2L, NOS3, NQO1, NR4A2, NRAS, NRP1 (includes EG:18186), PARP1, PAWR, PBK, PCNA, PDCD4, PELP1, PHB, PIK3CB, PINK1, PKMYT1, PLAU, PLAUR, PLK1, PMAIP1, PPP1R13L, PPP1R15A, PPP2CA, PRDM1, PRKCA, PRKDC, PRNP, PRPF19, PTTG1, RAD21, RAD23B, RAF1, REPS2, RHOC, RPA1, RRM1, RUNX1, S100A4, SAT1, SDC1 (includes EG:20969), SEMA3B, SIVA1, SKP2 (includes EG:27401), SLPI, SMOX, SND1, SOD2, SOX9, SPHK2, SRPK1, SRPX, SRSF1, STAT1, STAT3, STMN1, STOML2, SYVN1, TACC3, TBK1, TCP1, TFRC, TGFA, TGM2, THOC1, TIAM1, TMBIM6, TMED10, TNFRSF10B, TNFRSF18, TNFRSF21, TNFRSF6B, TNFSF14, TOP2A, TOPBP1, TPD52, TRIAP1, TRIM28, TSG101, TTF1, TTK, TXN (includes EG:116484), TXNDC17, TYMP, TYMS, UNC5B, VEGFA, VHL, WEE1, WNT16, XBP1 (includes EG:140614), XPC, XPO1, XPR1, YBX1, YY1, ZNF148

480

Cell Death apoptosis apoptosis 1.75E-08 A4GALT, ABCE1, ACIN1, ACVR1, ADAM17, ADM, AHSA1, 388 AIFM1, AKAP12, ALDH2, ALOX5, ANGPTL4, ANXA1, APOPT1, ASNS, ATAD2, ATF3, ATF6, ATG5 (includes EG:11793), AURKA, AXL, AZI2, B2M, BACH2, BAG3, BARD1, BCL2L1, BCL3, BCL6, BCLAF1, BEX2, BIK, BIRC3, BIRC5, BLM, BMP4, BNIP3, BNIP3L, BRCA1, BRCA2, BSG (includes EG:12215), BTG1, C15orf63, C1QBP, CAPNS1, CAPRIN2, CASP3, CASP6, CAT, CAV1, CCNB1, CCNE1, CCT2, CD24, CD55, CD99, CDC20 (includes EG:107995), CDC25A, CDC25C, CDC42, CDC45, CDC7 (includes EG:12545), CDCA2, CDCP1, CDH1, CDK1, CDK2, CDK6, CDK8, CDKN1A, CEBPB (includes EG:1051), CEBPD, CHEK1, CHEK2, CKAP2, COPS5, CR2, CRABP2, CREB1, CRLF1, CSE1L, CSF2RA, CSNK2A1, CTCF (includes EG:10664), CTNNBL1, CTSB, CUL4B, CXCL1, CYB5A (includes EG:109672), CYBA, CYFIP2, DDIT3, DDIT4, DDN, DDR1, DDX17, DDX47, DEPDC1, DEPTOR, DHRS2, DIRAS3, DKK1, DNAJB1, DNM1L, DSG2, DUSP6, DUT, EGR1, EHF, EIF2AK3, EIF4G2, EIF5A, EPAS1, ERN1, ESPL1, F2R, FAF1 (includes EG:11124), FANCA, FASN, FBXO32, FGF19, FGFR3, FHL2, FLNB, FOS, FOSB, FOXO3, FOXO4, FTH1 (includes EG:14319), FUBP1, FXR1, GABPB1, GADD45A, GAL, GDF15, GRN, GSN, HAUS1, HBEGF, HDAC4, HERPUD1, HES1 (includes EG:15205), HEY1, HIST1H1C, HLA-G, HMGB1, HMMR, HNRNPA1, HNRNPC, HOXA5, HRAS, HSF1 (includes EG:15499), HSP90AA1, HSP90AB1, HSPA1A/HSPA1B, HSPA4, HSPA5, HSPA8, HSPD1, HUWE1, HYOU1, ICAM3, ID1, ID3 (includes EG:15903), IER3, IFIH1, IGF2R, IGFBP3, IGFBP6, IL15 (includes EG:16168), IL17RD, IL18 (includes EG:16173), IL32, IL8, ILK, ING2, IRF1 (includes EG:16362), ITGA5, ITGAV, ITGB1, ITGB3BP, ITGB4, ITPR3, JAK1 (includes EG:16451), JUN, JUND, JUP, KEAP1, KIAA1967, KIF14, KLF2, KLF4, KLF5, KLF6, KLF9, LCN2, LEP, LEPR, LGALS1, LGALS3, LGALS3BP, LGMN, LIF, LIG1, LILRB1, LMNB1, LMO2, LTBR, MAD2L1, MALT1, MAP2K4, MAP3K7 (includes EG:172842), MAP4K1, MAPK1, MAPK3, MAPKAP1, MAPT, MC1R, MCM10 (includes EG:307126), MCM2, MDC1, MELK, MOAP1, MSH2, MSRB2, MST1R, MSX1, MT2A, MTA2, MYB, MZF1, NAE1, NCL, NCOA3, NDC80, NDRG1, NDUFS3, NFKB2, NFKBIA, NFKBIB, NFKBIZ, NME1 (includes EG:18102), NME2, NOC2L, NOS3, NQO1, NR4A2, NRAS, NRP1 (includes EG:18186), NT5E, NUP62, ODC1, P2RX4, PARP1, PAWR, PBK, PCNA, PDCD4,

481

PELP1, PHB, PHLDA1, PIK3CB, PINK1, PKMYT1, PLAU, PLAUR, PLK1, PMAIP1, PNPT1, PPP1R13L, PPP1R15A, PPP2CA, PRAME, PRDM1, PRDX1, PRDX3, PRDX5, PRELID1, PRKCA, PRKDC, PRKRIR, PRNP, PRPF19, PSME3, PTGS1, PTTG1, RAC2, RAD21, RAD23B, RAF1, REPS2, RHOC, RNF130, RPA1, RPS6KA2, RPS6KA3, RRM1, RUNX1, S100A4, SAT1, SCRIB, SDC1 (includes EG:20969), SEMA3B, SET, SHC1 (includes EG:20416), SIVA1, SKP2 (includes EG:27401), SLPI, SMAD6, SMOX, SNCG, SND1, SOD2, SOX9, SPHK2, SRPK1, SRPX, SRSF1, SRXN1, STAT1, STAT3, STK3, STMN1, STOML2, SYVN1, TACC3, TAF9, TBK1, TCP1, TERF2, TFRC, TGFA, TGFBR2, TGM2, THOC1, TIAL1, TIAM1, TIMP1, TMBIM6, TMED10, TNFAIP1, TNFRSF10B, TNFRSF12A, TNFRSF18, TNFRSF21, TNFRSF6B, TNFSF14, TNFSF15, TOP2A, TOPBP1, TP53INP1, TPD52, TPP2, TRIAP1, TRIB3, TRIM24, TRIM28, TSG101, TSPO, TTF1, TTK, TXN (includes EG:116484), TXNDC17, TYMP, TYMS, UBA3 (includes EG:117553), UBQLN1, UBR4, UNC5B, UNG, USP7, UTP11L, UXT, VEGFA, VHL, WEE1, WNT10B, WNT16, XBP1 (includes EG:140614), XPC, XPO1, XPR1, YARS, YBX1, YWHAQ, YY1, ZAK, ZNF148 Cell Death apoptosis apoptosis of breast cancer cell 3.79E-06 ADM, ATG5 (includes EG:11793), B2M, BAG3, BARD1, 73 lines BCL2L1, BEX2, BIK, BIRC3, BIRC5, BNIP3, BNIP3L, BRCA1, CASP3, CASP6, CAV1, CDH1, CDK1, CDKN1A, CHEK1, CRABP2, CSE1L, CTCF (includes EG:10664), CYB5A (includes EG:109672), DDIT4, DIRAS3, FBXO32, FOXO3, FTH1 (includes EG:14319), GADD45A, HOXA5, HSPA5, HSPD1, IER3, IGFBP3, ILK, ITGB3BP, ITGB4, JUN, KLF5, LGALS3, MAPK1, MAPK3, MOAP1, NCOA3, NFKBIA, NME1 (includes EG:18102), NOS3, NQO1, PARP1, PDCD4, PELP1, PHB, PIK3CB, PINK1, PLK1, PMAIP1, PRKCA, PRNP, PTTG1, SDC1 (includes EG:20969), SIVA1, SND1, SOD2, SPHK2, SRPK1, STAT1, TBK1, TFRC, THOC1, TNFRSF10B, TYMS, XBP1 (includes EG:140614) Cell Death apoptosis apoptosis of prostate cancer cell 1.48E-03 ABCE1, AKAP12, ALOX5, BCL2L1, BIRC3, BIRC5, BRCA1, 42 lines CASP3, CAT, CAV1, CREB1, DIRAS3, EGR1, EHF, FASN, FOS, FOXO3, GADD45A, GDF15, HBEGF, HSPA1A/HSPA1B, HSPD1, ID1, IGFBP3, ITGB1, LGALS1, MAP3K7 (includes EG:172842), NCOA3, PAWR, PLAU, PLAUR, PMAIP1, PRKCA, SPHK2, STAT3, TGFA, TMBIM6, TMED10, TNFRSF10B, TPD52, TYMP, YY1 Cell Death apoptosis apoptosis of lymphoma cell 2.95E-03 A4GALT, BACH2, BCL2L1, BIK, BIRC3, BIRC5, CASP3, 33

482

lines CAT, CD24, CD55, CDKN1A, CHEK2, DUT, EIF2AK3, ERN1, FUBP1, HNRNPA1, HNRNPC, IL15 (includes EG:16168), JAK1 (includes EG:16451), LMNB1, NCL, NFKB2, NFKBIA, PMAIP1, RAD23B, RUNX1, STAT3, TGM2, TIAM1, TNFRSF10B, TNFRSF6B, XBP1 (includes EG:140614) Cell Death apoptosis apoptosis of neuroblastoma cell 3.41E-03 ADM, AIFM1, BCL2L1, BIRC5, CASP3, CDC42, CDKN1A, 23 lines DDIT3, DNAJB1, GAL, HSPA1A/HSPA1B, IGFBP3, ITGB1, LEP, NFKBIB, PARP1, PINK1, PRNP, SYVN1, TGM2, TXN (includes EG:116484), VEGFA, XPR1 Cell Death apoptosis apoptosis of vascular 5.70E-03 ADM, ALDH2, ATF3, AXL, BCL2L1, CASP3, CAV1, DDIT3, 19 endothelial cells FOXO3, HSPA5, HSPD1, IL18 (includes EG:16173), IL8, KLF5, MAPK1, MAPK3, PMAIP1, TNFRSF10B, VEGFA Cell Death apoptosis apoptosis of lung cancer cell 1.74E-02 AIFM1, ATAD2, BCL2L1, BIRC3, BIRC5, BRCA2, CASP3, 38 lines CDCP1, CDKN1A, CEBPB (includes EG:1051), CREB1, FOXO3, GADD45A, HSF1 (includes EG:15499), ID3 (includes EG:15903), IGFBP3, IGFBP6, KLF6, LCN2, LGALS3, MAP3K7 (includes EG:172842), MDC1, MTA2, PARP1, PLK1, PPP2CA, REPS2, RRM1, SEMA3B, SKP2 (includes EG:27401), SRSF1, STAT3, TBK1, TNFRSF10B, TRIAP1, TTF1, XPC, YBX1 Cell Death apoptosis apoptosis of embryonic cells 2.28E-02 BMP4, NRP1 (includes EG:18186), TNFRSF6B, TNFSF14 4 Cell Death apoptosis apoptosis of cancer cells 3.09E-02 B2M, BCL2L1, BIK, BIRC5, BRCA1, CASP3, CAT, CD24, 29 CDC45, CDH1, CDKN1A, CHEK1, CTSB, DDIT3, FOXO4, IL15 (includes EG:16168), ITGAV, LGALS1, MCM2, NCOA3, NR4A2, PARP1, PHLDA1, PLK1, PMAIP1, SEMA3B, TNFRSF10B, TXN (includes EG:116484), VEGFA Cell Death apoptosis apoptosis of tumor cells 3.17E-02 B2M, BCL2L1, BIK, BIRC5, BRCA1, CASP3, CAT, CD24, 34 CDC45, CDH1, CDK2, CDKN1A, CHEK1, CTSB, DDIT3, FOXO4, HBEGF, IL15 (includes EG:16168), ITGAV, LGALS1, MCM2, NCOA3, NR4A2, PARP1, PHLDA1, PLK1, PMAIP1, PPP2CA, SEMA3B, STAT3, TGFBR2, TNFRSF10B, TXN (includes EG:116484), VEGFA Cell Death apoptosis apoptosis of cultured 3.21E-02 BCL2L1, PARP1 2 osteosarcoma cells Cell Death cell viability cell viability of myeloma cell 3.66E-05 AURKB, BCL2L1, CARS, CKAP5, CRBN, EIF3C/EIF3CL, 27 lines EIF4A3, KIF11, KIF18A, KIFC2, NDC80, NUF2, PLK1, PSMA1, PSMA3, PSMA4, PSMA6, PSMC3, PSMC4, PSMC5, RRM1, SLC25A23, SNRPA1, STAT3, TPMT, WEE1, XPO1 Cell Death cell viability cell viability of tumor cell lines 4.37E-04 ABCC3, ADAM17, AIFM1, ANLN, ATF6, ATG3 (includes 145 EG:171415), AURKAIP1, AURKB, AXL, BCL2L1, BCL6,

483

BEX2, BIRC5, BLM, BNIP3, BRCA1, BRCA2, BUB1B, CAMK2N1, CARS, CASP3, CAT, CAV1, CCNA2, CCNB1, CDH1, CDK2, CDK6, CDK8, CDKN1A, CEBPB (includes EG:1051), CEBPD, CHEK1, CHEK2, CKAP5, CLDN3, CRBN, CREB1, DCK, DPP4, DUSP19, DUSP5, DUSP6, EIF3C/EIF3CL, EIF4A3, ELF3, FANCG, FGFR3, FOXM1, FTH1 (includes EG:14319), GCLC, GDF15, H6PD, HBEGF, HOXA5, HRAS, HSPA5, ID2, IGFBP3, IL8, ILK, INPP1, INPP5J, ITGB1, JAK1 (includes EG:16451), KIF11, KIF18A, KIFC1, KIFC2, KLF5, LGALS3, LIF, LIG1, MAD2L1, MMS22L, MSH2, MYB, NCSTN, NDC80, NFKB2, NRAS, NRP1 (includes EG:18186), NUF2, PARK7, PARP1, PBK, PBX1, PCNA, PLAU, PLAUR, PLK1, PPAP2A, PPM1G, PPM1M, PPP1CC, PPP1R8, PPP2CA, PPP2R1B, PRKCA, PRKDC, PRNP, PRPF19, PSMA1, PSMA3, PSMA4, PSMA6, PSMC3, PSMC4, PSMC5, PTP4A2, RAF1, RIF1 (includes EG:295602), RPA1, RRM1, RRM2, S100A4, SFR1, SHFM1, SLC25A23, SLPI, SNRPA1, SOD2, SQSTM1, STAT3, STX8, SYVN1, TBC1D9, TBK1, TCP1, TGFBR2, TGM2, THOC1, TIMP2 (includes EG:21858), TPMT, TRIM28, TXN (includes EG:116484), TYMP, TYMS, VEGFA, VHL, WEE1, XPC, XPO1, YBX1, ZFP36 Cell Death cell viability cell viability 7.22E-04 ABCC3, ADAM17, AIFM1, ANLN, ANTXR1, ATF6, ATG3 180 (includes EG:171415), AURKAIP1, AURKB, AXL, BCL2L1, BCL6, BEX2, BIRC5, BLM, BMP4, BNIP3, BRCA1, BRCA2, BUB1B, CALB2, CAMK2N1, CARS, CASP3, CAT, CAV1, CCNA2, CCNB1, CDC7 (includes EG:12545), CDH1, CDK2, CDK2AP1, CDK6, CDK8, CDKN1A, CEBPB (includes EG:1051), CEBPD, CHEK1, CHEK2, CKAP5, CLDN3, CRBN, CREB1, DCK, DDIT3, DPP4, DUSP19, DUSP5, DUSP6, EIF3C/EIF3CL, EIF4A3, ELF3, FANCD2, FANCG, FGFR3, FOXM1, FTH1 (includes EG:14319), GCLC, GDF15, H6PD, HBEGF, HERPUD1, HMGB1, HOXA5, HRAS, HSPA5, HYOU1, ID2, IGFBP3, IL15 (includes EG:16168), IL8, ILK, INPP1, INPP5J, IRF9, ITGAV, ITGB1, JAK1 (includes EG:16451), KIF11, KIF18A, KIFC1, KIFC2, KLF5, LEP, LGALS3, LIF, LIG1, MAD2L1, MAPK1, MAPK3, MAPT, METAP2, MGST1, MMS22L, MSH2, MVP, MYB, NCSTN, NDC80, NDRG1, NFIL3, NFKB2, NFKBIA, NRAS, NRP1 (includes EG:18186), NUF2, PARK7, PARP1, PBK, PBX1, PCNA, PLAU, PLAUR, PLK1, POR, PPAP2A, PPM1G, PPM1M, PPP1CC, PPP1R8, PPP2CA, PPP2R1B, PRDX6, PRKCA, PRKDC, PRNP, PRPF19, PSMA1, PSMA3, PSMA4,

484

PSMA6, PSMC3, PSMC4, PSMC5, PTP4A2, RAC2, RAF1, RASD2, RIF1 (includes EG:295602), RPA1, RRM1, RRM2, RUNX1, S100A4, SETMAR, SFR1, SHFM1, SLC25A23, SLC9A1, SLPI, SND1, SNRPA1, SOD2, SQSTM1, STAT2, STAT3, STMN1, STX8, SYVN1, TAF1B, TBC1D9, TBK1, TCP1, TGFBR2, TGM2, THOC1, TIMP1, TIMP2 (includes EG:21858), TPMT, TRIM28, TXN (includes EG:116484), TYMP, TYMS, VEGFA, VHL, WEE1, XPC, XPO1, YBX1, ZFP36 Cell Death cell viability cell viability of kidney cell 2.95E-02 ABCC3, BIRC5, CAV1, CEBPD, HYOU1, IRF9, ITGAV, 12 lines MAPT, NFKBIA, RASD2, SETMAR, STAT2 Cell Death survival cell survival 8.00E-04 ABCC3, ADAM17, AIFM1, ANLN, ANTXR1, ATF4, ATF6, 187 ATG3 (includes EG:171415), AURKAIP1, AURKB, AXL, BCL2L1, BCL6, BEX2, BIRC5, BLM, BMP4, BNIP3, BRCA1, BRCA2, BUB1B, CALB2, CAMK2N1, CARS, CASP3, CASP6, CAT, CAV1, CCNA2, CCNB1, CDC7 (includes EG:12545), CDH1, CDK2, CDK2AP1, CDK6, CDK8, CDKN1A, CEBPB (includes EG:1051), CEBPD, CHEK1, CHEK2, CKAP5, CLDN3, CRBN, CREB1, DCK, DDIT3, DPP4, DUSP19, DUSP5, DUSP6, EIF3C/EIF3CL, EIF4A3, ELF3, FANCD2, FANCG, FGFR3, FOXM1, FTH1 (includes EG:14319), GCLC, GDF15, H6PD, HBEGF, HERPUD1, HMGB1, HOXA5, HRAS, HSPA5, HYOU1, ID2, IGFBP3, IL15 (includes EG:16168), IL27RA, IL8, ILK, INPP1, INPP5J, IRF9, ITGA5, ITGAV, ITGB1, JAK1 (includes EG:16451), KIF11, KIF18A, KIFC1, KIFC2, KLF5, LEP, LGALS3, LIF, LIG1, MAD2L1, MAPK1, MAPK3, MAPT, METAP2, MGST1, MMS22L, MSH2, MVP, MYB, NCSTN, NDC80, NDRG1, NFIL3, NFKB2, NFKBIA, NRAS, NRP1 (includes EG:18186), NUF2, PARK7, PARP1, PBK, PBX1, PCNA, PLAU, PLAUR, PLK1, POR, PPAP2A, PPM1G, PPM1M, PPP1CC, PPP1R8, PPP2CA, PPP2R1B, PRDX6, PRKCA, PRKDC, PRNP, PRPF19, PSMA1, PSMA3, PSMA4, PSMA6, PSMC3, PSMC4, PSMC5, PTP4A2, RAC2, RAD51C, RAF1, RASD2, RIF1 (includes EG:295602), RPA1, RRM1, RRM2, RUNX1, S100A4, SETMAR, SFR1, SHFM1, SLC25A23, SLC9A1, SLPI, SND1, SNRPA1, SOD2, SQSTM1, STAT2, STAT3, STMN1, STX8, SYVN1, TAF1B, TBC1D9, TBK1, TCP1, TGFBR2, TGM2, THOC1, TIMP1, TIMP2 (includes EG:21858), TOP2A, TOPBP1, TPMT, TRIM28, TXN (includes EG:116484), TYMP, TYMS, VEGFA, VHL, WEE1, XPC, XPO1, YBX1, ZFP36 Cell Death anoikis anoikis of RPE cells 1.03E-03 BCL2L1, MAPK1, PLAU, PLAUR 4

485

Cell Death anoikis anoikis 2.96E-03 BCL2L1, CAV1, CD99, CDCP1, GRN, ILK, ITGAV, ITGB1, 18 LGALS3, MAPK1, NCOA3, NR4A2, PLAU, PLAUR, PRKCA, SLPI, TIMP1, TNFRSF10B Cell Death anoikis anoikis of tumor cell lines 7.28E-03 CAV1, CD99, CDCP1, GRN, ILK, ITGAV, LGALS3, MAPK1, 13 NCOA3, NR4A2, PRKCA, SLPI, TNFRSF10B Cell Death colony survival colony survival of tumor cell 2.99E-02 BCL2L1, CDKN1A, CHEK2, FANCG, MSH2, PARP1, SLPI 7 lines

Appendix 12. 2 The top molecular and cellular functions associated with differentially expressed genes in the FPGS-inhibited HCT116 colon cancer cells No. of Category Function Function Annotation P-value Genes Genes Cell Death cell death cell death of tumor cell lines 4.02E-09 ASNS, ATF3, ATF4, BARD1, BID, BIK, BIRC3, BMP4, 62 BNIP3L, BRCA1, BTG1, CASP2, CDC25A, CDC25C, CDKN1A, CEBPB (includes EG:1051), CHMP5, CRABP2, DDIT3, DDIT4, DHCR24, DNAJC15, EGR1, EHF, EPAS1, FAS, FBXO32, GADD45A, GDF15, HSPA5, ID1, ID3 (includes EG:15903), IER3, IGFBP6, IL8, IP6K2, ITGB3BP, JUP, KLF4, KLF9, LCN2, LGALS1, LGALS3, LSMD1, LTBR, MAPK3, MCM10 (includes EG:307126), MSH2, NDRG1, PINK1, PLAGL1, PLAU, PMAIP1, PRAME, RAD23B, RRM2B, S100A4, SEMA3A, STAT1, TNFRSF10B, TNFRSF6B, YARS Cell Death cell death cell death of breast cancer cell 8.43E-09 BARD1, BID, BIK, BIRC3, BNIP3L, BRCA1, CASP2, 25 lines CDKN1A, CHMP5, CRABP2, DDIT4, DNAJC15, FAS, FBXO32, GADD45A, HSPA5, IER3, ITGB3BP, LGALS3, MAPK3, MSH2, PINK1, PMAIP1, STAT1, TNFRSF10B Cell Death cell death cell death 2.20E-08 ASNS, ATF3, ATF4, ATF6, AXIN2, BARD1, BID, BIK, 93 BIRC3, BMP4, BNIP3L, BRCA1, BTG1, CA2, CALB2, CARS, CASP2, CDC25A, CDC25C, CDKN1A, CEBPB (includes EG:1051), CHMP5, CRABP2, CST3, DDB2, DDIT3, DDIT4, DHCR24, DHRS2, DNAJC15, DUSP6, EGR1, EHF, ELF3, EPAS1, F2RL1, FAS, FBXO32, FOXO4, GADD45A, GAS6, GDF15, HERPUD1, HSPA5, ID1, ID3 (includes EG:15903), IER3, IGFBP6, IL8, IP6K2, IRF9, ISG15, ITGB3BP, JUP, KLF4, KLF9, LCN2, LGALS1, LGALS3, LSMD1, LTBR, MAPK3, MCM10 (includes EG:307126), MECOM, MSH2, NBN,

486

NDRG1, NEK3, NFIL3, PINK1, PLAGL1, PLAU, PMAIP1, PRAME, PRDX5, PRKACB, PROCR, RAD23B, RRM2B, S100A4, SEMA3A, STAT1, STX8, TNFRSF10B, TNFRSF6B, TP53INP1, TRIB3, TRIM24, TXNRD1, ULBP1, UNG, YARS, ZFP36 Cell Death cell death cell death of endothelial cells 1.34E-06 ATF3, DDIT3, F2RL1, FAS, FOXO4, GAS6, HSPA5, IL8, 12 MAPK3, PMAIP1, PROCR, TNFRSF10B Cell Death cell death cell death of vascular 1.58E-06 ATF3, DDIT3, F2RL1, FAS, GAS6, HSPA5, IL8, MAPK3, 10 endothelial cells PMAIP1, TNFRSF10B Cell Death cell death cell death of melanoma cell 8.85E-06 BIK, CDKN1A, DDIT3, EGR1, FAS, GADD45A, HSPA5, IL8, 10 lines PMAIP1, TNFRSF10B Cell Death cell death cell death of fibroblasts 9.92E-06 ATF3, BID, BIK, BIRC3, BMP4, BRCA1, CDKN1A, FAS, 9 TNFRSF10B Cell Death cell death cell death of connective tissue 3.11E-05 ATF3, ATF6, BID, BIK, BIRC3, BMP4, BRCA1, CDKN1A, 12 cells FAS, NBN, TNFRSF10B, TNFRSF6B Cell Death cell death cell death of cancer cells 3.93E-05 BID, BIK, BMP4, BRCA1, CASP2, CDKN1A, DDIT3, FAS, 13 FOXO4, IL8, LGALS1, PMAIP1, TNFRSF10B Cell Death cell death cell death of ovarian cancer cell 6.81E-05 BARD1, CDKN1A, DDIT3, DNAJC15, FAS, IL8, IP6K2, 10 lines PMAIP1, TNFRSF10B, YARS Cell Death cell death cell death of prostate cancer 1.33E-04 BIRC3, BRCA1, DDIT3, EGR1, EHF, FAS, GADD45A, 13 cell lines GDF15, ID1, LGALS1, PLAU, PMAIP1, TNFRSF10B Cell Death cell death cell death of colon cancer cell 1.50E-04 ATF3, BID, BIK, CASP2, CDKN1A, DDIT3, FAS, GADD45A, 16 lines GDF15, LGALS3, LSMD1, LTBR, NDRG1, STAT1, TNFRSF10B, TNFRSF6B Cell Death cell death cell death of lung cancer cell 1.78E-04 BIRC3, CASP2, CDKN1A, CEBPB (includes EG:1051), DDIT3, 13 lines FAS, GADD45A, ID3 (includes EG:15903), IGFBP6, LCN2, LGALS3, RRM2B, TNFRSF10B Cell Death cell death cell death of lymphoma cell 2.50E-04 BIK, BIRC3, CASP2, CDKN1A, FAS, LGALS1, LGALS3, 11 lines PMAIP1, RAD23B, TNFRSF10B, TNFRSF6B Cell Death cell death cell death of kidney cell lines 2.55E-04 ATF6, BID, BIK, BIRC3, BNIP3L, CASP2, CHMP5, FAS, 15 HSPA5, IER3, IP6K2, PMAIP1, TNFRSF10B, TP53INP1, TRIB3 Cell Death cell death cell death of epithelial cells 3.20E-04 BID, BIK, BIRC3, BNIP3L, CASP2, CHMP5, FAS, HSPA5, 16 IER3, IL8, IP6K2, PLAU, PMAIP1, TNFRSF10B, TP53INP1, TRIB3 Cell Death cell death cell death of embryonic cell 3.61E-04 BID, BIK, BIRC3, BNIP3L, CASP2, CHMP5, FAS, HSPA5, 14

487

lines IER3, IP6K2, PMAIP1, TNFRSF10B, TP53INP1, TRIB3 Cell Death cell death cell death of 3.80E-04 ATF4, CDKN1A, PMAIP1, TNFRSF10B 4 rhabdomyosarcoma cell lines Cell Death cell death cell death of epithelial cell lines 4.77E-04 BID, BIK, BIRC3, BNIP3L, CASP2, CHMP5, FAS, HSPA5, 14 IER3, IP6K2, PMAIP1, TNFRSF10B, TP53INP1, TRIB3 Cell Death cell death cell death of fibrosarcoma cell 7.96E-04 BIRC3, CDKN1A, FAS, PMAIP1, STAT1, TNFRSF10B 6 lines Cell Death cell death cell death of pancreatic cancer 1.21E-03 ASNS, DDIT3, FAS, IL8, S100A4, TNFRSF10B 6 cell lines Cell Death cell death cell death of breast cell lines 1.23E-03 BID, BRCA1, CDKN1A, FAS, ID1 5 Cell Death cell death cell death of cervical cancer 2.85E-03 BID, BNIP3L, CASP2, CDKN1A, CHMP5, DDIT3, FAS, 15 cell lines GADD45A, IER3, LSMD1, MCM10 (includes EG:307126), PINK1, PRAME, STAT1, TNFRSF10B Cell Death cell death cell death of brain cancer cell 2.98E-03 BIRC3, BMP4, DHCR24, EPAS1, FAS, SEMA3A, TNFRSF10B, 8 lines TNFRSF6B Cell Death cell death cell death of immune cells 3.43E-03 BMP4, CST3, DDIT3, FAS, IL8, IP6K2, KLF4, LGALS1, 13 LGALS3, NBN, NFIL3, PMAIP1, TNFRSF6B Cell Death cell death cell death of cytotoxic T cells 4.90E-03 CST3, FAS 2 Cell Death cell death cell death of hematopoietic cell 6.16E-03 BIRC3, DDIT3, FAS, LGALS1, LGALS3, NBN 6 lines Cell Death cell death cell death of lymphoblastoid 7.03E-03 CASP2, CDKN1A, FAS, MSH2 4 cell lines Cell Death cell death cell death of leukocyte cell 7.99E-03 DDIT3, FAS, LGALS1, LGALS3, NBN 5 lines Cell Death cell death cell death of leukemia cell lines 7.99E-03 BID, BIK, BIRC3, BTG1, CASP2, CDKN1A, DDIT3, FAS, 13 KLF4, LGALS1, LGALS3, TNFRSF10B, TNFRSF6B Cell Death cell death cell death of hematopoietic 9.49E-03 BMP4, FAS, IP6K2, LGALS1 4 progenitor cells Cell Death cell death cell death of neuroglia 1.29E-02 FAS, GAS6, IL8 3 Cell Death cell death cell death of astrocytes 1.31E-02 FAS, IL8 2 Cell Death cell death cell death of squamous cell 1.47E-02 BID, FAS, JUP, STAT1 4 carcinoma cell lines Cell Death cell death cell death of liver cells 1.64E-02 FAS, IER3, TNFRSF6B 3 Cell Death cell death cell death of lymphoblasts 1.66E-02 IP6K2, LGALS1 2

488

Cell Death cell death cell death of primary effusion 2.27E-02 FAS 1 lymphoma cells Cell Death cell death cell death of pro-B 2.27E-02 FAS 1 lymphocytes Cell Death necrosis necrosis 4.39E-09 ASNS, ATF3, ATF4, ATF6, BARD1, BID, BIK, BIRC3, BMP4, 74 BNIP3L, BRCA1, BTG1, CASP2, CDC25A, CDC25C, CDKN1A, CEBPB (includes EG:1051), CHMP5, CRABP2, CST3, DDIT3, DDIT4, DHCR24, DNAJC15, EGR1, EHF, EPAS1, F2RL1, FAS, FBXO32, FOXO4, GADD45A, GAS6, GDF15, HERPUD1, HSPA5, ID1, ID3 (includes EG:15903), IER3, IGFBP6, IL8, IP6K2, ITGB3BP, JUP, KLF4, KLF9, LCN2, LGALS1, LGALS3, LSMD1, LTBR, MAPK3, MCM10 (includes EG:307126), MSH2, NBN, NDRG1, NFIL3, PINK1, PLAGL1, PLAU, PMAIP1, PRAME, PROCR, RAD23B, RRM2B, S100A4, SEMA3A, STAT1, TNFRSF10B, TNFRSF6B, TP53INP1, TRIB3, UNG, YARS Cell Death apoptosis apoptosis of breast cancer cell 8.97E-09 BARD1, BID, BIK, BIRC3, BNIP3L, BRCA1, CASP2, 23 lines CDKN1A, CHMP5, CRABP2, DDIT4, FAS, FBXO32, GADD45A, HSPA5, IER3, ITGB3BP, LGALS3, MAPK3, PINK1, PMAIP1, STAT1, TNFRSF10B Cell Death apoptosis apoptosis 6.28E-08 ASNS, ATF3, ATF6, BARD1, BID, BIK, BIRC3, BMP4, 72 BNIP3L, BRCA1, BTG1, CASP2, CDC25A, CDC25C, CDKN1A, CEBPB (includes EG:1051), CHMP5, CRABP2, DDIT3, DDIT4, DHRS2, DNAJC15, DUSP6, EGR1, EHF, EPAS1, FAS, FBXO32, FOXO4, GADD45A, GAS6, GDF15, HERPUD1, HSPA5, ID1, ID3 (includes EG:15903), IER3, IGFBP6, IL8, IP6K2, ITGB3BP, JUP, KLF4, KLF9, LCN2, LGALS1, LGALS3, LTBR, MAPK3, MCM10 (includes EG:307126), MECOM, MSH2, NBN, NDRG1, PINK1, PLAGL1, PLAU, PMAIP1, PRAME, PRDX5, PROCR, RAD23B, S100A4, SEMA3A, STAT1, TNFRSF10B, TNFRSF6B, TP53INP1, TRIB3, TRIM24, UNG, YARS Cell Death apoptosis apoptosis of tumor cell lines 1.30E-07 ASNS, ATF3, BARD1, BID, BIK, BIRC3, BMP4, BNIP3L, 52 BRCA1, CASP2, CDC25A, CDKN1A, CEBPB (includes EG:1051), CHMP5, CRABP2, DDIT3, DDIT4, DNAJC15, EGR1, EHF, EPAS1, FAS, FBXO32, GADD45A, GDF15, HSPA5, ID1, ID3 (includes EG:15903), IER3, IGFBP6, IL8, IP6K2, ITGB3BP, JUP, KLF4, KLF9, LCN2, LGALS1, LGALS3, MAPK3, MCM10 (includes EG:307126), NDRG1, PINK1, PLAGL1, PLAU, PMAIP1, RAD23B, S100A4,

489

SEMA3A, STAT1, TNFRSF10B, TNFRSF6B Cell Death apoptosis apoptosis of endothelial cells 4.59E-06 ATF3, DDIT3, FAS, FOXO4, GAS6, HSPA5, IL8, MAPK3, 11 PMAIP1, PROCR, TNFRSF10B Cell Death apoptosis apoptosis of vascular 6.52E-06 ATF3, DDIT3, FAS, GAS6, HSPA5, IL8, MAPK3, PMAIP1, 9 endothelial cells TNFRSF10B Cell Death apoptosis apoptosis of kidney cell lines 2.87E-05 ATF6, BID, BIK, BIRC3, BNIP3L, CASP2, CHMP5, FAS, 14 HSPA5, IER3, PMAIP1, TNFRSF10B, TP53INP1, TRIB3 Cell Death apoptosis apoptosis of melanoma cell 3.05E-05 BIK, CDKN1A, DDIT3, EGR1, FAS, GADD45A, HSPA5, 9 lines PMAIP1, TNFRSF10B Cell Death apoptosis apoptosis of embryonic cell 4.21E-05 BID, BIK, BIRC3, BNIP3L, CASP2, CHMP5, FAS, HSPA5, 13 lines IER3, PMAIP1, TNFRSF10B, TP53INP1, TRIB3 Cell Death apoptosis apoptosis of cancer cells 5.94E-05 BID, BIK, BRCA1, CASP2, CDKN1A, DDIT3, FAS, FOXO4, 11 LGALS1, PMAIP1, TNFRSF10B Cell Death apoptosis apoptosis of epithelial cell lines 6.30E-05 BID, BIK, BIRC3, BNIP3L, CASP2, CHMP5, FAS, HSPA5, 13 IER3, PMAIP1, TNFRSF10B, TP53INP1, TRIB3 Cell Death apoptosis apoptosis of prostate cancer cell 1.57E-04 BIRC3, BRCA1, EGR1, EHF, FAS, GADD45A, GDF15, ID1, 12 lines LGALS1, PLAU, PMAIP1, TNFRSF10B Cell Death apoptosis apoptosis of fibroblasts 2.04E-04 ATF3, BIK, BIRC3, BMP4, CDKN1A, FAS, TNFRSF10B 7 Cell Death apoptosis apoptosis of lymphoid organ 4.88E-04 BMP4, CDKN1A, FAS, LGALS1 4 Cell Death apoptosis apoptosis of lung cancer cell 6.90E-04 BIRC3, CASP2, CDKN1A, CEBPB (includes EG:1051), FAS, 11 lines GADD45A, ID3 (includes EG:15903), IGFBP6, LCN2, LGALS3, TNFRSF10B Cell Death apoptosis apoptosis of connective tissue 7.87E-04 ATF3, BIK, BIRC3, BMP4, CDKN1A, FAS, TNFRSF10B, 8 cells TNFRSF6B Cell Death apoptosis apoptosis of colon cancer cell 9.30E-04 ATF3, BID, BIK, CASP2, CDKN1A, DDIT3, FAS, GADD45A, 13 lines GDF15, LGALS3, NDRG1, STAT1, TNFRSF10B Cell Death apoptosis apoptosis of lymphoma cell 1.21E-03 BIK, BIRC3, CASP2, CDKN1A, FAS, PMAIP1, RAD23B, 9 lines TNFRSF10B, TNFRSF6B Cell Death apoptosis apoptosis of fibrosarcoma cell 1.77E-03 BIRC3, CDKN1A, FAS, PMAIP1, STAT1 5 lines Cell Death apoptosis apoptosis of carcinoma cell 2.16E-03 CASP2, DDIT3, DNAJC15, FAS, HSPA5, ID3 (includes 9 lines EG:15903), IGFBP6, LCN2, TNFRSF10B Cell Death apoptosis apoptosis of acute lymphoid 2.98E-03 CASP2, FAS 2 leukemia blast cells

490

Cell Death apoptosis apoptosis of endocrine cell 2.98E-03 LCN2, LGALS3 2 lines Cell Death apoptosis apoptosis of breast cell lines 4.46E-03 BID, CDKN1A, FAS, ID1 4 Cell Death apoptosis apoptosis of cervical cancer cell 5.12E-03 BID, BNIP3L, CASP2, CDKN1A, CHMP5, DDIT3, FAS, 12 lines GADD45A, IER3, MCM10 (includes EG:307126), PINK1, TNFRSF10B Cell Death apoptosis apoptosis of carcinoma cells 5.19E-03 DDIT3, FAS, FOXO4 3 Cell Death apoptosis apoptosis of thyroid tumor cell 6.20E-03 DDIT3, FAS, HSPA5 3 lines Cell Death apoptosis apoptosis of bladder cancer cell 7.31E-03 CDKN1A, FAS, LGALS3 3 lines Cell Death apoptosis apoptosis of fibroblast-like 7.31E-03 FAS, TNFRSF10B, TNFRSF6B 3 synoviocytes Cell Death apoptosis apoptosis of ovarian cancer cell 7.48E-03 BARD1, CDKN1A, DNAJC15, FAS, IP6K2, PMAIP1 6 lines Cell Death apoptosis apoptosis of leukemia cell lines 7.67E-03 BID, BIK, BIRC3, CASP2, CDKN1A, DDIT3, FAS, KLF4, 12 LGALS1, LGALS3, TNFRSF10B, TNFRSF6B Cell Death apoptosis apoptosis of PBMCs 9.98E-03 FAS, KLF4 2 Cell Death apoptosis apoptosis of embryonic cells 9.98E-03 BMP4, TNFRSF6B 2 Cell Death apoptosis apoptosis of liver cell lines 9.98E-03 FAS, IER3 2 Cell Death apoptosis apoptosis of lung cancer cells 9.98E-03 BID, TNFRSF10B 2 Cell Death apoptosis apoptosis of microvascular 1.29E-02 FAS, HSPA5, TNFRSF10B 3 endothelial cells Cell Death apoptosis apoptosis of airway epithelial 1.31E-02 BIK, FAS 2 cells Cell Death apoptosis apoptosis of 1.31E-02 FAS, TNFRSF10B 2 cholangiocarcinoma cell lines Cell Death apoptosis apoptosis of bone cancer cell 1.31E-02 BIK, BRCA1, CDKN1A, FAS, PLAGL1, PMAIP1, TNFRSF10B 7 lines Cell Death apoptosis apoptosis of epithelial cells 1.58E-02 BIK, FAS, IL8, PLAU, TNFRSF10B 5 Cell Death apoptosis apoptosis of hepatoma cell lines 1.64E-02 CDC25A, CDKN1A, DDIT3, FAS, STAT1, TNFRSF10B 6 Cell Death apoptosis apoptosis of pancreatic cancer 1.73E-02 ASNS, FAS, IL8, S100A4 4 cell lines

491

Cell Death apoptosis apoptosis of mesothelioma cell 2.04E-02 BID, FAS 2 lines Cell Death apoptosis apoptosis of thymocytes 2.04E-02 BMP4, LGALS1 2 Cell Death apoptosis apoptosis of hematopoietic cell 2.12E-02 BIRC3, DDIT3, FAS, LGALS3, NBN 5 lines Cell Death apoptosis apoptosis of lymphoblastoid 2.26E-02 CASP2, CDKN1A, FAS 3 cell lines Cell Death apoptosis apoptosis of T lymphoblasts 2.27E-02 LGALS1 1 Cell Death apoptosis apoptosis of limb bud cells 2.27E-02 BMP4 1 Cell Death apoptosis apoptosis of male germ cells 2.27E-02 FAS 1 Cell Death apoptosis apoptosis of recent thymic 2.27E-02 FAS 1 emigrants Cell Death apoptosis apoptosis of regulatory T 2.27E-02 FAS 1 lymphocytes Cell Death killing killing of cells 2.71E-05 BRCA1, FAS, HSPA5, ITGB3BP, LGALS1, LGALS3, NBN, 9 PLAU, TNFRSF10B Cell Death killing killing of tumor cell lines 1.20E-04 BRCA1, FAS, HSPA5, ITGB3BP, PLAU, TNFRSF10B 6 Cell Death killing killing of thymocytes 5.12E-04 LGALS1, LGALS3 2 Cell Death killing killing of breast cancer cell 8.76E-04 BRCA1, ITGB3BP, TNFRSF10B 3 lines Cell Death killing killing of brain cancer cell lines 1.51E-03 HSPA5, PLAU 2 Cell Death cell viability cell viability of lymphoblastoid 1.23E-03 CASP2, CDKN1A, NBN 3 cell lines Cell Death cell viability cell viability 1.51E-03 ATF6, BMP4, BRCA1, CA2, CALB2, CARS, CASP2, 32 CDKN1A, CEBPB (includes EG:1051), DDIT3, DUSP6, ELF3, FAS, GAS6, GDF15, HERPUD1, HSPA5, IL8, IRF9, LGALS3, MAPK3, MSH2, NBN, NDRG1, NEK3, NFIL3, PLAU, PRKACB, RRM2B, S100A4, STX8, ZFP36 Cell Death cell viability cell viability of vascular 7.24E-03 DDIT3, HSPA5 2 endothelial cells Cell Death cell viability cell viability of hematopoietic 1.46E-02 BMP4, IL8, NFIL3 3 progenitor cells Cell Death cell viability cell viability of tumor cell lines 1.59E-02 ATF6, BRCA1, CA2, CARS, CASP2, CDKN1A, CEBPB 23 (includes EG:1051), DUSP6, ELF3, FAS, GDF15, HSPA5, IL8, LGALS3, MSH2, NBN, NEK3, PLAU, PRKACB, RRM2B,

492

S100A4, STX8, ZFP36 Cell Death cell viability cell viability of breast cancer 2.39E-02 BRCA1, CA2, CDKN1A, ELF3, GDF15, LGALS3 6 cell lines Cell Death survival cell survival 1.60E-03 ATF4, ATF6, BMP4, BRCA1, CA2, CALB2, CARS, CASP2, 33 CDKN1A, CEBPB (includes EG:1051), DDIT3, DUSP6, ELF3, FAS, GAS6, GDF15, HERPUD1, HSPA5, IL8, IRF9, LGALS3, MAPK3, MSH2, NBN, NDRG1, NEK3, NFIL3, PLAU, PRKACB, RRM2B, S100A4, STX8, ZFP36 Cell Death survival survival of mesangial cells 2.27E-02 FAS 1 Cell Death survival survival of pro-B lymphocytes 2.27E-02 NFIL3 1 Cell Death fragmentation fragmentation of genomic DNA 7.24E-03 FAS, STAT1 2 Cell Death fragmentation fragmentation of DNA 2.09E-02 BIK, CASP2, CDKN1A, FAS, S100A4, STAT1 6 Cell Death colony survival colony survival of endometrial 2.27E-02 MSH2 1 cancer cell lines Cell Death cytolysis cytolysis of carcinoma cell 2.27E-02 TNFRSF10B 1 lines Cell Death cytolysis cytolysis of lung cancer cell 2.27E-02 TNFRSF10B 1 lines Cell Death cytotoxicity cytotoxicity of melanoma cell 2.27E-02 FAS 1 lines Cell Death maturation maturation of apoptotic bodies 2.27E-02 CASP2 1 Cell Cycle premature premature senescence of tumor 6.78E-07 BMP4, CDKN1A, DDB2, EHF, ID1 5 senescence cell lines Cell Cycle premature premature senescence of 2.98E-03 BMP4, CDKN1A 2 senescence carcinoma cell lines Cell Cycle premature premature senescence of lung 2.98E-03 BMP4, CDKN1A 2 senescence cancer cell lines Cell Cycle cell cycle arrest in cell cycle progression 1.05E-06 BMP4, BRCA1, CDC25A, CDKN1A, CEBPB (includes 18 progression EG:1051), EHF, FOXO4, GADD45A, ID1, ID3 (includes EG:15903), IL8, IRF7, LGALS3, NBN, PLAGL1, S100A4, STAT1, TP53INP1 Cell Cycle cell cycle cell cycle progression 4.32E-04 ASNS, ATF3, BMP4, BRCA1, CASP2, CDC25A, CDC25C, 30 progression CDCA5, CDKN1A, CEBPB (includes EG:1051), DDB2, DTL, EHF, FAS, FBXO5, FOXO4, GADD45A, ID1, ID3 (includes EG:15903), IER3, IL8, IRF7, LGALS3, MAPK3, MECOM,

493

NBN, PLAGL1, S100A4, STAT1, TP53INP1 Cell Cycle cell cycle arrest in cell cycle progression 1.95E-03 BMP4, BRCA1, CDKN1A, CEBPB (includes EG:1051), EHF, 8 progression of tumor cell lines LGALS3, PLAGL1, S100A4 Cell Cycle cell cycle cell cycle progression of cancer 2.79E-03 CDKN1A, FAS, GADD45A 3 progression cells Cell Cycle cell cycle cell cycle progression of tumor 3.19E-03 BMP4, BRCA1, CASP2, CDC25C, CDKN1A, CEBPB (includes 11 progression cell lines EG:1051), EHF, IER3, LGALS3, PLAGL1, S100A4 Cell Cycle cell cycle delay in cell cycle progression 7.24E-03 CASP2, CDKN1A 2 progression of lymphoblastoid cell lines Cell Cycle cell cycle cell cycle progression of 2.26E-02 CDC25C, CDKN1A, IER3 3 progression cervical cancer cell lines Cell Cycle cell cycle arrest in cell cycle progression 2.27E-02 BMP4 1 progression of myeloma cell lines Cell Cycle cell cycle arrest in cell cycle progression 2.27E-02 GADD45A 1 progression of ovarian cancer cells Cell Cycle cell cycle arrest in cell cycle progression 2.27E-02 S100A4 1 progression of pancreatic cancer cell lines Cell Cycle cell cycle arrest in cell cycle progression 2.27E-02 CDKN1A 1 progression of synovial fibroblasts Cell Cycle cell cycle cell cycle progression of colon 2.27E-02 CDKN1A 1 progression carcinoma cells Cell Cycle cell cycle delay in cell cycle progression 2.27E-02 CDKN1A 1 progression of colon cancer cell lines Cell Cycle interphase interphase of fibroblasts 1.91E-06 CCNE2, CDC25C, CDKN1A, GADD45A, ID3 (includes 6 EG:15903), NBN Cell Cycle interphase interphase 5.17E-05 BMP4, BRCA1, CCNE2, CDC25A, CDC25C, CDCA5, 26 CDKN1A, DDIT3, DTL, FBXO5, FOXO4, GADD45A, GDF15, ID1, ID3 (includes EG:15903), KDM5B, KLF4, LGALS1, LGALS3, MCM10 (includes EG:307126), MSH2, MXD4, NBN, PLAU, RRM2B, STAT1 Cell Cycle interphase arrest in interphase of breast 7.20E-05 BRCA1, CDC25A, CDC25C, CDKN1A, GDF15, LGALS1, 7 cancer cell lines LGALS3 Cell Cycle interphase arrest in interphase of 1.58E-04 CDC25C, CDKN1A, GADD45A, NBN 4 fibroblasts Cell Cycle interphase interphase of breast cancer cell 2.16E-04 BRCA1, CDC25A, CDC25C, CDKN1A, GDF15, LGALS1, 8

494

lines LGALS3, MSH2 Cell Cycle interphase arrest in interphase 2.44E-04 BMP4, BRCA1, CDC25A, CDC25C, CDKN1A, DDIT3, 17 FBXO5, GADD45A, GDF15, KDM5B, KLF4, LGALS1, LGALS3, MSH2, NBN, PLAU, RRM2B Cell Cycle interphase arrest in interphase of colon 1.08E-03 BRCA1, CDKN1A, GADD45A, KLF4, LGALS1 5 cancer cell lines Cell Cycle interphase arrest in interphase of tumor 1.49E-03 BRCA1, CDC25A, CDC25C, CDKN1A, DDIT3, GADD45A, 13 cell lines GDF15, KLF4, LGALS1, LGALS3, MSH2, PLAU, RRM2B Cell Cycle interphase interphase of colon cancer cell 1.62E-03 BRCA1, CDKN1A, GADD45A, KLF4, LGALS1, MXD4 6 lines Cell Cycle interphase interphase of tumor cell lines 2.05E-03 BRCA1, CDC25A, CDC25C, CDKN1A, DDIT3, GADD45A, 16 GDF15, KLF4, LGALS1, LGALS3, MCM10 (includes EG:307126), MSH2, MXD4, PLAU, RRM2B, STAT1 Cell Cycle interphase interphase of breast cell lines 3.49E-03 CDKN1A, ID1, KDM5B 3 Cell Cycle interphase entry into interphase of 4.90E-03 CDKN1A, ID3 (includes EG:15903) 2 fibroblasts Cell Cycle interphase re-entry into interphase 5.19E-03 CDC25A, CDC25C, CDKN1A 3 Cell Cycle interphase arrest in interphase of cancer 1.66E-02 CDKN1A, LGALS1 2 cells Cell Cycle interphase re-entry into interphase of 2.04E-02 CDC25A, CDC25C 2 tumor cell lines Cell Cycle G2/M phase G2/M phase 3.11E-05 BRCA1, CDC25A, CDC25C, CDKN1A, DTL, GADD45A, 12 KDM5B, LGALS1, MSH2, NBN, PLAU, RRM2B Cell Cycle G2/M phase G2/M phase of tumor cell lines 3.71E-03 BRCA1, CDC25A, CDC25C, CDKN1A, MSH2 5 Cell Cycle G2/M phase G2/M phase of breast cancer 6.20E-03 BRCA1, CDC25C, MSH2 3 cell lines Cell Cycle G2/M phase arrest in G2/M phase 1.14E-02 BRCA1, CDC25C, CDKN1A, KDM5B 4 Cell Cycle G2/M phase arrest in G2/M phase of breast 2.46E-02 BRCA1, CDC25C 2 cancer cell lines Cell Cycle G1 phase G1 phase of breast cancer cell 1.20E-04 BRCA1, CDC25A, CDKN1A, GDF15, LGALS1, LGALS3 6 lines Cell Cycle G1 phase arrest in G1 phase of breast 1.39E-04 BRCA1, CDC25A, GDF15, LGALS1, LGALS3 5 cancer cell lines Cell Cycle G1 phase G1 phase 8.15E-04 BRCA1, CCNE2, CDC25A, CDC25C, CDCA5, CDKN1A, 15 DDIT3, FBXO5, FOXO4, GADD45A, GDF15, KLF4, LGALS1,

495

LGALS3, NBN Cell Cycle G1 phase arrest in G1 phase of colon 2.61E-03 BRCA1, CDKN1A, KLF4, LGALS1 4 cancer cell lines Cell Cycle G1 phase G1 phase of fibroblasts 2.98E-03 CCNE2, CDKN1A 2 Cell Cycle G1 phase arrest in G1 phase of tumor cell 7.24E-03 BRCA1, CDC25A, CDKN1A, DDIT3, GDF15, KLF4, LGALS1, 8 lines LGALS3 Cell Cycle G1 phase arrest in G1 phase 1.16E-02 BRCA1, CDC25A, CDKN1A, DDIT3, FBXO5, GDF15, KLF4, 9 LGALS1, LGALS3 Cell Cycle G1 phase G1 phase of tumor cell lines 1.66E-02 BRCA1, CDC25A, CDC25C, CDKN1A, DDIT3, GDF15, KLF4, 9 LGALS1, LGALS3 Cell Cycle G1 phase entry into G1 phase of 2.27E-02 CDKN1A 1 fibroblasts Cell Cycle G1 phase re-entry into G1 phase of 2.27E-02 CDC25C 1 neuroblastoma cell lines Cell Cycle G2/M phase arrest in G2/M phase transition 1.94E-04 CDC25C, CDKN1A, GADD45A, LGALS1, PLAU, RRM2B 6 transition Cell Cycle G2/M phase arrest in G2/M phase transition 1.51E-03 CDKN1A, PLAU 2 transition of brain cancer cell lines Cell Cycle G2/M phase arrest in G2/M phase transition 2.98E-03 CDC25C, GADD45A 2 transition of fibroblasts Cell Cycle G2/M phase arrest in G2/M phase transition 3.01E-03 CDKN1A, GADD45A, PLAU, RRM2B 4 transition of tumor cell lines Cell Cycle G2/M phase initiation of G2/M phase 2.27E-02 CDKN1A 1 transition transition of brain cancer cell lines Cell Cycle S phase S phase of fibroblasts 8.76E-04 CDKN1A, ID3 (includes EG:15903), NBN 3 Cell Cycle S phase S phase 1.29E-03 BRCA1, CDC25A, CDC25C, CDKN1A, FBXO5, ID1, ID3 11 (includes EG:15903), LGALS1, MCM10 (includes EG:307126), MXD4, NBN Cell Cycle S phase arrest in S phase 5.03E-03 BRCA1, CDKN1A, LGALS1, NBN 4 Cell Cycle S phase exit from S phase of colon 7.24E-03 CDKN1A, MXD4 2 cancer cell lines Cell Cycle S phase S phase of breast cell lines 9.98E-03 CDKN1A, ID1 2 Cell Cycle S phase entry into S phase 1.78E-02 CDC25A, CDKN1A, FBXO5, ID3 (includes EG:15903), MCM10 5

496

(includes EG:307126) Cell Cycle S phase arrest in S phase of fibroblasts 2.27E-02 NBN 1 Cell Cycle S phase re-entry into S phase of 2.27E-02 CDKN1A 1 fibroblasts Cell Cycle G2 phase arrest in G2 phase 8.80E-04 BRCA1, CDC25C, CDKN1A, GADD45A, KDM5B, LGALS1, 9 MSH2, PLAU, RRM2B Cell Cycle G2 phase arrest in G2 phase of fibroblasts 1.23E-03 CDC25C, CDKN1A, GADD45A 3 Cell Cycle G2 phase G2 phase of tumor cell lines 1.71E-03 BRCA1, CDC25A, CDC25C, CDKN1A, GADD45A, MSH2, 8 PLAU, RRM2B Cell Cycle G2 phase arrest in G2 phase of tumor cell 2.74E-03 BRCA1, CDC25C, CDKN1A, GADD45A, MSH2, PLAU, 7 lines RRM2B Cell Cycle G2 phase arrest in G2 phase of colon 2.79E-03 BRCA1, CDKN1A, GADD45A 3 cancer cell lines Cell Cycle G2 phase G2 phase of cervical cancer cell 1.29E-02 CDC25A, CDC25C, GADD45A 3 lines Cell Cycle G2 phase delay in G2 phase of colon 2.27E-02 CDKN1A 1 carcinoma cells Cell Cycle G2 phase re-entry into G2 phase of 2.27E-02 CDC25C 1 cervical cancer cell lines Cell Cycle G1/S phase transition arrest in G1/S phase transition 1.51E-03 CDKN1A, KLF4 2 of colon cancer cell lines Cell Cycle G1/S phase transition arrest in G1/S phase transition 1.83E-02 CDC25A, CDKN1A, KLF4 3 of tumor cell lines Cell Cycle G1/S phase transition G1/S phase 2.19E-02 CCNE2, CDC25A, CDCA5, CDKN1A, GADD45A, KLF4, NBN 7 Cell Cycle mitosis mitosis of breast cancer cell 3.49E-03 BMP4, BRCA1, CDKN1A 3 lines Cell Cycle mitosis entry into mitosis 7.31E-03 CDC25A, CDC25C, CDKN1A 3 Cell Cycle mitosis mitosis of tumor cell lines 1.01E-02 BMP4, BRCA1, CDC25A, CDC25C, CDKN1A, FBXO5 6 Cell Cycle mitosis entry into mitosis of cervical 1.66E-02 CDC25A, CDC25C 2 cancer cell lines Cell Cycle mitosis delay in mitosis of breast 2.27E-02 BRCA1 1 cancer cell lines Cell Cycle senescence senescence of breast cancer cell 1.31E-02 EHF, ID1 2 lines

497

Cell Cycle senescence senescence of cells 1.56E-02 BMP4, BRCA1, CDKN1A, DDB2, EHF, ID1 6 Cell Cycle DNA damage DNA damage checkpoint 1.46E-02 FBXO6, NBN, RPS27L 3 checkpoint Cell Cycle G0 phase arrest in G0 phase of fibroblasts 2.27E-02 CDKN1A 1 Cell Cycle polyploidy polyploidy of bladder cancer 2.27E-02 NDRG1 1 cell lines Cell Cycle polyploidy polyploidy of colon cancer cell 2.27E-02 NDRG1 1 lines Cell Cycle polyploidy polyploidy of mammary cells 2.27E-02 NDRG1 1 Cell Morphology transmembrane transmembrane potential of 7.21E-06 BARD1, BID, CASP2, CDKN1A, CHMP5, DDIT3, FAS, 13 potential mitochondria HERPUD1, LGALS1, NDRG1, PINK1, PMAIP1, TNFRSF10B Cell Morphology blebbing blebbing of fibroblast cell lines 5.12E-04 FAS, TNFRSF10B 2 Cell Morphology blebbing blebbing of kidney cell lines 2.98E-03 FAS, TNFRSF10B 2 Cell Morphology autophagy autophagy of breast cancer cell 1.66E-03 ATF4, BRCA1, TNFRSF10B 3 lines Cell Morphology autophagy autophagy of tumor cell lines 5.47E-03 ATF4, BNIP3L, BRCA1, FAS, PINK1, TNFRSF10B, ULK1 7 Cell Morphology autophagy autophagy of hepatoma cell 9.98E-03 FAS, TNFRSF10B 2 lines Cell Morphology autophagy autophagy of prostate cancer 9.98E-03 BNIP3L, TNFRSF10B 2 cell lines Cell Morphology mitochondrial mitochondrial membrane 1.66E-03 FAS, PMAIP1, TNFRSF10B 3 membrane potential potential Cell Morphology permeability permeability of endothelial 5.19E-03 IL8, PLAU, PROCR 3 cells Cell Morphology permeability permeability of vascular 2.46E-02 IL8, PLAU 2 endothelial cells Cell Morphology morphology morphology of mitochondria 9.88E-03 BID, PINK1, PMAIP1 3 Cell Morphology morphology morphology of bladder cancer 2.27E-02 CDKN1A 1 cell lines Cell Morphology morphology morphology of microtubules 2.27E-02 CHMP1B 1 Cell Morphology morphology morphology of ovarian cancer 2.27E-02 PLAGL1 1 cells Cell Morphology tubulation tubulation of cells 1.40E-02 BMP4, CLIC4, ID1, ID3 (includes EG:15903), IL8 5

498

Cell Morphology budding budding of cells 2.27E-02 BMP4 1 Cell Morphology budding budding of mitochondria 2.27E-02 LGALS1 1 Cell Morphology depolarization depolarization of leukemia cell 2.27E-02 FAS 1 lines Cell Morphology height height of colon cancer cell lines 2.27E-02 BST2 1 Cell Morphology micronucleation micronucleation of breast 2.27E-02 BRCA1 1 cancer cell lines Cell Morphology shape shape of neutrophils 2.27E-02 IL8 1 Cell Morphology shrinkage shrinkage of leukemia cell lines 2.27E-02 FAS 1 Cell Morphology structural integrity structural integrity of 2.27E-02 LGALS3 1 mitochondria Cellular Function and transmembrane transmembrane potential of 7.21E-06 BARD1, BID, CASP2, CDKN1A, CHMP5, DDIT3, FAS, 13 Maintenance potential mitochondria HERPUD1, LGALS1, NDRG1, PINK1, PMAIP1, TNFRSF10B Cellular Function and endoplasmic endoplasmic reticulum stress 2.98E-05 ATF4, ATF6, DDIT3, HSPA5 4 Maintenance reticulum stress response of cervical cancer cell response lines Cellular Function and endoplasmic endoplasmic reticulum stress 5.19E-05 ATF4, ATF6, DDIT3, HSPA5, SLC38A2 5 Maintenance reticulum stress response of cells response Cellular Function and autophagy autophagy of breast cancer cell 1.66E-03 ATF4, BRCA1, TNFRSF10B 3 Maintenance lines Cellular Function and autophagy autophagy of tumor cell lines 5.47E-03 ATF4, BNIP3L, BRCA1, FAS, PINK1, TNFRSF10B, ULK1 7 Maintenance Cellular Function and autophagy autophagy of hepatoma cell 9.98E-03 FAS, TNFRSF10B 2 Maintenance lines Cellular Function and autophagy autophagy of prostate cancer 9.98E-03 BNIP3L, TNFRSF10B 2 Maintenance cell lines Cellular Function and mitochondrial mitochondrial membrane 1.66E-03 FAS, PMAIP1, TNFRSF10B 3 Maintenance membrane potential potential Cellular Function and permeability permeability of cellular 2.18E-03 FAS, IL8, PMAIP1 3 Maintenance membrane Cellular Function and permeability permeability of endothelial 5.19E-03 IL8, PLAU, PROCR 3 Maintenance cells Cellular Function and permeability permeability of vascular 2.46E-02 IL8, PLAU 2

499

Maintenance endothelial cells Cellular Function and homeostasis cellular homeostasis 4.22E-03 ATF4, BARD1, BID, BMP4, BNIP3L, BRCA1, CASP2, 23 Maintenance CDKN1A, CHMP5, DDIT3, F2RL1, FAS, HERPUD1, IL8, LGALS1, NDRG1, PINK1, PLAU, PMAIP1, PROCR, RGS2 (includes EG:19735), TNFRSF10B, ULK1 Cellular Function and function function of mitochondria 4.90E-03 PINK1, SACS 2 Maintenance Cellular Function and function function of natural killer cells 2.27E-02 CLEC2D 1 Maintenance Cellular Function and efflux efflux of inorganic cation 9.98E-03 F2RL1, FAS 2 Maintenance Cellular Function and efflux efflux of K+ 2.27E-02 FAS 1 Maintenance Cellular Function and colony survival colony survival of endometrial 2.27E-02 MSH2 1 Maintenance cancer cell lines Cellular Function and depolarization depolarization of leukemia cell 2.27E-02 FAS 1 Maintenance lines Cellular Function and differentiation arrest in differentiation of 2.27E-02 BMP4 1 Maintenance thymocytes Cellular Function and disappearance disappearance of astral 2.27E-02 NDRG1 1 Maintenance microtubules Cellular Function and endocytosis endocytosis by keratinocytes 2.27E-02 F2RL1 1 Maintenance Cellular Function and morphology morphology of microtubules 2.27E-02 CHMP1B 1 Maintenance Cellular Function and preservation preservation of cells 2.27E-02 CDKN1A 1 Maintenance Cellular Function and production production of olfactory receptor 2.27E-02 BMP4 1 Maintenance neurons Cellular Compromise endoplasmic endoplasmic reticulum stress 2.98E-05 ATF4, ATF6, DDIT3, HSPA5 4 reticulum stress response of cervical cancer cell response lines Cellular Compromise endoplasmic endoplasmic reticulum stress 5.19E-05 ATF4, ATF6, DDIT3, HSPA5, SLC38A2 5 reticulum stress response of cells response

500

Cellular Compromise stress response stress response of cells 3.33E-05 ALDH3A2, ATF4, ATF6, CDKN1A, DDIT3, HSPA5, SLC38A2 7 Cellular Compromise stress response stress response of tumor cell 1.73E-04 ATF4, ATF6, CDKN1A, DDIT3, HSPA5 5 lines Cellular Compromise stress response stress response of embryonic 1.31E-02 ALDH3A2, SLC38A2 2 cell lines Cellular Compromise stress response stress response of epithelial cell 1.31E-02 ALDH3A2, SLC38A2 2 lines Cellular Compromise stress response stress response of kidney cell 2.04E-02 ALDH3A2, SLC38A2 2 lines Cellular Compromise damage damage of mitochondria 1.66E-03 CASP2, CDKN1A, LGALS3 3 Cellular Compromise condensation condensation of cytoplasm 2.27E-02 FAS 1 Cellular Compromise destabilization destabilization of myosin 2.27E-02 S100A4 1 filaments Cellular Compromise disappearance disappearance of astral 2.27E-02 NDRG1 1 microtubules Cellular Compromise disassembly disassembly of microfilaments 2.27E-02 FAS 1 Cellular Compromise disintegration disintegration of nucleoli 2.27E-02 FAS 1 Cellular Compromise micronucleation micronucleation of breast 2.27E-02 BRCA1 1 cancer cell lines Cellular Compromise oxidative stress oxidative stress response of 2.27E-02 ALDH3A2 1 response embryonic cell lines Cellular Compromise oxidative stress oxidative stress response of 2.27E-02 ALDH3A2 1 response epithelial cell lines Cellular Compromise shrinkage shrinkage of leukemia cell lines 2.27E-02 FAS 1

Appendix 12. 3 The top molecular and cellular functions associated with differentially expressed genes in the FPGS- overexpressed MDA-MB-435 breast cancer cells No. of Category Function Function Annotation P-value Genes Genes Cell Death cell death cell death 5.08E-11 ABCC4 (includes EG:10257), ABCC5, ADM, AGTRAP, AHR, 338 AKAP12, AKR1B1, ALDH1A1, ALDH3B1, ALKBH8, ANKRD1, ANTXR1, ANTXR2, APIP, ARMC10, ASAH1,

501

ATF3, ATG12 (includes EG:361321), ATG4B, ATP7A, AXL, B2M, B4GALT5, BAD, BAG3, BCHE, BCL2A1, BCL2L1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BLID, BNIP3L, BTG1, C15orf63, C5, C9orf80, CABLES1, CADM1, CALB2, CAMK2D, CAMK2N1, CAPN3, CARS, CASP1, CASP2, CASP4, CASP9 (includes EG:100140945), CBL, CCL2, CCND1, CCND3, CD44 (includes EG:100330801), CD55, CD74, CD9, CD96, CDC45, CDK2, CDK20, CDK2AP1, CDK5, CDK5R1, CDKN1B, CDKN2A, CDKN2C, CDKN2D, CEBPD, CERS6, CIB1, CKAP5, CLCF1, CNN2, CUL4B, CUL9, CYB5A (includes EG:109672), CYCS, CYP2J2, CYR61, DAP, DCT, DCTN3, DDIT3, DDIT4, DEFB1 (human), DEFB103A/DEFB103B, DEK, DEPTOR, DKK1, DLC1, DLST, DNAJB2, DPF2, DUSP19, DUSP5, DUT, E2F3, EDNRB, EGR1, EMP1, EMP3, EPAS1, ERCC5, EXOC2, FASTK, FHL2, FKBP1A, FKBP5, FOS, FOSB, FOXM1, FST, GADD45A, GADD45B, GCLC, GLO1, GLRX, GPC1, GPI, GPR37, GPR65, GSTM1, HBEGF, HDAC1, HDAC9, HERPUD1, HIST1H1C, HK2, HLA-DRB4, HOXA5, HOXC6, HSD17B10, HSP90AB1, HSPB8, HTATIP2, HTRA1, ID1, ID2, ID3 (includes EG:15903), ID4, IER3, IFI6, IFIH1, IFNAR2, IGFBP6, IGFBP7, IKBIP, IL24, IL4R, IL7, IL7R, IL8, ILK, ING3, IRF1 (includes EG:16362), IRF4, IRF9, IRS1, ISG15, ITGA5, ITGB1, ITPK1, JUN, KCNMA1, KLF4, KLF6, KLF9, LAMP1, LAMP2, LAPTM4B, LEPR, LGALS1, LGALS3, LPAR1, LRPAP1, MAOA, MCM10 (includes EG:307126), MECOM, MED14, MET, MINPP1, MITF, MLLT11, MSRB2, MT1X, MT2A, MTDH, MTFP1, MUC1, MVP, MX1, MYB, NAA35, NAMPT, NBN, NCOA3, NFATC1, NFIL3, NFKB2, NFKBIB, NPC1, NQO1, NR4A2, NRP1 (includes EG:18186), NT5E, NTN4, NUAK1, NUPR1, ODC1, OPA1, PAK2, PARVA, PAXIP1, PBK, PCBP2, PCGF2, PCNA, PEA15, PHB, PHLDA1, PHLDA2, PIK3C3, PKM2, PLAUR, PLSCR1, PMAIP1, PPAP2A, PPM1M, PPP1R15A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R4, PPP2R5C, PPT1, PRDX3, PRKACB, PRKAR1A, PRKCZ, PRKDC, PRKRIR, PRMT2, PRUNE2, PSMA1, PSME3, PTGR1, PTGS1, PTN, PTPN22, PTPRM, RAB32, RAC2, RASSF1, RBM17, RELB, REPS2, RHOT2, RPS6KA3, RSF1 (includes EG:233532), S100A1, S100A4, SAT1, SCARB1, SCG2, SDC1 (includes EG:20969), SEMA3A, SERPINB2, SGK1, SH3BGRL3, SH3RF1, SHC1 (includes EG:20416), SHFM1, SIRPA, SIVA1, SKP2 (includes EG:27401), SLC11A2, SLC29A1, SLC29A2, SLC3A2, SLC5A8, SNAI2,

502

SNCA, SND1, SOD2, SORT1, SOX4, SPARC, SPC25 (includes EG:100144563), SPHK1, SPP1 (includes EG:20750), SPRY2, SRPK1, SSTR2, STAT1, STAT3, STC1, STMN1, SUB1 (includes EG:10923), SYF2 (includes EG:170933), TFAP2A, TFAP2C, TGFA, TGFB1I1, TGFBR3, THBS2, TICAM1, TIMP3, TMED10, TMSB10/TMSB4X, TMX1, TNFRSF10B, TNFRSF21, TNFSF13B, TPD52, TPD52L1, TPK1, TPMT, TRIB2, TSG101, TXNDC5, TYMP, TYMS, UBA7, UBQLN1, URI1, USP11, VHL, WAS, XAF1, XBP1 (includes EG:140614), YWHAZ, ZAK, ZFP36, ZFYVE16, ZMYM2, ZNF443 Cell Death cell death cell death of tumor cell lines 1.78E-10 ABCC4 (includes EG:10257), ADM, AKAP12, AKR1B1, 201 ANKRD1, ARMC10, ATF3, ATG12 (includes EG:361321), B2M, BAD, BAG3, BCHE, BCL2A1, BCL2L1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BNIP3L, BTG1, C15orf63, C5, C9orf80, CABLES1, CASP1, CASP2, CASP4, CASP9 (includes EG:100140945), CCL2, CCND1, CCND3, CD44 (includes EG:100330801), CD55, CDK5, CDK5R1, CDKN1B, CDKN2A, CDKN2C, CDKN2D, CEBPD, CERS6, CIB1, CNN2, CUL9, CYB5A (includes EG:109672), CYCS, CYP2J2, CYR61, DCT, DCTN3, DDIT3, DDIT4, DEFB1 (human), DEK, DKK1, DUT, E2F3, EGR1, EPAS1, EXOC2, FKBP1A, FKBP5, FOS, FOSB, FST, GADD45A, GADD45B, GLO1, GLRX, GPC1, GPI, GPR37, GPR65, GSTM1, HBEGF, HDAC1, HK2, HLA-DRB4, HOXA5, HOXC6, HSP90AB1, HSPB8, HTATIP2, HTRA1, ID1, ID3 (includes EG:15903), IER3, IFI6, IFNAR2, IGFBP6, IGFBP7, IL24, IL7, IL8, ILK, IRF1 (includes EG:16362), IRF4, ITGB1, ITPK1, JUN, KLF4, KLF6, KLF9, LAMP1, LAMP2, LGALS1, LGALS3, MAOA, MCM10 (includes EG:307126), MET, MITF, MSRB2, MT1X, MT2A, MTFP1, MUC1, MYB, NAA35, NAMPT, NCOA3, NFKB2, NFKBIB, NQO1, NR4A2, NRP1 (includes EG:18186), NUAK1, NUPR1, PAK2, PARVA, PBK, PCBP2, PCNA, PEA15, PHB, PIK3C3, PKM2, PLAUR, PMAIP1, PPP1R15A, PPP2R2A, PPT1, PRKAR1A, PRKCZ, PRKDC, RAB32, RASSF1, RBM17, RELB, REPS2, S100A4, SAT1, SDC1 (includes EG:20969), SEMA3A, SGK1, SH3RF1, SHC1 (includes EG:20416), SIRPA, SIVA1, SKP2 (includes EG:27401), SLC11A2, SLC29A1, SLC29A2, SLC3A2, SLC5A8, SNCA, SND1, SOD2, SOX4, SPARC, SPC25 (includes EG:100144563), SPHK1, SPP1 (includes EG:20750), SPRY2, SRPK1, SSTR2, STAT1, STAT3, STMN1, TFAP2A, TFAP2C, TGFA, TGFBR3, TICAM1, TIMP3, TMED10, TMSB10/TMSB4X, TNFRSF10B, TNFRSF21, TNFSF13B, TPD52, TSG101, TYMP, TYMS, UBA7, UBQLN1, VHL,

503

XAF1, XBP1 (includes EG:140614), YWHAZ, ZFYVE16 Cell Death cell death cell death of melanoma cell 1.92E-06 BIRC2, CASP4, CASP9 (includes EG:100140945), CDKN1B, 23 lines CDKN2A, DCT, DDIT3, DEK, EGR1, GADD45A, GSTM1, HDAC1, HSPB8, IL24, IL8, PBK, PMAIP1, PPP1R15A, SAT1, SPHK1, STAT3, TNFRSF10B, XAF1 Cell Death cell death cell death of breast cancer cell 2.18E-05 ABCC4 (includes EG:10257), ADM, B2M, BAD, BAG3, 53 lines BCL2L1, BIRC2, BIRC3, BNIP3L, CASP1, CASP2, CASP9 (includes EG:100140945), CCND1, CD44 (includes EG:100330801), CYB5A (includes EG:109672), CYCS, CYR61, DDIT4, EXOC2, GADD45A, HK2, HOXA5, HSPB8, IER3, IL24, ILK, JUN, LGALS3, MUC1, NCOA3, NQO1, PEA15, PHB, PIK3C3, PMAIP1, PRKDC, SDC1 (includes EG:20969), SH3RF1, SIVA1, SLC29A1, SLC5A8, SND1, SOD2, SPHK1, SRPK1, STAT1, STMN1, TFAP2A, TFAP2C, TGFBR3, TNFRSF10B, TYMS, XBP1 (includes EG:140614) Cell Death cell death cell death of prostate cancer 2.36E-05 AKAP12, BAD, BCL2L1, BIRC2, BIRC3, CASP1, CASP9 36 cell lines (includes EG:100140945), CCL2, CDK5, CDK5R1, CDKN2A, DDIT3, EGR1, FOS, FST, GADD45A, HBEGF, HOXC6, HSPB8, ID1, IGFBP7, IL24, ITGB1, LGALS1, MET, NCOA3, NFKB2, PLAUR, PMAIP1, SH3RF1, STAT3, TGFA, TMED10, TNFRSF10B, TPD52, TYMP Cell Death cell death cell death of epithelial cell 6.22E-05 ALDH3B1, BAD, BCL2A1, BCL2L1, BCL6, BIRC2, BIRC3, 42 lines BNIP3L, CASP1, CASP2, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CDKN2A, CUL4B, CYCS, DLST, EMP1, EMP3, GPR37, HK2, HSPB8, IER3, IL24, IRF1 (includes EG:16362), IRS1, ITGB1, MET, NAMPT, PAK2, PEA15, PMAIP1, PPP1R15A, PRDX3, PRKDC, PRMT2, RASSF1, SPHK1, SPP1 (includes EG:20750), TGFB1I1, TICAM1, TMX1, TNFRSF10B Cell Death cell death cell death of cervical cancer 1.49E-04 BAD, BCL2A1, BCL2L1, BIRC2, BNIP3L, C15orf63, C9orf80, 51 cell lines CASP1, CASP2, CASP4, CCND3, CDKN1B, CDKN2A, CDKN2C, CIB1, DCTN3, DDIT3, EXOC2, FOS, FST, GADD45A, GADD45B, IER3, IRF1 (includes EG:16362), ITPK1, JUN, MCM10 (includes EG:307126), MUC1, NAA35, NR4A2, PAK2, PARVA, PCBP2, PIK3C3, PKM2, PPP2R2A, RAB32, RASSF1, RBM17, SGK1, SNCA, SOD2, SPC25 (includes EG:100144563), SPP1 (includes EG:20750), STAT1, TFAP2A, TICAM1, TIMP3, TNFRSF10B, TNFRSF21, UBQLN1 Cell Death cell death cell death of epithelial cells 2.53E-04 ALDH1A1, ALDH3B1, BAD, BCL2A1, BCL2L1, BCL6, 46

504

BIRC2, BIRC3, BNIP3L, CASP1, CASP2, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CDKN2A, CUL4B, CYCS, DLST, EMP1, EMP3, GPR37, HK2, HSPB8, IER3, IL24, IL8, IRF1 (includes EG:16362), IRS1, ITGB1, MET, NAMPT, PAK2, PEA15, PLAUR, PMAIP1, PPP1R15A, PRDX3, PRKDC, PRMT2, RASSF1, SPHK1, SPP1 (includes EG:20750), TGFB1I1, TICAM1, TIMP3, TMX1, TNFRSF10B Cell Death cell death cell death of embryonic cell 3.05E-04 ALDH3B1, BAD, BCL2A1, BCL2L1, BCL6, BIRC2, BIRC3, 39 lines BNIP3L, CASP1, CASP2, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CUL4B, CYCS, DLST, EMP1, EMP3, GPR37, HK2, IER3, IL24, IRF1 (includes EG:16362), IRS1, ITGB1, NAMPT, PAK2, PEA15, PMAIP1, PPP1R15A, PRDX3, PRKDC, PRMT2, RASSF1, SPHK1, SPP1 (includes EG:20750), TGFB1I1, TICAM1, TMX1, TNFRSF10B Cell Death cell death cell death of kidney cells 3.76E-04 ALDH3B1, BAD, BCL2A1, BCL2L1, BCL6, BIRC2, BIRC3, 42 BLID, BNIP3L, CASP1, CASP2, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CDKN1B, CUL4B, CYCS, DLST, EMP1, EMP3, GPR37, HK2, IER3, IL24, IRF1 (includes EG:16362), IRS1, ITGB1, NAMPT, PAK2, PEA15, PMAIP1, PPP1R15A, PRDX3, PRKDC, PRMT2, RASSF1, SOD2, SPHK1, SPP1 (includes EG:20750), TGFB1I1, TICAM1, TMX1, TNFRSF10B Cell Death cell death cell death of muscle 4.36E-04 ADM, APIP, BCL2L1, CASP1, CASP9 (includes 15 EG:100140945), CYCS, HSPB8, IL8, PMAIP1, PTN, S100A1, STAT3, TIMP3, TNFRSF10B, XAF1 Cell Death cell death cell death of kidney cell lines 8.21E-04 ALDH3B1, BAD, BCL2A1, BCL2L1, BCL6, BIRC2, BIRC3, 40 BLID, BNIP3L, CASP1, CASP2, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CUL4B, CYCS, DLST, EMP1, EMP3, GPR37, HK2, IER3, IL24, IRF1 (includes EG:16362), IRS1, ITGB1, NAMPT, PAK2, PEA15, PMAIP1, PPP1R15A, PRDX3, PRKDC, PRMT2, RASSF1, SPHK1, SPP1 (includes EG:20750), TGFB1I1, TICAM1, TMX1, TNFRSF10B Cell Death cell death cell death of muscle cells 9.55E-04 ADM, APIP, BCL2L1, CASP1, CASP9 (includes 14 EG:100140945), CYCS, HSPB8, PMAIP1, PTN, S100A1, STAT3, TIMP3, TNFRSF10B, XAF1 Cell Death cell death cell death of myeloid 1.12E-03 CDKN2A, PCBP2, SLC29A2 3 progenitor cells Cell Death cell death cell death of neuroblastoma 1.36E-03 ADM, BAD, BCHE, BCL2L1, CASP4, CASP9 (includes 24 cell lines EG:100140945), CDKN2D, CUL9, CYCS, DDIT3, FKBP1A, GLRX, GPC1, GPR37, HDAC1, ITGB1, MAOA, NFKBIB,

505

PPT1, SLC11A2, SNCA, SPHK1, SPP1 (includes EG:20750), TNFRSF10B Cell Death cell death cell death of tumor cells 2.29E-03 B2M, B4GALT5, BAD, BCL2L1, CASP1, CASP2, CASP9 31 (includes EG:100140945), CCND3, CD74, CDC45, CDK2, CDKN1B, CDKN2A, DDIT3, HBEGF, HSPB8, IL24, IL7, IL8, LGALS1, LPAR1, MUC1, NCOA3, NR4A2, PHLDA1, PMAIP1, SNAI2, STAT3, TFAP2A, TNFRSF10B, TNFSF13B Cell Death cell death cell death of immune cells 3.35E-03 BAD, BCL2L1, BCL6, C5, CASP4, CASP9 (includes 39 EG:100140945), CDKN1B, CDKN2A, DDIT3, HLA-DRB4, IFIH1, IL7, IL7R, IL8, IRF4, ITGB1, JUN, KLF4, LEPR, LGALS1, LGALS3, MVP, MX1, NAMPT, NBN, NFATC1, NFIL3, PCBP2, PMAIP1, PPP2R2B, PRKCZ, RAC2, SHC1 (includes EG:20416), SIVA1, SLC29A2, SOD2, SPP1 (includes EG:20750), STAT3, TNFSF13B Cell Death cell death cell death of leukocyte cell 3.50E-03 BAD, BCL2L1, CASP9 (includes EG:100140945), DDIT3, HLA- 13 lines DRB4, IFIH1, IRF4, JUN, LGALS1, LGALS3, NBN, PRKCZ, SHC1 (includes EG:20416) Cell Death cell death cell death of hematopoietic cell 7.27E-03 BAD, BCL2L1, BIRC3, CASP9 (includes EG:100140945), 15 lines DDIT3, HLA-DRB4, IFIH1, IRF4, JUN, LGALS1, LGALS3, NBN, PRKCZ, SHC1 (includes EG:20416), TRIB2 Cell Death cell death cell death of ovarian cancer 1.04E-02 BCL2L1, BIRC2, CASP1, CASP9 (includes EG:100140945), 18 cell lines CUL9, DDIT3, HTRA1, IL8, KLF6, PMAIP1, PRKDC, RBM17, SLC3A2, SPARC, TGFA, TMSB10/TMSB4X, TNFRSF10B, TSG101 Cell Death cell death cell death of fibrosarcoma cell 1.04E-02 BCL2A1, BCL2L1, BIRC2, BIRC3, IFNAR2, NAMPT, NFKB2, 11 lines PMAIP1, SOD2, STAT1, TNFRSF10B Cell Death cell death cell death of connective tissue 1.12E-02 ADM, ATF3, BCL2L1, BIRC2, BIRC3, BLID, CASP4, CASP9 22 cells (includes EG:100140945), CD44 (includes EG:100330801), CDKN1B, CYR61, GLO1, GPI, IFNAR2, ITGB1, LRPAP1, NBN, NFKB2, NPC1, SNAI2, TIMP3, TNFRSF10B Cell Death cell death cell death of fibroblast cell 1.53E-02 BCL2L1, BLID, CASP9 (includes EG:100140945), CDKN1B, 10 lines CYR61, IFNAR2, ITGB1, NBN, NFKB2, TNFRSF10B Cell Death cell death cell death of pancreatic cancer 1.86E-02 BCL2L1, C5, CDKN2A, DDIT3, EXOC2, GLRX, IL24, IL8, 11 cell lines S100A4, SSTR2, TNFRSF10B Cell Death cell death cell death of cancer cells 2.27E-02 B2M, B4GALT5, BCL2L1, CASP1, CASP2, CASP9 (includes 24 EG:100140945), CD74, CDC45, CDKN1B, DDIT3, HSPB8, IL24, IL7, IL8, LGALS1, LPAR1, MUC1, NCOA3, NR4A2, PHLDA1, PMAIP1, TFAP2A, TNFRSF10B, TNFSF13B

506

Cell Death cell death cell death of myeloma cell 2.42E-02 B2M, BCL2L1, CASP9 (includes EG:100140945), CDKN1B, 12 lines HDAC1, HLA-DRB4, IFI6, IRF4, KLF9, PMAIP1, RELB, SDC1 (includes EG:20969) Cell Death cell death cell death of leukemia cell lines 2.77E-02 B2M, BAD, BCL2A1, BCL2L1, BIRC3, BTG1, C5, CASP2, 38 CASP9 (includes EG:100140945), CD55, CDKN1B, CDKN2A, CEBPD, CYCS, DDIT3, FKBP5, GLO1, HLA-DRB4, IL7, ITGB1, JUN, KLF4, LGALS1, LGALS3, MET, MSRB2, MUC1, PAK2, PRKCZ, SHC1 (includes EG:20416), SLC29A2, SOD2, SPHK1, STMN1, TGFA, TNFRSF10B, TNFSF13B, UBA7 Cell Death cell death initiation of cell death 3.31E-02 BAD, BNIP3L, CASP4, CDKN1B, CDKN2A, CDKN2C, 20 CYCS, DLC1, DPF2, EXOC2, HTATIP2, IKBIP, MX1, PMAIP1, PRMT2, PRUNE2, SIVA1, TNFRSF10B, TPD52L1, ZNF443 Cell Death cell death cell death of lymphoma cell 3.87E-02 BCL2L1, BIRC3, CASP2, CASP4, CASP9 (includes 21 lines EG:100140945), CD55, CNN2, DUT, GLO1, GPR65, HLA- DRB4, LGALS1, LGALS3, NFKB2, PMAIP1, PRKCZ, SOD2, STAT3, TNFRSF10B, XBP1 (includes EG:140614), YWHAZ Cell Death apoptosis apoptosis 1.10E-09 ADM, AHR, AKAP12, AKR1B1, ALDH1A1, ANKRD1, APIP, 250 ARMC10, ASAH1, ATF3, AXL, B2M, B4GALT5, BAD, BAG3, BCL2A1, BCL2L1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BLID, BNIP3L, BTG1, C15orf63, C5, CAPN3, CASP1, CASP2, CASP4, CASP9 (includes EG:100140945), CBL, CCND1, CCND3, CD44 (includes EG:100330801), CD55, CD74, CDC45, CDK2, CDK5, CDK5R1, CDKN1B, CDKN2A, CDKN2C, CDKN2D, CEBPD, CERS6, CIB1, CLCF1, CNN2, CUL4B, CUL9, CYB5A (includes EG:109672), CYCS, CYP2J2, CYR61, DAP, DCT, DDIT3, DDIT4, DEFB1 (human), DEPTOR, DKK1, DLC1, DPF2, DUT, E2F3, EDNRB, EGR1, EPAS1, ERCC5, EXOC2, FASTK, FHL2, FKBP1A, FKBP5, FOS, FOSB, FST, GADD45A, GADD45B, GLO1, GLRX, GPR65, HBEGF, HDAC1, HERPUD1, HIST1H1C, HK2, HOXA5, HOXC6, HSD17B10, HSP90AB1, HSPB8, HTATIP2, HTRA1, ID1, ID3 (includes EG:15903), IER3, IFI6, IFIH1, IFNAR2, IGFBP6, IGFBP7, IKBIP, IL24, IL4R, IL7, IL7R, IL8, ILK, ING3, IRF1 (includes EG:16362), IRF4, IRS1, ITGA5, ITGB1, ITPK1, JUN, KCNMA1, KLF4, KLF6, KLF9, LEPR, LGALS1, LGALS3, LPAR1, LRPAP1, MAOA, MCM10 (includes EG:307126), MECOM, MET, MITF, MLLT11, MSRB2, MT2A, MTDH, MTFP1, MUC1, MX1, MYB, NAMPT, NBN, NCOA3, NFATC1, NFKB2, NFKBIB, NQO1,

507

NR4A2, NRP1 (includes EG:18186), NT5E, NTN4, NUPR1, ODC1, OPA1, PAK2, PARVA, PBK, PCBP2, PCNA, PEA15, PHB, PHLDA1, PHLDA2, PIK3C3, PKM2, PLAUR, PLSCR1, PMAIP1, PPP1R15A, PPP2R2A, PPP2R2B, PPP2R4, PPT1, PRDX3, PRKAR1A, PRKCZ, PRKDC, PRKRIR, PRMT2, PRUNE2, PSME3, PTGS1, PTN, RAB32, RAC2, RASSF1, REPS2, RHOT2, RPS6KA3, S100A1, S100A4, SAT1, SCARB1, SCG2, SDC1 (includes EG:20969), SEMA3A, SGK1, SH3BGRL3, SH3RF1, SHC1 (includes EG:20416), SIRPA, SIVA1, SKP2 (includes EG:27401), SLC5A8, SNAI2, SNCA, SND1, SOD2, SORT1, SOX4, SPARC, SPHK1, SPP1 (includes EG:20750), SPRY2, SRPK1, SSTR2, STAT1, STAT3, STMN1, SUB1 (includes EG:10923), SYF2 (includes EG:170933), TFAP2A, TGFA, TGFBR3, TICAM1, TIMP3, TMED10, TMSB10/TMSB4X, TMX1, TNFRSF10B, TNFRSF21, TNFSF13B, TPD52, TPD52L1, TRIB2, TSG101, TYMP, TYMS, UBA7, UBQLN1, URI1, VHL, XAF1, XBP1 (includes EG:140614), YWHAZ, ZAK, ZFYVE16, ZNF443 Cell Death apoptosis apoptosis of tumor cell lines 5.43E-08 ADM, AKAP12, AKR1B1, ANKRD1, ARMC10, ATF3, B2M, 166 BAD, BAG3, BCL2A1, BCL2L1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BNIP3L, C15orf63, C5, CASP1, CASP2, CASP4, CASP9 (includes EG:100140945), CCND1, CCND3, CD44 (includes EG:100330801), CD55, CDK5, CDK5R1, CDKN1B, CDKN2A, CDKN2C, CDKN2D, CEBPD, CERS6, CNN2, CUL9, CYB5A (includes EG:109672), CYCS, CYP2J2, CYR61, DCT, DDIT3, DDIT4, DEFB1 (human), DKK1, DUT, E2F3, EGR1, EPAS1, EXOC2, FKBP5, FOS, FOSB, FST, GADD45A, GADD45B, GLO1, GLRX, GPR65, HBEGF, HDAC1, HK2, HOXA5, HOXC6, HSP90AB1, HSPB8, HTATIP2, HTRA1, ID1, ID3 (includes EG:15903), IER3, IFI6, IFNAR2, IGFBP6, IGFBP7, IL24, IL7, IL8, ILK, IRF1 (includes EG:16362), IRF4, ITGB1, ITPK1, JUN, KLF4, KLF6, KLF9, LGALS1, LGALS3, MAOA, MCM10 (includes EG:307126), MET, MITF, MSRB2, MT2A, MTFP1, MUC1, MYB, NCOA3, NFKB2, NFKBIB, NQO1, NR4A2, NRP1 (includes EG:18186), NUPR1, PAK2, PARVA, PBK, PCBP2, PCNA, PEA15, PHB, PIK3C3, PLAUR, PMAIP1, PPP1R15A, PPP2R2A, PRKAR1A, PRKCZ, PRKDC, RAB32, RASSF1, REPS2, S100A4, SAT1, SDC1 (includes EG:20969), SEMA3A, SGK1, SH3RF1, SIRPA, SIVA1, SKP2 (includes EG:27401), SLC5A8, SNCA, SND1, SOD2, SOX4, SPARC, SPHK1, SPP1 (includes EG:20750), SPRY2, SRPK1, SSTR2, STAT1, STAT3, STMN1, TGFA, TGFBR3, TICAM1, TIMP3, TMED10,

508

TMSB10/TMSB4X, TNFRSF10B, TNFRSF21, TNFSF13B, TPD52, TSG101, TYMP, TYMS, UBA7, VHL, XAF1, XBP1 (includes EG:140614), YWHAZ, ZFYVE16 Cell Death apoptosis apoptosis of melanoma cell 2.33E-05 BIRC2, CASP4, CASP9 (includes EG:100140945), CDKN1B, 20 lines CDKN2A, DCT, DDIT3, EGR1, GADD45A, HDAC1, HSPB8, IL24, PBK, PMAIP1, PPP1R15A, SAT1, SPHK1, STAT3, TNFRSF10B, XAF1 Cell Death apoptosis apoptosis of breast cancer cell 2.58E-05 ADM, B2M, BAD, BAG3, BCL2L1, BIRC2, BIRC3, BNIP3L, 47 lines CASP1, CASP2, CASP9 (includes EG:100140945), CCND1, CD44 (includes EG:100330801), CYB5A (includes EG:109672), CYCS, CYR61, DDIT4, EXOC2, GADD45A, HK2, HOXA5, HSPB8, IER3, IL24, ILK, JUN, LGALS3, MUC1, NCOA3, NQO1, PEA15, PHB, PIK3C3, PMAIP1, SDC1 (includes EG:20969), SH3RF1, SIVA1, SLC5A8, SND1, SOD2, SPHK1, SRPK1, STAT1, TGFBR3, TNFRSF10B, TYMS, XBP1 (includes EG:140614) Cell Death apoptosis apoptosis of prostate cancer 5.65E-05 AKAP12, BAD, BCL2L1, BIRC2, BIRC3, CASP9 (includes 32 cell lines EG:100140945), CDK5, CDK5R1, CDKN2A, EGR1, FOS, FST, GADD45A, HBEGF, HOXC6, HSPB8, ID1, IGFBP7, IL24, ITGB1, LGALS1, MET, NCOA3, PLAUR, PMAIP1, SH3RF1, STAT3, TGFA, TMED10, TNFRSF10B, TPD52, TYMP Cell Death apoptosis apoptosis of epithelial cell lines 1.59E-04 BAD, BCL2A1, BCL2L1, BCL6, BIRC2, BIRC3, BNIP3L, 32 CASP1, CASP2, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CDKN2A, CUL4B, CYCS, HSPB8, IER3, IL24, IRF1 (includes EG:16362), IRS1, ITGB1, MET, PAK2, PEA15, PMAIP1, PPP1R15A, PRDX3, PRMT2, SPHK1, SPP1 (includes EG:20750), TICAM1, TMX1, TNFRSF10B Cell Death apoptosis apoptosis of tumor cells 1.86E-04 B2M, B4GALT5, BAD, BCL2L1, CASP1, CASP2, CASP9 29 (includes EG:100140945), CCND3, CDC45, CDK2, CDKN1B, CDKN2A, DDIT3, HBEGF, HSPB8, IL24, IL7, LGALS1, LPAR1, MUC1, NCOA3, NR4A2, PHLDA1, PMAIP1, SNAI2, STAT3, TFAP2A, TNFRSF10B, TNFSF13B Cell Death apoptosis apoptosis of embryonic cell 8.80E-04 BAD, BCL2A1, BCL2L1, BCL6, BIRC2, BIRC3, BNIP3L, 29 lines CASP1, CASP2, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CUL4B, CYCS, IER3, IL24, IRF1 (includes EG:16362), IRS1, ITGB1, PAK2, PEA15, PMAIP1, PPP1R15A, PRDX3, PRMT2, SPHK1, SPP1 (includes EG:20750), TICAM1, TMX1, TNFRSF10B Cell Death apoptosis apoptosis of muscle 9.23E-04 ADM, BCL2L1, CASP1, CASP9 (includes EG:100140945), 13 CYCS, HSPB8, IL8, PMAIP1, PTN, S100A1, STAT3, TIMP3,

509

XAF1 Cell Death apoptosis apoptosis of cervical cancer 1.61E-03 BAD, BCL2L1, BIRC2, BNIP3L, C15orf63, CASP1, CASP2, 38 cell lines CASP4, CCND3, CDKN1B, CDKN2A, CDKN2C, DDIT3, EXOC2, FOS, FST, GADD45A, GADD45B, IER3, IRF1 (includes EG:16362), ITPK1, JUN, MCM10 (includes EG:307126), MUC1, NR4A2, PAK2, PARVA, PCBP2, PIK3C3, PPP2R2A, RAB32, RASSF1, SGK1, SNCA, TICAM1, TIMP3, TNFRSF10B, TNFRSF21 Cell Death apoptosis apoptosis of cardiomyocytes 1.79E-03 ADM, BCL2L1, CASP1, HSPB8, PTN, S100A1 6 Cell Death apoptosis apoptosis of muscle cells 2.04E-03 ADM, BCL2L1, CASP1, CASP9 (includes EG:100140945), 12 CYCS, HSPB8, PMAIP1, PTN, S100A1, STAT3, TIMP3, XAF1 Cell Death apoptosis apoptosis of kidney cell lines 2.15E-03 BAD, BCL2A1, BCL2L1, BCL6, BIRC2, BIRC3, BLID, 30 BNIP3L, CASP1, CASP2, CASP9 (includes EG:100140945), CD44 (includes EG:100330801), CUL4B, CYCS, IER3, IL24, IRF1 (includes EG:16362), IRS1, ITGB1, PAK2, PEA15, PMAIP1, PPP1R15A, PRDX3, PRMT2, SPHK1, SPP1 (includes EG:20750), TICAM1, TMX1, TNFRSF10B Cell Death apoptosis apoptosis of cancer cells 3.38E-03 B2M, B4GALT5, BCL2L1, CASP1, CASP2, CASP9 (includes 22 EG:100140945), CDC45, CDKN1B, DDIT3, HSPB8, IL24, IL7, LGALS1, LPAR1, MUC1, NCOA3, NR4A2, PHLDA1, PMAIP1, TFAP2A, TNFRSF10B, TNFSF13B Cell Death apoptosis apoptosis of fibrosarcoma cell 1.64E-02 BCL2A1, BCL2L1, BIRC2, BIRC3, IFNAR2, NFKB2, 9 lines PMAIP1, SOD2, STAT1 Cell Death apoptosis apoptosis of vascular smooth 2.56E-02 CASP9 (includes EG:100140945), CYCS, PMAIP1, STAT3, 5 muscle cells XAF1 Cell Death apoptosis apoptosis of fibroblast cell 2.86E-02 BCL2L1, BLID, CASP9 (includes EG:100140945), CYR61, 8 lines IFNAR2, ITGB1, NFKB2, TNFRSF10B Cell Death apoptosis apoptosis of leukemia cell lines 3.07E-02 B2M, BAD, BCL2A1, BCL2L1, BIRC3, C5, CASP2, CASP9 34 (includes EG:100140945), CD55, CDKN1B, CDKN2A, CEBPD, CYCS, DDIT3, FKBP5, GLO1, IL7, ITGB1, JUN, KLF4, LGALS1, LGALS3, MET, MSRB2, MUC1, PAK2, PRKCZ, SOD2, SPHK1, STMN1, TGFA, TNFRSF10B, TNFSF13B, UBA7 Cell Death apoptosis apoptosis of kidney cancer cell 3.25E-02 BCL2L1, DEFB1 (human), EPAS1, PRKAR1A, STAT3, VHL 6 lines Cell Death apoptosis apoptosis of pancreatic cancer 3.45E-02 BCL2L1, C5, CDKN2A, EXOC2, GLRX, IL24, IL8, S100A4, 9 cell lines SSTR2

510

Cell Death apoptosis apoptosis of vascular 3.86E-02 ADM, ATF3, AXL, BCL2L1, CASP1, CASP9 (includes 11 endothelial cells EG:100140945), CYCS, DDIT3, IL8, PMAIP1, TNFRSF10B Cell Death apoptosis apoptosis of leukocyte cell 3.92E-02 BAD, BCL2L1, CASP9 (includes EG:100140945), DDIT3, IFIH1, 10 lines IRF4, LGALS3, NBN, PRKCZ, SHC1 (includes EG:20416) Cell Death apoptosis apoptosis of RPE cells 4.20E-02 BCL2L1, PLAUR, TIMP3 3 Cell Death apoptosis apoptosis of ovarian cancer cell 4.83E-02 BCL2L1, BIRC2, CASP1, CASP9 (includes EG:100140945), 13 lines CUL9, HTRA1, KLF6, PMAIP1, PRKDC, SPARC, TGFA, TMSB10/TMSB4X, TSG101 Cell Death apoptosis apoptosis of myeloma cell lines 4.92E-02 B2M, BCL2L1, CASP9 (includes EG:100140945), CDKN1B, 10 HDAC1, IFI6, IRF4, KLF9, PMAIP1, SDC1 (includes EG:20969) Cell Death necrosis necrosis 3.27E-09 ABCC4 (includes EG:10257), ADM, AKAP12, AKR1B1, 243 ALDH1A1, ALDH3B1, ANKRD1, ANTXR1, ANTXR2, APIP, ARMC10, ATF3, ATG12 (includes EG:361321), AXL, B2M, B4GALT5, BAD, BAG3, BCHE, BCL2A1, BCL2L1, BCL3, BCL6, BHLHE40, BIRC2, BIRC3, BLID, BNIP3L, BTG1, C15orf63, C5, C9orf80, CABLES1, CASP1, CASP2, CASP4, CASP9 (includes EG:100140945), CCL2, CCND1, CCND3, CD44 (includes EG:100330801), CD55, CD74, CDC45, CDK2, CDK5, CDK5R1, CDKN1B, CDKN2A, CDKN2C, CDKN2D, CEBPD, CERS6, CIB1, CLCF1, CNN2, CUL4B, CUL9, CYB5A (includes EG:109672), CYCS, CYP2J2, CYR61, DCT, DCTN3, DDIT3, DDIT4, DEFB1 (human), DEK, DKK1, DLST, DNAJB2, DUT, E2F3, EGR1, EMP1, EMP3, EPAS1, EXOC2, FKBP1A, FKBP5, FOS, FOSB, FST, GADD45A, GADD45B, GLO1, GLRX, GPC1, GPI, GPR37, GPR65, GSTM1, HBEGF, HDAC1, HDAC9, HERPUD1, HK2, HLA-DRB4, HOXA5, HOXC6, HSD17B10, HSP90AB1, HSPB8, HTATIP2, HTRA1, ID1, ID3 (includes EG:15903), IER3, IFI6, IFIH1, IFNAR2, IGFBP6, IGFBP7, IL24, IL7, IL7R, IL8, ILK, IRF1 (includes EG:16362), IRF4, IRS1, ITGB1, ITPK1, JUN, KLF4, KLF6, KLF9, LAMP1, LAMP2, LEPR, LGALS1, LGALS3, LPAR1, LRPAP1, MAOA, MCM10 (includes EG:307126), MET, MITF, MSRB2, MT1X, MT2A, MTFP1, MUC1, MVP, MX1, MYB, NAA35, NAMPT, NBN, NCOA3, NFATC1, NFIL3, NFKB2, NFKBIB, NPC1, NQO1, NR4A2, NRP1 (includes EG:18186), NUAK1, NUPR1, PAK2, PARVA, PBK, PCBP2, PCNA, PEA15, PHB, PHLDA1, PIK3C3, PKM2, PLAUR, PMAIP1, PPP1R15A, PPP2R2A, PPP2R2B, PPT1, PRDX3, PRKAR1A, PRKCZ, PRKDC, PRMT2, PTN, RAB32, RAC2, RASSF1, RBM17, RELB, REPS2, S100A1, S100A4, SAT1, SDC1

511

(includes EG:20969), SEMA3A, SGK1, SH3RF1, SHC1 (includes EG:20416), SIRPA, SIVA1, SKP2 (includes EG:27401), SLC11A2, SLC29A1, SLC29A2, SLC3A2, SLC5A8, SNAI2, SNCA, SND1, SOD2, SOX4, SPARC, SPC25 (includes EG:100144563), SPHK1, SPP1 (includes EG:20750), SPRY2, SRPK1, SSTR2, STAT1, STAT3, STMN1, TFAP2A, TFAP2C, TGFA, TGFB1I1, TGFBR3, TICAM1, TIMP3, TMED10, TMSB10/TMSB4X, TMX1, TNFRSF10B, TNFRSF21, TNFSF13B, TPD52, TSG101, TYMP, TYMS, UBA7, UBQLN1, VHL, XAF1, XBP1 (includes EG:140614), YWHAZ, ZFYVE16 Cell Death cell viability cell viability of tumor cell lines 1.93E-03 AGTRAP, ASAH1, ATG4B, AXL, BAD, BCL2A1, BCL2L1, 88 BCL6, CADM1, CAMK2D, CAMK2N1, CARS, CASP1, CASP2, CBL, CCL2, CCND1, CD44 (includes EG:100330801), CDK2, CDK20, CDKN1B, CDKN2A, CEBPD, CKAP5, CUL9, DCT, DUSP19, DUSP5, FKBP5, FOXM1, GCLC, GPC1, GSTM1, HBEGF, HOXA5, HTATIP2, ID2, ID4, IL7, IL8, ILK, ITGB1, LGALS3, MED14, MET, MINPP1, MTDH, MYB, NBN, NFKB2, NRP1 (includes EG:18186), NUAK1, NUPR1, PBK, PCGF2, PCNA, PLAUR, PPAP2A, PPM1M, PPP2R1B, PPP2R2B, PPP2R5C, PRKACB, PRKDC, PSMA1, PTN, PTPN22, PTPRM, RELB, RSF1 (includes EG:233532), S100A4, SERPINB2, SGK1, SHFM1, SNCA, SOD2, SPHK1, SPP1 (includes EG:20750), STAT3, STC1, TNFSF13B, TPK1, TPMT, TYMP, TYMS, USP11, VHL, ZFP36 Cell Death cell viability cell viability 2.41E-03 AGTRAP, ALKBH8, ANTXR1, ASAH1, ATG4B, ATP7A, 109 AXL, B4GALT5, BAD, BCL2A1, BCL2L1, BCL6, C5, CADM1, CALB2, CAMK2D, CAMK2N1, CARS, CASP1, CASP2, CBL, CCL2, CCND1, CD44 (includes EG:100330801), CD74, CDK2, CDK20, CDK2AP1, CDKN1B, CDKN2A, CEBPD, CKAP5, CUL9, DCT, DDIT3, DUSP19, DUSP5, FKBP5, FOXM1, GCLC, GPC1, GSTM1, HBEGF, HERPUD1, HOXA5, HTATIP2, ID2, ID4, IL7, IL8, ILK, IRF9, ITGB1, LGALS3, MED14, MET, MINPP1, MTDH, MVP, MX1, MYB, NBN, NFIL3, NFKB2, NRP1 (includes EG:18186), NTN4, NUAK1, NUPR1, PBK, PCGF2, PCNA, PLAUR, PPAP2A, PPM1M, PPP2R1B, PPP2R2B, PPP2R5C, PRKACB, PRKDC, PSMA1, PTGR1, PTN, PTPN22, PTPRM, RAC2, RELB, RSF1 (includes EG:233532), S100A4, SERPINB2, SGK1, SHFM1, SNCA, SND1, SOD2, SPHK1, SPP1 (includes EG:20750), STAT3, STC1, STMN1, THBS2, TNFSF13B, TPK1, TPMT, TXNDC5, TYMP, TYMS, USP11, VHL, ZFP36 Cell Death cell viability cell viability of breast cancer 1.15E-02 BCL2L1, CCND1, CD44 (includes EG:100330801), CDK2, 18

512

cell lines CDKN2A, CEBPD, FKBP5, FOXM1, HOXA5, ID2, ID4, ILK, LGALS3, PBK, SOD2, SPHK1, STAT3, USP11 Cell Death cell viability cell viability of stomach cancer 1.46E-02 CD44 (includes EG:100330801), ITGB1, MET, SPP1 (includes 4 cell lines EG:20750) Cell Death cell viability cell viability of brain cancer 1.73E-02 CDKN2A, MET, NRP1 (includes EG:18186), PCGF2, PLAUR, 8 cell lines PTPRM, STAT3, STC1 Cell Death cell viability cell viability of melanoma cell 1.97E-02 ASAH1, AXL, DCT, GCLC, GSTM1 5 lines Cell Death survival cell survival 2.71E-03 AGTRAP, ALKBH8, ANTXR1, ASAH1, ATG4B, ATP7A, 113 AXL, B4GALT5, BAD, BCL2A1, BCL2L1, BCL6, C5, CADM1, CALB2, CAMK2D, CAMK2N1, CARS, CASP1, CASP2, CBL, CCL2, CCND1, CD44 (includes EG:100330801), CD74, CDK2, CDK20, CDK2AP1, CDKN1B, CDKN2A, CEBPD, CKAP5, CUL9, DCT, DDIT3, DEFB103A/DEFB103B, DUSP19, DUSP5, FKBP5, FOXM1, GCLC, GPC1, GSTM1, HBEGF, HERPUD1, HOXA5, HTATIP2, ID2, ID4, IL7, IL8, ILK, IRF9, ITGA5, ITGB1, LAPTM4B, LGALS3, MED14, MET, MINPP1, MTDH, MVP, MX1, MYB, NBN, NFIL3, NFKB2, NRP1 (includes EG:18186), NTN4, NUAK1, NUPR1, PAXIP1, PBK, PCGF2, PCNA, PLAUR, PPAP2A, PPM1M, PPP2R1B, PPP2R2B, PPP2R5C, PRKACB, PRKDC, PSMA1, PTGR1, PTN, PTPN22, PTPRM, RAC2, RELB, RSF1 (includes EG:233532), S100A4, SERPINB2, SGK1, SHFM1, SNCA, SND1, SOD2, SPHK1, SPP1 (includes EG:20750), STAT3, STC1, STMN1, THBS2, TNFSF13B, TPK1, TPMT, TXNDC5, TYMP, TYMS, USP11, VHL, ZFP36 Cell Death survival survival of chronic 3.01E-02 CD74, IL8 2 lymphocytic leukemia B cells Cell Death survival survival of pre-B lymphocytes 3.01E-02 NFIL3, TNFSF13B 2 Cell Death killing killing of B-lymphocyte 4.13E-03 EXOC2, NBN, RALA 3 derived cell lines Cell Death killing killing of thymocytes 1.08E-02 LGALS1, LGALS3 2 Cell Death inhibition inhibition of apoptosis 1.03E-02 ACAA2, ANXA5, BCL2A1, BCL2L1, BIRC3, CCL2, 24 CDKN2D, GCLC, GLO1, HDAC1, HTATIP2, IFI6, MUC1, OPA1, PEA15, PRKCZ, SERPINB2, SIVA1, SNCA, SOCS2, SPHK1, TMX1, TXNDC5, YWHAZ Cell Death colony survival colony survival of lymphoma 1.08E-02 BCL2L1, CCND1 2 cell lines

513

Cell Death anoikis anoikis 1.62E-02 BCL2L1, CASP9 (includes EG:100140945), CDKN2A, HTRA1, 11 ILK, ITGB1, LGALS3, NCOA3, NR4A2, PLAUR, TNFRSF10B Cellular Movement cell movement cell movement of tumor cell 4.18E-06 AGK, ANKS1A, ARFGEF1, ARPC1B, AXL, BCAR3, C5, 99 lines CCL2, CCL20, CD151, CD44 (includes EG:100330801), CD9, CDK14, CDK5, CDK7, CDKN1B, CDKN2A, CYP2J2, CYR61, DEFB103A/DEFB103B, DLC1, DPYSL2, EGR1, ENPP2, EPS8, FOXM1, FSCN1, FYN, GPI, HAS2, HTATIP2, HTRA1, ID1, IGFBP6, IGSF8, IL8, ILK, IRS1, ITGA1, ITGB1, JUN, KLF4, LAMB3, LAMC2, LCP1, LGALS1, LGALS3, LPAR1, LRPAP1, MET, MUC1, MX1, MYB, MYO10, NCOA3, NES, NOV, NRP1 (includes EG:18186), PAK2, PBK, PHB, PLAUR, PLXNA1, PODXL, PPFIA1, PREX1, PRKCZ, PRUNE, PTN, PTPRM, RASSF1, RPS6KA3, RPS6KA5, SDC1 (includes EG:20969), SDCBP, SEMA3A, SEPT9, SHC1 (includes EG:20416), SIRPA, SIVA1, SNAI2, SOD2, SPARC, SPHK1, SPP1 (includes EG:20750), SPRY2, SPTLC1, ST3GAL5, STARD13, STAT3, STMN1, SYNM, TFAP2A, TFAP2C, TGFA, THBS2, TMSB10/TMSB4X, WBP2, ZYX Cellular Movement cell movement cell movement 1.31E-04 ADM, AGK, ALCAM, ALPP/ALPPL2, ANKS1A, ARFGEF1, 152 ARHGAP24, ARPC1B, ASAP1, ASAP2, AXL, BCAR3, C5, CBL, CCL2, CCL20, CCL3L1/CCL3L3, CCND1, CD151, CD44 (includes EG:100330801), CD63, CD9, CDK14, CDK5, CDK7, CDKN1B, CDKN2A, CIB1, CLIC4, CNN2, CYP2J2, CYR61, DEFB1 (human), DEFB103A/DEFB103B, DEK, DKK1, DLC1, DPYSL2, EDNRB, EGR1, ENPP2, EPS8, FOXM1, FSCN1, FYN, GPI, HAS2, HBEGF, HTATIP2, HTRA1, ID1, ID2, ID3 (includes EG:15903), IGFBP6, IGSF8, IL7, IL8, ILK, IRS1, ITGA1, ITGA3, ITGA5, ITGB1, ITGB1BP1, JAM3, JUN, KCNMA1, KLF4, LAMB3, LAMC2, LCP1, LGALS1, LGALS3, LPAR1, LRPAP1, MET, MUC1, MX1, MYB, MYO10, NARS, NCOA3, NES, NEXN, NOV, NRP1 (includes EG:18186), NTN4, PAK2, PBK, PHB, PLAUR, PLXNA1, PODXL, PODXL2, PON2, PPAP2A, PPFIA1, PREX1, PRKCZ, PRUNE, PTN, PTPRF, PTPRM, RALA, RAP2A, RASSF1, ROPN1B, RPS6KA3, RPS6KA5, S100A4, SCARB1, SCG2, SDC1 (includes EG:20969), SDC4, SDCBP, SEMA3A, SEPT9, SERPINA3, SHC1 (includes EG:20416), SIRPA, SIVA1, SNAI2, SOD2, SORT1, SP100, SPA17, SPAG9, SPARC, SPHK1, SPP1 (includes EG:20750), SPRY2, SPTLC1, SRPX2, ST3GAL5, STARD13, STAT3, STC1, STMN1, SYNM, TFAP2A, TFAP2C, TGFA, THBS2, TIMP3, TMSB10/TMSB4X, TNS3, TUBB2B, VHL, WAS, WBP2, ZNF217, ZYX

514

Cellular Movement cell movement cell movement of gonadal cell 3.51E-04 ASAP1, CD9, CIB1, ITGB1, ITGB1BP1, PLAUR, PODXL2, 8 lines PTN Cellular Movement cell movement cell movement of prostate 1.12E-03 AGK, FSCN1, ID1, IGSF8, IL8, IRS1, LAMB3, LCP1, 16 cancer cell lines LGALS3, MX1, NCOA3, NES, PAK2, PBK, PHB, PTN Cellular Movement cell movement cell movement of breast cancer 1.39E-03 ANKS1A, ARPC1B, AXL, BCAR3, CD151, CYP2J2, GPI, IL8, 32 cell lines ITGB1, JUN, LPAR1, LRPAP1, MET, NRP1 (includes EG:18186), PAK2, PLAUR, PLXNA1, PRKCZ, SDC1 (includes EG:20969), SDCBP, SEMA3A, SEPT9, SIVA1, SNAI2, SPARC, SPHK1, SPP1 (includes EG:20750), STAT3, TFAP2A, TFAP2C, TGFA, WBP2 Cellular Movement cell movement cell movement of carcinoma 1.84E-03 CYR61, DLC1, HTATIP2, ID1, IL8, IRS1, ITGB1, LGALS1, 16 cell lines MET, NOV, PODXL, PTN, RASSF1, RPS6KA3, STMN1, TGFA Cellular Movement cell movement cell movement of lung cancer 7.65E-03 CD9, CDKN2A, HTATIP2, ID1, IRS1, ITGB1, MET, PODXL, 13 cell lines PTN, RASSF1, RPS6KA3, STMN1, TGFA Cellular Movement cell movement cell movement of brain cancer 1.91E-02 CDKN2A, EGR1, ENPP2, FYN, LPAR1, NRP1 (includes 13 cell lines EG:18186), PLAUR, PTN, PTPRM, SEMA3A, SHC1 (includes EG:20416), SIRPA, SYNM Cellular Movement cell movement cell movement of mammary 2.10E-02 CBL, CD44 (includes EG:100330801), ITGB1, LGALS3 4 tumor cells Cellular Movement cell movement cell movement of eosinophils 2.25E-02 ALPP/ALPPL2, C5, CCL2, IL8, ITGB1, LGALS3, PLAUR, 9 SCG2, SIRPA Cellular Movement invasion invasion of cells 4.60E-06 ADM, ASAP1, ATF3, AXL, B4GALT5, BCAR3, CBL, CCL2, 85 CCND1, CD151, CD44 (includes EG:100330801), CD9, CDK14, CDK5, CDKN1B, CYP2J2, DKK1, DLC1, EDNRB, ENPP2, FKBP1A, FOXM1, FSCN1, FST, FXYD5, GBP1, GJB1, GPI, HAS2, HBEGF, HTATIP2, ID1, ID2, IER3, IL13RA2, IL8, ILK, ITGA1, ITGA3, ITGA5, ITGB1, JAM3, JUN, KCNMA1, KLF4, LCP1, LGALS1, LGALS3, LPAR1, MET, MITF, MMP1 (includes EG:300339), MUC1, MX1, MYH10, NAMPT, NCOA3, NES, NOV, NRP1 (includes EG:18186), NUAK1, PKM2, PLAUR, PODXL, SDC4, SDCBP, SEPT9, SKP2 (includes EG:27401), SOD2, SP100, SPARC, SPHK1, SPP1 (includes EG:20750), SPRY2, SSTR2, ST8SIA1, STAT3, STMN1, TFAP2A, TFAP2C, TGFA, THBS2, TIMP3, TMSB10/TMSB4X, VHL Cellular Movement invasion invasion of tumor cell lines 1.76E-05 ADM, ASAP1, ATF3, AXL, BCAR3, CBL, CCL2, CD151, 73 CD44 (includes EG:100330801), CD9, CDK14, CDK5, CDKN1B, CYP2J2, DKK1, DLC1, ENPP2, FOXM1, FSCN1, FXYD5,

515

GJB1, GPI, HAS2, HBEGF, HTATIP2, ID1, ID2, IL13RA2, IL8, ITGA3, ITGA5, ITGB1, JAM3, JUN, KLF4, LCP1, LGALS1, LGALS3, LPAR1, MET, MITF, MMP1 (includes EG:300339), MUC1, MX1, NAMPT, NCOA3, NES, NOV, NRP1 (includes EG:18186), NUAK1, PKM2, PLAUR, PODXL, SDC4, SDCBP, SEPT9, SKP2 (includes EG:27401), SOD2, SP100, SPARC, SPHK1, SPP1 (includes EG:20750), SPRY2, SSTR2, ST8SIA1, STAT3, STMN1, TFAP2A, TFAP2C, TGFA, TIMP3, TMSB10/TMSB4X, VHL Cellular Movement invasion invasion of tumor-infiltrating 1.12E-03 CCL2, IL8, ITGA1 3 lymphocytes Cellular Movement invasion invasion of colon cancer cell 6.05E-03 ATF3, AXL, CD44 (includes EG:100330801), FSCN1, HAS2, 12 lines KLF4, MET, NRP1 (includes EG:18186), NUAK1, STAT3, TGFA, TMSB10/TMSB4X Cellular Movement invasion invasion of lung cancer cell 6.05E-03 ASAP1, AXL, CD44 (includes EG:100330801), DKK1, 12 lines HTATIP2, ID1, ITGB1, MET, SSTR2, ST8SIA1, STAT3, STMN1 Cellular Movement invasion invasion of prostate cancer cell 1.11E-02 FSCN1, IL8, LCP1, LGALS3, MMP1 (includes EG:300339), 12 lines MX1, NCOA3, NES, PLAUR, PODXL, SPP1 (includes EG:20750), SPRY2 Cellular Movement invasion invasion of endothelial cells 2.56E-02 CD151, EDNRB, GBP1, SP100, THBS2 5 Cellular Movement invasion invasion of kidney cancer cell 2.84E-02 GJB1, MUC1, VHL 3 lines Cellular Movement invasion invasion of carcinoma cell lines 3.38E-02 ASAP1, DKK1, DLC1, FSCN1, HTATIP2, ID1, ITGB1, 12 LGALS1, MET, SPP1 (includes EG:20750), STMN1, TIMP3 Cellular Movement invasion invasion of breast cancer cell 4.14E-02 ASAP1, BCAR3, CBL, CD44 (includes EG:100330801), 21 lines CDKN1B, FXYD5, ID1, ID2, ITGA3, ITGA5, JUN, MET, MUC1, NAMPT, PLAUR, PODXL, SDCBP, SEPT9, SP100, SPARC, SPP1 (includes EG:20750) Cellular Movement invasion invasion of squamous cell 4.26E-02 CD44 (includes EG:100330801), CYP2J2, FSCN1, HBEGF, 7 carcinoma cell lines PLAUR, SPP1 (includes EG:20750), TGFA Cellular Movement invasion invasion of hepatoma cell lines 4.66E-02 CDK14, CYP2J2, DLC1, LPAR1, MMP1 (includes EG:300339), 6 SPP1 (includes EG:20750) Cellular Movement migration migration of tumor cell lines 2.61E-04 AGK, ANKS1A, ARFGEF1, AXL, BCAR3, C5, CCL2, CCL20, 74 CD151, CD44 (includes EG:100330801), CDK5, CDK7, CDKN1B, CDKN2A, CYP2J2, CYR61, DPYSL2, EGR1, ENPP2, FOXM1, FSCN1, FYN, HAS2, HTATIP2, ID1, IGFBP6, IGSF8, IL8, IRS1, ITGB1, JUN, KLF4, LAMB3,

516

LAMC2, LGALS1, LGALS3, LPAR1, MET, MUC1, MYO10, NCOA3, NES, NOV, NRP1 (includes EG:18186), PAK2, PHB, PLAUR, PLXNA1, PODXL, PPFIA1, PREX1, PTN, PTPRM, RASSF1, RPS6KA5, SDC1 (includes EG:20969), SDCBP, SEMA3A, SHC1 (includes EG:20416), SIRPA, SNAI2, SOD2, SPHK1, SPP1 (includes EG:20750), SPRY2, STAT3, STMN1, SYNM, TFAP2A, TFAP2C, TGFA, THBS2, WBP2, ZYX Cellular Movement migration migration of gonadal cell lines 3.62E-04 ASAP1, CIB1, ITGB1, ITGB1BP1, PLAUR, PTN 6 Cellular Movement migration migration of carcinoma cell 2.01E-03 CYR61, HTATIP2, ID1, IL8, IRS1, ITGB1, LGALS1, MET, 14 lines NOV, PODXL, PTN, RASSF1, STMN1, TGFA Cellular Movement migration migration of macrophages 3.24E-03 C5, CCL2, DEFB1 (human), DEFB103A/DEFB103B, LGALS3 5 Cellular Movement migration migration of cells 4.63E-03 ADM, AGK, ALCAM, ALPP/ALPPL2, ANKS1A, ARFGEF1, 125 ARHGAP24, ASAP1, ASAP2, AXL, BCAR3, C5, CBL, CCL2, CCL20, CCL3L1/CCL3L3, CD151, CD44 (includes EG:100330801), CD9, CDK5, CDK7, CDKN1B, CDKN2A, CIB1, CLIC4, CNN2, CYP2J2, CYR61, DEFB1 (human), DEFB103A/DEFB103B, DEK, DKK1, DLC1, DPYSL2, EDNRB, EGR1, ENPP2, FOXM1, FSCN1, FYN, HAS2, HBEGF, HTATIP2, ID1, ID2, ID3 (includes EG:15903), IGFBP6, IGSF8, IL7, IL8, ILK, IRS1, ITGA1, ITGA3, ITGA5, ITGB1, ITGB1BP1, JAM3, JUN, KCNMA1, KLF4, LAMB3, LAMC2, LGALS1, LGALS3, LPAR1, MET, MUC1, MYO10, NARS, NCOA3, NES, NEXN, NOV, NRP1 (includes EG:18186), NTN4, PAK2, PHB, PLAUR, PLXNA1, PODXL, PON2, PPAP2A, PPFIA1, PREX1, PRKCZ, PTN, PTPRF, PTPRM, RAP2A, RASSF1, RPS6KA5, S100A4, SCARB1, SCG2, SDC1 (includes EG:20969), SDC4, SDCBP, SEMA3A, SERPINA3, SHC1 (includes EG:20416), SIRPA, SNAI2, SOD2, SORT1, SP100, SPAG9, SPHK1, SPP1 (includes EG:20750), SPRY2, STAT3, STC1, STMN1, SYNM, TFAP2A, TFAP2C, TGFA, THBS2, TIMP3, TNS3, TUBB2B, VHL, WAS, WBP2, ZYX Cellular Movement migration migration of prostate cancer 7.10E-03 AGK, FSCN1, ID1, IGSF8, IL8, LAMB3, LGALS3, NCOA3, 12 cell lines NES, PAK2, PHB, PTN Cellular Movement migration migration of leukocyte cell 1.03E-02 CCL2, CD44 (includes EG:100330801), DEFB1 (human), 6 lines DEFB103A/DEFB103B, IL8, RAP2A Cellular Movement migration migration of lung cancer cell 1.78E-02 CDKN2A, HTATIP2, ID1, IRS1, ITGB1, PODXL, PTN, 10 lines RASSF1, STMN1, TGFA Cellular Movement migration migration of brain cancer cell 2.41E-02 CDKN2A, EGR1, ENPP2, FYN, LPAR1, PLAUR, PTN, 11

517

lines PTPRM, SHC1 (includes EG:20416), SIRPA, SYNM Cellular Movement migration migration of Langerhans cells 2.84E-02 C5, CCL20, CD44 (includes EG:100330801) 3 Cellular Movement migration migration of thymocytes 2.84E-02 ITGA3, ITGB1, SEMA3A 3 Cellular Movement migration arrest in migration of 3.01E-02 C5, IL8 2 eosinophils Cellular Movement migration migration of eosinophils 4.05E-02 ALPP/ALPPL2, C5, IL8, PLAUR, SIRPA 5 Cellular Movement migration migration of skin cancer cell 4.20E-02 CD151, IGSF8, LAMC2 3 lines Cellular Movement migration migration of antigen presenting 4.45E-02 C5, CCL2, CCL20, CD44 (includes EG:100330801), DEFB1 9 cells (human), DEFB103A/DEFB103B, LGALS3, NARS, WAS Cellular Movement migration migration of squamous cell 4.95E-02 CD44 (includes EG:100330801), CYP2J2, JUN, MET, ZYX 5 carcinoma cell lines Cellular Movement chemoattraction chemoattraction of 1.46E-03 CCL2, CCL3L1/CCL3L3, DEK, IL8 4 mononuclear leukocytes Cellular Movement chemoattraction chemoattraction of phagocytes 3.24E-03 CCL2, CCL20, CCL3L1/CCL3L3, DEK, IL8 5 Cellular Movement chemoattraction chemoattraction of myeloid 1.46E-02 CCL2, CCL3L1/CCL3L3, DEK, IL8 4 cells Cellular Movement chemoattraction chemoattraction of cells 2.67E-02 CCL2, CCL20, CCL3L1/CCL3L3, DEK, IL8, SCG2 6 Cellular Movement chemoattraction chemoattraction of T 3.01E-02 DEK, IL8 2 lymphocytes Cellular Movement chemoattraction chemoattraction of monocytes 3.01E-02 CCL2, CCL3L1/CCL3L3 2 Cellular Movement cell rolling cell rolling of eosinophils 3.13E-03 C5, IL8, ITGB1, LGALS3 4 Cellular Movement cell rolling arrest in cell rolling of 1.76E-02 C5, IL8, ITGB1 3 eosinophils Cellular Movement chemotaxis chemotaxis of monocyte- 4.13E-03 C5, DEFB103A/DEFB103B, SPHK1 3 derived macrophages Cellular Movement chemotaxis chemotaxis of memory T 2.84E-02 CCL2, DEFB1 (human), DEFB103A/DEFB103B 3 lymphocytes Cellular Movement chemotaxis chemotaxis of breast cancer 2.90E-02 ITGB1, JUN, PRKCZ, SPHK1 4 cell lines Cellular Movement late cytokinesis late cytokinesis of tumor cell 1.08E-02 KIF20B, SEPT9 2 lines Cellular Movement recruitment recruitment of macrophages 1.08E-02 CCL2, IL8 2

518

Cellular Movement recruitment recruitment of phagocytes 4.20E-02 C5, CCL2, IL8 3 Cellular Movement transmigration transmigration of vascular 1.08E-02 ID1, ID3 (includes EG:15903) 2 endothelial cells Cellular Movement transmigration transmigration of granulocytes 1.71E-02 ALPP/ALPPL2, C5, IL8, JAM3, LGALS1, SIRPA 6 Cellular Movement transmigration transmigration of eosinophils 3.01E-02 ALPP/ALPPL2, SIRPA 2 Cellular Movement homing homing of tumor cell lines 2.22E-02 C5, CCL2, CD151, DEFB103A/DEFB103B, IL8, ITGA1, 16 ITGB1, JUN, MYB, NRP1 (includes EG:18186), PLAUR, PRKCZ, SEMA3A, SNAI2, SPHK1, SPTLC1 Cellular Movement chemorepulsion chemorepulsion of brain cancer 3.01E-02 NRP1 (includes EG:18186), SEMA3A 2 cell lines Cellular Growth and proliferation proliferation of cells 6.69E-06 ABCC5, ADM, AGK, AK2, AKR1C1/AKR1C2, AKR1C3, 326 Proliferation ALDH1A1, APOD, ARHGAP24, ARL2BP, ARL3, ARMC10, ASAH1, ASH2L, ATF3, ATP5G1, ATPIF1, AXL, BAD, BAMBI, BCAR3, BCL2A1, BCL2L1, BCL3, BCL6, BHLHE40, BIRC2, BST2, BTG1, C14orf169, C5, C6orf108, CABLES1, CAMK2N1, CASP1, CASP2, CASP9 (includes EG:100140945), CBX7, CCL2, CCL20, CCL3L1/CCL3L3, CCND1, CCND3, CD151, CD33, CD44 (includes EG:100330801), CD55, CD63, CD74, CD83, CD9, CDC16, CDC45, CDCA7, CDK14, CDK2, CDK20, CDK2AP1, CDK5, CDK5R1, CDK7, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CEBPD, CENPB, CITED1, CKS2, CLCF1, CLIP1, CPSF4, CSDA (includes EG:56449), CTDSPL, CTNNBIP1, CUL1, CYP2J2, CYR61, DAP, DDIT3, DEFB1 (human), DKK1, DLC1, DLST, DMTF1, DNAJB2, DUSP5, E2F3, ECM1 (includes EG:100332249), EDNRB, EEF1A1, EGR1, EIF4G1, EIF5A2, EMP1, EMP3, EPAS1, EPS8, ERRFI1, FADS1, FBXO4, FGF13, FGFRL1, FHL2, FKBP5, FOS, FOXM1, FSCN1, FST, FZD7, GADD45A, GBP1, GHR, GPC1, GPI, GPNMB, GSTM1, HAS2, HBEGF, HDAC1, HGS, HILPDA, HK2, HLA-DPB1, HNRNPAB, HNRNPU, HOXA5, HOXC6, HSPB8, ID1, ID2, ID3 (includes EG:15903), IER3, IFIT3, IFITM1, IGFBP6, IGFBP7, IL13RA2, IL1RL1, IL24, IL4R, IL7, IL7R, IL8, ILK, IRF1 (includes EG:16362), IRF4, IRS1, ISG15, ITGA1, ITGA3, ITGA5, ITGB1, JUN, KAT2B, KCNMA1, KCNN4, KIAA0101, KLF4, KLF6, LCP1, LDHA, LEPR, LGALS1, LGALS3, LTBP3, LTBP4, LZTS1, MAFF, MAGED2, MAPRE2, MCM3, MCM7, MECOM, MET, MGAT4B, MINA, MT1A, MT2A, MTCH1, MTPN, MTUS1, MUC1, MVP, MX1, MXI1, MYB, MYOF, NAA35, NAMPT, NCK2, NCOA3, NES, NEU1, NFKB2,

519

NFKBIB, NOL8, NOV, NQO1, NR4A2, NRP1 (includes EG:18186), NTN4, NUPR1, ODC1, PBK, PCGF2, PCNA, PDIA5, PDS5B, PEG10, PFKFB3, PGK1, PHB, PHLDA1, PHLDA2, PIK3C3, PKM2, PLAUR, PLCE1, PLIN2, PLIN3, PLP1 (includes EG:18823), POLA1, POLR2J, PPP1R15A, PPP2R5C, PPT1, PRDX3, PRKAR1A, PRKCSH, PRKCZ, PRKRIR, PRPF4B, PSME2, PTGS1, PTN, PTPN22, PTPRM, PTPRR, QPCT, RAC2, RALA, RARRES3, RASSF1, RBL1, RELB, RNF14, RPS6KA3, SAT1, SCG2, SEC61A1, SEPT9, SERPINB2, SERTAD2, SF3B2, SF3B3, SGK1, SHC1 (includes EG:20416), SIRPA, SKP2 (includes EG:27401), SLC29A2, SLC3A2, SMARCD3, SNAI2, SND1, SOD2, SOX2, SOX4, SPARC, SPHK1, SPP1 (includes EG:20750), SPRY2, SS18, SSR1, SSTR2, ST8SIA1, STARD13, STAT1, STAT3, STC1, STMN1, SYNM, TAF1D, TBC1D8, TBRG4, TFAP2A, TFAP2C, TGFA, TGFB1I1, TGFBI, TGFBR3, THBS2, TIMELESS, TIMP3, TMSB10/TMSB4X, TNFRSF10B, TNFRSF21, TNFSF13B, TPD52, TPMT, TRIB2, TRIM33, TSG101, TYMS, TYR, TYRP1, UBE2L6, UBE3A, UBIAD1, URI1, USP4, VHL, VMP1, WAS, WBP2, WDR6, XBP1 (includes EG:140614), YY1AP1, ZBED1, ZBTB17, ZFP36, ZMAT3, ZMYM2, ZNF217, ZYX Cellular Growth and proliferation proliferation of prostate cancer 1.66E-04 ADM, AGK, ALDH1A1, BCL2L1, CBX7, CCND1, CD44 39 Proliferation cell lines (includes EG:100330801), CDKN1B, CDKN2A, CEBPD, CTNNBIP1, DEFB1 (human), FKBP5, FSCN1, FST, HBEGF, HGS, HOXC6, IGFBP7, IL24, IL8, ITGB1, LCP1, LGALS3, LZTS1, MET, NCOA3, PTN, RASSF1, RNF14, SAT1, SHC1 (includes EG:20416), SND1, STAT3, TGFA, TGFB1I1, TPD52, URI1, XBP1 (includes EG:140614) Cellular Growth and proliferation proliferation of ovarian cancer 1.11E-03 ATF3, CCND1, CD44 (includes EG:100330801), CDKN2A, 19 Proliferation cell lines CLIP1, DLC1, E2F3, FST, HAS2, HBEGF, LGALS1, PRKAR1A, SKP2 (includes EG:27401), SOD2, SPARC, STAT3, TGFA, TMSB10/TMSB4X, TSG101 Cellular Growth and proliferation proliferation of pulmonary 1.12E-03 ITGA5, ITGB1, VHL 3 Proliferation fibroblasts Cellular Growth and proliferation proliferation of lung cells 1.12E-03 CCND1, CCND3, HLA-DPB1, ITGA5, ITGB1, VHL 6 Proliferation Cellular Growth and proliferation proliferation of tumor cell lines 1.23E-03 ADM, AGK, AKR1C3, ALDH1A1, ARL2BP, ASAH1, ATF3, 168 Proliferation AXL, BAD, BCAR3, BCL2L1, BCL6, BIRC2, C14orf169, CABLES1, CAMK2N1, CASP2, CBX7, CCL2, CCL20, CCND1, CCND3, CD151, CD44 (includes EG:100330801),

520

CDC16, CDK14, CDK2, CDK2AP1, CDK5, CDK5R1, CDKN1B, CDKN2A, CDKN2B, CEBPD, CENPB, CITED1, CLIP1, CTNNBIP1, CYP2J2, CYR61, DEFB1 (human), DKK1, DLC1, DMTF1, DUSP5, E2F3, EEF1A1, EGR1, EPS8, ERRFI1, FGFRL1, FKBP5, FOS, FOXM1, FSCN1, FST, GADD45A, GHR, GPC1, GSTM1, HAS2, HBEGF, HDAC1, HGS, HK2, HOXC6, HSPB8, ID1, ID2, IER3, IGFBP7, IL24, IL7, IL8, ILK, IRF1 (includes EG:16362), IRS1, ITGA1, ITGA5, ITGB1, JUN, KIAA0101, KLF4, LCP1, LDHA, LGALS1, LGALS3, LZTS1, MECOM, MET, MINA, MT1A, MT2A, MTUS1, MUC1, MXI1, MYB, NAA35, NCOA3, NFKBIB, NOV, NR4A2, NRP1 (includes EG:18186), PBK, PCGF2, PCNA, PDS5B, PFKFB3, PIK3C3, PKM2, PLAUR, PPP2R5C, PPT1, PRKAR1A, PRPF4B, PTN, PTPN22, PTPRR, RALA, RARRES3, RASSF1, RBL1, RNF14, SAT1, SEPT9, SERPINB2, SGK1, SHC1 (includes EG:20416), SKP2 (includes EG:27401), SLC3A2, SND1, SOD2, SOX2, SOX4, SPARC, SPHK1, SPP1 (includes EG:20750), SPRY2, SS18, SSTR2, ST8SIA1, STARD13, STAT1, STAT3, STMN1, SYNM, TFAP2A, TFAP2C, TGFA, TGFB1I1, THBS2, TIMP3, TMSB10/TMSB4X, TNFRSF10B, TNFSF13B, TPD52, TPMT, TRIB2, TSG101, TYMS, UBIAD1, URI1, VHL, VMP1, WBP2, XBP1 (includes EG:140614), YY1AP1, ZFP36 Cellular Growth and proliferation proliferation of liver cells 2.10E-03 C5, DLC1, LGALS1, LGALS3, PTN, SKP2 (includes 7 Proliferation EG:27401), TGFA Cellular Growth and proliferation proliferation of lymphoblastoid 2.29E-03 ARL2BP, C14orf169, CASP2, CENPB, FKBP5, GHR, LDHA, 10 Proliferation cell lines PCNA, SOD2, TPMT Cellular Growth and proliferation proliferation of breast cancer 2.60E-03 ASAH1, BAD, BCAR3, CCND1, CD151, CD44 (includes 52 Proliferation cell lines EG:100330801), CDC16, CDKN2A, CEBPD, CITED1, CYR61, DLC1, EEF1A1, FOXM1, GPC1, HAS2, HBEGF, HSPB8, ID1, ID2, IER3, IL24, IRF1 (includes EG:16362), IRS1, ITGA5, JUN, LGALS1, LGALS3, MET, MYB, NCOA3, NRP1 (includes EG:18186), PBK, PDS5B, PLAUR, PRPF4B, RALA, RARRES3, SAT1, SEPT9, SKP2 (includes EG:27401), SOD2, SOX2, SPHK1, SPP1 (includes EG:20750), STAT3, TFAP2A, TFAP2C, TGFA, TYMS, WBP2, XBP1 (includes EG:140614) Cellular Growth and proliferation proliferation of hepatocytes 3.24E-03 C5, DLC1, PTN, SKP2 (includes EG:27401), TGFA 5 Proliferation Cellular Growth and proliferation proliferation of pancreatic 8.03E-03 ADM, CCND1, CDK5, CDKN1B, FOXM1, HBEGF, 14 Proliferation cancer cell lines KIAA0101, LGALS1, MET, MTUS1, NCOA3, PTN, SOD2, STAT3

521

Cellular Growth and proliferation proliferation of 9.52E-03 ID1, ID2, ID3 (includes EG:15903) 3 Proliferation adenocarcinoma cells Cellular Growth and proliferation proliferation of tumor cells 1.05E-02 BAMBI, CASP1, CCND1, CD44 (includes EG:100330801), 27 Proliferation CDKN2A, CYR61, DDIT3, EGR1, FOS, FOXM1, FST, ID1, ID2, ID3 (includes EG:15903), IL13RA2, ILK, LZTS1, MUC1, NR4A2, ODC1, PEG10, PLAUR, SKP2 (includes EG:27401), SLC3A2, SSTR2, TNFSF13B, ZMAT3 Cellular Growth and proliferation proliferation of carcinoma cells 2.56E-02 ID1, ID2, ID3 (includes EG:15903), ILK, PLAUR 5 Proliferation Cellular Growth and proliferation proliferation of fibroblast-like 3.86E-02 CCL2, CD44 (includes EG:100330801), KCNMA1, TNFRSF10B 4 Proliferation synoviocytes Cellular Growth and proliferation proliferation of epithelial cell 4.01E-02 ABCC5, CASP9 (includes EG:100140945), CCND1, CDK2, 18 Proliferation lines CDK2AP1, CDKN2A, CYR61, DLST, EGR1, FSCN1, ITGA5, MET, PLAUR, PTN, QPCT, RARRES3, RASSF1, STAT3 Cellular Growth and proliferation proliferation of cervical cancer 4.16E-02 BIRC2, CABLES1, CDKN1B, CDKN2A, FOS, GADD45A, 22 Proliferation cell lines HGS, HSPB8, IRF1 (includes EG:16362), ITGA1, MECOM, NAA35, NR4A2, PFKFB3, RBL1, SPP1 (includes EG:20750), SPRY2, STAT3, TFAP2A, TFAP2C, TGFA, ZFP36 Cellular Growth and proliferation proliferation of kidney cancer 4.66E-02 MET, RASSF1, STAT3, TGFA, VHL, VMP1 6 Proliferation cell lines Cellular Growth and colony formation colony formation 1.79E-04 ALDH1A1, ANKRD1, ATF3, BAMBI, BCL2L1, CABLES1, 53 Proliferation CADM1, CASP9 (includes EG:100140945), CCL3L1/CCL3L3, CCND1, CDCA7, CDKN1B, CDKN2A, CYP2J2, CYR61, DLC1, EPAS1, FOS, GADD45A, GADD45B, HAS2, IFIH1, IGFBP7, IL7, KLF4, KLF6, LAPTM4B, LGALS3, LZTS1, MECOM, MET, MT1X, NCOA3, NUPR1, PBK, PCGF2, PHB, PHLDA1, PPP1R15A, PRKAR1A, PTN, RASSF1, RPS6KA5, SIVA1, SLC5A8, SOD2, SPARC, SPHK1, SPRY2, TGFB1I1, TMSB10/TMSB4X, TPD52, YY1AP1 Cellular Growth and colony formation colony formation of cells 2.82E-04 ALDH1A1, ANKRD1, ATF3, BAMBI, BCL2L1, CABLES1, 52 Proliferation CADM1, CASP9 (includes EG:100140945), CCL3L1/CCL3L3, CCND1, CDCA7, CDKN1B, CDKN2A, CYP2J2, CYR61, DLC1, EPAS1, FOS, GADD45A, GADD45B, HAS2, IFIH1, IGFBP7, IL7, KLF4, KLF6, LAPTM4B, LZTS1, MECOM, MET, MT1X, NCOA3, NUPR1, PBK, PCGF2, PHB, PHLDA1, PPP1R15A, PRKAR1A, PTN, RASSF1, RPS6KA5, SIVA1, SLC5A8, SOD2, SPARC, SPHK1, SPRY2, TGFB1I1, TMSB10/TMSB4X, TPD52, YY1AP1 Cellular Growth and colony formation colony formation of tumor cell 1.05E-03 ALDH1A1, ANKRD1, ATF3, BCL2L1, CABLES1, CADM1, 38

522

Proliferation lines CASP9 (includes EG:100140945), CDKN1B, CDKN2A, CYP2J2, CYR61, DLC1, EPAS1, FOS, GADD45A, GADD45B, HAS2, IGFBP7, KLF4, KLF6, LZTS1, MECOM, MT1X, NCOA3, NUPR1, PBK, PCGF2, PHB, PRKAR1A, PTN, RASSF1, SIVA1, SLC5A8, SOD2, SPARC, TMSB10/TMSB4X, TPD52, YY1AP1 Cellular Growth and colony formation colony formation of lung 6.52E-03 CADM1, CASP9 (includes EG:100140945), CYR61, DLC1, 9 Proliferation cancer cell lines GADD45A, GADD45B, KLF6, RASSF1, SOD2 Cellular Growth and colony formation colony formation of breast 8.29E-03 BCL2L1, CDKN1B, CYP2J2, DLC1, EPAS1, NCOA3, NUPR1, 12 Proliferation cancer cell lines PBK, PCGF2, PHB, SIVA1, SPARC Cellular Growth and colony formation colony formation of 3.01E-02 CDKN2A, HAS2 2 Proliferation fibrosarcoma cell lines Cellular Growth and colony formation colony formation of hepatoma 4.20E-02 ANKRD1, GADD45B, YY1AP1 3 Proliferation cell lines Cellular Growth and formation formation of chondrocytes 1.08E-02 ITGA5, ITGB1 2 Proliferation Cellular Growth and growth arrest in growth of kidney 1.08E-02 STAT3, VHL 2 Proliferation cancer cell lines Cellular Growth and growth arrest in growth of tumor cell 1.50E-02 CBX7, CCND1, CDK2, CDKN1B, CDKN2A, CDKN2B, IRF1 15 Proliferation lines (includes EG:16362), KLF4, MUC1, RBL1, SKP2 (includes EG:27401), SPHK1, STAT1, STAT3, VHL Cellular Growth and growth arrest in growth of cells 2.68E-02 CBX7, CCND1, CDK2, CDKN1B, CDKN2A, CDKN2B, EGR1, 18 Proliferation IRF1 (includes EG:16362), KLF4, MUC1, NRP1 (includes EG:18186), RBL1, SKP2 (includes EG:27401), SPHK1, STAT1, STAT3, VHL, ZBTB17 Cellular Growth and cytostasis cytostasis of smooth muscle 3.01E-02 ID1, ID2 2 Proliferation cells Cellular Growth and cytostasis cytostasis 3.44E-02 ABCC5, BCL6, BHLHE40, CCND1, CCND3, CDKN1B, 25 Proliferation CDKN2A, CDKN2C, CDKN2D, CYR61, GPI, HDAC1, ID1, ID2, IRF1 (includes EG:16362), KLF6, MECOM, MXI1, NQO1, PPP1R15A, PRKAR1A, STAT1, TRIM33, UBE3A, ZNF217 Cellular Growth and outgrowth outgrowth of tumor cell lines 3.01E-02 CCND1, CYR61 2 Proliferation Cell Cycle interphase initiation of interphase 2.91E-05 BAD, CDKN1B, CDKN2A, E2F3, ITGB1, SKP2 (includes 7 EG:27401), VHL Cell Cycle interphase initiation of interphase of 2.11E-04 BAD, CDKN1B, CDKN2A, ITGB1, VHL 5 tumor cell lines

523

Cell Cycle interphase initiation of interphase of 4.13E-03 BAD, CDKN1B, CDKN2A 3 breast cancer cell lines Cell Cycle interphase interphase 1.13E-02 AGK, AHR, BAD, BCL3, BHLHE40, BLID, BRSK1, 67 CAMK2N1, CCND1, CCND3, CD44 (includes EG:100330801), CDK2, CDK2AP1, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CEBPD, CYR61, DDIT3, E2F3, FOXM1, GADD45A, HAS2, HBEGF, HINFP, ID1, ID2, ID3 (includes EG:15903), IL24, ITGA5, ITGB1, KLF4, KLF6, KRT7, LGALS1, LGALS3, LZTS1, MCM10 (includes EG:307126), MCM7, MET, MT1A, MTDH, MXI1, MYB, NBN, NCOA3, NES, PLAUR, POLA1, PPP2CB, PPP2R2A, RASSF1, RBL1, SHC1 (includes EG:20416), SKP2 (includes EG:27401), SOX2, SPRY2, SSTR2, STAT1, TBRG4, TGFA, TIMELESS, TPD52L1, TYMS, VHL Cell Cycle interphase interphase of prostate cancer 1.15E-02 AGK, CCND1, CDKN1B, IL24, NCOA3, SHC1 (includes 7 cell lines EG:20416), SKP2 (includes EG:27401) Cell Cycle interphase interphase of tumor cell lines 1.44E-02 AGK, AHR, BAD, BHLHE40, BLID, CAMK2N1, CCND1, 45 CCND3, CDK2, CDKN1B, CDKN2A, CDKN2B, CEBPD, CYR61, DDIT3, FOXM1, GADD45A, HAS2, IL24, ITGA5, ITGB1, KLF4, LGALS1, LGALS3, MCM10 (includes EG:307126), MET, MT1A, MTDH, MXI1, MYB, NCOA3, PLAUR, POLA1, PPP2CB, PPP2R2A, RASSF1, RBL1, SHC1 (includes EG:20416), SKP2 (includes EG:27401), SOX2, SPRY2, STAT1, TGFA, TYMS, VHL Cell Cycle interphase interphase of melanoma cell 1.48E-02 CCND1, CCND3, CDKN1B, CDKN2B, ITGB1 5 lines Cell Cycle interphase arrest in interphase of colon 2.44E-02 CAMK2N1, CDKN1B, CDKN2A, GADD45A, ITGA5, KLF4, 8 cancer cell lines LGALS1, TYMS Cell Cycle interphase interphase of colon cancer cell 2.73E-02 CAMK2N1, CDKN1B, CDKN2A, GADD45A, ITGA5, KLF4, 11 lines LGALS1, MT1A, MXI1, TGFA, TYMS Cell Cycle interphase interphase of leukemia cell 4.66E-02 CDK2, CDKN1B, CDKN2B, CEBPD, MYB, STAT1 6 lines Cell Cycle S phase S phase of prostate cancer cell 5.14E-04 AGK, CCND1, CDKN1B, SHC1 (includes EG:20416), SKP2 5 lines (includes EG:27401) Cell Cycle S phase initiation of S phase 5.14E-04 CDKN1B, CDKN2A, E2F3, ITGB1, SKP2 (includes EG:27401) 5 Cell Cycle S phase S phase 1.46E-03 AGK, BHLHE40, CAMK2N1, CCND1, CCND3, CDK2AP1, 30 CDKN1B, CDKN2A, E2F3, FOXM1, HBEGF, HINFP, ID1, ID3 (includes EG:15903), ITGB1, LGALS1, LZTS1, MCM10

524

(includes EG:307126), MCM7, MET, MXI1, NBN, NCOA3, POLA1, RBL1, SHC1 (includes EG:20416), SKP2 (includes EG:27401), TGFA, TIMELESS, TYMS Cell Cycle S phase arrest in S phase 3.33E-03 AGK, CAMK2N1, CCND1, CDKN2A, LGALS1, MET, NBN, 9 RBL1, TYMS Cell Cycle S phase initiation of S phase of tumor 9.52E-03 CDKN1B, CDKN2A, ITGB1 3 cell lines Cell Cycle S phase arrest in S phase of cancer cells 1.08E-02 CDKN2A, LGALS1 2 Cell Cycle S phase arrest in S phase of prostate 1.08E-02 AGK, CCND1 2 cancer cell lines Cell Cycle S phase entry into S phase of bone 1.08E-02 BHLHE40, CDKN2A 2 cancer cell lines Cell Cycle S phase S phase of cancer cells 1.76E-02 CDKN2A, LGALS1, LZTS1 3 Cell Cycle S phase entry into S phase of cervical 1.76E-02 CDKN1B, FOXM1, MCM10 (includes EG:307126) 3 cancer cell lines Cell Cycle S phase entry into S phase 1.91E-02 BHLHE40, CCND1, CCND3, CDKN1B, CDKN2A, FOXM1, 13 HBEGF, HINFP, ID3 (includes EG:15903), ITGB1, MCM10 (includes EG:307126), NCOA3, SKP2 (includes EG:27401) Cell Cycle S phase S phase of melanoma cell lines 3.01E-02 CCND1, ITGB1 2 Cell Cycle S phase arrest in S phase of epithelial 3.01E-02 MET, NBN 2 cell lines Cell Cycle S phase entry into S phase of lung cell 3.01E-02 CDKN2A, NCOA3 2 lines Cell Cycle S phase initiation of S phase of breast 3.01E-02 CDKN1B, CDKN2A 2 cancer cell lines Cell Cycle cell cycle arrest in cell cycle progression 1.23E-03 AHR, C15orf63, CCND1, CD44 (includes EG:100330801), 36 progression CDK14, CDK2, CDK7, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CUL1, E2F2, FOXM1, GADD45A, GDF11, ID1, ID3 (includes EG:15903), IL8, IRF7, KAT2B, LGALS3, NBN, PPP1R15A, RASSF1, S100A4, SKP2 (includes EG:27401), SOX2, SSBP2, STAT1, TBRG4, TSG101, WDR6, XBP1 (includes EG:140614), ZAK Cell Cycle cell cycle cell cycle progression 1.35E-03 AHR, ANAPC11, ATF3, AXL, BCL6, BHLHE40, BIRC2, 96 progression BORA, BRD7, C15orf63, CAMK2N1, CASP2, CBL, CCND1, CCND3, CCNO, CD44 (includes EG:100330801), CDC16, CDK14, CDK2, CDK5, CDK7, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CHRDL1, CKAP5, CLIP1, CUL1,

525

CYR61, DEK, E2F2, FOS, FOXM1, FYN, GADD45A, GAS1 (includes EG:14451), GDF11, GPI, HAUS6, HBEGF, ID1, ID3 (includes EG:15903), IER3, IL7, IL8, IRF1 (includes EG:16362), IRF7, KAT2B, KLF6, LGALS3, MAD2L1BP, MECOM, MXI1, NAMPT, NBN, ORC6 (includes EG:23594), PCGF2, PCNA, PHB, PPP1R15A, PPP2R5C, PRKAR1A, PTN, RASSF1, RBL1, S100A4, SEPT9, SHC1 (includes EG:20416), SKP2 (includes EG:27401), SOX2, SPARC, SPC25 (includes EG:100144563), SPHK1, SPRY2, SSBP2, STAT1, STAT3, TAF1D, TBRG4, TFE3, TGFA, TM4SF1, TRIM33, TSC22D1, TSG101, VHL, WDR6, XBP1 (includes EG:140614), YY1AP1, ZAK, ZBTB17, ZC3HC1, ZNF365 Cell Cycle cell cycle cell cycle progression of 4.13E-03 CCND1, E2F2, SKP2 (includes EG:27401) 3 progression hepatocytes Cell Cycle cell cycle cell cycle progression of T cell 1.08E-02 CDKN1B, IL7 2 progression acute lymphoblastic leukemia cells Cell Cycle cell cycle cell cycle progression of 2.90E-02 CCND1, CDKN2A, RBL1, SKP2 (includes EG:27401) 4 progression fibroblasts Cell Cycle cell cycle arrest in cell cycle progression 3.01E-02 CCND1, E2F2 2 progression of hepatocytes Cell Cycle cell cycle arrest in cell cycle progression 3.01E-02 AHR, CDK14 2 progression of neuroblastoma cell lines Cell Cycle cell cycle arrest in cell cycle progression 3.01E-02 CDKN1B, CDKN2A 2 progression of skin cancer cell lines Cell Cycle cell cycle cell cycle progression of 3.01E-02 S100A4, STAT3 2 progression pancreatic cancer cell lines Cell Cycle cell cycle cell cycle progression of cancer 3.86E-02 CCND1, CDKN1B, GADD45A, IL7 4 progression cells Cell Cycle cell cycle cell cycle progression of brain 4.97E-02 CCND1, CD44 (includes EG:100330801), CDKN1B, CDKN2C 4 progression cancer cell lines Cell Cycle sub-G1 phase arrest in sub-G1 phase of tumor 1.93E-03 BLID, CDK2, CDKN1B, CDKN2B, HAS2 5 cell lines Cell Cycle sub-G1 phase arrest in sub-G1 phase of 3.01E-02 CDKN1B, CDKN2B 2 melanoma cell lines Cell Cycle G1/S phase G1/S phase transition of tumor 6.08E-03 AHR, BAD, CCND1, CCND3, CDKN1B, CDKN2A, FOXM1, 14 transition cell lines ITGB1, KLF4, MET, MT1A, NCOA3, SOX2, SPRY2

526

Cell Cycle G1/S phase G1/S phase 7.71E-03 AHR, BAD, CCND1, CCND3, CDKN1B, CDKN2A, CDKN2B, 22 transition CDKN2C, CDKN2D, E2F3, FOXM1, GADD45A, HINFP, ID2, ITGB1, KLF4, MET, MT1A, NBN, NCOA3, SOX2, SPRY2 Cell Cycle G1/S phase G1/S phase transition of 1.08E-02 CCND3, ITGB1 2 transition melanoma cell lines Cell Cycle G1/S phase G1/S phase transition of breast 2.56E-02 BAD, CCND1, CDKN1B, CDKN2A, SOX2 5 transition cancer cell lines Cell Cycle G1 phase G1 phase of breast cancer cell 7.98E-03 BAD, BHLHE40, CCND1, CDKN1B, CDKN2A, LGALS1, 9 lines LGALS3, RASSF1, SOX2 Cell Cycle G1 phase G1 phase 1.70E-02 AHR, BAD, BCL3, BHLHE40, CCND1, CCND3, CDK2, 38 CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CEBPD, CYR61, DDIT3, E2F3, FOXM1, GADD45A, HINFP, ID2, ITGA5, ITGB1, KLF4, KLF6, LGALS1, LGALS3, MET, MT1A, NBN, NCOA3, RASSF1, RBL1, SKP2 (includes EG:27401), SOX2, SPRY2, SSTR2, TBRG4, TYMS Cell Cycle G1 phase arrest in G1 phase of breast 2.67E-02 BHLHE40, CCND1, CDKN1B, LGALS1, LGALS3, RASSF1 6 cancer cell lines Cell Cycle G1 phase G1 phase of colon cancer cell 3.09E-02 CDKN1B, CDKN2A, ITGA5, KLF4, LGALS1, MT1A, TYMS 7 lines Cell Cycle G1 phase G1 phase of tumor cell lines 3.23E-02 AHR, BAD, BHLHE40, CCND1, CCND3, CDK2, CDKN1B, 26 CDKN2A, CDKN2B, CYR61, DDIT3, FOXM1, ITGA5, ITGB1, KLF4, LGALS1, LGALS3, MET, MT1A, NCOA3, RASSF1, RBL1, SKP2 (includes EG:27401), SOX2, SPRY2, TYMS Cell Cycle G1 phase G1 phase of melanoma cell 4.20E-02 CCND3, CDKN1B, ITGB1 3 lines Cell Cycle G1 phase G1 phase of ovarian cancer cell 4.20E-02 CCND1, CDKN2A, SKP2 (includes EG:27401) 3 lines Cell Cycle G1 phase arrest in G1 phase of colon 4.66E-02 CDKN1B, CDKN2A, ITGA5, KLF4, LGALS1, TYMS 6 cancer cell lines Cell Cycle mitogenesis mitogenesis of melanoma cell 9.52E-03 AXL, TFE3, TRIM33 3 lines Cell Cycle mitogenesis mitogenesis 2.67E-02 AXL, CBL, CYR61, FYN, HBEGF, PTN, SHC1 (includes 13 EG:20416), SPARC, SPHK1, SPRY2, TFE3, TGFA, TRIM33 Cell Cycle mitogenesis mitogenesis of tumor cell lines 2.86E-02 AXL, FYN, SHC1 (includes EG:20416), SPHK1, SPRY2, TFE3, 8 TGFA, TRIM33

527

Cell Cycle late cytokinesis late cytokinesis of tumor cell 1.08E-02 KIF20B, SEPT9 2 lines Cell Cycle DNA damage DNA damage checkpoint 2.67E-02 CCND1, FBXO6, HINFP, NBN, TIPRL, ZAK 6 checkpoint Cell Cycle morphology morphology of mitotic spindle 4.20E-02 FOXM1, ORC6 (includes EG:23594), RASSF1 3 Cell-To-Cell Signaling binding binding of prostate cancer cell 7.96E-05 CD44 (includes EG:100330801), HAS2, ITGA3, ITGA5, ITGB1, 8 and Interaction lines LGALS3, NRP1 (includes EG:18186), PTPRM Cell-To-Cell Signaling binding binding of keratinocytes 3.01E-02 ITGA3, ITGB1 2 and Interaction Cell-To-Cell Signaling binding binding of skin cell lines 3.01E-02 CYR61, ITGB1 2 and Interaction Cell-To-Cell Signaling binding binding of embryonic cell lines 3.91E-02 ABCA1, ITGB1, PLAUR, SCARB1, SDC4, SORT1 6 and Interaction Cell-To-Cell Signaling attachment attachment of cells 1.35E-03 CD44 (includes EG:100330801), EGR1, ILK, ITGA1, ITGA3, 12 and Interaction ITGA5, ITGB1, LRPAP1, SPP1 (includes EG:20750), SPRY2, TGFBI, VHL Cell-To-Cell Signaling attachment attachment of tumor cell lines 2.67E-02 CD44 (includes EG:100330801), EGR1, ITGA3, ITGA5, ITGB1, 6 and Interaction SPRY2 Cell-To-Cell Signaling attachment attachment of fibroblasts 3.01E-02 LRPAP1, TGFBI 2 and Interaction Cell-To-Cell Signaling attachment attachment of smooth muscle 3.01E-02 ITGA1, ITGB1 2 and Interaction cells Cell-To-Cell Signaling chemoattraction chemoattraction of 1.46E-03 CCL2, CCL3L1/CCL3L3, DEK, IL8 4 and Interaction mononuclear leukocytes Cell-To-Cell Signaling chemoattraction chemoattraction of phagocytes 3.24E-03 CCL2, CCL20, CCL3L1/CCL3L3, DEK, IL8 5 and Interaction Cell-To-Cell Signaling chemoattraction chemoattraction of myeloid 1.46E-02 CCL2, CCL3L1/CCL3L3, DEK, IL8 4 and Interaction cells Cell-To-Cell Signaling chemoattraction chemoattraction of cells 2.67E-02 CCL2, CCL20, CCL3L1/CCL3L3, DEK, IL8, SCG2 6 and Interaction Cell-To-Cell Signaling chemoattraction chemoattraction of T 3.01E-02 DEK, IL8 2 and Interaction lymphocytes Cell-To-Cell Signaling chemoattraction chemoattraction of monocytes 3.01E-02 CCL2, CCL3L1/CCL3L3 2 and Interaction Cell-To-Cell Signaling adhesion adhesion of lymphatic system 9.52E-03 CCL20, ITGA5, ITGB1 3

528 and Interaction cells Cell-To-Cell Signaling adhesion adhesion of cell-associated 9.68E-03 CDKN2A, COL13A1, ECM2 (includes EG:1842), ITGA11, 9 and Interaction matrix ITGA3, ITGB1, ITGB1BP1, PPFIA1, SORBS1 Cell-To-Cell Signaling adhesion adhesion of glomerular cells 1.08E-02 CCL2, IL8 2 and Interaction Cell-To-Cell Signaling adhesion adhesion of mesothelial cells 1.08E-02 HBEGF, ITGA3 2 and Interaction Cell-To-Cell Signaling adhesion adhesion of kidney cells 1.62E-02 CADM1, CCL2, HBEGF, IL8, ITGA3, ITGB1, PLAUR, RRAS, 11 and Interaction SOD2, TIMP3, VHL Cell-To-Cell Signaling adhesion adhesion of fibrosarcoma cell 2.84E-02 ITGA5, ITGB1, JAM3 3 and Interaction lines Cell-To-Cell Signaling adhesion adhesion of stomach cancer 2.84E-02 CYR61, ITGA3, ITGB1 3 and Interaction cell lines Cell-To-Cell Signaling adhesion adhesion of cervical cancer cell 4.05E-02 FOS, ITGB1, MECOM, PAK2, RASSF1 5 and Interaction lines Cell-To-Cell Signaling formation formation of fibrillar adhesions 1.08E-02 ITGB1, VHL 2 and Interaction Cell-To-Cell Signaling recruitment recruitment of macrophages 1.08E-02 CCL2, IL8 2 and Interaction Cell-To-Cell Signaling recruitment recruitment of phagocytes 4.20E-02 C5, CCL2, IL8 3 and Interaction Cell-To-Cell Signaling activation activation of leukocyte cell 1.48E-02 B2M, HLA-A, LGALS3, SOCS2, VAMP4 5 and Interaction lines Cell-To-Cell Signaling response response of embryonic cell 2.90E-02 BIRC2, BIRC3, IFIH1, IRAK2 4 and Interaction lines Cell-To-Cell Signaling response response of epithelial cell lines 2.90E-02 BIRC2, BIRC3, IFIH1, IRAK2 4 and Interaction Cell-To-Cell Signaling response response of kidney cell lines 2.90E-02 BIRC2, BIRC3, IFIH1, IRAK2 4 and Interaction Cell-To-Cell Signaling sensitization sensitization of cells 2.90E-02 BCL2L1, CASP9 (includes EG:100140945), STAT1, TYMS 4 and Interaction Cell-To-Cell Signaling sensitization sensitization of tumor cells 3.01E-02 BCL2L1, CASP9 (includes EG:100140945) 2 and Interaction

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Appendix 12. 4 The top molecular and cellular functions associated with differentially expressed genes in the FPGS-inhibited MDA-MB-435 breast cancer cells No. of Category Function Function Annotation P-value Genes Genes Cell Death cell death cell death of breast cancer cell 3.06E-04 ADM, AKT1, BCL2L1, BID, BIRC2, BIRC3, BNIP3, CCND1, 28 lines CDK1, CEACAM1 (includes others), CHMP5, CYR61, DAB2, FASN, FTH1 (includes EG:14319), GADD45A, HIF1A, HSPB8, JUN, MUC1, NQO1, PSEN1, SIVA1, SND1, SOD2, TGFBR3, TRADD, UGCG Cell Death cell death cell death of T lymphocytes 5.26E-04 AKT1, AKTIP, BCL2L1, CDK1, EZR, FTH1 (includes 12 EG:14319), IL7, RAC2, SIVA1, SPP1 (includes EG:20750), STOML2, ZC3H8 Cell Death cell death cell death of brain cancer cell 1.86E-03 AKT1, BCL2L1, BIRC2, BIRC3, CDK1, DKK1, FTH1 (includes 13 lines EG:14319), HIF1A, MITF, PSEN1, SEC61G, SNCA, SOD2 Cell Death cell death cell death of cervical cancer 1.98E-03 AKT1, BCL2L1, BID, BIRC2, C15orf63, C9orf80, CDK1, 26 cell lines CHMP5, DCTN3, FLNB, GADD45A, ITPK1, JUN, MAPKAP1, MUC1, NAA35, NEK6, PKM2, SEC61G, SGK1, SNCA, SOD2, SPC25 (includes EG:100144563), SPP1 (includes EG:20750), TRIM32, UBQLN1 Cell Death cell death cell death of lymphocytes 2.95E-03 AKT1, AKTIP, BCL2L1, CDK1, EZR, FCAR, FTH1 (includes 14 EG:14319), HLA-DRB4, IL7, RAC2, SIVA1, SPP1 (includes EG:20750), STOML2, ZC3H8 Cell Death cell death cell death of tumor cell lines 1.01E-02 ADM, AGPAT2, AKAP12, AKR1B1, AKT1, BCHE, BCL2L1, 79 BHLHE40, BID, BIRC2, BIRC3, BNIP3, C15orf63, C9orf80, CCND1, CD55, CDK1, CDKN2D, CEACAM1 (includes others), CHMP5, CREB3L2, CSF2RA, CYP2J2, CYR61, DAB2, DCTN3, DKK1, ETS1, FASN, FKBP1A, FLNB, FTH1 (includes EG:14319), GADD45A, GLRX, HIF1A, HLA-DRB4, HOXC6, HSPB8, HTRA1, IFI6, IL7, ITPK1, JUN, KLF9, MAPKAP1, MITF, MT1X, MT2A, MUC1, MYB, NAA35, NEK6, NQO1, NUAK1, NUPR1, PKM2, PSEN1, RAD23B, S100A4, SEC61G, SGK1, SIVA1, SNCA, SND1, SOD2, SPC25 (includes EG:100144563), SPP1 (includes EG:20750), SSTR2, STOML2, TGFBR3, TMED10, TMEM123 (includes EG:114908), TNFAIP3, TRADD, TRIM32, TTF1, UBQLN1, UGCG, VHL Cell Death cell death cell death of neuroblastoma cell 3.15E-02 ADM, AKT1, BCHE, BCL2L1, CDKN2D, CREB3L2, FKBP1A, 11 lines GLRX, SNCA, SPP1 (includes EG:20750), UGCG Cell Death cell death cell death of blood cells 3.32E-02 AKT1, AKTIP, BCL2L1, CDK1, CSF2RA, EZR, FCAR, FTH1 19

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(includes EG:14319), HLA-DRB4, IL7, JUN, MVP, MX1, RAC2, SIVA1, SOD2, SPP1 (includes EG:20750), STOML2, ZC3H8 Cell Death cell death cell death of immune cells 4.33E-02 AKT1, AKTIP, BCL2L1, CDK1, EZR, FCAR, FTH1 (includes 18 EG:14319), HLA-DRB4, IL7, JUN, MVP, MX1, RAC2, SIVA1, SOD2, SPP1 (includes EG:20750), STOML2, ZC3H8 Cell Death apoptosis apoptosis of breast cancer cell 3.44E-04 ADM, AKT1, BCL2L1, BID, BIRC2, BIRC3, BNIP3, CCND1, 25 lines CDK1, CEACAM1 (includes others), CHMP5, CYR61, DAB2, FTH1 (includes EG:14319), GADD45A, HIF1A, HSPB8, JUN, MUC1, NQO1, SIVA1, SND1, SOD2, TGFBR3, UGCG Cell Death apoptosis apoptosis of T lymphocytes 2.50E-03 AKT1, AKTIP, BCL2L1, EZR, IL7, RAC2, SIVA1, SPP1 10 (includes EG:20750), STOML2, ZC3H8 Cell Death apoptosis apoptosis of cardiomyocytes 3.62E-03 ADM, BCL2L1, HSPB8, S100A1 4 Cell Death apoptosis apoptosis of neurons 7.00E-03 BCL2L1, BID, GLRX, HERPUD1, NAE1, PSEN1, SNCA, 8 SOD2 Cell Death apoptosis apoptosis of cervical cancer cell 2.32E-02 AKT1, BCL2L1, BID, BIRC2, C15orf63, CDK1, CHMP5, 18 lines FLNB, GADD45A, ITPK1, JUN, MAPKAP1, MUC1, NEK6, SEC61G, SGK1, SNCA, TRIM32 Cell Death apoptosis apoptosis of tumor cell lines 2.37E-02 ADM, AGPAT2, AKAP12, AKR1B1, AKT1, BCL2L1, 66 BHLHE40, BID, BIRC2, BIRC3, BNIP3, C15orf63, CCND1, CD55, CDK1, CDKN2D, CEACAM1 (includes others), CHMP5, CSF2RA, CYP2J2, CYR61, DAB2, DKK1, ETS1, FASN, FLNB, FTH1 (includes EG:14319), GADD45A, GLRX, HIF1A, HOXC6, HSPB8, HTRA1, IFI6, IL7, ITPK1, JUN, KLF9, MAPKAP1, MITF, MT2A, MUC1, MYB, NEK6, NQO1, NUPR1, PSEN1, RAD23B, S100A4, SEC61G, SGK1, SIVA1, SNCA, SND1, SOD2, SPP1 (includes EG:20750), SSTR2, STOML2, TGFBR3, TMED10, TNFAIP3, TRADD, TRIM32, TTF1, UGCG, VHL Cell Death apoptosis apoptosis 2.93E-02 ADM, AGPAT2, AKAP12, AKR1B1, AKT1, AKTIP, BCL2L1, 98 BHLHE40, BID, BIRC2, BIRC3, BNIP3, C15orf63, CAPN3, CBL, CCND1, CD55, CD74, CDK1, CDKN2D, CEACAM1 (includes others), CHMP5, CSF2RA, CYP2J2, CYR61, DAB2, DEPTOR, DKK1, DPF2, ERCC5, ETS1, EZR, FASN, FKBP1A, FLNB, FTH1 (includes EG:14319), GADD45A, GLRX, HERPUD1, HEY1, HIF1A, HOXC6, HSPB8, HTRA1, IFI6, IL7, ITPK1, JUN, KCNMA1, KLF9, LPAR1, MAPKAP1, MITF, MT2A, MUC1, MX1, MYB, NAE1, NEK6, NME3, NQO1, NT5E, NUPR1, ODC1, PHLDA1, PKM2, PLSCR1, PPP2R4, PRKRIR, PSEN1, RAC2, RAD23B, S100A1, S100A4, SEC61G,

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SGK1, SH3BGRL3, SIVA1, SNCA, SND1, SOD2, SORT1, SPP1 (includes EG:20750), SSTR2, STOML2, TGFBR3, TMED10, TMEM123 (includes EG:114908), TNFAIP3, TPD52L1, TRADD, TRIM32, TTF1, UBQLN1, UGCG, URI1, VHL, ZC3H8 Cell Death apoptosis apoptosis of endometrial cancer 3.05E-02 ADM, CYR61 2 cell lines Cell Death apoptosis apoptosis of melanoma cells 3.28E-02 ETS1, HSPB8, PHLDA1 3 Cell Death apoptosis apoptosis of brain cancer cell 4.33E-02 AKT1, BCL2L1, CDK1, DKK1, FTH1 (includes EG:14319), 8 lines MITF, PSEN1, SEC61G Cell Death apoptosis apoptosis of endothelioma cell 4.81E-02 MYB 1 lines Cell Death colony survival colony survival of lymphoma 2.31E-03 BCL2L1, CCND1 2 cell lines Cell Death inhibition inhibition of apoptosis 8.03E-03 AKT1, BCL2L1, BFAR, BIRC3, BNIP3, CDK1, CDKN2D, 14 IFI6, MUC1, PSEN1, SIVA1, SNCA, SUPV3L1, TNFAIP3 Cell Death cytolysis cytolysis of prostate cancer cell 1.30E-02 CD55, MUC1 2 lines Cell Death activation-induced activation-induced cell death of 1.37E-02 AKT1, RAC2, SIVA1 3 cell death T lymphocytes Cell Death activation-induced activation-induced cell death 2.27E-02 ADM, AKT1, RAC2, SIVA1 4 cell death Cell Death necrosis necrosis 1.67E-02 ACP1 (includes EG:11431), ADM, AGPAT2, AKAP12, AKR1B1, 98 AKT1, AKTIP, BCHE, BCL2L1, BHLHE40, BID, BIRC2, BIRC3, BNIP3, C15orf63, C9orf80, CCND1, CD55, CD74, CDK1, CDKN2D, CEACAM1 (includes others), CHMP5, CREB3L2, CSF2RA, CYP2J2, CYR61, DAB2, DCTN3, DKK1, DLST, ETS1, EZR, FASN, FCAR, FKBP1A, FLNB, FTH1 (includes EG:14319), GADD45A, GLRX, HERPUD1, HIF1A, HK1, HLA-DRB4, HOXC6, HSPB8, HTRA1, IFI6, IL7, ITPK1, JUN, KLF9, LPAR1, MAPKAP1, MITF, MT1X, MT2A, MUC1, MVP, MX1, MYB, NAA35, NAE1, NDUFAB1, NEK6, NQO1, NUAK1, NUPR1, PHLDA1, PKM2, PMP22, PSEN1, RAC2, RAD23B, S100A1, S100A4, SEC61G, SGK1, SIVA1, SNCA, SND1, SOD2, SPC25 (includes EG:100144563), SPP1 (includes EG:20750), SSTR2, STOML2, TGFB1I1, TGFBR3, TMED10, TMEM123 (includes EG:114908), TNFAIP3, TRADD, TRIM32, TTF1, UBQLN1, UGCG, VHL, ZC3H8

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Cell Death cell viability cell viability of keratinocyte 4.81E-02 BCL2L1 1 cancer cell lines Cell Death cytotoxicity cytotoxicity of decidual natural 4.81E-02 CEACAM1 (includes others) 1 killer cells Cell Death cytotoxicity cytotoxicity of hepatoma cell 4.81E-02 SGK1 1 lines Cell Death degeneration degeneration of striatonigral 4.81E-02 SNCA 1 neurons Cell Death opsonization opsonization of neuroblastoma 4.81E-02 CD55 1 cell lines Cell Death osteonecrosis osteonecrosis 4.81E-02 ACP1 (includes EG:11431) 1 Cell Death permeability permeability of leukemia cell 4.81E-02 TMEM123 (includes EG:114908) 1 lines Cell Death self-renewal self-renewal of leukemia cell 4.81E-02 MUC1 1 lines Cell Death survival survival of natural killer-22 4.81E-02 IL7 1 cells Cellular Assembly and development development of mitochondria 1.99E-03 PPARGC1A, STOML2, SUPV3L1 3 Organization Cellular Assembly and development development of fibrillar 4.81E-02 SNCA 1 Organization inclusions Cellular Assembly and opening opening of cellular membrane 6.72E-03 BCL2L1, BID 2 Organization Cellular Assembly and adhesion adhesion of cell-associated 6.89E-03 COL13A1, ITGA10, ITGA11, PPFIA1, SGCE, TSC1 6 Organization matrix Cellular Assembly and biogenesis biogenesis of mitochondria 1.30E-02 PPARGC1A, STOML2 2 Organization Cellular Assembly and formation formation of cilia 1.39E-02 FOPNL, TMEM67, TTC8, VHL 4 Organization Cellular Assembly and quantity quantity of nucleus 2.10E-02 ADM, AKT1 2 Organization Cellular Assembly and quantity quantity of perikaryon 4.81E-02 SNCA 1 Organization Cellular Assembly and permeability permeability of mitochondrial 3.05E-02 BCL2L1, BNIP3 2 Organization membrane

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Cellular Assembly and association association of actin 4.81E-02 FXYD5 1 Organization cytoskeleton Cellular Assembly and association association of plasma 4.81E-02 ABCA1 1 Organization membrane Cellular Assembly and binding binding of Golgi membranes 4.81E-02 TMED10 1 Organization Cellular Assembly and binding binding of myosin filaments 4.81E-02 FHL1 (includes EG:14199) 1 Organization Cellular Assembly and ciliogenesis ciliogenesis of kidney cancer 4.81E-02 VHL 1 Organization cell lines Cellular Assembly and conversion conversion of Golgi apparatus 4.81E-02 GOLGA5 1 Organization Cellular Assembly and conversion conversion of Golgi stacks 4.81E-02 GOLGA5 1 Organization Cellular Assembly and disruption disruption of fibrils 4.81E-02 SNCA 1 Organization Cellular Assembly and disruption disruption of vesicle aggregates 4.81E-02 SNCA 1 Organization Cellular Assembly and morphology morphology of nuclear bodies 4.81E-02 ETS1 1 Organization Cellular Assembly and movement movement of lipid bilayer 4.81E-02 PLSCR1 1 Organization Cellular Assembly and presence presence of focal adhesions 4.81E-02 VHL 1 Organization Cellular Assembly and production production of cellular inclusion 4.81E-02 SNCA 1 Organization bodies Cellular Assembly and production production of cytoplasmic 4.81E-02 SNCA 1 Organization aggregates Cellular Assembly and redistribution redistribution of F-actin 4.81E-02 TNIK 1 Organization Cellular Assembly and volume volume of Golgi apparatus 4.81E-02 GOLGA5 1 Organization Cell Cycle S phase arrest in S phase of prostate 2.31E-03 AGK, CCND1 2 cancer cell lines Cell Cycle S phase arrest in S phase of ovarian 4.81E-02 CDK1 1

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cancer cell lines Cell Cycle S phase entry into S phase of melanoma 4.81E-02 CCND1 1 cell lines Cell Cycle cell cycle cell cycle progression of tumor 3.62E-03 CCND1, FASN, GADD45A, IL7 4 progression cells Cell Cycle cell cycle cell cycle progression of cancer 2.21E-02 CCND1, GADD45A, IL7 3 progression cells Cell Cycle cell cycle arrest in cell cycle progression 3.05E-02 FASN, GADD45A 2 progression of tumor cells Cell Cycle cell cycle arrest in cell cycle progression 4.81E-02 GADD45A 1 progression of ovarian cancer cells Cell Cycle cell cycle arrest in cell cycle progression 4.81E-02 S100A4 1 progression of pancreatic cancer cell lines Cell Cycle cell cycle exit from cell cycle progression 4.81E-02 VHL 1 progression of kidney cancer cell lines Cell Cycle G2/M phase arrest in G2/M phase transition 6.72E-03 CCND1, CDK1 2 transition of breast cancer cell lines Cell Cycle senescence senescence of liver cell lines 6.72E-03 FASN, SREBF1 (includes EG:176574) 2 Cell Cycle G0 phase initiation of G0 phase of kidney 4.81E-02 VHL 1 cancer cell lines Cell Cycle G1 phase G1 phase of uterine cell lines 4.81E-02 CCND1 1 Cell Cycle G2/M phase G2/M phase of leukemia cell 4.81E-02 MYB 1 lines Cell Cycle late G1 phase late G1 phase of colon cancer 4.81E-02 MT1A 1 cell lines Cell Cycle mitosis initiation of mitosis 4.81E-02 CDK1 1 Cell Cycle ploidy ploidy of chromosomes 4.81E-02 HIF1A 1 Cell Cycle spindle checkpoint spindle checkpoint of breast 4.81E-02 HIF1A 1 cancer cell lines Cell Cycle sub-G1 phase sub-G1 phase of hepatoma cell 4.81E-02 E2F5 1 lines Cellular Compromise damage damage of embryonic cell lines 2.31E-03 NDUFAB1, SOD2 2 Cellular Compromise damage damage of epithelial cell lines 2.31E-03 NDUFAB1, SOD2 2

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Cellular Compromise damage damage of kidney cell lines 6.72E-03 NDUFAB1, SOD2 2 Cellular Compromise damage damage of tumor cell lines 3.05E-02 ADM, SPP1 (includes EG:20750) 2 Cellular Compromise oxidative stress oxidative stress response of 3.05E-02 NQO1, VHL 2 response tumor cell lines Cellular Compromise oxidative stress oxidative stress response of 3.28E-02 MUC1, NQO1, VHL 3 response cells Cellular Compromise oxidative stress oxidative stress response of 4.81E-02 VHL 1 response kidney cancer cell lines Cellular Compromise degeneration degeneration of striatonigral 4.81E-02 SNCA 1 neurons Cellular Compromise destabilization destabilization of myosin 4.81E-02 S100A4 1 filaments Cellular Compromise development development of fibrillar 4.81E-02 SNCA 1 inclusions Cellular Compromise disruption disruption of fibrils 4.81E-02 SNCA 1 Cellular Compromise disruption disruption of vesicle aggregates 4.81E-02 SNCA 1 Cellular Compromise injury injury of embryonic cell lines 4.81E-02 SOD2 1 Cellular Compromise injury injury of epithelial cell lines 4.81E-02 SOD2 1 Cellular Function and colony survival colony survival of lymphoma 2.31E-03 BCL2L1, CCND1 2 Maintenance cell lines Cellular Function and autophagy autophagy of neuroblastoma 1.30E-02 RAB1A (includes EG:178620), SNCA 2 Maintenance cell lines Cellular Function and autophagy autophagy of lung cell lines 4.81E-02 BNIP3 1 Maintenance Cellular Function and formation formation of cilia 1.39E-02 FOPNL, TMEM67, TTC8, VHL 4 Maintenance Cellular Function and transmembrane transmembrane potential 1.48E-02 BCL2L1, BID, BNIP3, CHMP5, HERPUD1, IFI6, IL7, 13 Maintenance potential KCNMA1, NDUFAB1, PAPOLA, SLC25A14, SOD2, STOML2 Cellular Function and transmembrane transmembrane potential of 2.16E-02 BCL2L1, BID, BNIP3, CHMP5, HERPUD1, IFI6, IL7, 12 Maintenance potential mitochondria NDUFAB1, PAPOLA, SLC25A14, SOD2, STOML2 Cellular Function and transmembrane transmembrane potential of 4.81E-02 BCL2L1 1 Maintenance potential lymphoma cell lines Cellular Function and permeability permeability of mitochondrial 3.05E-02 BCL2L1, BNIP3 2 Maintenance membrane

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Cellular Function and permeability permeability of leukemia cell 4.81E-02 TMEM123 (includes EG:114908) 1 Maintenance lines Cellular Function and ciliogenesis ciliogenesis of kidney cancer 4.81E-02 VHL 1 Maintenance cell lines Cellular Function and colony formation colony formation of 4.81E-02 IL7 1 Maintenance thymocytes Cellular Function and endocytosis endocytosis of virus 4.81E-02 CBL 1 Maintenance Cellular Function and function function of melanocytes 4.81E-02 DKK1 1 Maintenance Cellular Function and mineralization mineralization of bone cancer 4.81E-02 SNCA 1 Maintenance cell lines Cellular Function and presence presence of focal adhesions 4.81E-02 VHL 1 Maintenance Cellular Function and respiration respiration of cultured 4.81E-02 BCL2L1 1 Maintenance osteosarcoma cells Cellular Function and self-renewal self-renewal of leukemia cell 4.81E-02 MUC1 1 Maintenance lines Cellular Function and single-stranded DNA single-stranded DNA break 4.81E-02 NAE1 1 Maintenance break repair repair of breast cell lines Cellular Function and single-stranded DNA single-stranded DNA break 4.81E-02 NAE1 1 Maintenance break repair repair of colon cancer cell lines Cellular Function and turnover turnover of Langerhans cells 4.81E-02 CCL20 1 Maintenance Cellular Function and vesiculation vesiculation of lysosome 4.81E-02 WDR48 1 Maintenance

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Appendix 13 The list of differentially expressed genes associated with the top networks in response to FPGS modulation

Appendix 13. 1 The top networks matched by the genes differentially expressed in the FPGS-overexpressed HCT116 cells

Focus No. Top Functions Score Genes in Network Genes 1 DNA Replication, 23 34 ↓ANKRD17*, ↓ANP32B, ↓BAG2, ↓CCNE1, ↓CCNF, ↑CD55, ↑CD97*, ↓CDC45, ↓CDC7 (includes Recombination, and EG:12545), ↓CSNK2A1*, ↑EGR2, ↑GCLC, ↓ IGFBP6, ↑KDELR1, ↓LMNB2, ↓ MCM2, ↓MCM3*, Repair, Cell Cycle, ↓MCM4*, ↓MCM6, ↓MCM7*, ↓MCMBP, ↑NCOA3, ↓ORC1 (includes EG:18392), ↓ORC3 Cellular Assembly and (includes EG:23595), ↓ORC5 (includes EG:26429)*, ↓ORC6 (includes EG:23594), P38 MAPK, ↓PBK, Organization ↓RANBP1, ↓RBMX, ↓RNF219*, ↑TRIB1, ↓TXNDC17, ↑UBR4, ↓XPO1 2 Molecular Transport, 21 33 ↓ABCD3, ↑ARL4A*, ↓CDK6, ↑CDKN2B*, ↑CITED2, ↓DHFR, ↑ELL2, ↑FAM46A, ↓GNPDA1, Small Molecule ↑HERC5, ↑HOMER3, ↑HSH2D, ↑ID3 (includes EG:15903), ↑IFIT2, ↑ISG20, ↑KLF4*, ↓LIME1, Biochemistry, Nucleic ↑LIPA, ↓LMNB1, ↓NME2, ↓NME1 (includes EG:18102)*, ↑OASL, ↓ODC1, ↓PA2G4, ↓PAF1 Acid Metabolism (includes EG:361531), ↓PGK1, ↓PRELID1, ↑PRIC285, Rb, ↑SAT1, ↑SERTAD1, ↓SHMT1, TCR, ↓TYMS, ↑ZNFX1 3 DNA Replication, 21 33 ↓ASF1B, ↓CBX3, ↓CHAF1A, ↓CHAF1B, ↑CRIP2, ↑E2F5, ↓ECT2, ↑EFNA1, ↑ENO3, ↓H2AFZ, Recombination, and Histone H1, Histone h3, ↓KIF14, ↓KIF23*, ↓KIF4A, ↓KIFC1, ↑MT1G, ↓NCAPD2, ↓NCAPD3, Repair, Cellular ↓NCAPG2, ↓NCAPG, ↓NCAPH, ↑POLD4, ↓PRC1 (includes EG:233406), ↓PRMT6, ↓RACGAP1*, Assembly and ↓SF3B2, ↓SF3B3, ↓SMC2*, ↓SMC4*, ↑SMYD3, ↓SNRPA1, ↓TCERG1*, ↓THRAP3, ↓VRK1 Organization, Cell Cycle 4 DNA Replication, 21 33 ↓ACD, ↓ATRIP*, ↓BARD1, ↓BCCIP*, ↓BRCA1*, ↓BRCA2, ↓BRCC3, ↓C9orf80*, ↓CCNB2, Recombination, and ↓CHEK2*, ↓CSTF1, ↓FANCB, ↓FANCD2*, ↓FANCE, ↓FANCI, ↓FANCL, ↓GABPB1, Repair, Developmental ↓H3F3A/H3F3B, ↑HES1 (includes EG:15205), I kappa b kinase, ↓IPO4, ↑LMO4*, ↑OBFC2A, Disorder, Hematological ↓OBFC2B, ↓RAD51C*, ↓RBBP8*, ↓RIF1 (includes EG:295602)*, ↓RMI1 (includes EG:306734), Disease RPA, ↓SHFM1*, ↑TERF2, ↑TERF2IP, ↑TINF2, ↑UBXN1, ↑XRCC2 5 Molecular Transport, 21 33 Actin, ↓ALYREF, ↑ANTXR1, ↓CCNC*, ↓CCT2, ↓CCT3, ↓CCT4, ↓CCT7*, ↓CCT8, ↓CCT6A, RNA Trafficking, Cell ↓CDK8, ↓DDX39A, ↓DKK1, ↓ELK4, ↓ERCC8, ↓ERH, ↑GSN, ↑HBG1, ↓HNRNPA1*, Death ↓HNRNPC, ↑KAT2B, ↑LRP5, ↓MED20, ↓MED6 (includes EG:10001), ↓MTA2, ↓PCBP1, ↓PKMYT1*, ↓RAD51AP1, RNA polymerase II, ↓SRRT, ↓TCP1*, ↓THOC1, ↓THOC3*, ↓THOC5*, ↓THOC6 Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers; ↑ upregulated; ↓ downregulated.

538

Appendix 13. 2 The top networks matched by the genes differentially expressed in the FPGS-inhibited HCT116 cells

Focus No. Top Functions Score Genes in Network Genes 1 Cell Morphology, Cellular 45 31 ↑ASNS*, ↑ATF3, ↑ATF4*, ↑CEBPB (includes EG:1051), ↑CTH, ↑DDIT3, ↑DDIT4, ↓EGR1, Function and Maintenance, ↑EHF, ↑ELF3, ↑FOXO4, ↑GARS, ↑GDF15, Gsk3, Hdac, ↑HERPUD1*, ↑ IER3, ↑KDM3A, Metabolic Disease ↑LCN2, MAP2K1/2, ↑MTHFD2*, ↑NDRG1, NFkB (complex), ↑PCK2*, ↓PPAP2B*, ↑PSAT1, ↑PSMB9, ↑SLC38A2, ↑STK40, ↓TANK*, ↑TAP1, ↑TNFRSF10B*, ↑TRIB3, ↑TRIM6, ↑TRIM8 2 Cell Cycle, DNA Replication, 36 27 26s Proteasome, ↑ACTA2, ↓ANKRD36B (includes others), ↓BARD1, ↓BMP4*, ↓BRCA1*, Recombination, and Repair, ↓BUB3 (includes EG:12237)*, ↓CDC25A, ↑CDC25C, ↑CDKN1A*, ↑CRABP2, Cyclin A, Cyclin Cellular Assembly and E, E2f, ↓EXO1 (includes EG:26909)*, ↓FBXO5*, Histone h4, ↑IGFBP6, IgG, ↑JUP*, ↑KDM5B, Organization ↑KLF4*, ↑LGALS1, ↓LGALS3, ↑LMO4*, ↑MBNL2*, ↓MSH2*, ↓NBN, P38 MAPK, ↓RAD23B, ↑STX8, TFIIH, ↑TP53I3, ↓TSPAN7*, ↑UBXN1 3 Cellular Assembly and 30 24 Akt, Ap1, BCR (complex), ↓BID, ↑BTG1, ↑CALB2, ↓CCNE2, CD3, ↓CDCA5, ↑DSC2, ↓DTL, Organization, Cellular ↓DUSP6*, ERK, estrogen receptor, ↑FAS, ↑GADD45A*, hCG, ↑IL8*, Jnk, Mapk, ↓MCM10 Function and Maintenance, (includes EG:307126), ↑MKNK2*, PDGF BB, Pkc(s), ↓PKIA, ↑PLAU, ↑PMAIP1, ↑RGS2 Cancer (includes EG:19735), ↑S100A4, ↓SACS*, ↓SLC2A3, ↑TJP3, ↑TOB1, ↓UNG, ↑ZFP36 4 Inflammatory Disease, 26 22 ↑ABCA1, ↑ATF6, ↑BIK, ↑CD97*, ↓DIAPH1, ↑EEF1A1, ERK1/2, ↑F2RL1*, ↑FBXO32, Neurological Disease, Skeletal ↑HSPA5, ↓HTR7, ↑IFI27, ↑IFIT1, IFN Beta, IFN TYPE 1, Igm, IL12 (complex), Interferon alpha, and Muscular Disorders ↑IRF7*, ↑IRF9, ↑ISG15, LDL, ↑MAPK3, PI3K (complex), PI3K (family), ↓PKD2 (includes EG:18764)*, ↑PRAME, ↑PROCR, Ras, Ras homolog, ↑STAT1*, TCR, ↑TRIOBP*, ↑TXNRD1, Vegf 5 DNA Replication, 16 16 ↑AP1S1, ↑BCL3, ↑CLTC, ↑CSE1L*, Ctbp, ↑DHRS2*, DICER1, ↓EBNA1BP2, ↑FAM3C*, Recombination, and Repair, ↑FAS, ↑HEY1, ↑HS3ST1, ↑LSMD1, NAA30, ↑NAA35, NPM1, OTUB1, ↑PLAUR*, ↑PMAIP1, Cell Morphology, Connective ↓PVRL3*, ↑RPS27L, ↑RPS6KA5, ↓RRM2, ↑RRM2B, ↑SHISA5, SIRT1, ↓SLC29A1*, ↓SRSF3, Tissue Development and ↑TNFRSF10B*, TOP1, ↓TOP2A, TP53 (includes EG:22059), ↓TRIM24*, UBE2D1, Ubiquitin Function

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers; ↑ upregulated; ↓ downregulated.

539

Appendix 13. 3 The top networks matched by the genes differentially expressed in the FPGS-overexpressed MDA-MB-435 breast cancer cells

Focus No. Top Functions Score Genes in Network Genes 1 Cancer, Reproductive System 31 34 ↑ARID5B, ↑ARMCX1*, ↓BAIAP2L1, ↑CCND1, ↓CDCA7, ↓CPNE3, ↓DNAJB6, ↓EIF5A2*, Disease, Nucleic Acid ↓ENPP2*, ↓FANCI, ↓FZD7, ↓GALNT11, ↑H2AFJ*, ↑HIST2H2BE (includes others), Metabolism ↓IDH3A, ↓KIAA1524, ↑KIF20B, ↑KLF9, ↑KRT80, ↑LINC00467*, ↓LOC339290, ↓MNS1, ↑MORC4, ↑NT5E, ↑PDLIM3, ↑PHGDH, ↓PTBP3*, ↓RBMS3, ↓SPC25 (includes EG:100144563), ↑SUGP2, TCF/LEF, ↑TMEM17, ↓TRIP13, ↑ ZBED5, ↓ZFP106 2 Infectious Disease, Cancer, 27 32 ↓ADIPOR1, ↑BIRC3*, ↑CAMK2D*, CaMKII, ↑CCL3L1/CCL3L3, ↑CDC42EP1, ↑ DDA1, Dermatological Diseases and ↓FASTKD1, ↓FASTKD5, ↑HLA-F, IKK (complex), NFkB (complex), ↓NGFRAP1, ↑PDLIM1, Conditions ↑PLCE1, ↑RELB, ↑RFTN1, ↑RIOK3, ↓RIPK4, ↑RRAS2, ↑RUSC2, ↑SDC4, ↑STK40, ↓TBC1D4, ↓TFAM, ↑TJP2*, ↓TMEM14C*, ↓TPMT, ↑TRIM8, ↑UBA7, ↓UBE2N, ↑UNC93B1, ↑USP11, ↑VMP1, ↑ZFP64 3 Cancer, Cardiovascular 27 32 Ap1, ↑ARHGAP22, ↑AXL, ↑CALB2, ↓CALD1, ↑CD68*, ↑COL13A1*, ↓COL4A5, System Development and ↑DNTTIP1, ↑EMP1, estrogen receptor, ↓FGF13, ↑FJX1, hCG, ↑HK2, ↑KRT7, ↑KRT15, Function, Organismal ↑KYNU*, ↑MT2A, ↓MTDH, ↑MUC1*, ↑PLAUR*, ↑PODXL, ↓QPCT, ↓RGS20, ↑S100A3, Development ↑S100A4*, ↓SND1, ↑SPARC, ↑SPRY2, ↑STC1, ↓TGFBR3, ↑TIMP3, ↑TM4SF1, ↑TMEM158 4 DNA Replication, 27 32 ↑BLID, ↓CASP2, ↑CASP9 (includes EG:100140945), caspase, ↓CCNC, ↑CDKN2A, ↑CDKN2C, Recombination, and Repair, ↑CSDA (includes EG:56449), ↓CYCS, E2f, ↓FEN1, ↑GADD45A*, ↑HCST, ↓KAT2B, Cancer, Cellular Response to ↓KIAA0101*, ↓MCM3*, ↓MCM4, ↓MCM7, ↓MED14, ↓MED20, ↓OPA1, ↓PCNA*, ↓POLB, Therapeutics ↓PPARGC1A, ↑PRKCZ, ↓RFC4*, ↓RFC5, RNA polymerase II, ↓SLC29A1*, ↓SNRPC, ↑SSTR2, ↑TAF10 (includes EG:216185), ↑TMEM126B*, ↑ZNF83, ↑ZYX 5 Molecular Transport, Small 25 31 ↓ADM, ↑AKAP12*, AMPK, ↓APOD, ↑ATG12 (includes EG:361321), ↑DAK, ↓EPAS1, ↑HLA- Molecule Biochemistry, DRB3 (includes others), ↓ID3 (includes EG:15903), ↑IFI6*, ↑IFIH1, ↑IFIT2, ↑IFIT3, ↑IL7, Infectious Disease ↑IL7R, ↓LAMP1, ↑NFE2L3, ↑NT5C2, ↑ODC1, ↓PCBP2, ↓PFKFB3, ↓PGK1, ↓PKM2, ↑PRKACB*, ↑SAT1, ↑SEMA3A, ↑SLC2A3, ↑SPAG4, ↑STC2, ↑STIM1, TCR, VAV, Vegf, ↑VHL*, ↑ZNFX1

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers; ↑ upregulated; ↓ downregulated.

540

Appendix 13. 4 The top networks matched by the genes differentially expressed in the FPGS-inhibited MDA-MB-435 breast cancer cells

Focus No. Top Functions Score Genes in Network Genes 1 Cell Cycle, Dermatological 41 33 ↑ACSL3*, ↓CCND1, ↓CD2BP2, ↓CDK5RAP2, ↑CENPN, ↑COX4I1, ↑DDX24, ↑EEF1A1, Diseases and Conditions, ↑ENPP2*, ↑FASN, ↑GPC6, Hedgehog, ↓HIST2H2BE (includes others), ↑IDH3A, ↑KDELR2, Lipid Metabolism ↑KRT80, ↓LAMA5, ↑LAMB2 (includes EG:16779), ↑MFSD6*, ↑MNS1, ↑MORC4, Nr1h, ↑NT5E, ↑PKM2, ↓PMPCA, ↑PPARGC1A, ↑SERF2, ↑SPC25 (includes EG:100144563), ↑SREBF1 (includes EG:176574), ↓STOM*, ↓SUGP2, ↑TK1, ↓TMEM17, ↓TMSB15A, ↓ZNF462 2 Immunological Disease, 36 31 ↓ACO1, ↑ADM, ↑AKT1, ↓AMPH, ↓CD74*, ↓CDC42EP1, ↑FTH1 (includes EG:14319), Neurological Disease, Skeletal ↓HLA-DMB, ↓HLA-DRA*, ↓HLA-F, ↑IFIT1, IFN Beta, ↑MLKL, ↓MUC1*, NFkB (complex), and Muscular Disorders ↓NUAK1*, ↓NUBP1, ↓PDLIM1, PI3K (family), Pkc(s), ↓PPA1, ↑PPP1CB, ↑RFTN1, ↓RFX5, ↓RRAS2, ↓SH3GLB2, ↑SNCA*, ↓SPP1 (includes EG:20750)*, ↓ TCEB2*, ↑TFG*, ↓TJP2*, ↑TRADD, ↑TSC22D3, ↓UNC93B1, ↓VHL* 3 Cell Death, Cellular Growth 30 28 14-3-3, ↓AKAP12*, ↓APOD, ↓ARHGDIB, ↑BCL2L1, ↓CBL, ↑CBY1*, ↑CCL20, CD3, ↓CD55, and Proliferation, Cancer ↓CD83*, ↑CDK1, ↓CNOT7, ↓ETS1*, ↓FBXL19-AS1, ↑FLNB, ↓GADD45A*, ↑HIF1A*, ↑HSPB8, ↑IFI44, Jnk, ↓NFE2L3, ↑NQO1, ↓ODC1, P38 MAPK, ↑PEG10*, ↑PGK1, PI3K (complex), ↓PRKACB*, ↓RAC2, TCR, ↑TMED10, ↑TYR, ↓UGCG, Vegf 4 Cellular Growth and 26 26 ↑AGK, Ap1, ↑BHLHE40, ↓CDC42EP5, Creb, Cyclin A, ↑CYP2J2, ↑CYR61, ↑DAB2, Proliferation, Cellular ↑DBNDD1, ↓DKK1, ERK1/2, ↑EZR, ↓GRK5, ↑HERPUD1*, ↓HTRA1, ↓IL7, ↓JUN, LDL, Movement, Neurological ↓LRP5, MAP2K1/2, Mapk, Mek, ↓MVP*, PDGF BB, ↓PHLDA1*, ↓PLIN2, ↑PSEN1, Disease ↑PTGER4, ↑S100A3, ↑S100A4*, ↑SGK1*, ↓SIGMAR1, ↓SOD2, ↓TFF2 5 Antimicrobial Response, Cell- 26 26 26s Proteasome, Actin, ↓ADCY9, Akt, ↑ARPC1A, ↑ATP1B1*, ↑ATP6V0D1, ↑ATP9A, BCR To-Cell Signaling and (complex), ↓BIRC2, ↓BIRC3*, ↓CAP2, caspase, ↓CCDC50, ↑COL15A1, ↓CTSC, ↑FCRLA, Interaction, Embryonic FSH, ↑HBG1, hCG, ↓HLA-E, ↓IFITM1, IgG, ↓KLF9, Lh, ↑LMAN1, ↑MITF, ↑P4HA2, Development ↓PDLIM3, ↑RAB1A (includes EG:178620), ↑RAB33B, ↓SSTR2, ↑TM4SF1, ↓TNFAIP3, ↓XPO6

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers; ↑ upregulated; ↓ downregulated.

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Appendix 14 Genes with altered promoter methylation and expression in response to FPGS modulation

Appendix 14. 1 Genes with altered promoter methylation and expression in the FPGS-overexpressed HCT116 colon cancer cells

DNA Gene Probe ID Methylation Expression Entrez Gene CpG Description Gene Chromosome Symbol β-Value Island Fold Change DNA Gene ID Difference (vs. Control) Methylation Expression (vs. Control)

Hypermethylated and Downregulated DEPDC1B DEP domain containing 1B 0.38 -2.74 cg03905094 ILMN_2103685 55789 5 No DEPDC1B DEP domain containing 1B 0.38 -2.19 cg03905094 ILMN_1814600 55789 5 No MSH2 mutS homolog 2, colon cancer, 0.31 -2.08 cg11311499 ILMN_1737413 4436 2 No nonpolyposis type 1 (E. coli) FANCE Fanconi anemia, complementation group E 0.45 -1.94 cg03030757 ILMN_2043452 2178 6 Yes MSH2 mutS homolog 2, colon cancer, 0.31 -1.94 cg11311499 ILMN_2203453 4436 2 No nonpolyposis type 1 (E. coli) ALDH1A3 aldehyde dehydrogenase 1 family, member 0.29 -1.93 cg23191950 ILMN_2139970 220 15 No A3 HNRNPM heterogeneous nuclear ribonucleoprotein M 0.20 -1.92 cg17477756 ILMN_1745385 4670 19 No BRCA1 breast cancer 1, early onset 0.24 -1.88 cg19088651 ILMN_1738027 672 17 No HNRNPM heterogeneous nuclear ribonucleoprotein M 0.20 -1.87 cg17477756 ILMN_3269405 4670 19 No (HNRNPM), transcript variant 1 HNRNPM heterogeneous nuclear ribonucleoprotein M 0.20 -1.79 cg17477756 ILMN_3192791 4670 19 No (HNRNPM), transcript variant 2 WEE1 WEE1 homolog (S. pombe) 0.20 -1.78 cg25876934 ILMN_1778561 7465 11 No LIME1 Lck interacting transmembrane adaptor 1 0.23 -1.75 cg06653796 ILMN_2183687 54923 20 No C16orf35 chromosome 16 open reading frame 35 0.21 -1.73 cg24399405 ILMN_2369580 8131 16 No BRCA1 breast cancer 1, early onset 0.24 -1.70 cg19088651 ILMN_2311089 672 17 No MNS1 meiosis-specific nuclear structural 1 0.61 -1.69 cg14797887 ILMN_2157240 55329 15 Yes ANAPC4 anaphase promoting complex subunit 4 0.33 -1.66 cg13918811 ILMN_1802973 29945 4 No

542

AMMECR1 Alport syndrome, mental retardation, 0.25 -1.64 cg00279797 ILMN_1779374 9949 X No midface hypoplasia and elliptocytosis chromosomal region gene 1 SLC25A46 solute carrier family 25, member 46 0.21 -1.63 cg07611177 ILMN_1720311 91137 5 No ALDH1A3 aldehyde dehydrogenase 1 family, member 0.29 -1.61 cg23191950 ILMN_1807439 220 15 No A3 FASTKD3 FAST kinase domains 3 0.31 -1.61 cg10300154 ILMN_2129859 79072 5 No FASTKD3 FAST kinase domains 3 0.31 -1.60 cg10300154 ILMN_1750160 79072 5 No COPS3 COP9 constitutive photomorphogenic 0.45 -1.55 cg13823791 ILMN_1770732 8533 17 No homolog subunit 3 (Arabidopsis) WNT3A wingless-type MMTV integration site 0.31 -1.54 cg01322134 ILMN_1815700 89780 1 Yes family, member 3A RERG RAS-like, estrogen-regulated, growth 0.29 -1.52 cg03028472 ILMN_1746359 85004 12 Yes inhibitor EFHD2 EF-hand domain family, member D2 0.21 -1.51 cg22269795 ILMN_1761463 79180 1 Yes IQCC IQ motif containing C 0.59 -1.50 cg01154193 ILMN_1774589 55721 1 Yes LIN9 lin-9 homolog (C. elegans) 0.44 -1.49 cg26199493 ILMN_2137084 286826 1 No CCDC109B coiled-coil domain containing 109B 0.23 -1.48 cg03005261 ILMN_1801766 55013 4 Yes SETMAR SET domain and mariner transposase fusion 0.24 -1.48 cg15384599 ILMN_1682404 6419 3 No gene PPWD1 peptidylprolyl isomerase domain and WD 0.22 -1.47 cg01403114 ILMN_2223380 23398 5 No repeat containing 1 NOC3L nucleolar complex associated 3 homolog (S. 0.23 -1.46 cg07270175 ILMN_2187727 64318 10 No cerevisiae) TTC4 tetratricopeptide repeat domain 4 0.35 -1.46 cg02255609 ILMN_1678140 7268 1 No VPS33A vacuolar protein sorting 33 homolog A (S. 0.27 -1.45 cg09924998 ILMN_1662316 65082 12 No cerevisiae) ARHGAP22 Rho GTPase activating protein 22 0.22 -1.43 cg20506783 ILMN_1676361 58504 10 No C8orf41 chromosome 8 open reading frame 41 0.27 -1.43 cg00792687 ILMN_1760400 80185 8 No C15orf44 chromosome 15 open reading frame 44 0.28 -1.38 cg22794078 ILMN_1795524 81556 15 No ALKBH1 alkB, alkylation repair homolog 1 (E. coli) 0.24 -1.35 cg20385229 ILMN_1758038 8846 14 No BNIP3 BCL2/adenovirus E1B 19kDa interacting 0.22 -1.32 cg22473973 ILMN_1724658 664 10 No protein 3

543

SLC25A5 solute carrier family 25 (mitochondrial 0.45 -1.31 cg10321196 ILMN_1774062 292 X No carrier; adenine nucleotide translocator), member 5 C16orf80 chromosome 16 open reading frame 80 0.22 -1.31 cg25771195 ILMN_2112599 29105 16 No MGST1 microsomal glutathione S-transferase 1 0.22 -1.31 cg11203041 ILMN_1781952 4257 12 No

Hypomethylated and Upregulated H1F0 H1 histone family, member 0 -0.33 3.52 cg07141002 ILMN_1757467 3005 22 Yes HCP5 HLA complex P5 -0.34 2.62 cg07971188 ILMN_1803945 10866 6 No REEP6 receptor accessory protein 6 -0.35 2.24 cg22759185 ILMN_1697460 92840 19 No ST6GALNAC2 ST6 (alpha-N-acetyl-neuraminyl-2, 3-beta- -0.22 2.20 cg08666623 ILMN_1658706 10610 17 No galactosyl-1, 3)-N-acetylgalactosaminide alpha-2, 6-sialyltransferase 2

HLA-DMA major histocompatibility complex, class II, -0.26 2.16 cg14833385 ILMN_1695311 3108 6 No DM alpha TRAPPC6A trafficking protein particle complex 6A -0.23 2.12 cg02713563 ILMN_1775703 79090 19 No OAF OAF homolog (Drosophila) -0.26 2.01 cg12572278 ILMN_1668345 220323 11 No SHC1 SHC (Src homology 2 domain containing) -0.22 1.97 cg06418219 ILMN_1721022 6464 1 No transforming protein 1 ARFGAP3 ADP-ribosylation factor GTPase activating -0.21 1.93 cg00079563 ILMN_1731287 26286 22 Yes protein 3 GPM6A glycoprotein M6A -0.21 1.91 cg19639622 ILMN_1716869 2823 4 No IRF1 interferon regulatory factor 1 -0.27 1.90 cg20287640 ILMN_1708375 3659 5 No FRAT2 frequently rearranged in advanced T-cell -0.37 1.87 cg27212359 ILMN_1788213 23401 10 Yes lymphomas 2 ACSF2 acyl-CoA synthetase family member 2 -0.43 1.81 cg14672994 ILMN_3236756 80221 17 Yes BSDC1 BSD domain containing 1 -0.21 1.66 cg18182038 ILMN_1734483 55108 1 Yes WNT16 wingless-type MMTV integration site -0.35 1.65 cg24849648 ILMN_1731964 51384 7 No family, member 16 KHNYN KH and NYN domain containing -0.23 1.64 cg13105904 ILMN_1654392 23351 14 No NLRP8 NLR family, pyrin domain containing 8 -0.37 1.60 cg22190114 ILMN_2075794 126205 19 No AIP aryl hydrocarbon receptor interacting -0.27 1.50 cg21747271 ILMN_2103841 9049 11 No

544

protein DDOST dolichyl-diphosphooligosaccharide-protein -0.20 1.50 cg03218019 ILMN_1734231 1650 1 Yes glycosyltransferase RALGPS1 Ral GEF with PH domain and SH3 binding -0.25 1.50 cg08433538 ILMN_1674135 9649 9 No motif 1 CGGBP1 CGG triplet repeat binding protein 1 -0.20 1.49 cg15379887 ILMN_1752631 8545 3 No ST6GALNAC4 ST6 (alpha-N-acetyl-neuraminyl-2, 3-beta- -0.33 1.48 cg25732252 ILMN_2413064 27090 9 No galactosyl-1, 3)-N-acetylgalactosaminide alpha-2, 6-sialyltransferase 4

COL7A1 collagen, type VII, alpha 1 (epidermolysis -0.20 1.47 cg16357381 ILMN_1751161 1294 3 Yes bullosa, dystrophic, dominant and recessive) CDK10 cyclin-dependent kinase (CDC2-like) 10 -0.25 1.47 cg04155793 ILMN_1741459 8558 16 No CDK10 cyclin-dependent kinase (CDC2-like) 10 -0.21 1.47 cg12119029 ILMN_1741459 8558 16 Yes KCNH6 potassium voltage-gated channel, subfamily -0.20 1.46 cg13520327 ILMN_2235785 81033 17 No H (eag-related), member 6 COPA coatomer protein complex, subunit alpha -0.36 1.43 cg08015496 ILMN_1811615 1314 1 No H6PD hexose-6-phosphate dehydrogenase (glucose -0.24 1.43 cg22601917 ILMN_1721136 9563 1 No 1-dehydrogenase) P4HA2 prolyl 4-hydroxylase, alpha polypeptide II -0.31 1.39 cg24117468 ILMN_1795778 8974 5 No PLBD2 mannose-6-phosphate protein p76 -0.21 1.35 cg17946995 ILMN_1734184 196463 12 No DIRAS3 DIRAS family, GTP-binding RAS-like 3 -0.30 1.34 cg21808053 ILMN_1688877 9077 1 No DSG2 desmoglein 2 -0.22 1.32 cg15259973 ILMN_3251672 1829 18 No

545

Appendix 14. 2 Genes with altered promoter methylation and expression in the FPGS-inhibited HCT116 colon cancer cells

DNA Gene Probe ID Methylation Expression Entrez Gene CpG Description Gene Chromosome Symbol β-Value Island Fold Change DNA Gene ID Difference (vs. Control) Methylation Expression (vs. Control)

Hypermethylated and Downregulated MECOM ecotropic viral integration site 1 0.27 -1.62 cg19848683 ILMN_1803367 2122 3 Yes FAM178A chromosome 10 open reading frame 6 0.27 -1.45 cg08177017 ILMN_1710207 55719 10 Yes

Hypomethylated and Upregulated ABCC2 ATP-binding cassette, sub-family C -0.26 1.56 cg17044311 ILMN_1676278 1244 10 No (CFTR/MRP), member 2 SKAP1 src kinase associated phosphoprotein 1 -0.21 1.51 cg12513481 ILMN_1751400 8631 17 No REEP6 receptor accessory protein 6 -0.43 1.37 cg22759185 ILMN_1697460 92840 19 No REEP6 receptor accessory protein 6 -0.42 1.37 cg02674804 ILMN_1697460 92840 19 No TTC33 tetratricopeptide repeat domain 33 -0.30 1.34 cg02588309 ILMN_1807088 23548 5 No SKAP1 src kinase associated phosphoprotein 1 -0.21 1.32 cg12513481 ILMN_2335604 8631 17 No

546

Appendix 14. 3 Genes with altered promoter methylation and expression in the FPGS-overexpressed MDA-MB-435 breast cancer cells

DNA Gene Probe ID Methylation Expression Entrez CpG Gene Symbol Description β Chromosome -Value Fold Change DNA Gene Gene ID Island Difference (vs. Control) Methylation Expression (vs. Control)

Hypermethylated and Downregulated TSPAN7 tetraspanin 7 0.61 -22.33 cg20450764 ILMN_2120695 7102 X No TYR tyrosinase (oculocutaneous albinism IA) 0.42 -20.19 cg03417466 ILMN_1788774 7299 11 No TSPAN7 tetraspanin 7 0.61 -7.59 cg20450764 ILMN_1809291 7102 X No GYG2 glycogenin 2 0.24 -4.24 cg03506684 ILMN_2319424 8908 X Yes NBL1 neuroblastoma, suppression of tumorigenicity 0.37 -2.94 cg19136075 ILMN_2405009 4681 1 No 1 GYG2 glycogenin 2 0.24 -2.84 cg03506684 ILMN_1684017 8908 X Yes PLSCR1 phospholipid scramblase 1 0.21 -2.62 cg20586531 ILMN_1745242 5359 3 No BNC2 basonuclin 2 0.35 -2.33 cg14613546 ILMN_1656373 54796 9 No PTGFRN prostaglandin F2 receptor negative regulator 0.38 -2.25 cg03752628 ILMN_2077905 5738 1 No PTGFRN prostaglandin F2 receptor negative regulator 0.38 -2.20 cg03752628 ILMN_1743130 5738 1 No GSTT1 glutathione S-transferase theta 1 0.47 -2.19 cg24330042 ILMN_1730054 2952 22 Yes NBL1 neuroblastoma, suppression of tumorigenicity 0.37 -2.17 cg19136075 ILMN_1789599 4681 1 No 1 GDPD5 glycerophosphodiester phosphodiesterase 0.52 -2.09 cg16393207 ILMN_1701643 81544 11 Yes domain containing 5 TMEM51 transmembrane protein 51 0.26 -2.01 cg05385377 ILMN_1674985 55092 1 Yes VDAC3 voltage-dependent anion channel 3 0.30 -1.84 cg17910564 ILMN_1729816 7419 8 No SOX10 SRY (sex determining region Y)-box 10 0.40 -1.83 cg06614002 ILMN_1653750 6663 22 No HAPLN3 hyaluronan and proteoglycan link protein 3 0.29 -1.82 cg08348496 ILMN_1654319 145864 15 Yes GPR143 G protein-coupled receptor 143 0.38 -1.80 cg11325578 ILMN_1756261 4935 X Yes HAS2 hyaluronan synthase 2 0.24 -1.79 cg19275050 ILMN_1792978 3037 8 No

547

MAGED2 melanoma antigen family D, 2 0.30 -1.77 cg00690049 ILMN_1683576 10916 X No IRF4 interferon regulatory factor 4 0.22 -1.75 cg25140370 ILMN_1754507 3662 6 No FKBP9L FK506 binding protein 9-like 0.25 -1.75 cg11808544 ILMN_2089977 360132 7 No TMSB15A thymosin beta 15a 0.25 -1.66 cg19062189 ILMN_1681737 11013 X Yes IGSF8 immunoglobulin superfamily, member 8 0.26 -1.61 cg02032115 ILMN_1730432 93185 1 No GREB1 growth regulation by estrogen in breast cancer 0.22 -1.59 cg10612997 ILMN_1721170 9687 2 No 1 BPHL biphenyl hydrolase-like (serine hydrolase) 0.22 -1.55 cg21182407 ILMN_1657746 670 6 No SFXN4 sideroflexin 4 0.32 -1.53 cg23274030 ILMN_2363361 119559 10 No ATP5J2 ATP synthase, H+ transporting, 0.37 -1.51 cg03665605 ILMN_2310621 9551 7 No mitochondrial F0 complex, subunit F2 XYLT2 xylosyltransferase II 0.22 -1.51 cg23430664 ILMN_1799815 64132 17 Yes XYLT2 xylosyltransferase II 0.21 -1.51 cg05105913 ILMN_1799815 64132 17 Yes ATP5G3 ATP synthase, H+ transporting, 0.55 -1.50 cg07428089 ILMN_1770466 518 2 Yes mitochondrial F0 complex, subunit C3 (subunit 9) ATP5G3 ATP synthase, H+ transporting, 0.27 -1.50 cg19713083 ILMN_1770466 518 2 Yes mitochondrial F0 complex, subunit C3 (subunit 9) GSTM2 glutathione S-transferase M2 (muscle) 0.20 -1.50 cg03070194 ILMN_2201580 2946 1 Yes ATP5J2 ATP synthase, H+ transporting, 0.37 -1.49 cg03665605 ILMN_2307883 9551 7 No mitochondrial F0 complex, subunit F2 ABCA2 ATP-binding cassette, sub-family A (ABC1), 0.53 -1.48 cg15428653 ILMN_1747627 20 9 Yes member 2 RNF14 ring finger protein 14 0.25 -1.47 cg04182865 ILMN_2351241 9604 5 No AFG3L2 AFG3 ATPase family gene 3-like 2 (yeast) 0.22 -1.46 cg03766998 ILMN_2066124 10939 18 No UBIAD1 UbiA prenyltransferase domain containing 1 0.24 -1.46 cg11858029 ILMN_1651872 29914 1 No FAM167B family with sequence similarity 167, member 0.38 -1.41 cg12278770 ILMN_1659856 84734 1 No B MAGED2 melanoma antigen family D, 2 0.30 -1.41 cg00690049 ILMN_1684984 10916 X No SLC23A2 solute carrier family 23 (nucleobase 0.21 -1.39 cg12821724 ILMN_1746578 9962 20 No transporters), member 2

548

CBR3 carbonyl reductase 3 0.21 -1.39 cg14564494 ILMN_1652237 874 21 No RHOT2 ras homolog gene family, member T2 0.33 -1.39 cg05135288 ILMN_1669310 89941 16 No CUL9 cullin 9 0.21 -1.34 cg13678049 ILMN_1806010 23113 6 No SND1 staphylococcal nuclease and tudor domain 0.21 -1.32 cg00893242 ILMN_1775111 27044 7 No containing 1 SNRK SNF related kinase 0.26 -1.32 cg04008913 ILMN_2107184 54861 3 No TYMP endothelial cell growth factor 1 (platelet- 0.36 -1.31 cg11654620 ILMN_2109708 1890 22 Yes derived) TMEM55A transmembrane protein 55A 0.35 -1.31 cg06688396 ILMN_1752117 55529 8 No HAUS6 HAUS augmin-like complex, subunit 6 0.31 -1.30 cg07664183 ILMN_1664139 54801 9 No

Hypomethylated and Upregulated IL24 interleukin 24 -0.23 12.91 cg06796611 ILMN_1774685 11009 1 No CNN3 calponin 3, acidic -0.36 7.16 cg26619317 ILMN_1782439 1266 1 Yes LAIR2 leukocyte-associated immunoglobulin-like -0.30 5.50 cg00269932 ILMN_1807491 3904 19 No receptor 2 LAIR2 leukocyte-associated immunoglobulin-like -0.30 5.18 cg00269932 ILMN_2323933 3904 19 No receptor 2 IL24 interleukin 24 -0.23 4.81 cg06796611 ILMN_2407799 11009 1 No OLFM1 olfactomedin 1 -0.24 4.58 cg27286999 ILMN_1742025 10439 9 Yes PTPN22 protein tyrosine phosphatase, non-receptor -0.38 3.21 cg14385738 ILMN_2246328 26191 1 No type 22 (lymphoid) PTPN22 protein tyrosine phosphatase, non-receptor -0.21 3.21 cg00916635 ILMN_2246328 26191 1 No type 22 (lymphoid) MX1 myxovirus (influenza virus) resistance 1, -0.44 3.13 cg22152328 ILMN_1662358 4599 21 Yes interferon-inducible protein p78 (mouse)

HLA-DPA1 major histocompatibility complex, class II, -0.64 2.82 cg13906813 ILMN_1772218 3113 6 No DP alpha 1 S100A4 S100 calcium binding protein A4 -0.37 2.79 cg26894575 ILMN_1684306 6275 1 No CD55 CD55 molecule, decay accelerating factor for -0.35 2.58 cg06792598 ILMN_1800540 1604 1 Yes complement (Cromer blood group)

549

C15orf52 chromosome 15 open reading frame 52 -0.26 2.53 cg16119128 ILMN_1775330 388115 15 No CDC42EP1 CDC42 effector protein (Rho GTPase -0.34 2.46 cg27553637 ILMN_1764927 11135 22 Yes binding) 1 ACSS2 acyl-CoA synthetase short-chain family -0.22 2.33 cg13368786 ILMN_2336595 55902 20 No member 2 PTPN22 protein tyrosine phosphatase, non-receptor -0.38 2.30 cg14385738 ILMN_1695640 26191 1 No type 22 (lymphoid) PTPN22 protein tyrosine phosphatase, non-receptor -0.21 2.30 cg00916635 ILMN_1695640 26191 1 No type 22 (lymphoid) DKK1 dickkopf homolog 1 (Xenopus laevis) -0.55 2.28 cg07684796 ILMN_1773337 22943 10 Yes RRAS related RAS viral (r-ras) oncogene homolog -0.29 2.27 cg17383958 ILMN_1780825 6237 19 Yes HBE1 hemoglobin, epsilon 1 -0.24 2.26 cg08970694 ILMN_1651358 3046 11 No ACSS2 acyl-CoA synthetase short-chain family -0.22 2.25 cg13368786 ILMN_1714197 55902 20 No member 2 IL24 interleukin 24 -0.23 2.07 cg06796611 ILMN_1725814 11009 1 No JAM3 junctional adhesion molecule 3 -0.27 2.04 cg24625128 ILMN_1769575 83700 11 Yes RRAS2 related RAS viral (r-ras) oncogene homolog 2 -0.24 2.01 cg03922337 ILMN_2077623 22800 11 No SOX2 SRY (sex determining region Y)-box 2 -0.30 2.00 cg01340005 ILMN_2177156 6657 3 No CTXN1 cortexin 1 -0.29 1.96 cg04351979 ILMN_1759766 404217 19 Yes FKBP11 FK506 binding protein 11, 19 kDa -0.76 1.87 cg21908259 ILMN_1787345 51303 12 Yes FKBP11 FK506 binding protein 11, 19 kDa -0.31 1.87 cg15147435 ILMN_1787345 51303 12 Yes ACSS2 acyl-CoA synthetase short-chain family -0.22 1.85 cg13368786 ILMN_1697510 55902 20 No member 2 ZCCHC6 zinc finger, CCHC domain containing 6 -0.28 1.85 cg05065037 ILMN_1779677 79670 9 No KCTD14 potassium channel tetramerisation domain -0.27 1.84 cg17272843 ILMN_1731044 65987 11 No containing 14

ZFP37 zinc finger protein 37 homolog (mouse) -0.32 1.79 cg03454353 ILMN_1793578 7539 9 No PPP2R2B protein phosphatase 2 (formerly 2A), -0.29 1.76 cg01112778 ILMN_2298365 5521 5 No regulatory subunit B, beta isoform SRPX2 sushi-repeat-containing protein, X-linked 2 -0.21 1.75 cg18727700 ILMN_1676213 27286 X No DDAH1 dimethylarginine dimethylaminohydrolase 1 -0.40 1.74 cg21589280 ILMN_1668507 23576 1 Yes

550

KRT80 keratin 80 -0.21 1.69 cg11051139 ILMN_1705814 144501 12 No HLA-DQB1 major histocompatibility complex, class II, -0.31 1.69 cg01889448 ILMN_1661266 3119 6 No DQ beta 1 PHLDA2 pleckstrin homology-like domain, family A, -0.20 1.68 cg01263716 ILMN_1671557 7262 11 Yes member 2 SDC1 syndecan 1 -0.29 1.65 cg11475332 ILMN_1815308 6382 2 Yes DAK dihydroxyacetone kinase 2 homolog (S. -0.59 1.63 cg25406518 ILMN_1678619 26007 11 No cerevisiae) PPFIA1 protein tyrosine phosphatase, receptor type, f -0.27 1.54 cg17913068 ILMN_1727050 8500 11 No polypeptide (PTPRF), interacting protein (liprin), alpha 1

RASSF1 Ras association (RalGDS/AF-6) domain -0.28 1.54 cg21554552 ILMN_1734205 11186 3 Yes family 1 MYL5 myosin, light chain 5, regulatory -0.25 1.53 cg18176712 ILMN_2203588 4636 4 No SDC4 syndecan 4 -0.27 1.51 cg10876928 ILMN_1663042 6385 20 No FBXO4 F-box protein 4 -0.40 1.49 cg10850119 ILMN_1656691 26272 5 No FBXO4 F-box protein 4 -0.28 1.49 cg22359217 ILMN_1656691 26272 5 No C12orf75 hypothetical protein -0.39 1.47 cg26940261 ILMN_1716382 387882 12 No HTRA1 HtrA serine peptidase 1 -0.32 1.46 cg10588377 ILMN_1676563 5654 10 Yes PTPRF protein tyrosine phosphatase, receptor type, -0.27 1.46 cg16570917 ILMN_2380163 5792 1 No F DNTTIP2 deoxynucleotidyltransferase, terminal, -0.23 1.46 cg22807700 ILMN_1708345 30836 1 No interacting protein 2 PLAUR plasminogen activator, urokinase receptor -0.36 1.46 cg06540636 ILMN_1691508 5329 19 No STX2 syntaxin 2 -0.43 1.44 cg08169325 ILMN_1747775 2054 12 No IL4R interleukin 4 receptor -0.38 1.44 cg03980304 ILMN_1652185 3566 16 Yes ANKRD1 ankyrin repeat domain 1 (cardiac muscle) -0.51 1.44 cg14558138 ILMN_1716264 27063 10 No STX2 syntaxin 2 -0.43 1.42 cg08169325 ILMN_2344216 2054 12 No HBG2 hemoglobin, gamma G -0.26 1.42 cg10920765 ILMN_2084825 3048 11 No RELB v-rel reticuloendotheliosis viral oncogene -0.22 1.41 cg02727285 ILMN_1811258 5971 19 No homolog B C15orf63 chromosome 15 open reading frame 63 -0.52 1.40 cg24125648 ILMN_2073543 25764 15 No

551

SNAPC2 small nuclear RNA activating complex, -0.29 1.39 cg24132694 ILMN_1698478 6618 19 No polypeptide 2, 45kDa

S100A4 S100 calcium binding protein A4 -0.37 1.37 cg26894575 ILMN_1688780 6275 1 No PTPN22 protein tyrosine phosphatase, non-receptor -0.38 1.37 cg14385738 ILMN_1715885 26191 1 No type 22 (lymphoid) PTPN22 protein tyrosine phosphatase, non-receptor -0.21 1.37 cg00916635 ILMN_1715885 26191 1 No type 22 (lymphoid) CIR1 corepressor interacting with RBPJ, 1 -0.23 1.36 cg14138171 ILMN_1671516 9541 2 No WAS Wiskott-Aldrich syndrome (eczema- -0.49 1.36 cg00078867 ILMN_1760027 7454 X No thrombocytopenia) POMT1 protein-O-mannosyltransferase 1 -0.34 1.34 cg27024922 ILMN_2305721 10585 9 No GBP3 guanylate binding protein 3 -0.20 1.33 cg23540651 ILMN_1725314 2635 1 No CD33 CD33 molecule -0.37 1.33 cg11122968 ILMN_1747622 945 19 No CD33 CD33 molecule -0.33 1.33 cg10129493 ILMN_1747622 945 19 No C13orf31 chromosome 13 open reading frame 31 -0.51 1.31 cg00056767 ILMN_1693431 144811 13 No

552

Appendix 14. 4 Genes with altered promoter methylation and expression in the FPGS-inhibited MDA-MB-435 breast cancer cells

DNA Gene Probe ID Methylation Expression Gene Entrez CpG Description β Chromosome Symbol -Value Fold Change DNA Gene Gene ID Island Difference (vs. Control) Methylation Expression (vs. Control)

Hypermethylated and Downregulated THBS2 thrombospondin 2 0.45 -5.25 cg21652958 ILMN_1678842 7058 6 No C1S complement component 1, s subcomponent 0.21 -3.26 cg05538432 ILMN_1781626 716 12 No HLA-DPA1 major histocompatibility complex, class II, 0.28 -2.87 cg13906813 ILMN_1772218 3113 6 No DP alpha 1 CAP2 CAP, adenylate cyclase-associated protein, 2 0.20 -2.55 cg23013864 ILMN_1691237 10486 6 No (yeast) CTSL2 cathepsin L2 0.36 -2.48 cg02936872 ILMN_1748352 1515 9 Yes LRP5 low density lipoprotein receptor-related 0.26 -2.02 cg21572997 ILMN_1702775 4041 11 Yes protein 5 FAHD1 fumarylacetoacetate hydrolase domain 0.22 -1.89 cg16270890 ILMN_1701457 81889 16 No containing 1 SCARF2 scavenger receptor class F, member 2 0.30 -1.88 cg23748737 ILMN_1655405 91179 22 Yes CDC42EP5 CDC42 effector protein (Rho GTPase 0.30 -1.78 cg03620376 ILMN_1774982 148170 19 No binding) 5 GBGT1 globoside alpha-1, 3-N- 0.33 -1.70 cg18089000 ILMN_1652906 26301 9 No acetylgalactosaminyltransferase 1

AMPH amphiphysin 0.26 -1.67 cg09966445 ILMN_1685834 273 7 No CTSF cathepsin F 0.38 -1.67 cg06817264 ILMN_1804955 8722 11 Yes TMEM132A transmembrane protein 132A 0.21 -1.65 cg15553522 ILMN_2317923 54972 11 Yes C15orf63 chromosome 15 open reading frame 63 0.37 -1.63 cg24125648 ILMN_2073543 25764 15 No FAHD1 fumarylacetoacetate hydrolase domain 0.22 -1.62 cg16270890 ILMN_2246661 81889 16 No containing 1 PDLIM3 PDZ and LIM domain 3 0.30 -1.61 cg02515725 ILMN_2230025 27295 4 No KLHDC8B kelch domain containing 8B 0.48 -1.61 cg10872212 ILMN_1695246 200942 3 Yes

553

C9orf78 chromosome 9 open reading frame 78 0.50 -1.58 cg17509612 ILMN_1697166 51759 9 No MYO1B myosin IB 0.23 -1.56 cg15096140 ILMN_2093027 4430 2 No TMSB15A thymosin beta 15a 0.32 -1.56 cg19062189 ILMN_1681737 11013 X Yes DNTTIP2 deoxynucleotidyltransferase, terminal, 0.28 -1.54 cg22807700 ILMN_1708345 30836 1 No interacting protein 2

PTGFRN prostaglandin F2 receptor negative regulator 0.29 -1.51 cg03752628 ILMN_2077905 5738 1 No RNPS1 RNA binding protein S1, serine-rich domain 0.37 -1.50 cg10889070 ILMN_1691843 10921 16 No SSTR2 somatostatin receptor 2 0.26 -1.49 cg18943599 ILMN_2152257 6752 17 No FKBP11 FK506 binding protein 11, 19 kDa 0.25 -1.46 cg21908259 ILMN_1787345 51303 12 Yes NFATC2IP nuclear factor of activated T-cells, 0.70 -1.45 cg26962295 ILMN_2379080 84901 16 No cytoplasmic, calcineurin-dependent 2 interacting protein RAC2 ras-related C3 botulinum toxin substrate 2 0.23 -1.43 cg14072120 ILMN_1709795 5880 22 No (rho family, small GTP binding protein Rac2)

TCEB2 transcription elongation factor B (SIII), 0.36 -1.42 cg07059052 ILMN_2377185 6923 16 No polypeptide 2 (18kDa, elongin B)

TCEB2 transcription elongation factor B (SIII), 0.36 -1.38 cg07059052 ILMN_1733927 6923 16 No polypeptide 2 (18kDa, elongin B)

CD83 CD83 molecule 0.29 -1.37 cg01288598 ILMN_2328666 9308 6 No CD83 CD83 molecule 0.29 -1.35 cg01288598 ILMN_1780582 9308 6 No OSBPL5 oxysterol binding protein-like 5 0.23 -1.34 cg20345446 ILMN_2307032 114879 11 No PTGFRN prostaglandin F2 receptor negative regulator 0.29 -1.33 cg03752628 ILMN_1743130 5738 1 No COQ4 coenzyme Q4 homolog (S. cerevisiae) 0.25 -1.32 cg14458731 ILMN_3234979 51117 9 No

Hypomethylated and Upregulated CNN3 calponin 3, acidic -0.62 2.19 cg02718725 ILMN_1782439 1266 1 Yes CNN3 calponin 3, acidic -0.41 2.19 cg26619317 ILMN_1782439 1266 1 Yes MT1E metallothionein 1E -0.24 1.90 cg20083730 ILMN_2173611 4493 16 No

554

LEPREL1 leprecan-like 1 -0.39 1.82 cg20270599 ILMN_1657373 55214 3 No MT1A metallothionein 1A -0.25 1.69 cg09137696 ILMN_1691156 4489 16 Yes RHEB Ras homolog enriched in brain -0.24 1.66 cg21134096 ILMN_1657949 6009 7 Yes PIR pirin (iron-binding nuclear protein) -0.31 1.59 cg09250703 ILMN_2383383 8544 X No PIR pirin (iron-binding nuclear protein) -0.31 1.59 cg19144013 ILMN_2383383 8544 X No ARHGAP15 Rho GTPase activating protein 15 -0.35 1.52 cg27365426 ILMN_2208413 55843 2 No ARHGAP15 Rho GTPase activating protein 15 -0.25 1.52 cg23627134 ILMN_2208413 55843 2 No CTSL1 cathepsin L1 -0.29 1.50 cg15242570 ILMN_1812995 1514 9 Yes TM4SF18 transmembrane 4 L six family member 18 -0.49 1.49 cg11761535 ILMN_1739170 116441 3 No PDIA5 protein disulfide isomerase family A, member -0.33 1.42 cg10098541 ILMN_1695763 10954 3 No 5 PIR pirin (iron-binding nuclear protein) -0.31 1.41 cg09250703 ILMN_1761247 8544 X No PIR pirin (iron-binding nuclear protein) -0.31 1.41 cg19144013 ILMN_1761247 8544 X No WDR72 WD repeat domain 72 -0.24 1.40 cg18613421 ILMN_1763196 256764 15 No ACYP2 acylphosphatase 2, muscle type -0.22 1.38 cg18951427 ILMN_2158705 98 2 No PID1 phosphotyrosine interaction domain -0.36 1.36 cg04743872 ILMN_1671891 55022 2 No containing 1

555

Appendix 15 The list of differentially methylated and expressed genes associated with the top networks in response to FPGS modulation

Appendix 15. 1 The top networks matched by the genes with altered expression and promoter methylation in the FPGS- overexpressed HCT116 colon cancer cells

Focus No. Top Functions Score Genes in Network Genes Hypermethylated and Downregulated 1 DNA Replication, Recombination, and 20 10 AHR, ALDH1A3*, ATF1 (includes EG:100040260), BARD1, BRCA1*, C16orf80, Repair, Cell Cycle, Vitamin and Mineral CCNB2, CCND1, CDC6 (includes EG:23834), CSK, CSTF1, CTSD, CYP1A1, Metabolism CYP1B1, E2F4, FEN1, HNRNPM*, KRT17, LGALS3, LIME1, LIN9, MGST1, MNS1, MSH2*, MSH6, ORC1 (includes EG:18392), RAD51, RBBP8, RBL2 (includes EG:100331892), SFPQ, SUZ12, TCR, TNF, UBE2N, WNT3A 2 Cellular Function and Maintenance, 2 1 CD24, DEPDC1B* Cellular Movement, Cell-To-Cell Signaling and Interaction 3 Tissue Morphology, DNA Replication, 2 1 COPS3, CUL4A Recombination, and Repair, Cell Morphology 4 DNA Replication, Recombination, and 2 1 SETMAR, SOX11 Repair, Digestive System Development and Function, Cell Death 5 Molecular Transport, Nucleic Acid 2 1 Nuclear factor 1, SLC25A5 Metabolism, Small Molecule Biochemistry Hypomethylated and Upregulated 1 Inflammatory Response, Cell Death, Cell- 18 9 ANXA5, ARFGAP3, BCL3, CDKN1A, COL7A1, CYBB, DIRAS3, EBI3, EDN1, To-Cell Signaling and Interaction ESR1, FRAT2, H6PD, HLA-DMA, IDO1, IFNA1/IFNA13, IFNG (includes EG:15978), IL27, IL12 (family), IRF1 (includes EG:16362), mir-22, PLA2G16, PRL, PSMB8, PSMB9, RARRES3, RET, SHC1 (includes EG:20416), STAT2, STAT3, TAP1, TAP2, TGFB1 (includes EG:21803), TRIM22, USP18, WNT16 2 Cellular Movement, Infectious Disease, 2 1 EZH2, HCP5 Cellular Development

556

3 Cell-To-Cell Signaling and Interaction, 2 1 CGGBP1, FMR1 Cellular Function and Maintenance, Connective Tissue Development and Function 4 Amino Acid Metabolism, Post- 2 1 FSH, Lh, P4HA2 Translational Modification, Small Molecule Biochemistry 5 Carbohydrate Metabolism, Cellular 2 1 COPA, SACM1L, TMED9 Assembly and Organization, DNA Replication, Recombination, and Repair

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

Appendix 15. 2 The top networks matched by the genes with altered expression and promoter methylation in the FPGS- inhibited HCT116 colon cancer cells

Focus No. Top Functions Score Genes in Network Genes Hypermethylated and Downregulated 1 Cancer, Hematological Disease, Cell 3 1 MAPK8, MECOM, mir-22, mir-449, miR-22-3p/miR-22 Morphology Hypomethylated and Upregulated 1 Cellular Movement, Hematological System 3 1 APBB1IP, FYB, FYN, PTK2B (includes EG:19229), RASSF5, SKAP1* Development and Function, Immune Cell Trafficking 2 Lipid Metabolism, Molecular Transport, 3 1 ABCC2, ABCC3, HNF1A, IRF3, NR1H4, NR1I2, NR1I3 Small Molecule Biochemistry

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

557

Appendix 15. 3 The top networks matched by the genes with altered expression and promoter methylation in the FPGS- overexpressed MDA-MB-435 breast cancer cells

Focus No. Top Functions Score Genes in Network Genes Hypermethylated and Downregulated

1 Cell Death, Cancer, Cellular Development 21 11 ACTA2, BACE1, BMP4, CANX, CD9, CUL7, CUL9, Erm, FBXW8, FKBP4, GBP1, GSTM2, HAS2, HYAL2, IGSF8, IRF4, LGALS3, LTBP1, MYC, NFkB (complex), NQO1, PTGFRN*, REL, SNRK, SOX4, SPIB, TGFB1 (includes EG:21803), TMSB15A, TNF, TNFRSF1B, TP73, TP53 (includes EG:22059), TSPAN7*, TYMP, TYR

2 Cellular Development, Cellular Growth 10 6 AR, CD44 (includes EG:100330801), CREB1, CSF2, CTNNB1, EGFR, EREG, ESR1, and Proliferation, Organismal Survival ETV1, G protein alphai, GPR143, GREB1, hCG, JAG1, KISS1, KLK3, LTBP1, MAPK1, MED1 (includes EG:19014), NCOR1, OTUB1, PARP1, PLSCR1, PTGS2, RNA polymerase II, RNF14, Shc, SMAD3, SMYD3, SND1, SP1, SP3, SRC, TGM2, TMEM55A

3 Cellular Assembly and Organization, RNA 2 1 ATP5G3*, RBM5 Post-Transcriptional Modification, Cellular Growth and Proliferation

4 Cell Morphology, Cellular Assembly and 2 1 HAPLN3, SMARCA4 Organization, Cellular Growth and Proliferation

5 Cellular Assembly and Organization, 2 1 HAUS6, HAUS8 Cellular Function and Maintenance, Cell Cycle

Hypomethylated and Upregulated

1 Cellular Function and Maintenance, 28 15 ACSS2*, CD28, CD33*, CEBPB (includes EG:1051), DDAH1, FOXP3, Gm-csf, Hematological System Development and HLA-DPA1, HLA-DQB1, IFNG (includes EG:15978), IgG, IL10, IL24*, IL27, IL12 Function, Cell-mediated Immune Response (complex), IL12A, IL12B, IL12RB2, IL23A, IL4 (includes EG:16189), IL4R, MX1, NFkB (complex), PHLDA2, PLAUR, RASSF2, RASSF5, RELB, RRAS2, RRAS, SBDS, SDC4, SPI1 (includes EG:20375), TGM2, WAS

558

2 Cancer, Gastrointestinal Disease, Gene 25 14 ANKRD1, CCND1, CD55, CEBPB (includes EG:1051), CUL1, DKK1, EGFR, Expression FBXO4, HBE1, Histone h3, HTRA1, KDM5A, KRT80, LAMC2, Lh, LRP6, MEIS1, PLAC1, POU2F1, PPP2R2B, PTPRF, RASSF1, RN7SK, S100A4*, SDC1 (includes EG:20969), SFRP1, SNAPC1, SNAPC2, SNAPC3, SNAPC4, SOX2, SPDEF, TFF3, TGFB1 (includes EG:21803), TIMP2 (includes EG:21858)

3 Cardiac Proliferation, Cardiovascular 2 1 DICER1, GBP3 System Development and Function, Cell Death

4 Cell Cycle, Cellular Growth and 2 1 LAIR1, LAIR2* Proliferation, Hematopoiesis

5 Infectious Disease, Cancer, Renal and 2 1 DAK, IFIH1 Urological Disease

Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

559

Appendix 15. 4 The top networks matched by the genes with altered expression and promoter methylation in the FPGS- inhibited MDA-MB-435 breast cancer cells Focus No. Top Functions Score Genes in Network Genes Hypermethylated and Downregulated 1 Cell Death, Cell Cycle, Cancer 27 13 AMPH, AR, BCL2, C1S, CAP2, CASP8, caspase, CD83*, CTSF, CTSL2, CUL2, CUL5, IGFBP3, LGALS3, Lh, MAPK1, MYO1B, NAMPT, NFATC2IP, NFkB (complex), OSCAR, PDLIM3, RAC2, RBX1, SOCS3, SOX11, SSTR2, TCEB1, TCEB2*, TLR8, TLR9, TMSB15A, TNFRSF10A, TP53 (includes EG:22059), TRAF2 2 Cellular Assembly and Organization, 2 1 CDC42EP5, ERK1/2 Cellular Function and Maintenance, Cell Signaling 3 Connective Tissue Development and 2 1 DKK1, LRP5 Function, Skeletal and Muscular System Development and Function, Tissue Development 4 Immunological Disease, Infectious 2 1 EBI3, HLA-DPA1, IL27 Disease, Cell Morphology 5 Cell-To-Cell Signaling and Interaction, 2 1 IGFBP3, NOTCH3, THBS2 Connective Tissue Development and Function, Cellular Movement Hypomethylated and Upregulated 1 Inflammatory Disease, Inflammatory 6 2 KDM5B, MT1E, PIR* Response, Organ Morphology 2 Carbohydrate Metabolism, Cellular 3 1 IL13, PID1 Compromise, Inflammatory Response 3 Tissue Development, Genetic Disorder, 3 1 HNRNPA2B1, TM4SF18 Metabolic Disease 4 Cellular Growth and Proliferation, Tumor 3 1 MTOR, RHEB Morphology, Cancer 5 Cell Cycle, Cellular Compromise, 3 1 CLDN7, MT1A, SATB1 Molecular Transport Bold indicates focus genes (genes mapped to their corresponding genes in the Ingenuity Pathway Knowledge Base); An asterisk (*) indicates that a given gene is represented in the microarray set with multiple identifiers.

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Appendix 16 The list of genes associated with the FPGS-specific gene expression analysis

Appendix 16. 1 The list of genes with altered expression associated with the function of FPGS in the FPGS-modulated HCT116 colon cancer cells

Fold Change (vs. Control) Gene Description Accession Probe_ID Symbol FPGS FPGS Overexpression Inhibition

Upregulated in FPGS Overexpression and Downregulated in FPGS Inhibition

DUSP6 2.63 -1.71 dual specificity phosphatase 6 NM_022652.2 ILMN_2396020 ENC1 1.41 -1.64 ectodermal-neural cortex (with BTB- NM_003633.1 ILMN_1779147 like domain) NFIB 1.42 -1.61 nuclear factor I/B NM_005596.2 ILMN_1778991 PPAP2B 1.33 -1.52 phosphatidic acid phosphatase type 2B NM_003713.3 ILMN_2388800 DUSP6 2.14 -1.50 dual specificity phosphatase 6 NM_001946.2 ILMN_1677466 LGALS3 2.10 -1.48 lectin, galactoside-binding, soluble, 3 NM_002306.1 ILMN_1803788 (galectin 3) LAMB1 2.48 -1.44 laminin, beta 1 NM_002291.1 ILMN_2214790 JARID2 1.35 -1.44 jumonji, AT rich interactive domain 2 NM_004973.2 ILMN_1764177 LOC400986 1.39 -1.44 PREDICTED: protein immuno-reactive XM_001126815.1 ILMN_1811117 with anti-PTH polyclonal antibodies TANK 1.32 -1.44 TRAF family member-associated NM_004180.2 ILMN_1715069 NFKB activator SUSD2 1.57 -1.41 sushi domain containing 2 NM_019601.3 ILMN_1693270 MLPH 1.35 -1.40 melanophilin NM_001042467.1 ILMN_1795342 PKD2 1.41 -1.40 polycystic kidney disease 2 (autosomal NM_000297.2 ILMN_2052891 dominant) LTBR 1.56 -1.40 lymphotoxin beta receptor (TNFR NM_002342.1 ILMN_1667476 superfamily, member 3) SEC23B 1.42 -1.38 Sec23 homolog B (S. cerevisiae) NM_032985.4 ILMN_2366246 EGR1 2.47 -1.33 early growth response 1 NM_001964.2 ILMN_1762899 ZIC2 1.62 -1.33 Zic family member 2 (odd-paired NM_007129.2 ILMN_1696339 homolog, Drosophila) NR2F1 1.79 -1.33 nuclear receptor subfamily 2, group F, NM_005654.4 ILMN_1786197 member 1 MCFD2 1.53 -1.32 multiple coagulation factor deficiency 2 NM_139279.3 ILMN_2202790 ILF3 1.36 -1.32 interleukin enhancer binding factor 3, NM_012218.2 ILMN_1698463 90kDa OSBPL5 1.36 -1.31 oxysterol binding protein-like 5 NM_020896.2 ILMN_1802151 TSPAN7 2.01 -1.31 tetraspanin 7 NM_004615.2 ILMN_2120695 MT1G 1.44 -1.31 metallothionein 1G NM_005950.1 ILMN_1715401 NFE2L1 2.21 -1.30 nuclear factor (erythroid-derived 2)-like NM_003204.1 ILMN_1739450 1 SERP1 1.42 -1.30 stress-associated endoplasmic reticulum NM_014445.3 ILMN_1706817

561

protein 1

Downregulated in FPGS Overexpression and Upregulated in FPGS Inhibition

ALDH1A3 -1.61 1.85 aldehyde dehydrogenase 1 family, NM_000693.2 ILMN_1807439 member A3 ALDH1A3 -1.93 1.80 aldehyde dehydrogenase 1 family, NM_000693.1 ILMN_2139970 member A3 GPR110 -1.58 1.70 G protein-coupled receptor 110 NM_153840.2 ILMN_2241124 IGFBP6 -1.59 1.64 insulin-like growth factor binding NM_002178.2 ILMN_1669362 protein 6 ALDH3A2 -1.34 1.49 aldehyde dehydrogenase 3 family, NM_001031806.1 ILMN_1667564 member A2 DLEU2L -1.31 1.49 deleted in lymphocytic leukemia 2-like NR_002771.1 ILMN_2143011 FBXO6 -1.42 1.48 F-box protein 6 NM_018438.4 ILMN_1701455 PPFIBP1 -1.64 1.43 PTPRF interacting protein, binding NM_003622.2 ILMN_1679460 protein 1 (liprin beta 1) RPL34 -2.46 1.43 ribosomal protein L34 NM_000995.2 ILMN_1706873 CDC25C -1.55 1.39 cell division cycle 25 homolog C (S. NM_001790.3 ILMN_2407619 pombe) RDM1 -1.42 1.37 RAD52 motif 1 NM_001034836.1 ILMN_2309534 OR51I1 -1.43 1.35 olfactory receptor, family 51, subfamily NM_001005288.1 ILMN_1815230 I, member 1 DDT -1.42 1.35 D-dopachrome tautomerase NM_001355.3 ILMN_1690982 MRPL52 -2.57 1.34 mitochondrial ribosomal protein L52 NM_181306.1 ILMN_2311041 OR51B5 -1.34 1.34 olfactory receptor, family 51, subfamily NM_001005567.1 ILMN_1657002 B, member 5 DENND1A -1.47 1.33 DENN/MADD domain containing 1A NM_024820.2 ILMN_1727315 DARS2 -1.80 1.32 aspartyl-tRNA synthetase 2, NM_018122.3 ILMN_1676191 mitochondrial BSCL2 -1.35 1.32 Bernardinelli-Seip congenital NM_032667.4 ILMN_1774596 lipodystrophy 2 (seipin) HSPA4L -1.58 1.32 heat shock 70kDa protein 4-like NM_014278.2 ILMN_1732468 C5orf51 -1.32 1.32 chromosome 5 open reading frame 51 NM_175921.4 ILMN_3237850 LSMD1 -1.62 1.32 LSM domain containing 1 NM_032356.3 ILMN_1691131 PTRH1 -1.31 1.31 peptidyl-tRNA hydrolase 1 homolog (S. NM_001002913.1 ILMN_1763842 cerevisiae)

562

Appendix 16. 2 The list of genes with altered expression associated with the function of FPGS in the FPGS-modulated MDA-MB-435 breast cancer cells

Fold Change (vs. Control) Gene Description Accession Probe_ID Symbol FPGS FPGS Overexpression Inhibition

Upregulated in FPGS Overexpression and Downregulated in FPGS Inhibition

HLA-DQA1 9.13 -5.75 PREDICTED: major histocompatibility XM_936128.2 ILMN_1808405 complex, class II, DQ alpha 1, transcript variant 10 THBS2 2.13 -5.25 thrombospondin 2 NM_003247.2 ILMN_1678842 C21orf34 8.79 -4.48 chromosome 21 open reading frame 34 NM_001005734.1 ILMN_1690703 HLA-DOA 4.12 -3.95 major histocompatibility complex, class NM_002119.3 ILMN_1659075 II, DO alpha C1S 1.59 -3.26 complement component 1, s NM_001734.2 ILMN_1781626 subcomponent HLA-DMB 3.19 -3.17 major histocompatibility complex, class NM_002118.3 ILMN_1761733 II, DM beta CTHRC1 4.19 -2.88 collagen triple helix repeat containing 1 NM_138455.2 ILMN_1725090 HLA-DPA1 2.82 -2.87 major histocompatibility complex, class NM_033554.2 ILMN_1772218 II, DP alpha 1 HLA-DRB4 4.04 -2.82 major histocompatibility complex, class NM_021983.4 ILMN_2159694 II, DR beta 4 HLA-DPB1 2.31 -2.76 major histocompatibility complex, class NM_002121.4 ILMN_1749070 II, DP beta 1 CHN1 2.18 -2.66 chimerin (chimaerin) 1 NM_001025201.1 ILMN_1678493 HLA-DRB6 3.35 -2.57 major histocompatibility complex, class NR_001298.1 ILMN_2066066 II, DR beta 6 (pseudogene) JAM3 2.04 -2.47 junctional adhesion molecule 3 NM_032801.3 ILMN_1769575 AKR1C3 2.07 -2.44 aldo-keto reductase family 1, member NM_003739.4 ILMN_1713124 C3 (3-alpha hydroxysteroid dehydrogenase, type II) CTHRC1 3.97 -2.30 collagen triple helix repeat containing 1 NM_138455.2 ILMN_2117508 HLA-DRA 2.47 -2.22 major histocompatibility complex, class NM_019111.3 ILMN_1689655 II, DR alpha FSCN1 1.57 -2.19 fascin homolog 1, actin-bundling NM_003088.2 ILMN_1808707 protein (Strongylocentrotus purpuratus) IFITM1 2.99 -2.08 interferon induced transmembrane NM_003641.3 ILMN_1801246 protein 1 (9-27) HTRA1 1.46 -2.08 HtrA serine peptidase 1 NM_002775.3 ILMN_1676563 HLA-DRB3 1.85 -2.07 major histocompatibility complex, class NM_022555.3 ILMN_1717261 II, DR beta 3. C1R 2.50 -2.03 complement component 1, r NM_001733.4 ILMN_1677198 subcomponent ZNF562 1.35 -2.00 zinc finger protein 562 NM_017656.2 ILMN_1672940 HIST1H2BD 2.11 -1.99 histone cluster 1, H2bd NM_138720.1 ILMN_1651496

563

GAS1 2.09 -1.97 growth arrest-specific 1 NM_002048.1 ILMN_1772910 HLA-DMA 1.69 -1.96 major histocompatibility complex, class NM_006120.2 ILMN_1695311 II, DM alpha HLA-B 2.95 -1.96 major histocompatibility complex, class NM_005514.5 ILMN_1778401 I, B GAS1 1.94 -1.93 growth arrest-specific 1 NM_002048.1 ILMN_2062701 AKAP12 1.66 -1.89 A kinase (PRKA) anchor protein NM_005100.2 ILMN_2308950 (gravin) 12 RAB3IL1 1.47 -1.89 RAB3A interacting protein (rabin3)-like NM_013401.2 ILMN_1741632 1 HLA-DRA 2.39 -1.89 major histocompatibility complex, class NM_019111.3 ILMN_2157441 II, DR alpha SCARF2 1.82 -1.88 scavenger receptor class F, member 2 NM_153334.3 ILMN_1655405 CATSPER1 4.12 -1.87 cation channel, sperm associated 1 NM_053054.2 ILMN_1789394 AHNAK 5.96 -1.84 AHNAK nucleoprotein NM_024060.2 ILMN_1752159 CD74 3.85 -1.82 CD74 molecule, major NM_004355.2 ILMN_2379644 histocompatibility complex, class II invariant chain CD74 3.91 -1.82 CD74 molecule, major NM_001025159.1 ILMN_1736567 histocompatibility complex, class II invariant chain TMEM173 1.54 -1.82 transmembrane protein 173 NM_198282.1 ILMN_2145116 ZBTB43 1.61 -1.81 zinc finger and BTB domain containing NM_014007.2 ILMN_1731113 43 MUC1 2.67 -1.80 mucin 1, cell surface associated NM_001044391.1 ILMN_1756992 KIAA1751 1.50 -1.78 KIAA1751 NM_001080484.1 ILMN_2415979 CDC42EP5 5.99 -1.78 CDC42 effector protein (Rho GTPase NM_145057.2 ILMN_1774982 binding) 5 NNMT 6.82 -1.76 nicotinamide N-methyltransferase NM_006169.2 ILMN_1715508 FZD4 2.82 -1.76 frizzled homolog 4 (Drosophila) NM_012193.2 ILMN_1743367 LOC283932 1.45 -1.76 hypothetical protein LOC283932 NM_175901.3 ILMN_1710954 C21orf34 2.53 -1.75 chromosome 21 open reading frame 34 NM_001005732.1 ILMN_1702226 FBXL11 1.46 -1.70 F-box and leucine-rich repeat protein 11 NM_012308.1 ILMN_3237966 GBGT1 1.70 -1.70 globoside alpha-1,3-N- NM_021996.3 ILMN_1652906 acetylgalactosaminyltransferase 1 ANKRD20A1 3.04 -1.70 ankyrin repeat domain 20 family, NM_032250.1 ILMN_2215824 member A1 JUN 2.85 -1.70 jun oncogene NM_002228.3 ILMN_1806023 FKBP14 1.57 -1.69 FK506 binding protein 14, 22 kDa NM_017946.2 ILMN_2150294 CD74 3.67 -1.68 CD74 molecule, major NM_001025159.1 ILMN_1761464 histocompatibility complex, class II invariant chain BIRC3 2.06 -1.68 baculoviral IAP repeat-containing 3 NM_182962.1 ILMN_2405684 TMEM42 2.83 -1.67 transmembrane protein 42 NM_144638.1 ILMN_1760245 SOD2 2.10 -1.67 2, mitochondrial NM_001024466.1 ILMN_2406501 TMEM132A 1.32 -1.65 transmembrane protein 132A NM_178031.2 ILMN_2317923

564

XRCC2 1.46 -1.65 X-ray repair complementing defective NM_005431.1 ILMN_2204909 repair in Chinese hamster cells 2 BIRC3 5.23 -1.65 baculoviral IAP repeat-containing 3 NM_001165.3 ILMN_1776181 NOL8 1.57 -1.64 nucleolar protein 8 NM_017948.4 ILMN_1689747 LMOD3 1.49 -1.64 leiomodin 3 (fetal) NM_198271.2 ILMN_1785703 RSF1 1.39 -1.64 remodeling and spacing factor 1 NM_016578.3 ILMN_1668834 ODC1 1.71 -1.64 ornithine decarboxylase 1 NM_002539.1 ILMN_1748591 C15orf63 1.40 -1.63 chromosome 15 open reading frame 63 NM_016400.2 ILMN_2073543 ZNF682 1.64 -1.62 zinc finger protein 682 NM_033196.2 ILMN_2313889 KCNG1 2.39 -1.62 potassium voltage-gated channel, NM_002237.3 ILMN_1673769 subfamily G, member 1 SHROOM4 1.32 -1.62 shroom family member 4 NM_020717.2 ILMN_2206188 NDE1 1.63 -1.61 nudE nuclear distribution gene E NM_017668.1 ILMN_1739805 homolog 1 (A. nidulans) PDLIM3 1.37 -1.61 PDZ and LIM domain 3 NM_014476.1 ILMN_2230025 HNRNPU 1.60 -1.60 heterogeneous nuclear NM_004501.3 ILMN_2370135 ribonucleoprotein U (scaffold attachment factor A) KLF9 2.12 -1.59 Kruppel-like factor 9 NM_001206.2 ILMN_1778523 ALPP 1.43 -1.58 alkaline phosphatase, placental (Regan NM_001632.3 ILMN_1693789 isozyme) TMEM136 2.05 -1.57 transmembrane protein 136 NM_174926.1 ILMN_1815346 TMEM17 1.44 -1.56 transmembrane protein 17 NM_198276.1 ILMN_2210386 MBD4 1.30 -1.55 methyl-CpG binding domain protein 4 NM_003925.1 ILMN_2055310 DNTTIP2 1.46 -1.54 deoxynucleotidyltransferase, terminal, NM_014597.3 ILMN_1708345 interacting protein 2 AKAP12 1.50 -1.54 A kinase (PRKA) anchor protein NM_144497.1 ILMN_1684836 (gravin) 12 SNORD3D 1.69 -1.54 small nucleolar RNA, C/D box 3D NR_006882.1 ILMN_3242315 ABCA1 1.51 -1.53 ATP-binding cassette, sub-family A NM_005502.2 ILMN_1766054 NINJ1 2.23 -1.53 ninjurin 1 NM_004148.3 ILMN_1815086 PPFIBP1 2.59 -1.53 PTPRF interacting protein, binding NM_003622.2 ILMN_1806320 protein 1 (liprin beta 1) RN7SK 1.83 -1.53 RNA, 7SK small nuclear NR_001445.1 ILMN_1739423 PALM2 1.65 -1.52 paralemmin 2 NM_001037293.1 ILMN_2369403 AHNAK 3.31 -1.52 AHNAK nucleoprotein NM_001620.1 ILMN_1792495 DMRT2 1.47 -1.52 doublesex and mab-3 related NM_006557.4 ILMN_1751785 transcription factor 2 PHLDA1 1.89 -1.52 pleckstrin homology-like domain, NM_007350.3 ILMN_1687978 family A, member 1 CYCSL1 1.34 -1.52 cytochrome c, somatic-like 1 NR_001561.1 ILMN_2061419 TMEM108 1.48 -1.52 transmembrane protein 108 NM_023943.1 ILMN_1669709 SNAPC1 1.43 -1.52 small nuclear RNA activating complex, NM_003082.2 ILMN_2093389 polypeptide 1, 43kDa C9orf85 1.48 -1.51 chromosome 9 open reading frame 85 NM_182505.3 ILMN_1678477

565

P8 1.73 -1.50 p8 protein (candidate of metastasis 1) NM_012385.1 ILMN_1810560 MUC1 2.01 -1.50 mucin 1, cell surface associated NM_001044390.1 ILMN_2371911 RAB30 1.34 -1.50 RAB30, member RAS oncogene family NM_014488.3 ILMN_2214355 DPF2 1.72 -1.50 D4, zinc and double PHD fingers family NM_006268.3 ILMN_1734317 2 C1R 2.02 -1.49 complement component 1, r NM_001733.4 ILMN_1764109 subcomponent IL7 1.75 -1.49 interleukin 7 NM_000880.2 ILMN_1705769 SSTR2 1.45 -1.49 somatostatin receptor 2 NM_001050.2 ILMN_2152257 UBQLN1 1.38 -1.49 ubiquilin 1 NM_053067.1 ILMN_2351611 SLU7 1.37 -1.48 SLU7 splicing factor homolog (S. NM_006425.4 ILMN_1685369 cerevisiae) GADD45A 3.28 -1.48 growth arrest and DNA-damage- NM_001924.2 ILMN_2052208 inducible, alpha TUT1 1.66 -1.48 terminal uridylyl transferase 1, U6 NM_022830.1 ILMN_1786108 snRNA-specific LRRFIP1 1.43 -1.48 leucine rich repeat (in FLII) interacting NM_004735.2 ILMN_2214997 protein 1 C19orf2 1.35 -1.47 chromosome 19 open reading frame 2 NM_134447.1 ILMN_2406892 PTGR1 2.26 -1.47 prostaglandin reductase 1 NM_012212.2 ILMN_2225537 CDC42EP1 2.46 -1.47 CDC42 effector protein (Rho GTPase NM_152243.1 ILMN_1764927 binding) 1 SF3B2 1.39 -1.46 splicing factor 3b, subunit 2, 145kDa NM_006842.2 ILMN_1775939 COL8A1 4.06 -1.46 collagen, type VIII, alpha 1 NM_020351.2 ILMN_1685433 KIAA1539 1.71 -1.46 KIAA1539 NM_025182.2 ILMN_1732609 BIRC2 1.81 -1.46 baculoviral IAP repeat-containing 2 NM_001166.3 ILMN_2182704 C16orf68 1.34 -1.46 chromosome 16 open reading frame 68 NM_024109.2 ILMN_1658290 PLDN 1.40 -1.46 pallidin homolog (mouse) NM_012388.2 ILMN_2105033 FKBP11 1.87 -1.46 FK506 binding protein 11, 19 kDa NM_016594.1 ILMN_1787345 HIST2H2BE 2.11 -1.45 histone cluster 2, H2be NM_003528.2 ILMN_1732071 DCTN3 1.37 -1.45 dynactin 3 (p22) NM_007234.3 ILMN_1762281 CIR1 1.36 -1.45 corepressor interacting with RBPJ, 1 NM_004882.3 ILMN_1671516 HPS5 2.30 -1.44 Hermansky-Pudlak syndrome 5 NM_007216.3 ILMN_1655312 EML3 1.67 -1.44 echinoderm microtubule associated NM_153265.2 ILMN_1772644 protein like 3 RRAS2 2.01 -1.44 related RAS viral (r-ras) oncogene NM_012250.3 ILMN_2077623 homolog 2 YPEL3 2.02 -1.44 yippee-like 3 (Drosophila) NM_031477.4 ILMN_1791147 MAGT1 1.50 -1.44 magnesium transporter 1 NM_032121.4 ILMN_1721349 ID4 1.36 -1.43 inhibitor of DNA binding 4, dominant NM_001546.2 ILMN_1721758 negative helix-loop-helix protein SPESP1 1.40 -1.43 sperm equatorial segment protein 1 NM_145658.2 ILMN_1668426 CITED1 1.95 -1.43 Cbp/p300-interacting transactivator, NM_004143.2 ILMN_1691641 with Glu/Asp-rich carboxy-terminal domain, 1

566

PLIN2 1.42 -1.43 perilipin 2 NM_001122.2 ILMN_2138765 COL13A1 4.53 -1.43 collagen, type XIII, alpha 1 NM_080815.2 ILMN_2311052 RAC2 4.02 -1.43 ras-related C3 botulinum toxin substrate NM_002872.3 ILMN_1709795 2 (rho family, small GTP binding protein Rac2) PTGR1 1.67 -1.43 prostaglandin reductase 1 NM_012212.2 ILMN_1704531 LOC400464 1.94 -1.42 similar to FLJ43276 protein NM_001013670.1 ILMN_2320480 FRMD6 1.50 -1.42 FERM domain containing 6 NM_152330.2 ILMN_1769282 HPS5 2.17 -1.42 Hermansky-Pudlak syndrome 5 NM_007216.3 ILMN_2411731 CCND1 2.26 -1.42 cyclin D1 NM_053056.2 ILMN_1688480 AK1 1.32 -1.42 adenylate kinase 1 NM_000476.1 ILMN_1779965 DDX50 1.46 -1.42 DEAD (Asp-Glu-Ala-Asp) box NM_024045.1 ILMN_1712320 polypeptide 50 UBXN1 1.51 -1.41 UBX domain protein 1 NM_015853.3 ILMN_1812769 ADAMTS9 1.71 -1.41 ADAM metallopeptidase with NM_182920.1 ILMN_1805543 thrombospondin type 1 motif, 9 CBL 1.46 -1.41 Cas-Br-M (murine) ecotropic retroviral NM_005188.2 ILMN_2181968 transforming sequence LOC440348 1.33 -1.40 similar to nuclear pore complex NM_001018059.2 ILMN_2147105 interacting protein SLC6A16 1.63 -1.40 solute carrier family 6, member 16 NM_014037.2 ILMN_1723287 NCOA7 1.92 -1.40 nuclear receptor coactivator 7 NM_181782.2 ILMN_1687768 NUPR1 1.71 -1.40 nuclear protein, transcriptional NM_001042483.1 ILMN_2404688 regulator, 1 ERCC5 1.96 -1.40 excision repair cross-complementing NM_000123.2 ILMN_1795495 rodent repair deficiency, complementation group 5 RFX5 1.65 -1.39 regulatory factor X, 5 (influences HLA NM_001025603.1 ILMN_1741200 class II expression) PPP2R4 1.43 -1.39 protein phosphatase 2A activator, NM_021131.3 ILMN_1729123 regulatory subunit 4 CREBZF 1.94 -1.39 CREB/ATF bZIP transcription factor NM_001039618.1 ILMN_1784847 ZNF521 1.52 -1.39 zinc finger protein 521 NM_015461.1 ILMN_2225548 MVP 2.69 -1.39 major vault protein NM_005115.3 ILMN_1803277 SOX2 2.00 -1.38 SRY (sex determining region Y)-box 2 NM_003106.2 ILMN_2177156 SLC25A25 1.43 -1.38 solute carrier family 25 (mitochondrial NM_052901.2 ILMN_1791728 carrier; phosphate carrier), member 25 COL8A1 3.42 -1.38 collagen, type VIII, alpha 1 NM_020351.2 ILMN_2402392 EML1 2.26 -1.38 echinoderm microtubule associated NM_004434.2 ILMN_1729455 protein like 1 C9orf85 1.33 -1.38 chromosome 9 open reading frame 85 NM_182505.3 ILMN_1806758 MAK10 1.41 -1.38 MAK10 homolog, amino-acid N- NM_024635.2 ILMN_2222651 acetyltransferase subunit, (S. cerevisiae) MVP 1.88 -1.38 major vault protein NM_017458.2 ILMN_2344373 CARS 2.13 -1.37 cysteinyl-tRNA synthetase NM_001014438.1 ILMN_1696066 HEY2 1.76 -1.37 hairy/enhancer-of-split related with NM_012259.1 ILMN_1682034

567

YRPW motif 2 MRGPRX3 1.73 -1.37 MAS-related GPR, member X3 NM_054031.2 ILMN_1773546 WDR74 1.47 -1.37 PREDICTED: WD repeat domain 74 XM_001125771.1 ILMN_1789775 ARL16 1.45 -1.37 ADP-ribosylation factor-like 16 NM_001040025.1 ILMN_2188119 OSBPL6 2.21 -1.36 oxysterol binding protein-like 6 NM_032523.2 ILMN_1756935 IARS 1.35 -1.36 isoleucyl-tRNA synthetase NM_002161.3 ILMN_1733956 NFE2L3 1.53 -1.36 nuclear factor (erythroid-derived 2)-like NM_004289.5 ILMN_2049766 3 NUAK1 1.69 -1.36 NUAK family, SNF1-like kinase, 1 NM_014840.2 ILMN_2079786 LOC374395 1.35 -1.36 similar to RIKEN cDNA 1810059G22 NM_199337.1 ILMN_1733757 SYNM 1.62 -1.36 synemin, intermediate filament protein NM_015286.5 ILMN_1712075 ACSM3 1.37 -1.35 acyl-CoA synthetase medium-chain NM_202000.2 ILMN_1662738 family member 3 FRMD3 1.87 -1.35 FERM domain containing 3 NM_174938.3 ILMN_1698725 HLA-DQB1 1.69 -1.35 major histocompatibility complex, class NM_002123.2 ILMN_1661266 II, DQ beta 1 SFRS14 1.56 -1.35 splicing factor, arginine/serine-rich 14 NM_001017392.2 ILMN_1772487 CAPN5 1.67 -1.35 calpain 5 NM_004055.4 ILMN_1737089 DOCK11 2.34 -1.35 dedicator of cytokinesis 11 NM_144658.3 ILMN_1765860 TAF1D 1.92 -1.35 TATA box binding protein (TBP)- NM_024116.2 ILMN_3235027 associated factor, RNA polymerase I, D, 41kDa PRKRIR 1.40 -1.35 protein-kinase, interferon-inducible NM_004705.2 ILMN_1655622 double stranded RNA dependent inhibitor, repressor of (P58 repressor) PRKACB 1.30 -1.35 protein kinase, cAMP-dependent, NM_002731.2 ILMN_1771523 catalytic, beta OSBPL5 1.71 -1.34 oxysterol binding protein-like 5 NM_145638.1 ILMN_2307032 PLEKHB2 1.73 -1.34 pleckstrin homology domain containing, NM_001031706.1 ILMN_1698323 family B (evectins) member 2 PRSS33 1.69 -1.34 protease, serine, 33 NM_152891.2 ILMN_1736831 HIATL2 1.35 -1.34 hippocampus abundant transcript-like 2 NR_002894.1 ILMN_3235410 CEP70 1.41 -1.34 centrosomal protein 70kDa NM_024491.2 ILMN_1741350 PPP2R1B 1.53 -1.34 protein phosphatase 2 (formerly 2A), NM_181699.2 ILMN_1779841 regulatory subunit A, beta isoform CD55 2.58 -1.34 CD55 molecule, decay accelerating NM_000574.2 ILMN_1800540 factor for complement (Cromer blood group) DDX10 1.52 -1.33 DEAD (Asp-Glu-Ala-Asp) box NM_004398.2 ILMN_1753249 polypeptide 10 DKK1 2.28 -1.33 dickkopf homolog 1 (Xenopus laevis) NM_012242.2 ILMN_1773337 ITGA11 1.43 -1.33 integrin, alpha 11 NM_012211.3 ILMN_2406084 COL13A1 5.71 -1.33 collagen, type XIII, alpha 1 NM_080805.2 ILMN_2370624 PPFIA1 1.54 -1.33 protein tyrosine phosphatase, receptor NM_177423.1 ILMN_1727050 type, f polypeptide (PTPRF), interacting protein (liprin), alpha 1

568

UNC93B1 1.40 -1.33 unc-93 homolog B1 (C. elegans) NM_030930.2 ILMN_2193591 DUSP19 1.34 -1.33 dual specificity phosphatase 19 NM_080876.2 ILMN_1722492 SNORA25 1.64 -1.33 small nucleolar RNA, H/ACA box 25 NR_003028.1 ILMN_1682038 ZCCHC7 1.34 -1.33 zinc finger, CCHC domain containing 7 NM_032226.2 ILMN_1744980 FADS1 1.39 -1.33 fatty acid desaturase 1 NM_013402.3 ILMN_1670134 H1F0 2.50 -1.33 H1 histone family, member 0 NM_005318.2 ILMN_1757467 TJP2 2.23 -1.33 tight junction protein 2 (zona occludens NM_201629.1 ILMN_1664978 2) SYTL2 5.58 -1.32 synaptotagmin-like 2 NM_206928.1 ILMN_1682929 VHL 1.47 -1.32 von Hippel-Lindau tumor suppressor NM_198156.1 ILMN_1738579 TGFB1I1 2.00 -1.32 transforming growth factor beta 1 NM_015927.3 ILMN_2389876 induced transcript 1 RPGR 1.90 -1.32 retinitis pigmentosa GTPase regulator NM_001023582.1 ILMN_1768097 KCNMA1 1.99 -1.32 potassium large conductance calcium- NM_002247.2 ILMN_2297765 activated channel, subfamily M, alpha member 1 HLA-F 1.84 -1.31 major histocompatibility complex, class NM_018950.1 ILMN_2186806 I, F HOXC6 1.42 -1.31 homeobox C6 NM_004503.3 ILMN_1794492 IFT74 1.95 -1.31 intraflagellar transport 74 homolog NM_001099222.1 ILMN_1777449 (Chlamydomonas) KIRREL 1.33 -1.31 kin of IRRE like (Drosophila) NM_018240.4 ILMN_1742781 VWA5A 2.37 -1.31 von Willebrand factor A domain NM_198315.2 ILMN_1682996 containing 5A ART3 2.03 -1.31 ADP-ribosyltransferase 3 NM_001179.3 ILMN_1693218 SYTL2 4.69 -1.31 synaptotagmin-like 2 NM_206929.1 ILMN_2336609 NUDT7 2.49 -1.31 nudix (nucleoside diphosphate linked NM_001105663.1 ILMN_3226181 moiety X)-type motif 7 AGTPBP1 1.63 -1.31 ATP/GTP binding protein 1 NM_015239.1 ILMN_1718071 NUAK1 1.70 -1.31 NUAK family, SNF1-like kinase, 1 NM_014840.2 ILMN_1689318 TAF1D 2.08 -1.31 TATA box binding protein (TBP)- NM_024116.2 ILMN_2063114 associated factor, RNA polymerase I, D, 41kDa RPGR 1.66 -1.31 retinitis pigmentosa GTPase regulator NM_000328.2 ILMN_2336803 DAK 1.63 -1.31 dihydroxyacetone kinase 2 homolog (S. NM_015533.3 ILMN_1678619 cerevisiae) ZCCHC6 1.85 -1.31 zinc finger, CCHC domain containing 6 NM_024617.2 ILMN_1779677 PDLIM1 1.32 -1.30 PDZ and LIM domain 1 NM_020992.2 ILMN_1788955 ZNF234 1.52 -1.30 zinc finger protein 234 NM_006630.1 ILMN_2103397 KYNU 3.37 -1.30 kynureninase (L-kynurenine hydrolase) NM_003937.2 ILMN_1746517 RHBDD1 1.49 -1.30 rhomboid domain containing 1 NM_032276.2 ILMN_1681543

Downregulated in FPGS Overexpression and Upregulated in FPGS Inhibition

BCHE -11.02 7.02 butyrylcholinesterase NM_000055.1 ILMN_2176592

569

BCHE -8.69 5.66 butyrylcholinesterase NM_000055.2 ILMN_1685641 TRIM48 -11.38 3.67 tripartite motif-containing 48 NM_024114.2 ILMN_1762021 ANKS1A -1.92 2.83 ankyrin repeat and sterile alpha motif NM_015245.2 ILMN_1813669 domain containing 1A SLITRK4 -1.80 2.60 SLIT and NTRK-like family, member 4 NM_173078.2 ILMN_2199768 ST3GAL5 -2.14 2.20 ST3 beta-galactoside alpha-2,3- NM_001042437.1 ILMN_1713496 sialyltransferase 5 AKR1B1 -1.50 2.04 aldo-keto reductase family 1, member NM_001628.2 ILMN_1701731 B1 (aldose reductase) DYNC1I1 -5.14 1.99 dynein, cytoplasmic 1, intermediate NM_004411.3 ILMN_1690397 chain 1 PLSCR1 -2.62 1.97 phospholipid scramblase 1 NM_021105.1 ILMN_1745242 MIR1974 -1.65 1.97 microRNA 1974 NR_031738.1 ILMN_3308961 PPARGC1A -4.23 1.95 peroxisome proliferator-activated NM_013261.3 ILMN_1750062 receptor gamma, coactivator 1 alpha CYB5R2 -1.50 1.91 cytochrome b5 reductase 2 NM_016229.3 ILMN_1739576 ITPK1 -2.68 1.90 inositol 1,3,4-triphosphate 5/6 kinase NM_014216.3 ILMN_1715674 SPOCK1 -1.44 1.85 sparc/osteonectin, cwcv and kazal-like NM_004598.3 ILMN_1746013 domains proteoglycan (testican) 1 ORC6L -1.52 1.82 origin recognition complex, subunit 6 NM_014321.2 ILMN_1731070 like (yeast) GPM6B -5.99 1.81 glycoprotein M6B NM_001001995.1 ILMN_1704665 GINS2 -2.06 1.81 GINS complex subunit 2 (Psf2 homolog) NM_016095.1 ILMN_1809590 ST3GAL5 -1.80 1.80 ST3 beta-galactoside alpha-2,3- NM_001042437.1 ILMN_2388701 sialyltransferase 5 COL16A1 -1.31 1.78 collagen, type XVI, alpha 1 NM_001856.3 ILMN_1684554 CPS1 -1.83 1.74 carbamoyl-phosphate synthetase 1, NM_001875.2 ILMN_1792748 mitochondrial CAPN3 -4.52 1.73 calpain 3, (p94) NM_024344.1 ILMN_1687971 LOC729384 -3.61 1.72 tripartite motif protein 49-like NM_001105522.1 ILMN_3242226 CAPN3 -4.55 1.71 calpain 3, (p94) NM_173087.1 ILMN_2332691 ACSL3 -1.65 1.70 acyl-CoA synthetase long-chain family NM_004457.3 ILMN_1666096 member 3 ADM -1.75 1.70 adrenomedullin NM_001124.1 ILMN_1708934 IMMP2L -1.46 1.68 IMP2 inner mitochondrial membrane NM_032549.2 ILMN_1809292 peptidase-like (S. cerevisiae) TMED10 -1.76 1.67 transmembrane emp24-like trafficking NM_006827.5 ILMN_1736585 protein 10 (yeast) PEG10 -1.52 1.66 paternally expressed 10 NM_001040152.1 ILMN_2297626 TMEM106C -1.62 1.66 transmembrane protein 106C NM_024056.2 ILMN_1692511 SLC16A10 -2.17 1.65 solute carrier family 16, member 10 NM_018593.3 ILMN_1782938 (aromatic amino acid transporter) NCAPG2 -1.46 1.63 non-SMC condensin II complex, subunit NM_017760.5 ILMN_2066756 G2 NETO2 -1.33 1.62 neuropilin (NRP) and tolloid (TLL)-like NM_018092.3 ILMN_1760849 2

570

CALU -1.61 1.62 calumenin NM_001219.2 ILMN_1727194 CAPZA2 -1.58 1.62 capping protein (actin filament) muscle NM_006136.2 ILMN_1768870 Z-line, alpha 2 CYP2J2 -1.66 1.61 cytochrome P450, family 2, subfamily J, NM_000775.2 ILMN_1758731 polypeptide 2 GYG2 -4.24 1.61 glycogenin 2 NM_003918.2 ILMN_2319424 ORC5L -1.86 1.59 origin recognition complex, subunit 5- NM_002553.2 ILMN_1705093 like (yeast) PIR -4.46 1.59 pirin (iron-binding nuclear protein) NM_001018109.1 ILMN_2383383 SLC37A3 -1.63 1.59 solute carrier family 37 (glycerol-3- NM_032295.2 ILMN_2307598 phosphate transporter), member 3 IVNS1ABP -1.42 1.58 influenza virus NS1A binding protein NM_006469.4 ILMN_1717877 NHLRC3 -1.76 1.58 NHL repeat containing 3 NM_001012754.2 ILMN_1698365 IDH3A -1.76 1.58 isocitrate dehydrogenase 3 (NAD+) NM_005530.2 ILMN_1698533 alpha ORC5L -1.70 1.58 origin recognition complex, subunit 5- NM_002553.2 ILMN_1688094 like (yeast) ARPC1B -1.76 1.56 actin related protein 2/3 complex, NM_005720.2 ILMN_2085760 subunit 1B, 41kDa BHLHB2 -1.57 1.55 basic helix-loop-helix domain NM_003670.1 ILMN_1768534 containing, class B, 2 SNCA -2.80 1.55 synuclein, alpha (non A4 component of NM_007308.1 ILMN_1701933 amyloid precursor) EEF1A1 -1.71 1.54 eukaryotic translation elongation factor NM_001402.5 ILMN_3251737 1 alpha 1 UBE3C -1.54 1.54 ubiquitin protein ligase E3C NM_014671.1 ILMN_2181363 LARGE -1.95 1.54 like-glycosyltransferase NM_004737.3 ILMN_1662038 BRD7 -1.32 1.53 bromodomain containing 7 NM_013263.2 ILMN_2082810 SIVA -1.32 1.53 CD27-binding (Siva) protein NM_006427.2 ILMN_1787248 DLST -1.37 1.52 dihydrolipoamide S-succinyltransferase NM_001933.3 ILMN_1773228 (E2 component of 2-oxo-glutarate complex) SPC25 -1.44 1.52 SPC25, NDC80 kinetochore complex NM_020675.3 ILMN_1814281 component, homolog (S. cerevisiae) ITIH5L -1.44 1.51 inter-alpha (globulin) inhibitor H5-like NM_198510.1 ILMN_1709177 GJB1 -1.42 1.51 gap junction protein, beta 1, 32kDa NM_000166.4 ILMN_1799535 RANBP10 -1.76 1.51 RAN binding protein 10 NM_020850.1 ILMN_1667306 ARL2BP -1.32 1.51 ADP-ribosylation factor-like 2 binding NM_012106.3 ILMN_1755391 protein TYR -20.19 1.50 tyrosinase (oculocutaneous albinism IA) NM_000372.4 ILMN_1788774 LMBR1 -1.31 1.49 limb region 1 homolog (mouse) NM_022458.3 ILMN_1764522 FXYD5 -2.01 1.48 FXYD domain containing ion transport NM_144779.1 ILMN_1704286 regulator 5 TRAPPC2L -1.35 1.48 trafficking protein particle complex 2- NM_016209.2 ILMN_1747058 like ENPP2 -1.65 1.48 ectonucleotide NM_001040092.1 ILMN_2373791 pyrophosphatase/phosphodiesterase 2

571

PGAM1 -1.32 1.48 phosphoglycerate mutase 1 (brain) NM_002629.2 ILMN_2112417 TK1 -1.35 1.48 thymidine kinase 1, soluble NM_003258.2 ILMN_1806037 FXYD5 -1.73 1.47 FXYD domain containing ion transport NM_014164.4 ILMN_2309848 regulator 5 MITF -3.76 1.46 microphthalmia-associated transcription NM_198158.1 ILMN_2304186 factor CKMT1A -2.38 1.46 creatine kinase, mitochondrial 1A NM_001015001.1 ILMN_1732066 SLC35A2 -1.44 1.46 solute carrier family 35 (UDP-galactose NM_005660.1 ILMN_1798885 transporter), member A2 PKM2 -1.33 1.45 pyruvate kinase, muscle NM_002654.3 ILMN_1672650 TNPO3 -1.49 1.45 transportin 3 NM_012470.2 ILMN_1683811 IRX6 -1.30 1.44 iroquois homeobox 6 NM_024335.2 ILMN_1758705 FBXL2 -1.30 1.44 F-box and leucine-rich repeat protein 2 NM_012157.2 ILMN_1688639 SPRYD5 -2.83 1.44 SPRY domain containing 5 NM_032681.1 ILMN_1753648 KCNAB1 -2.93 1.44 potassium voltage-gated channel, NM_003471.2 ILMN_1744968 shaker-related subfamily, beta member 1 C18orf51 -3.48 1.44 chromosome 18 open reading frame 51 NM_001044369.1 ILMN_1670718 TAF1C -1.36 1.43 TATA box binding protein (TBP)- NM_139353.1 ILMN_1671839 associated factor, RNA polymerase I, C, 110kDa HNRNPU -1.51 1.43 heterogeneous nuclear ribonucleoprotein NM_031844.2 ILMN_1743677 U (scaffold attachment factor A) NCAPH2 -1.33 1.43 non-SMC condensin II complex, subunit NM_152299.2 ILMN_1715908 H2 PDIA5 -1.63 1.42 protein disulfide isomerase family A, NM_006810.2 ILMN_1695763 member 5 S100A1 -1.59 1.41 S100 calcium binding protein A1 NM_006271.1 ILMN_1653494 PIR -4.51 1.41 pirin (iron-binding nuclear protein) NM_001018109.1 ILMN_1761247 GSTT1 -2.19 1.41 glutathione S-transferase theta 1 NM_000853.1 ILMN_1730054 SCRG1 -2.69 1.40 scrapie responsive protein 1 NM_007281.1 ILMN_1726204 GPM6B -4.82 1.40 glycoprotein M6B NM_001001995.1 ILMN_1735438 SLC37A3 -1.57 1.39 solute carrier family 37 (glycerol-3- NM_207113.1 ILMN_1779979 phosphate transporter), member 3 C18orf51 -3.47 1.39 chromosome 18 open reading frame 51 NM_001044369.1 ILMN_2173500 AGK -1.53 1.39 acylglycerol kinase NM_018238.2 ILMN_1772645 NUP93 -1.45 1.38 nucleoporin 93kDa NM_014669.2 ILMN_2196569 SIVA1 -1.33 1.38 SIVA1, apoptosis-inducing factor NM_021709.2 ILMN_3300972 SND1 -1.32 1.38 staphylococcal nuclease and tudor NM_014390.2 ILMN_1775111 domain containing 1 CDC16 -2.20 1.38 cell division cycle 16 homolog (S. NM_001078645.1 ILMN_2256765 cerevisiae) SOX10 -1.83 1.38 SRY (sex determining region Y)-box 10 NM_006941.3 ILMN_1653750 FKBP1A -1.72 1.38 FK506 binding protein 1A, 12kDa NM_054014.1 ILMN_1702237 PTPLA -2.33 1.38 protein tyrosine phosphatase-like NM_014241.3 ILMN_1725791 (proline instead of catalytic arginine), member A

572

FAHD2B -1.38 1.37 fumarylacetoacetate hydrolase domain NM_199336.1 ILMN_2146657 containing 2B TRIM2 -1.40 1.37 tripartite motif-containing 2 NM_015271.2 ILMN_1745079 ARPC1A -1.92 1.37 actin related protein 2/3 complex, NM_006409.2 ILMN_1759915 subunit 1A, 41kDa MOSPD3 -1.33 1.37 motile sperm domain containing 3 NM_023948.4 ILMN_2356909 UGDH -1.71 1.37 UDP-glucose dehydrogenase NM_003359.2 ILMN_1729563 PAPOLA -1.81 1.37 poly(A) polymerase alpha NM_032632.3 ILMN_1798354 SHFM1 -1.75 1.36 split hand/foot malformation NM_006304.1 ILMN_1794505 (ectrodactyly) type 1 POLD2 -1.34 1.36 polymerase (DNA directed), delta 2, NM_001127218.1 ILMN_3305304 regulatory subunit 50kDa SLC25A13 -2.08 1.36 solute carrier family 25, member 13 NM_014251.1 ILMN_1668012 (citrin) HNRNPAB -1.77 1.36 heterogeneous nuclear ribonucleoprotein NM_031266.2 ILMN_1696485 A/B PIGB -1.80 1.36 phosphatidylinositol glycan anchor NM_004855.4 ILMN_1733311 biosynthesis, class B DBNDD1 -1.31 1.36 dysbindin (dystrobrevin binding protein NM_001042610.1 ILMN_2374352 1) domain containing 1 CDC16 -2.17 1.36 cell division cycle 16 homolog (S. NM_001078645.1 ILMN_2339796 cerevisiae) STX7 -1.60 1.36 syntaxin 7 NM_003569.1 ILMN_1792518 C18orf10 -1.62 1.35 chromosome 18 open reading frame 10 NM_015476.2 ILMN_2207328 MNS1 -1.65 1.35 meiosis-specific nuclear structural 1 NM_018365.1 ILMN_2157240 TMEM189 -1.55 1.35 transmembrane protein 189 NM_199129.1 ILMN_2162989 RHBDF2 -1.42 1.34 rhomboid 5 homolog 2 (Drosophila) NM_001005498.2 ILMN_1691717 SLC35B4 -1.69 1.34 solute carrier family 35, member B4 NM_032826.3 ILMN_1697959 PDIA4 -1.66 1.34 protein disulfide isomerase family A, NM_004911.3 ILMN_1815261 member 4 ST7 -2.18 1.34 suppression of tumorigenicity 7 NM_021908.2 ILMN_1746137 ST8SIA1 -1.67 1.34 ST8 alpha-N-acetyl-neuraminide alpha- NM_003034.2 ILMN_1664859 2,8-sialyltransferase 1 WDR61 -2.22 1.33 WD repeat domain 61 NM_025234.1 ILMN_1665887 RNF175 -2.13 1.33 ring finger protein 175 NM_173662.2 ILMN_1741281 NQO1 -1.48 1.33 NAD(P)H dehydrogenase, quinone 1 NM_000903.2 ILMN_1720282 BAMBI -2.01 1.32 BMP and activin membrane-bound NM_012342.2 ILMN_1691410 inhibitor homolog (Xenopus laevis) H2AFZ -1.69 1.32 H2A histone family, member Z NM_002106.3 ILMN_1707858 SELT -1.58 1.32 selenoprotein T NM_016275.3 ILMN_1746368 PGM1 -1.85 1.32 phosphoglucomutase 1 NM_002633.2 ILMN_1800659 PGK1 -1.59 1.32 phosphoglycerate kinase 1 NM_000291.2 ILMN_2216852 SORT1 -1.53 1.32 sortilin 1 NM_002959.4 ILMN_1707077 GPD2 -1.41 1.32 glycerol-3-phosphate dehydrogenase 2 NM_001083112.1 ILMN_1723139 (mitochondrial)

573

QDPR -1.51 1.31 quinoid dihydropteridine reductase NM_000320.1 ILMN_1672443 ST6GALNAC -2.81 1.31 ST6 (alpha-N-acetyl-neuraminyl-2,3- NM_152996.1 ILMN_2127379 3 beta-galactosyl-1, 3)-N- acetylgalactosaminide alpha-2,6- sialyltransferase 3 ME2 -1.66 1.31 malic enzyme 2, NAD(+)-dependent, NM_002396.3 ILMN_1675186 mitochondrial TSGA14 -1.33 1.30 testis specific, 14 NM_018718.1 ILMN_2223805 CHD9 -1.42 1.30 chromodomain helicase DNA binding NM_025134.4 ILMN_1762972 protein 9 MYB -1.41 1.30 v-myb myeloblastosis viral oncogene NM_005375.2 ILMN_1711894 homolog (avian)

574

Appendix 17 Genes with altered expression associated with both GGH and FPGS

Appendix 17. 1 The top molecular and cellular functions associated with the GGH- and FPGS-specific gene expression

Appendix 17. 1. 1 The top molecular and cellular functions associated with the GGH- and FPGS-specific gene expression in HCT116 colon cancer cells

GGH Overexpression and FPGS Inhibition GGH Inhibition and FPGS Overexpression

No. of No. of Category P-value Category P-value Genes Genes Downregulated Upregulated Cell Cycle 6.27E-04 - 2 Cell Death 1.84E-05 - 19 4.12E-02 4.56E-02 Cell Death 6.27E-04 - 2 Cellular Development 8.31E-05 - 9 3.27E-02 4.84E-02 Cell Morphology 6.27E-04 - 2 Cell Cycle 2.97E-04 - 6 3.46E-02 4.97E-02 Cell-To-Cell Signaling and 6.27E-04 - 3 Cellular Growth and 4.05E-04 - 12 Interaction 1.37E-02 Proliferation 4.84E-02 Cellular Development 6.27E-04 - 3 Carbohydrate 5.41E-04 - 5 2.73E-02 Metabolism 4.56E-02

Upregulated Downregulated Amino Acid Metabolism 4.48E-04 - 1 Cell Cycle 3.62E-05 - 9 1.34E-03 4.19E-02 Cellular Movement 4.48E-04 - 3 Cellular Growth and 1.09E-04 - 13 3.57E-02 Proliferation 3.26E-02 Small Molecule 4.48E-04 - 2 DNA Replication, 1.29E-04 - 10 Biochemistry 8.04E-03 Recombination, and 3.97E-02 Repair Cell Morphology 8.96E-04 - 2 Cellular Assembly and 1.83E-04 - 6 6.26E-03 Organization 3.08E-02 Cellular Function and 8.96E-04 - 1 Cell Death 7.34E-04 - 10 Maintenance 1.87E-02 4.14E-02

575

Appendix 17. 1. 2 The top molecular and cellular functions associated with the GGH- and FPGS-specific gene expression in MDA-MB-435 breast cancer cells

GGH Overexpression and FPGS Inhibition GGH Inhibition and FPGS Overexpression

No. of No. of Category P-value Category P-value Genes Genes Downregulated Upregulated Cell-To-Cell Signaling and 1.88E-03 - 4 Cell Death 1.55E-11 - 39 Interaction 4.60E-02 2.10E-02 Cellular Assembly and 1.88E-03 - 2 Cellular Growth and 1.44E-08 - 37 Organization 2.60E-02 Proliferation 2.10E-02 Cellular Movement 1.88E-03 - 1 Cellular Movement 1.92E-07 - 23 4.42E-02 2.10E-02 DNA Replication, 1.88E-03 - 2 Cellular Development 1.91E-06 - 25 Recombination, and Repair 2.24E-02 2.10E-02 Drug Metabolism 1.88E-03 - 2 Cell-To-Cell Signaling 8.09E-06 - 16 2.05E-02 and Interaction 2.10E-02

Upregulated Downregulated Cell Death 4.95E-04 - 10 Amino Acid 2.91E-03 - 2 3.41E-02 Metabolism 1.97E-02 Cellular Growth and 6.07E-04 - 10 Carbohydrate 2.91E-03 - 1 Proliferation 4.38E-02 Metabolism 2.91E-03 Cell Cycle 3.67E-03 - 7 Cellular Assembly and 2.91E-03 - 5 3.97E-02 Organization 4.00E-02 Cell Morphology 3.67E-03 - 2 Cellular Function and 2.91E-03 - 2 2.90E-02 Maintenance 1.45E-02 Cell-To-Cell Signaling and 3.67E-03 - 5 Cellular Movement 2.91E-03 - 4 Interaction 3.91E-02 4.78E-02

576

Appendix 17. 2 The top networks matched by the genes with the GGH- and FPGS- specific altered expression

Appendix 17. 2. 1 The top networks matched by the genes with the GGH- and FPGS- specific altered expression in HCT116 colon cancer cells

GGH Overexpression and FPGS Inhibition GGH Inhibition and FPGS Overexpression

Focus Focus No Top Functions Score Top Functions Score Genes Genes Downregulated Upregulated 1 Cancer, Cardiovascular System 19 8 Cellular Growth and 23 14 Development and Function, Cell Proliferation, Cell Death, Tumor Cycle Morphology 2 Cellular Assembly and 3 1 Cellular Assembly and 21 13 Organization, Hair and Skin Organization, Cellular Function Development and Function, and Maintenance, Cell Cycle Dermatological Diseases and Conditions 3 Connective Tissue Disorders, 21 13 Genetic Disorder, Skeletal and Muscular Disorders 4 Tissue Development, Genetic 2 1 Disorder, Metabolic Disease 5 Nucleic Acid Metabolism, 2 1 Small Molecule Biochemistry, Cell Signaling Upregulated Downregulated 1 Cell Death, Inflammatory 12 5 Cell Death, Cell Cycle, Cancer 24 12 Response, Cellular Movement 2 Cell Cycle, Connective Tissue 16 9 Development and Function, Cellular Assembly and Organization 3 Cancer, Neurological Disease, 2 1 Dermatological Diseases and Conditions

577

Appendix 17. 2. 2 The top networks matched by the genes with the GGH- and FPGS- specific altered expression in MDA-MB-435 breast cancer cells

GGH Overexpression and FPGS Inhibition GGH Inhibition and FPGS Overexpression

Focus Focus No Top Functions Score Top Functions Score Genes Genes Downregulated Upregulated 1 Cell Death, Cellular Growth and 22 11 Cellular Growth and 40 23 Proliferation, Connective Tissue Proliferation, Cell Death, Gene Development and Function Expression 2 Cell Death, Cellular Growth and 6 3 Cell Death, Cellular Movement, 40 23 Proliferation, Cancer Cellular Growth and Proliferation 3 Cell Morphology, Cellular 2 1 Cell-To-Cell Signaling and 12 10 Compromise, Cellular Growth Interaction, Cellular Growth and and Proliferation Proliferation, Connective Tissue Development and Function 4 Tissue Development, 2 1 Gastrointestinal Disease, 12 10 Connective Tissue Development Hepatic System Disease, and Function, Skeletal and Infectious Disease Muscular System Development and Function 5 Cellular Function and 2 1 Cell Death, Endocrine System 9 8 Maintenance, Cellular Development and Function, Movement, Cell-To-Cell Lipid Metabolism Signaling and Interaction Upregulated Downregulated 1 Metabolic Disease, Cancer, 21 13 Cellular Movement, Skeletal 28 15 Cellular Movement and Muscular System Development and Function, Hair and Skin Development and Function 2 Cellular Growth and 19 12 Cancer, Dermatological 16 10 Proliferation, Organ Diseases and Conditions, Cell Morphology, Reproductive Death System Development and Function 3 Cell Death, Cellular Movement, 19 12 Cell Morphology, Cellular 2 1 Cellular Development Compromise, Cellular Growth and Proliferation 4 Gene Expression, Cellular 17 11 Molecular Transport, Protein 2 1 Function and Maintenance, Cell Synthesis, Protein Trafficking Cycle 5 Cancer, Hepatic System 2 1 Cell Cycle, Genetic Disorder, 2 1 Disease, Cellular Movement Hematological Disease

578

Appendix 17. 3 The list of genes with altered expression associated with the function of GGH and FPGS

Appendix 17. 3. 1 Comparison between GGH overexpression and FPGS inhibition in HCT116 colon cancer cells

Fold Change (vs. Control) Gene Description Accession Probe_ID Symbol GGH FPGS Overexpression Inhibition

Downregulated in both GGH Overexpression and FPGS Inhibition

PVRL3 -3.86 -1.71 poliovirus receptor-related 3 NM_015480.1 ILMN_2188521 PVRL3 -3.25 -1.64 poliovirus receptor-related 3 NM_015480.1 ILMN_1727633 SUSD2 -2.27 -1.41 sushi domain containing 2 NM_019601.3 ILMN_1693270 BMP4 -2.19 -2.57 bone morphogenetic protein 4 NM_130851.1 ILMN_1740900 NR2F2 -1.89 -2.00 nuclear receptor subfamily 2, group F, NM_021005.2 ILMN_2094360 member 2 SLC2A3 -1.70 -1.49 solute carrier family 2 (facilitated NM_006931.1 ILMN_1775708 glucose transporter), member 3 CNTNAP2 -1.69 -1.57 contactin associated protein-like 2 NM_014141.4 ILMN_1690223 FAM111A -1.65 -1.40 family with sequence similarity 111, NM_022074.2 ILMN_2410038 member A BMP4 -1.64 -1.58 bone morphogenetic protein 4 NM_001202.2 ILMN_1709734 SACS -1.52 -1.38 spastic ataxia of Charlevoix-Saguenay NM_014363.3 ILMN_2131523 (sacsin) ADAM19 -1.48 -1.39 ADAM metallopeptidase domain 19 NM_033274.2 ILMN_1713751 (meltrin beta) SEMA3A -1.42 -1.46 sema domain, immunoglobulin NM_006080.2 ILMN_1765641 domain (Ig), short basic domain, secreted, (semaphorin) 3A FBXO5 -1.35 -1.40 F-box protein 5 NM_012177.2 ILMN_1710676 MKX -1.34 -1.32 mohawk homeobox NM_173576.1 ILMN_1681780 MLPH -1.32 -1.40 melanophilin NM_001042467.1 ILMN_1795342 UBE1DC1 -1.30 -1.31 ubiquitin-activating enzyme E1- NM_024818.2 ILMN_2408450 domain containing 1

Upregulated in both GGH Overexpression and FPGS Inhibition

GDF15 2.44 3.47 growth differentiation factor 15 NM_004864.1 ILMN_2188862 LCN2 1.93 2.72 lipocalin 2 NM_005564.3 ILMN_1692223 PLAU 1.89 1.55 plasminogen activator, urokinase NM_002658.2 ILMN_1656057 ANXA10 1.62 2.36 annexin A10 NM_007193.3 ILMN_1699421 CTH 1.60 1.38 cystathionase (cystathionine gamma- NM_153742.3 ILMN_2305112 lyase) HIST1H2BD 1.48 1.56 histone cluster 1, H2bd NM_138720.1 ILMN_1651496

579

CA2 1.39 1.80 carbonic anhydrase II NM_000067.1 ILMN_1662795 TRNP1 1.38 2.02 TMF1-regulated nuclear protein 1 NM_001013642.2 ILMN_1695946 CA2 1.36 1.79 carbonic anhydrase II NM_000067.1 ILMN_2199439 CYP4F11 1.31 1.54 cytochrome P450, family 4, subfamily NM_021187.2 ILMN_1719883 F, polypeptide 11 FAM84B 1.31 1.32 family with sequence similarity 84, NM_174911.3 ILMN_1670807 member B

Appendix 17. 3. 2 Comparison between GGH inhibition and FPGS overexpression in HCT116 colon cancer cells

Fold Change (vs. Control) Gene Description Accession Probe_ID Symbol GGH FPGS Inhibition Overexpression

Upregulated in both GGH Inhibition and FPGS Overexpression

IFI27 3.91 1.97 interferon, alpha-inducible protein 27 NM_005532.3 ILMN_2058782 TNFRSF6B 2.49 11.17 tumor necrosis factor receptor NM_032945.2 ILMN_2331231 superfamily, member 6b, decoy CALB2 2.47 2.47 calbindin 2 NM_007088.2 ILMN_1748840 TNFRSF6B 2.18 7.09 tumor necrosis factor receptor NM_003823.2 ILMN_1661825 superfamily, member 6b, decoy TNFRSF6B 2.16 8.81 tumor necrosis factor receptor NM_032945.2 ILMN_2331232 superfamily, member 6b, decoy FAT1 2.04 1.74 FAT tumor suppressor homolog 1 NM_005245.3 ILMN_3247578 (Drosophila) HIST1H2B 1.99 2.92 histone cluster 1, H2bd NM_138720.1 ILMN_1651496 D LEMD1 1.91 3.61 LEM domain containing 1 NM_001001552.3 ILMN_1785444 HIST1H2B 1.87 1.99 histone cluster 1, H2bk NM_080593.1 ILMN_1796179 K CHPF 1.81 2.36 chondroitin polymerizing factor NM_024536.4 ILMN_1731353 COL6A1 1.79 2.12 collagen, type VI, alpha 1 NM_001848.2 ILMN_1732151 CLDND1 1.77 1.44 claudin domain containing 1 NM_001040181.1 ILMN_1710326 FAT1 1.76 1.72 FAT tumor suppressor homolog 1 NM_005245.3 ILMN_1754795 (Drosophila) VPS41 1.72 1.45 vacuolar protein sorting 41 (yeast) NM_014396.2 ILMN_1703379 ADARB1 1.72 1.44 adenosine deaminase, RNA-specific, NM_001112.2 ILMN_1679797 B1 (RED1 homolog rat) SRPX 1.72 1.48 sushi-repeat-containing protein, X- NM_006307.3 ILMN_1709486 linked CRABP2 1.72 1.44 cellular retinoic acid binding protein 2 NM_001878.2 ILMN_1690170 CLDND1 1.71 1.44 claudin domain containing 1 NM_001040181.1 ILMN_2352563 SCARNA16 1.66 1.98 small Cajal body-specific RNA 16 NR_003013.1 ILMN_3237446

580

TSPAN7 1.66 2.01 tetraspanin 7 NM_004615.2 ILMN_2120695 CDKN1A 1.63 2.79 cyclin-dependent kinase inhibitor 1A NM_000389.2 ILMN_1784602 (p21, Cip1) CXADR 1.62 1.94 coxsackie virus and adenovirus NM_001338.3 ILMN_1796925 receptor SKAP1 1.59 2.60 src kinase associated phosphoprotein NM_003726.3 ILMN_1751400 1 ALDH5A1 1.57 1.56 aldehyde dehydrogenase 5 family, NM_001080.3 ILMN_2372403 member A1 RPS23 1.56 1.44 ribosomal protein S23 NM_001025.4 ILMN_1772459 SERPINB1 1.55 2.53 serpin peptidase inhibitor, clade B NM_030666.2 ILMN_1679133 (ovalbumin), member 1 TRNP1 1.55 3.47 TMF1-regulated nuclear protein 1 NM_001013642.2 ILMN_1695946 GRIN1 1.55 1.48 glutamate receptor, ionotropic, N- NM_021569.2 ILMN_1805404 methyl D-aspartate 1 TMEM30A 1.52 1.32 transmembrane protein 30A NM_018247.2 ILMN_1735680 SAT1 1.52 3.23 spermidine/spermine N1- NM_002970.1 ILMN_1753342 acetyltransferase 1 MAP4K1 1.49 1.56 mitogen-activated protein kinase NM_001042600.1 ILMN_1665943 kinase kinase kinase 1 TTC3 1.48 2.19 tetratricopeptide repeat domain 3 NM_003316.3 ILMN_1728605 SEL1L3 1.48 1.72 sel-1 suppressor of lin-12-like 3 (C. NM_015187.3 ILMN_1797822 elegans) ANKS1A 1.47 1.54 ankyrin repeat and sterile alpha motif NM_015245.2 ILMN_1813669 domain containing 1A HEBP1 1.47 1.30 heme binding protein 1 NM_015987.3 ILMN_1802557 AKR1C3 1.47 2.15 aldo-keto reductase family 1, member NM_003739.4 ILMN_1713124 C3 (3-alpha hydroxysteroid dehydrogenase, type II) CYB5A 1.47 1.31 cytochrome b5 type A (microsomal) NM_001914.2 ILMN_1714167 TAP1 1.46 1.74 transporter 1, ATP-binding cassette, NM_000593.5 ILMN_1751079 sub-family B (MDR/TAP) IRF9 1.46 3.94 interferon regulatory factor 9 NM_006084.4 ILMN_1745471 GAA 1.46 1.92 glucosidase, alpha; acid NM_001079804.1 ILMN_2410783 SMYD3 1.46 1.69 SET and MYND domain containing 3 NM_022743.1 ILMN_1741954 XPR1 1.45 1.31 xenotropic and polytropic retrovirus NM_004736.2 ILMN_1798030 receptor MLEC 1.45 1.33 malectin NM_014730.2 ILMN_1657495 ID2 1.44 2.55 inhibitor of DNA binding 2, dominant NM_002166.4 ILMN_2086095 negative helix-loop-helix protein PPP1R14C 1.44 1.55 protein phosphatase 1, regulatory NM_030949.2 ILMN_1664855 (inhibitor) subunit 14C ID2 1.43 2.88 inhibitor of DNA binding 2, dominant NM_002166.4 ILMN_1793990 negative helix-loop-helix protein AKR1A1 1.42 1.30 aldo-keto reductase family 1, member NM_153326.1 ILMN_2380771 A1 (aldehyde reductase) PHLDA1 1.42 1.91 pleckstrin homology-like domain, NM_007350.3 ILMN_3251550

581

family A, member 1 APOBEC3F 1.40 1.37 apolipoprotein B mRNA editing NM_145298.5 ILMN_1710726 enzyme, catalytic polypeptide-like 3F SKAP1 1.39 1.57 src kinase associated phosphoprotein NM_001075099.1 ILMN_2335604 1 HNRNPU 1.38 1.71 heterogeneous nuclear NM_031844.2 ILMN_1743677 ribonucleoprotein U (scaffold attachment factor A) TCFL5 1.38 1.51 transcription factor-like 5 (basic NM_006602.2 ILMN_1814247 helix-loop-helix) TMC6 1.37 2.52 transmembrane channel-like 6 NM_007267.5 ILMN_1794677 PYGL 1.37 1.65 phosphorylase, glycogen, liver NM_002863.3 ILMN_1696187 CKMT1A 1.36 1.48 creatine kinase, mitochondrial 1A NM_001015001.1 ILMN_1732066 TPD52 1.36 1.35 tumor protein D52 NM_005079.2 ILMN_2381064 TSC22D3 1.35 4.43 TSC22 domain family, member 3 NM_198057.2 ILMN_1748124 HIST1H1C 1.35 1.86 histone cluster 1, H1c NM_005319.3 ILMN_1757406 CLDN15 1.35 1.51 claudin 15 NM_014343.1 ILMN_1682226 KDM5B 1.34 2.37 lysine (K)-specific demethylase 5B NM_006618.3 ILMN_1755727 SOX9 1.34 1.78 SRY (sex determining region Y)-box NM_000346.2 ILMN_1805466 9 (campomelic dysplasia, autosomal sex-reversal) PSMB9 1.33 1.36 proteasome (prosome, macropain) NM_002800.4 ILMN_2376108 subunit, beta type, 9 (large multifunctional peptidase 2) TERF2 1.33 1.55 telomeric repeat binding factor 2 NM_005652.2 ILMN_1768488 NDRG1 1.32 4.95 N-myc downstream regulated gene 1 NM_006096.2 ILMN_1809931 SULF2 1.32 1.58 sulfatase 2 NM_018837.2 ILMN_1667460 NEU1 1.32 1.76 sialidase 1 (lysosomal sialidase) NM_000434.2 ILMN_1763144 HIST1H2B 1.32 1.31 histone cluster 1, H2bd NM_138720.1 ILMN_1758623 D FGFR3 1.32 1.48 fibroblast growth factor receptor 3 NM_022965.1 ILMN_1723123 (achondroplasia, thanatophoric dwarfism) NAPRT1 1.32 1.97 nicotinate phosphoribosyltransferase NM_145201.3 ILMN_1710752 domain containing 1 RAC2 1.30 1.62 ras-related C3 botulinum toxin NM_002872.3 ILMN_1709795 substrate 2 (rho family, small GTP binding protein Rac2) ABCG1 1.30 1.64 ATP-binding cassette, sub-family G NM_016818.2 ILMN_1794782 (WHITE), member 1 RPS6KA2 1.30 1.89 ribosomal protein S6 kinase, 90kDa, NM_001006932.1 ILMN_1702501 polypeptide 2

Downregulated in both GGH Inhibition and FPGS Overexpression ZBED2 -3.48 -1.54 zinc finger, BED-type containing 2 NM_024508.3 ILMN_1651365 NCAPD3 -2.07 -1.47 non-SMC condensin II complex, NM_015261.2 ILMN_1683441 subunit D3 TMPO -1.76 -1.90 thymopoietin NM_003276.1 ILMN_1677747

582

CDK6 -1.67 -1.74 cyclin-dependent kinase 6 NM_001259.5 ILMN_1802615 BMP4 -1.67 -1.93 bone morphogenetic protein 4 NM_001202.2 ILMN_1709734 FBXO5 -1.66 -1.92 F-box protein 5 NM_012177.2 ILMN_1710676 KIAA1731 -1.64 -1.58 KIAA1731 NM_033395.1 ILMN_3235104 IGFBP6 -1.64 -1.59 insulin-like growth factor binding NM_002178.2 ILMN_1669362 protein 6 PHF19 -1.62 -1.89 PHD finger protein 19 NM_001009936.1 ILMN_1756676 CKLF -1.62 -1.61 chemokine-like factor NM_001040139.1 ILMN_2414027 KIAA0101 -1.58 -3.19 KIAA0101 NM_014736.4 ILMN_2285996 TOPBP1 -1.58 -1.96 topoisomerase (DNA) II binding NM_007027.2 ILMN_1684929 protein 1 PLEKHB2 -1.57 -1.42 pleckstrin homology domain NM_001031706.1 ILMN_1698323 containing, family B (evectins) member 2 TMEM136 -1.56 -1.49 transmembrane protein 136 NM_174926.1 ILMN_1815346 MCM2 -1.56 -2.08 minichromosome maintenance NM_004526.2 ILMN_1681503 complex component 2 CKLF -1.54 -1.50 chemokine-like factor NM_001040138.1 ILMN_1712389 ZNF680 -1.54 -1.54 zinc finger protein 680 NM_178558.3 ILMN_1730888 BMP4 -1.54 -2.17 bone morphogenetic protein 4 NM_130851.1 ILMN_1740900 DEF8 -1.53 -1.89 differentially expressed in FDCP 8 NM_017702.2 ILMN_1656718 homolog (mouse) RERG -1.49 -1.52 RAS-like, estrogen-regulated, growth NM_032918.1 ILMN_1746359 inhibitor FANCL -1.47 -1.40 Fanconi anemia, complementation NM_018062.2 ILMN_1754045 group L FTSJ2 -1.46 -1.38 FtsJ homolog 2 (E. coli) NM_013393.1 ILMN_1815933 SGOL1 -1.45 -2.25 shugoshin-like 1 (S. pombe) NM_138484.2 ILMN_1730825 ANXA2 -1.44 -2.35 annexin A2 NM_001002857.1 ILMN_1711899 HIST1H4C -1.43 -1.76 histone cluster 1, H4c NM_003542.3 ILMN_2075334 CDC42 -1.43 -1.38 cell division cycle 42 (GTP binding NM_001039802.1 ILMN_1675156 protein, 25kDa) ATAD2 -1.43 -2.22 ATPase family, AAA domain NM_014109.2 ILMN_2048700 containing 2 GMCL1 -1.43 -1.53 germ cell-less homolog 1 NM_178439.3 ILMN_2194627 (Drosophila) KIF5B -1.43 -1.59 kinesin family member 5B NM_004521.1 ILMN_1788160 NGDN -1.43 -1.69 neuroguidin, EIF4E binding protein NM_015514.1 ILMN_1690049 PRKRIR -1.43 -1.40 protein-kinase, interferon-inducible NM_004705.2 ILMN_1655622 double stranded RNA dependent inhibitor, repressor of (P58 repressor) ALG8 -1.41 -1.39 asparagine-linked glycosylation 8, NM_024079.4 ILMN_1685413 alpha-1,3-glucosyltransferase homolog (S. cerevisiae) SDF2L1 -1.41 -1.42 stromal cell-derived factor 2-like 1 NM_022044.2 ILMN_1749213 PAWR -1.41 -1.51 PRKC, apoptosis, WT1, regulator NM_002583.2 ILMN_1806907

583

USP48 -1.39 -1.62 ubiquitin specific peptidase 48 NM_001032730.1 ILMN_1738572 ISY1 -1.38 -1.67 ISY1 splicing factor homolog (S. NM_020701.1 ILMN_1781099 cerevisiae) KISS1R -1.37 -1.39 KISS1 receptor NM_032551.3 ILMN_1673521 RRP7A -1.36 -1.72 ribosomal RNA processing 7 NM_015703.3 ILMN_1688178 homolog A (S. cerevisiae) GABPB2 -1.35 -1.39 GA binding protein transcription NM_016655.3 ILMN_1761147 factor, beta subunit 2 SFMBT1 -1.34 -1.36 Scm-like with four mbt domains 1 NM_016329.2 ILMN_1741585 ALDH1A3 -1.34 -1.93 aldehyde dehydrogenase 1 family, NM_000693.1 ILMN_2139970 member A3 RNU4ATAC -1.34 -1.31 RNA, U4atac small nuclear (U12- NR_023343.1 ILMN_3240594 dependent splicing) THOC4 -1.31 -3.38 THO complex 4 NM_005782.2 ILMN_2364062

Appendix 17. 3. 3 Comparison between GGH overexpression and FPGS inhibition in MDA-MB-435 breast cancer cells

Fold Change (vs. Control) Gene Description Accession Probe_ID Symbol GGH FPGS Overexpression Inhibition

Downregulated in both GGH Overexpression and FPGS Inhibition

APOD -5.41 -1.73 apolipoprotein D NM_001647.2 ILMN_1780170 SPP1 -4.51 -1.66 secreted phosphoprotein 1 NM_001040058.1 ILMN_2374449 SPP1 -4.13 -1.77 secreted phosphoprotein 1 NM_000582.2 ILMN_1651354 CTSL2 -3.78 -2.48 cathepsin L2 NM_001333.2 ILMN_1748352 MCOLN2 -3.58 -1.41 mucolipin 2 NM_153259.2 ILMN_1660462 NBL1 -3.20 -1.53 neuroblastoma, suppression of NM_005380.4 ILMN_2405009 tumorigenicity 1 C7orf52 -2.76 -2.20 chromosome 7 open reading frame 52 NM_198571.1 ILMN_2198859 NBL1 -2.48 -1.83 neuroblastoma, suppression of NM_182744.1 ILMN_1789599 tumorigenicity 1 ADD3 -2.27 -1.62 adducin 3 (gamma) NM_001121.2 ILMN_1814526 AIF1L -2.19 -4.57 allograft inflammatory factor 1-like NM_031426.2 ILMN_3246401 TP53TG3 -2.14 -1.45 TP53 target 3 NM_016212.2 ILMN_2159152 CART1 -2.13 -1.30 cartilage paired-class homeoprotein 1 NM_006982.1 ILMN_1724540 LOC729264 -2.10 -1.47 PREDICTED: similar to TP53TG3 XM_001133677.1 ILMN_1744252 protein, transcript variant 2 VGF -2.05 -1.64 VGF nerve growth factor inducible NM_003378.2 ILMN_1757497 CNBD1 -2.03 -1.50 cyclic nucleotide binding domain NM_173538.2 ILMN_3307813 containing 1 PTGFRN -2.03 -1.51 prostaglandin F2 receptor negative NM_020440.2 ILMN_2077905 regulator

584

LAPTM4B -2.00 -1.53 lysosomal protein transmembrane 4 beta NM_018407.4 ILMN_2101832 PTGFRN -1.97 -1.33 prostaglandin F2 receptor negative NM_020440.2 ILMN_1743130 regulator TMSB15A -1.96 -1.56 thymosin beta 15a NM_021992.2 ILMN_1681737 LAPTM4B -1.91 -1.32 lysosomal protein transmembrane 4 beta NM_018407.4 ILMN_1680196 TMEM51 -1.91 -1.38 transmembrane protein 51 NM_018022.1 ILMN_1674985 AIF1L -1.88 -2.67 allograft inflammatory factor 1-like NM_031426.2 ILMN_1770725 RBPMS2 -1.88 -2.07 RNA binding protein with multiple NM_194272.1 ILMN_1808238 splicing 2 TMEM67 -1.80 -1.32 transmembrane protein 67 NM_153704.3 ILMN_2054053 ACP5 -1.80 -1.30 acid phosphatase 5, tartrate resistant NM_001611.2 ILMN_2078599 ABCA2 -1.73 -1.34 ATP-binding cassette, sub-family A NM_212533.2 ILMN_1747627 (ABC1), member 2 DOCK10 -1.72 -1.55 dedicator of cytokinesis 10 NM_014689.2 ILMN_1702301 AP1S2 -1.71 -1.40 adaptor-related protein complex 1, sigma NM_003916.3 ILMN_1766411 2 subunit DEPDC6 -1.68 -1.55 DEP domain containing 6 NM_022783.1 ILMN_1756685 UGCG -1.63 -1.42 UDP-glucose ceramide NM_003358.1 ILMN_1736939 glucosyltransferase FAHD1 -1.62 -1.89 fumarylacetoacetate hydrolase domain NM_001018104.1 ILMN_1701457 containing 1 SNHG7 -1.61 -1.33 small nucleolar RNA host gene 7 (non- NR_003672.2 ILMN_3227023 protein coding) ZDHHC23 -1.60 -1.41 zinc finger, DHHC-type containing 23 NM_173570.2 ILMN_1736901 SHANK3 -1.54 -1.38 SH3 and multiple ankyrin repeat NM_001080420.1 ILMN_2317581 domains 3 C9orf58 -1.53 -1.65 chromosome 9 open reading frame 58 NM_001002260.1 ILMN_2323508 AP1S2 -1.50 -1.49 adaptor-related protein complex 1, sigma NM_003916.3 ILMN_2120273 2 subunit MGC39900 -1.50 -1.48 hypothetical protein MGC39900 NM_194324.1 ILMN_1731640 POP1 -1.47 -1.35 processing of precursor 1, ribonuclease NM_015029.1 ILMN_1768273 P/MRP subunit (S. cerevisiae) KREMEN2 -1.46 -1.47 kringle containing transmembrane NM_024507.2 ILMN_2382290 protein 2 MAK16 -1.44 -1.42 MAK16 homolog (S. cerevisiae) NM_032509.2 ILMN_1777139 FAHD1 -1.41 -1.62 fumarylacetoacetate hydrolase domain NM_031208.1 ILMN_2246661 containing 1 CCDC50 -1.40 -1.44 coiled-coil domain containing 50 NM_174908.2 ILMN_2302118 CD83 -1.37 -1.35 CD83 molecule NM_001040280.1 ILMN_1780582 TNFAIP3 -1.36 -1.31 tumor necrosis factor, alpha-induced NM_006290.2 ILMN_1702691 protein 3 FNBP1 -1.36 -1.36 formin binding protein 1 NM_015033.2 ILMN_1797342 APTX -1.36 -1.33 aprataxin NM_175069.1 ILMN_2317348 MAPKAP1 -1.36 -1.34 mitogen-activated protein kinase NM_001006618.1 ILMN_2268068 associated protein 1 LAMA5 -1.33 -1.44 laminin, alpha 5 NM_005560.3 ILMN_1773567

585

UQCRC2 -1.31 -1.56 ubiquinol-cytochrome c reductase core NM_003366.2 ILMN_1718853 protein II KIAA0114 -1.31 -1.30 KIAA0114 NR_024031.1 ILMN_3248882 CD83 -1.30 -1.37 CD83 molecule NM_004233.3 ILMN_2328666

Upregulated in both GGH Overexpression and FPGS Inhibition

S100A4 13.13 4.04 S100 calcium binding protein A4 NM_019554.2 ILMN_1684306 S100A4 10.51 3.83 S100 calcium binding protein A4 NM_019554.2 ILMN_1688780 CNN3 5.93 2.19 calponin 3, acidic NM_001839.2 ILMN_1782439 SERPINA3 5.74 1.40 serpin peptidase inhibitor, clade A NM_001085.4 ILMN_1788874 (alpha-1 antiproteinase, antitrypsin), member 3 CPVL 4.11 2.54 carboxypeptidase, vitellogenic-like NM_031311.3 ILMN_2400759 MT1X 4.09 2.09 metallothionein 1X NM_005952.2 ILMN_1775170 MT1E 3.63 1.90 metallothionein 1E NM_175617.3 ILMN_2173611 HAPLN1 3.53 6.06 hyaluronan and proteoglycan link protein NM_001884.2 ILMN_2210519 1 FAM133A 3.50 1.82 family with sequence similarity 133, NM_173698.1 ILMN_1781742 member A CYR61 3.50 1.34 cysteine-rich, angiogenic inducer, 61 NM_001554.3 ILMN_2188264 CA5B 3.48 1.51 carbonic anhydrase VB, mitochondrial NM_007220.3 ILMN_1672807 CPVL 3.46 2.27 carboxypeptidase, vitellogenic-like NM_019029.2 ILMN_1682928 PCOLCE2 3.38 1.31 procollagen C-endopeptidase enhancer 2 NM_013363.2 ILMN_1746888 S100A3 3.35 1.94 S100 calcium binding protein A3 NM_002960.1 ILMN_1712545 HBE1 3.11 1.52 hemoglobin, epsilon 1 NM_005330.3 ILMN_1651358 HAPLN1 3.11 6.12 hyaluronan and proteoglycan link protein NM_001884.2 ILMN_1678812 1 KDELR3 2.99 2.18 KDEL (Lys-Asp-Glu-Leu) endoplasmic NM_006855.2 ILMN_1722820 reticulum protein retention receptor 3 CD96 2.98 1.71 CD96 molecule NM_198196.2 ILMN_1711573 CXorf26 2.97 5.01 chromosome X open reading frame 26 NM_016500.3 ILMN_1768176 KDELR3 2.91 2.43 KDEL (Lys-Asp-Glu-Leu) endoplasmic NM_016657.1 ILMN_1713901 reticulum protein retention receptor 3 FAM46C 2.90 1.77 family with sequence similarity 46, NM_017709.3 ILMN_1713266 member C ATP9A 2.90 1.38 ATPase, class II, type 9A NM_006045.1 ILMN_2089073 MTE 2.85 1.94 metallothionein E NM_175621.2 ILMN_2136089 CTNNBIP1 2.81 1.38 catenin, beta interacting protein 1 NM_020248.2 ILMN_1688103 MT1A 2.74 1.69 metallothionein 1A NM_005946.2 ILMN_1691156 CD96 2.73 1.58 CD96 molecule NM_005816.4 ILMN_2415786 FCRLA 2.67 2.50 Fc receptor-like A NM_032738.3 ILMN_1691071 MT2A 2.64 1.51 metallothionein 2A NM_005953.2 ILMN_1686664 ITGA10 2.62 1.80 integrin, alpha 10 NM_003637.3 ILMN_1700144 GLRX 2.61 1.37 (thioltransferase) NM_002064.1 ILMN_1737308

586

MRGPRX4 2.61 1.40 MAS-related GPR, member X4 NM_054032.2 ILMN_1722227 MT1G 2.54 2.05 metallothionein 1G NM_005950.1 ILMN_1715401 CCL20 2.50 1.77 chemokine (C-C motif) ligand 20 NM_004591.1 ILMN_1657234 CLIC4 2.42 1.51 chloride intracellular channel 4 NM_013943.1 ILMN_2063584 S100A16 2.33 1.33 S100 calcium binding protein A16 NM_080388.1 ILMN_1728049 KDELR3 2.25 2.06 KDEL (Lys-Asp-Glu-Leu) endoplasmic NM_016657.1 ILMN_1798952 reticulum protein retention receptor 3 ATP1B1 2.25 1.65 ATPase, Na+/K+ transporting, beta 1 NM_001677.3 ILMN_1658071 polypeptide DACT3 2.24 1.33 dapper, antagonist of beta-catenin, NM_145056.1 ILMN_1733851 homolog 3 (Xenopus laevis) CLIC4 2.24 1.50 chloride intracellular channel 4 NM_013943.1 ILMN_2063586 SPRY1 2.16 1.85 sprouty homolog 1, antagonist of FGF NM_005841.1 ILMN_2329914 signaling (Drosophila) SH3BGRL3 2.16 1.32 SH3 domain binding glutamic acid-rich NM_031286.3 ILMN_1737163 protein like 3 TUBB2B 2.16 1.34 tubulin, beta 2B NM_178012.3 ILMN_1680874 CLIC4 2.11 1.58 chloride intracellular channel 4 NM_013943.1 ILMN_1671250 NUCB2 2.07 1.43 nucleobindin 2 NM_005013.2 ILMN_1655913 HIF1A 2.03 1.83 hypoxia-inducible factor 1, alpha subunit NM_181054.1 ILMN_2379788 (basic helix-loop-helix transcription factor) ARHGAP15 2.02 1.52 Rho GTPase activating protein 15 NM_018460.2 ILMN_2208413 KLRC3 2.01 1.35 killer cell lectin-like receptor subfamily NM_002261.2 ILMN_2386790 C, member 3 HBG2 1.99 1.76 hemoglobin, gamma G NM_000184.2 ILMN_2084825 ATP1B1 1.99 1.91 ATPase, Na+/K+ transporting, beta 1 NM_001001787.1 ILMN_2407824 polypeptide IL1RAPL1 1.99 3.41 interleukin 1 receptor accessory protein- NM_014271.2 ILMN_2160428 like 1 HBG1 1.96 1.87 hemoglobin, gamma A NM_000559.2 ILMN_1796678 RFTN1 1.96 1.35 raftlin, lipid raft linker 1 NM_015150.1 ILMN_1800787 CCDC109B 1.94 1.34 coiled-coil domain containing 109B NM_017918.3 ILMN_1801766 BFSP1 1.94 1.71 beaded filament structural protein 1, NM_001195.2 ILMN_1712608 filensin LDHA 1.87 1.43 lactate dehydrogenase A NM_005566.3 ILMN_3251145 BBS2 1.86 1.31 Bardet-Biedl syndrome 2 NM_031885.2 ILMN_1767612 ZHX2 1.81 1.31 zinc fingers and homeoboxes 2 NM_014943.3 ILMN_2184966 TM4SF1 1.81 1.35 transmembrane 4 L six family member 1 NM_014220.2 ILMN_1770338 SPRY1 1.80 1.77 sprouty homolog 1, antagonist of FGF NM_199327.1 ILMN_1691860 signaling (Drosophila) VAMP5 1.80 1.40 vesicle-associated membrane protein 5 NM_006634.2 ILMN_1809467 (myobrevin) CDKN2D 1.78 1.31 cyclin-dependent kinase inhibitor 2D NM_079421.2 ILMN_1748883 (p19, inhibits CDK4) SRPX2 1.77 1.76 sushi-repeat-containing protein, X-linked NM_014467.2 ILMN_1676213

587

2 MORC4 1.77 1.38 MORC family CW-type zinc finger 4 NM_024657.2 ILMN_1795463 COTL1 1.75 1.65 coactosin-like 1 (Dictyostelium) NM_021149.2 ILMN_1788283 ITIH5L 1.74 1.51 inter-alpha (globulin) inhibitor H5-like NM_198510.1 ILMN_1709177 ABCB9 1.73 1.71 ATP-binding cassette, sub-family B NM_019624.2 ILMN_2343048 (MDR/TAP), member 9 HIF1A 1.73 1.77 hypoxia-inducible factor 1, alpha subunit NM_001530.2 ILMN_1681283 (basic helix-loop-helix transcription factor) HIF1A 1.71 1.84 hypoxia-inducible factor 1, alpha subunit NM_001530.2 ILMN_1763260 (basic helix-loop-helix transcription factor) LEPREL1 1.66 1.82 leprecan-like 1 NM_018192.2 ILMN_1657373 DDAH1 1.64 1.46 dimethylarginine NM_012137.2 ILMN_1668507 dimethylaminohydrolase 1 OPN3 1.62 1.31 opsin 3 NM_014322.2 ILMN_1716988 WBSCR27 1.60 1.36 Williams Beuren syndrome chromosome NM_152559.2 ILMN_1719170 region 27 LOXL3 1.59 1.67 lysyl oxidase-like 3 NM_032603.2 ILMN_1733515 ABCB9 1.57 1.49 ATP-binding cassette, sub-family B NM_019624.2 ILMN_2343047 (MDR/TAP), member 9 SSBP2 1.54 1.61 single-stranded DNA binding protein 2 NM_012446.2 ILMN_1711608 CEACAM1 1.53 1.92 carcinoembryonic antigen-related cell NM_001024912.1 ILMN_1716815 adhesion molecule 1 (biliary glycoprotein) KRCC1 1.45 1.31 lysine-rich coiled-coil 1 NM_016618.1 ILMN_1745620 NT5E 1.44 1.53 5'-nucleotidase, ecto (CD73) NM_002526.1 ILMN_1697220 P4HA2 1.44 1.54 prolyl 4-hydroxylase, alpha polypeptide NM_001017973.1 ILMN_1795778 II SPG3A 1.44 1.47 spastic paraplegia 3A (autosomal NM_181598.2 ILMN_2381476 dominant) ZNHIT1 1.44 1.30 zinc finger, HIT type 1 NM_006349.2 ILMN_1741491 FAM62B 1.43 1.55 family with sequence similarity 62 (C2 NM_020728.1 ILMN_2057573 domain containing) member B ELMO1 1.42 1.35 engulfment and cell motility 1 NM_014800.9 ILMN_1784320 INSIG2 1.42 1.38 insulin induced gene 2 NM_016133.2 ILMN_1676629 PSMA1 1.41 1.30 proteasome (prosome, macropain) NM_002786.2 ILMN_1691809 subunit, alpha type, 1 C10orf33 1.41 1.79 chromosome 10 open reading frame 33 NM_032709.1 ILMN_1684497 LOC151162 1.40 1.51 hypothetical LOC151162 NR_024275.1 ILMN_3238058 TFPI 1.40 1.38 tissue factor pathway inhibitor NM_006287.4 ILMN_1707124 (lipoprotein-associated coagulation inhibitor) HNRNPU 1.39 1.43 heterogeneous nuclear ribonucleoprotein NM_004501.3 ILMN_2370135 U (scaffold attachment factor A) ACYP2 1.39 1.38 acylphosphatase 2, muscle type NM_138448.2 ILMN_2158705 GPC6 1.37 1.90 glypican 6 NM_005708.2 ILMN_1805216

588

TMEM106 1.36 1.66 transmembrane protein 106C NM_024056.2 ILMN_1692511 C CREB3L2 1.33 1.72 cAMP responsive element binding NM_194071.2 ILMN_1751097 protein 3-like 2 SRI 1.32 1.49 sorcin NM_198901.1 ILMN_1699525 ROPN1L 1.31 1.37 ropporin 1-like NM_031916.2 ILMN_1696466 CETN2 1.30 1.37 centrin, EF-hand protein, 2 NM_004344.1 ILMN_1695645

Appendix 17. 3. 4 Comparison between GGH inhibition and FPGS overexpression in MDA-MB-435 breast cancer cells

Fold Change (vs. Control) Gene Description Accession Probe_ID Symbol GGH FPGS Inhibition Overexpression

Upregulated in both GGH Inhibition and FPGS Overexpression

CXorf26 10.42 2.37 chromosome X open reading frame 26 NM_016500.3 ILMN_1768176 CCL20 4.88 1.75 chemokine (C-C motif) ligand 20 NM_004591.1 ILMN_1657234 PRSS7 4.66 1.76 protease, serine, 7 (enterokinase) NM_002772.1 ILMN_1695969 PRSS7 4.41 1.70 protease, serine, 7 (enterokinase) NM_002772.1 ILMN_2220845 IL8 4.15 4.57 interleukin 8 NM_000584.2 ILMN_2184373 LAIR2 3.57 5.18 leukocyte-associated immunoglobulin- NM_021270.2 ILMN_2323933 like receptor 2 FAM133A 3.45 4.61 family with sequence similarity 133, NM_173698.1 ILMN_1781742 member A LAIR2 3.43 5.50 leukocyte-associated immunoglobulin- NM_002288.3 ILMN_1807491 like receptor 2 IL8 3.35 2.52 interleukin 8 NM_000584.2 ILMN_1666733 RFTN1 3.11 1.45 raftlin, lipid raft linker 1 NM_015150.1 ILMN_1800787 SPRR2E 3.00 1.80 small proline-rich protein 2E NM_001024209.2 ILMN_2211018 MMP1 2.69 2.27 matrix metallopeptidase 1 (interstitial NM_002421.2 ILMN_1726448 collagenase) SPRR2D 2.66 6.26 small proline-rich protein 2D NM_006945.3 ILMN_2191967 ID1 2.51 1.40 inhibitor of DNA binding 1, dominant NM_181353.1 ILMN_1664861 negative helix-loop-helix protein CASP1 2.38 2.22 caspase 1, apoptosis-related cysteine NM_033294.2 ILMN_2326509 peptidase (interleukin 1, beta, convertase) DYNLT3 2.34 2.64 dynein, light chain, Tctex-type 3 NM_006520.1 ILMN_1681890 GBP1 2.30 1.37 guanylate binding protein 1, interferon- NM_002053.1 ILMN_2148785 inducible, 67kDa ID2 2.24 1.49 inhibitor of DNA binding 2, dominant NM_002166.4 ILMN_2086095 negative helix-loop-helix protein NCRNA00161 2.21 3.81 non-protein coding RNA 161 NR_026553.1 ILMN_3297789

589

HBEGF 2.21 1.60 heparin-binding EGF-like growth NM_001945.1 ILMN_2121408 factor (HBEGF) PLCXD3 2.12 2.11 phosphatidylinositol-specific NM_001005473.1 ILMN_1798841 phospholipase C, X domain containing 3 EMP1 2.09 2.26 epithelial membrane protein 1 NM_001423.1 ILMN_1801616 AKR1C3 2.06 2.07 aldo-keto reductase family 1, member NM_003739.4 ILMN_1713124 C3 (3-alpha hydroxysteroid dehydrogenase, type II) GNG11 2.06 2.82 guanine nucleotide binding protein (G NM_004126.3 ILMN_1782419 protein), gamma 11 PNLIPRP3 2.05 1.51 pancreatic lipase-related protein 3 NM_001011709.1 ILMN_1678655 SPRR2F 2.04 4.27 small proline-rich protein 2F NM_001014450.1 ILMN_1674367 MT1X 2.01 5.00 metallothionein 1X NM_005952.2 ILMN_1775170 SMAGP 1.96 2.06 small cell adhesion glycoprotein NM_001033873.1 ILMN_1804415 MT1G 1.94 3.18 metallothionein 1G NM_005950.1 ILMN_1715401 APCDD1L 1.93 3.94 adenomatosis polyposis coli down- NM_153360.1 ILMN_1689431 regulated 1-like CASP1 1.90 1.67 caspase 1, apoptosis-related cysteine NM_033294.2 ILMN_2326512 peptidase (interleukin 1, beta, convertase) ERRFI1 1.89 3.15 ERBB receptor feedback inhibitor 1 NM_018948.2 ILMN_1665510 C17orf91 1.89 1.60 chromosome 17 open reading frame 91 NM_001001870.1 ILMN_2390310 C1orf94 1.87 2.74 chromosome 1 open reading frame 94 NM_032884.2 ILMN_1793263 CALB2 1.86 1.49 calbindin 2 NM_007088.2 ILMN_1748840 S100A16 1.85 3.25 S100 calcium binding protein A16 NM_080388.1 ILMN_1728049 MTE 1.84 3.14 metallothionein E NM_175621.2 ILMN_2136089 C21orf100 1.84 3.15 PREDICTED: chromosome 21 open XR_017892.1 ILMN_1667790 reading frame 100 SGK1 1.84 1.46 serum/glucocorticoid regulated kinase NM_005627.3 ILMN_3305938 1 MRGPRX4 1.83 2.53 MAS-related GPR, member X4 NM_054032.2 ILMN_1722227 DUSP10 1.83 1.30 dual specificity phosphatase 10 NM_144729.1 ILMN_2401878 CD68 1.82 1.54 CD68 antigen NM_001251.1 ILMN_1714861 LDHA 1.81 1.41 lactate dehydrogenase A NM_005566.3 ILMN_3251145 KIAA0367 1.81 1.53 KIAA0367 NM_015225.1 ILMN_1810628 ITPRIP 1.80 3.18 inositol 1,4,5-triphosphate receptor NM_033397.2 ILMN_1805192 interacting protein NT5E 1.80 1.89 5'-nucleotidase, ecto (CD73) NM_002526.1 ILMN_1697220 STC1 1.78 1.68 stanniocalcin 1 NM_003155.2 ILMN_1758164 CYR61 1.77 5.62 cysteine-rich, angiogenic inducer, 61 NM_001554.3 ILMN_2188264 CPVL 1.75 1.35 carboxypeptidase, vitellogenic-like NM_019029.2 ILMN_1682928 DUSP5 1.74 1.76 dual specificity phosphatase 5 NM_004419.3 ILMN_1656501 SAA2 1.72 1.75 serum amyloid A2 NM_030754.2 ILMN_1728262 SGK 1.72 1.59 serum/glucocorticoid regulated kinase NM_005627.2 ILMN_1702487

590

FAIM 1.72 1.38 Fas apoptotic inhibitory molecule NM_001033032.1 ILMN_2351548 SGK1 1.72 1.63 serum/glucocorticoid regulated kinase NM_005627.3 ILMN_3229324 1 MT1E 1.72 5.62 metallothionein 1E NM_175617.3 ILMN_2173611 FRMD4A 1.71 1.53 FERM domain containing 4A NM_018027.3 ILMN_1678961 FHOD3 1.71 2.02 formin homology 2 domain containing NM_025135.2 ILMN_1761322 3 CPVL 1.68 1.67 carboxypeptidase, vitellogenic-like NM_031311.3 ILMN_2400759 ITPRIP 1.68 2.44 inositol 1,4,5-triphosphate receptor NM_033397.2 ILMN_3239181 interacting protein ZYX 1.67 1.66 zyxin NM_003461.4 ILMN_1701875 FGFRL1 1.67 3.82 fibroblast growth factor receptor-like 1 NM_021923.3 ILMN_1795865 LDLR 1.66 1.94 low density lipoprotein receptor NM_000527.2 ILMN_2053415 (familial hypercholesterolemia) PCLO 1.66 1.45 piccolo (presynaptic cytomatrix NM_033026.5 ILMN_3230160 protein) CCND1 1.65 2.26 cyclin D1 NM_053056.2 ILMN_1688480 FHL2 1.64 1.46 four and a half LIM domains 2 NM_201557.2 ILMN_1668411 MX1 1.62 3.13 myxovirus (influenza virus) resistance NM_002462.2 ILMN_1662358 1, interferon-inducible protein p78 (mouse) CDC42EP2 1.62 1.36 CDC42 effector protein (Rho GTPase NM_006779.2 ILMN_1652777 binding) 2 MT1A 1.61 3.10 metallothionein 1A NM_005946.2 ILMN_1691156 KLF4 1.61 3.56 Kruppel-like factor 4 (gut) NM_004235.3 ILMN_2137789 TMEM158 1.59 2.30 transmembrane protein 158 NM_015444.2 ILMN_1792455 IFI6 1.58 1.80 interferon, alpha-inducible protein 6 NM_022872.2 ILMN_2347798 DYNLT3 1.57 1.58 dynein, light chain, Tctex-type 3 NM_006520.1 ILMN_2162564 C1orf133 1.56 1.48 chromosome 1 open reading frame 133 NR_024337.1 ILMN_3241665 CLCF1 1.56 1.60 cardiotrophin-like cytokine factor 1 NM_013246.2 ILMN_1661197 FGFRL1 1.55 1.62 fibroblast growth factor receptor-like 1 NM_001004358.1 ILMN_2348367 LRRCC1 1.53 1.80 leucine rich repeat and coiled-coil NM_033402.3 ILMN_2384807 domain containing 1 ABHD2 1.53 1.73 abhydrolase domain containing 2 NM_152924.3 ILMN_2403446 SAMD9 1.52 1.38 sterile alpha motif domain containing 9 NM_017654.2 ILMN_1814305 PHLDA2 1.52 1.68 pleckstrin homology-like domain, NM_003311.3 ILMN_1671557 family A, member 2 PALM2 1.52 1.65 paralemmin 2 NM_001037293.1 ILMN_2369403 PPAP2B 1.50 1.30 phosphatidic acid phosphatase type 2B NM_003713.3 ILMN_2388800 C10orf32 1.50 1.52 chromosome 10 open reading frame 32 NM_144591.1 ILMN_2151056 WBSCR27 1.49 1.94 Williams Beuren syndrome NM_152559.2 ILMN_1719170 chromosome region 27 ITGA5 1.49 1.77 integrin, alpha 5 (fibronectin receptor, NM_002205.2 ILMN_1792679 alpha polypeptide) MLLT11 1.49 1.31 myeloid/lymphoid or mixed-lineage NM_006818.3 ILMN_1759097 leukemia (trithorax homolog,

591

Drosophila); translocated to, 11 FJX1 1.48 1.39 four jointed box 1 (Drosophila) NM_014344.2 ILMN_1746465 KYNU 1.48 2.68 kynureninase (L-kynurenine hydrolase) NM_001032998.1 ILMN_1737514 FAM20C 1.48 3.36 family with sequence similarity 20, NM_020223.2 ILMN_1712684 member C BCL2L1 1.48 1.39 BCL2-like 1 NM_138578.1 ILMN_1654118 CASP4 1.47 1.50 caspase 4, apoptosis-related cysteine NM_001225.3 ILMN_1678454 peptidase C21orf7 1.47 2.25 chromosome 21 open reading frame 7 NM_020152.2 ILMN_1699071 TIMP3 1.46 1.90 TIMP metallopeptidase inhibitor 3 NM_000362.4 ILMN_1701461 ALKBH8 1.46 1.43 alkB, alkylation repair homolog 8 (E. NM_138775.1 ILMN_1779190 coli) TGFB1I1 1.46 2.00 transforming growth factor beta 1 NM_015927.3 ILMN_2389876 induced transcript 1 HBE1 1.45 2.26 hemoglobin, epsilon 1 NM_005330.3 ILMN_1651358 UBQLN1 1.45 1.38 ubiquilin 1 NM_053067.1 ILMN_2351611 ABHD2 1.45 1.83 abhydrolase domain containing 2 NM_152924.3 ILMN_1723662 FAIM 1.44 1.48 Fas apoptotic inhibitory molecule NM_001033030.1 ILMN_1690101 LARP6 1.44 3.14 La ribonucleoprotein domain family, NM_197958.1 ILMN_1663401 member 6 FOSB 1.43 1.82 FBJ murine osteosarcoma viral NM_006732.1 ILMN_1751607 oncogene homolog B NCRNA00161 1.42 2.07 non-protein coding RNA 161 NR_026552.1 ILMN_3230631 ZCCHC6 1.42 1.85 zinc finger, CCHC domain containing NM_024617.2 ILMN_1779677 6 IFT20 1.42 1.33 intraflagellar transport 20 homolog NM_174887.2 ILMN_3235216 (Chlamydomonas) FCRLA 1.41 1.76 Fc receptor-like A NM_032738.3 ILMN_1691071 FRMD3 1.41 1.87 FERM domain containing 3 NM_174938.3 ILMN_1698725 RASSF1 1.41 1.54 Ras association (RalGDS/AF-6) NM_170712.1 ILMN_1734205 domain family 1 CLDN14 1.41 1.39 claudin 14 NM_012130.2 ILMN_1661194 C18orf19 1.40 1.57 chromosome 18 open reading frame 19 NM_152352.1 ILMN_1658110 BCL2A1 1.40 1.84 BCL2-related protein A1 NM_004049.2 ILMN_1769229 HBG1 1.40 1.36 hemoglobin, gamma A NM_000559.2 ILMN_1796678 DUS3L 1.40 1.48 dihydrouridine synthase 3-like (S. NM_020175.1 ILMN_1723235 cerevisiae) VWA5A 1.40 2.37 von Willebrand factor A domain NM_014622.4 ILMN_1764769 containing 5A SP100 1.40 1.66 SP100 nuclear antigen NM_001080391.1 ILMN_2390586 TFPI 1.39 2.25 tissue factor pathway inhibitor NM_001032281.2 ILMN_1701032 (lipoprotein-associated coagulation inhibitor) IFI6 1.39 1.39 interferon, alpha-inducible protein 6 NM_022873.2 ILMN_1687384 MT2A 1.39 3.55 metallothionein 2A NM_005953.2 ILMN_1686664 AEBP1 1.38 1.48 AE binding protein 1 NM_001129.3 ILMN_1736178

592

ZNF35 1.38 1.79 zinc finger protein 35 NM_003420.3 ILMN_1692100 DKK1 1.38 2.28 dickkopf homolog 1 (Xenopus laevis) NM_012242.2 ILMN_1773337 SLITRK6 1.38 1.38 SLIT and NTRK-like family, member NM_032229.2 ILMN_1813197 6 DDAH1 1.38 1.74 dimethylarginine NM_012137.2 ILMN_1668507 dimethylaminohydrolase 1 CDKN2B 1.38 1.60 cyclin-dependent kinase inhibitor 2B NM_078487.2 ILMN_2376723 (p15, inhibits CDK4) PLAUR 1.37 1.46 plasminogen activator, urokinase NM_001005376.1 ILMN_2374340 receptor AKAP12 1.37 1.50 A kinase (PRKA) anchor protein NM_144497.1 ILMN_1684836 (gravin) 12 KYNU 1.36 3.37 kynureninase (L-kynurenine hydrolase) NM_003937.2 ILMN_1746517 CYP4F11 1.35 1.35 cytochrome P450, family 4, subfamily NM_021187.2 ILMN_1719883 F, polypeptide 11 AKAP12 1.35 1.66 A kinase (PRKA) anchor protein NM_005100.2 ILMN_2308950 (gravin) 12 ZNFX1 1.35 1.67 zinc finger, NFX1-type containing 1 NM_021035.2 ILMN_1745148 SCARF2 1.35 1.82 scavenger receptor class F, member 2 NM_153334.3 ILMN_1655405 DEFB103A 1.33 1.52 defensin, beta 103A NM_018661.3 ILMN_2132515 C6orf150 1.32 1.45 chromosome 6 open reading frame 150 NM_138441.2 ILMN_1706645 CATSPER1 1.32 4.12 cation channel, sperm associated 1 NM_053054.2 ILMN_1789394 SOD2 1.32 2.10 superoxide dismutase 2, mitochondrial NM_001024465.1 ILMN_2336781 ZBTB44 1.32 1.51 zinc finger and BTB domain containing NM_014155.3 ILMN_2080637 44 KLF4 1.31 2.38 Kruppel-like factor 4 (gut) NM_004235.3 ILMN_1779857 PPP1R15A 1.31 2.70 protein phosphatase 1, regulatory NM_014330.2 ILMN_1659936 (inhibitor) subunit 15A OAF 1.30 1.91 OAF homolog (Drosophila) NM_178507.2 ILMN_1668345 MRGPRX3 1.30 1.73 MAS-related GPR, member X3 NM_054031.2 ILMN_1773546 CLIC4 1.30 3.09 chloride intracellular channel 4 NM_013943.1 ILMN_1671250

Downregulated in both GGH Inhibition and FPGS Overexpression

GGH -2.49 -1.61 gamma-glutamyl hydrolase (conjugase, NM_003878.1 ILMN_1681754 folylpolygammaglutamyl hydrolase) AIF1L -2.10 -3.41 allograft inflammatory factor 1-like NM_031426.2 ILMN_3246401 CTSL2 -2.04 -2.20 cathepsin L2 NM_001333.2 ILMN_1748352 LRRC20 -1.81 -1.30 leucine rich repeat containing 20 NM_018239.2 ILMN_1690523 PDGFRL -1.76 -1.57 platelet-derived growth factor receptor-like NM_006207.1 ILMN_1680339 NBL1 -1.75 -2.17 neuroblastoma, suppression of NM_182744.1 ILMN_1789599 tumorigenicity 1 MYH10 -1.73 -1.94 myosin, heavy chain 10, non-muscle NM_005964.1 ILMN_1815154 PCOLCE -1.69 -1.36 procollagen C-endopeptidase enhancer NM_002593.2 ILMN_1707070 MITF -1.68 -3.76 microphthalmia-associated transcription NM_198158.1 ILMN_2304186 factor

593

AIF1L -1.68 -2.28 allograft inflammatory factor 1-like NM_031426.2 ILMN_1770725 RNF150 -1.67 -1.33 ring finger protein 150 NM_020724.1 ILMN_1767446 ST8SIA1 -1.65 -1.65 ST8 alpha-N-acetyl-neuraminide alpha-2,8- NM_003034.2 ILMN_2048011 sialyltransferase 1 PRMT2 -1.64 -1.58 protein arginine methyltransferase 2 NM_206962.1 ILMN_2259119 CXXC5 -1.64 -2.54 CXXC finger 5 NM_016463.7 ILMN_3307729 MCOLN2 -1.64 -1.56 mucolipin 2 NM_153259.2 ILMN_1660462 LYST -1.62 -1.46 lysosomal trafficking regulator NM_000081.2 ILMN_1675956 TGFBR3 -1.62 -1.58 transforming growth factor, beta receptor NM_003243.2 ILMN_1784287 III RBPMS2 -1.61 -1.69 RNA binding protein with multiple splicing NM_194272.1 ILMN_1808238 2 LPAR1 -1.58 -1.40 lysophosphatidic acid receptor 1 NM_057159.2 ILMN_1701441 TFAP2A -1.57 -2.32 transcription factor AP-2 alpha (activating NM_001042425.1 ILMN_1765574 enhancer binding protein 2 alpha) PAQR8 -1.57 -1.43 progestin and adipoQ receptor family NM_133367.3 ILMN_1694589 member VIII TP53TG3 -1.57 -1.46 TP53 target 3 NM_016212.2 ILMN_2159152 RFX7 -1.52 -1.43 regulatory factor X, 7 NM_022841.5 ILMN_3245625 ZFP106 -1.52 -1.63 zinc finger protein 106 homolog (mouse) NM_022473.1 ILMN_2043265 DEPDC6 -1.51 -1.72 DEP domain containing 6 NM_022783.1 ILMN_1756685 GYG2 -1.50 -4.24 glycogenin 2 NM_003918.2 ILMN_2319424 ST8SIA1 -1.50 -1.67 ST8 alpha-N-acetyl-neuraminide alpha-2,8- NM_003034.2 ILMN_1664859 sialyltransferase 1 LOC729264 -1.48 -1.54 PREDICTED: similar to TP53TG3 protein, XM_001133677.1 ILMN_1744252 transcript variant 2 FARS2 -1.47 -1.44 phenylalanyl-tRNA synthetase 2, NM_006567.3 ILMN_1793732 mitochondrial RHOBTB3 -1.47 -1.30 Rho-related BTB domain containing 3 NM_014899.3 ILMN_1744949 HNRNPU -1.47 -1.51 heterogeneous nuclear ribonucleoprotein U NM_004501.3 ILMN_2370135 (scaffold attachment factor A) ADD3 -1.47 -2.09 adducin 3 (gamma) NM_001121.2 ILMN_1814526 PTGFRN -1.46 -2.25 prostaglandin F2 receptor negative NM_020440.2 ILMN_2077905 regulator RPL17 -1.45 -1.31 ribosomal protein L17 NM_000985.2 ILMN_1655422 PTGFRN -1.45 -2.20 prostaglandin F2 receptor negative NM_020440.2 ILMN_1743130 regulator PLP1 -1.42 -1.70 proteolipid protein 1 NM_199478.1 ILMN_2414135 SGCD -1.42 -1.63 sarcoglycan, delta (35kDa dystrophin- NM_000337.4 ILMN_1763457 associated glycoprotein) LAMP2 -1.41 -1.43 lysosomal-associated membrane protein 2 NM_002294.1 ILMN_1673282 TDRD7 -1.41 -2.04 tudor domain containing 7 NM_014290.1 ILMN_1705241 NBL1 -1.40 -2.94 neuroblastoma, suppression of NM_005380.4 ILMN_2405009 tumorigenicity 1 ERP29 -1.40 -1.50 endoplasmic reticulum protein 29 NM_006817.3 ILMN_2251184

594

TMED10 -1.40 -1.76 transmembrane emp24-like trafficking NM_006827.5 ILMN_1736585 protein 10 (yeast) TAPBP -1.40 -1.39 TAP binding protein (tapasin) NM_003190.3 ILMN_1742450 SIPA1L2 -1.39 -2.38 signal-induced proliferation-associated 1 NM_020808.3 ILMN_1732923 like 2 C4orf18 -1.39 -4.49 chromosome 4 open reading frame 18 NM_016613.5 ILMN_1761941 TFAP2A -1.38 -2.74 transcription factor AP-2 alpha (activating NM_001032280.2 ILMN_2374115 enhancer binding protein 2 alpha) FAM53B -1.38 -1.43 family with sequence similarity 53, member NM_014661.3 ILMN_1704571 B ZNF318 -1.38 -1.31 zinc finger protein 318 NM_014345.1 ILMN_1792305 MYO1B -1.37 -1.76 myosin IB NM_012223.2 ILMN_2093027 GPNMB -1.37 -1.95 glycoprotein (transmembrane) nmb NM_002510.2 ILMN_2407389 SGK3 -1.37 -1.33 serum/glucocorticoid regulated kinase NM_001033578.1 ILMN_1747020 family, member 3 PLEKHH1 -1.36 -2.79 pleckstrin homology domain containing, NM_020715.2 ILMN_1699254 family H (with MyTH4 domain) member 1 ITIH5L -1.36 -1.44 inter-alpha (globulin) inhibitor H5-like NM_198510.1 ILMN_1709177 NIN -1.36 -1.79 ninein (GSK3B interacting protein) NM_020921.3 ILMN_1724753 ICK -1.36 -1.52 intestinal cell (MAK-like) kinase NM_016513.3 ILMN_1709882 HOXA5 -1.35 -1.44 homeobox A5 NM_019102.2 ILMN_1753613 PEG10 -1.35 -1.52 paternally expressed 10 NM_001040152.1 ILMN_2297626 QDPR -1.35 -1.51 quinoid dihydropteridine reductase NM_000320.1 ILMN_1672443 RAB6B -1.35 -2.23 RAB6B, member RAS oncogene family NM_016577.3 ILMN_1752299 FNBP1 -1.35 -1.46 formin binding protein 1 NM_015033.2 ILMN_1797342 RAB11FIP1 -1.34 -1.73 RAB11 family interacting protein 1 (class I) NM_001002814.1 ILMN_1692219 CGGBP1 -1.34 -1.68 CGG triplet repeat binding protein 1 NM_001008390.1 ILMN_1752631 KIAA1598 -1.34 -1.76 KIAA1598 NM_018330.4 ILMN_1805992 CRTAP -1.32 -1.66 cartilage associated protein NM_006371.3 ILMN_1720484 SLC27A5 -1.32 -1.60 solute carrier family 27 (fatty acid NM_012254.1 ILMN_1725366 transporter), member 5 C7orf52 -1.31 -1.52 chromosome 7 open reading frame 52 NM_198571.1 ILMN_2198859 SHANK3 -1.31 -1.37 SH3 and multiple ankyrin repeat domains 3 NM_001080420.1 ILMN_2317581 C9orf58 -1.31 -1.77 chromosome 9 open reading frame 58 NM_001002260.1 ILMN_2323508 ACSL3 -1.31 -1.65 acyl-CoA synthetase long-chain family NM_004457.3 ILMN_1666096 member 3 MGC39900 -1.31 -1.34 hypothetical protein MGC39900 NM_194324.1 ILMN_1731640 CRTAP -1.30 -1.75 cartilage associated protein NM_006371.3 ILMN_1665235 SLC39A10 -1.30 -1.66 solute carrier family 39 (zinc transporter), NM_020342.1 ILMN_1656129 member 10