CROSSTALK BETWEEN INSULIN AND WNT SIGNALING PATHWAYS

by

JANE SUN

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology University of Toronto

©Copyright by Jane Sun 2009 CROSSTALK BETWEEN INSULIN AND WNT SIGNALING PATHWAYS

Jane Sun

Doctor of Philosophy, 2009 Department of Laboratory Medicine and Pathobiology Faculty of Medicine University of Toronto

Abstract

Type II diabetes and hyperinsulinemia are associated with increased risks of developing colorectal (CRC). Detailed mechanisms underlying this correlation, however, are yet to be explored. The present study demonstrates that insulin increases the expression of proto- c- and cyclin D1 via both translational and transcriptional mechanisms. We show here that insulin stimulates c-Myc translation via an Akt/PKB-dependent mechanism involving the mTOR signaling pathway. More importantly, we show for the first time that transcriptional stimulation of c-Myc and cyclin D1 expression by insulin involves a novel Akt/PKB- independent signal crosstalk between insulin and canonical Wnt signaling pathways.

We then identified p21-activated kianse 1 (PAK-1) as a novel mediator for insulin and Wnt/beta- (-cat) molecular crosstalk, involving MEK/ERK signaling. Furthermore, we found that insulin treatment leads to increased -cat phosphorylation at Ser675, and this is associated with increased -cat nuclear content and increased -cat interaction with Tcf/Lef-binding elements (TBEs) of the c-Myc gene promoter. Lastly, we demonstrated that insulin signaling directly

ii alters the expression levels of components of the Wnt signaling pathway, including fizzled homology 4 (Fdz-4) and TCF7L2 (=TCF-4). This study not only demonstrated the existence of signaling crosstalk between insulin and canonical

Wnt signaling pathways at multiple levels, it reveals molecular mechanisms for observed correlation between CRC and hyperinsulinemia. The growing evidence implicating PAK-1 in various human tumorigenesis has emerged PAK-1 as a potential therapeutic target. Our discovery of PAK-1 functioning as a novel central mediator for insulin and Wnt signaling crosstalk in intestinal cells suggests that PAK-

1 may potentially be a good target candidate for treating patients with CRC, especially those who have Type II diabetes or experience hyperinsulinemia.

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Acknowledgements

I would like to thank my supervisor Dr. Tianru Jin for his continuous support and guidance throughout this project. I would also like to extend my gratitude toward members of my Supervisory Committee, Dr. Donald Branch, Dr. Theodore

Brown and Dr. I. George Fantus, whose enthusiasm and constant encouragement made the completion of this research project an enjoyable experience. Thanks to all the past and present members of the Jin lab who helped me overcome the technical challenges during my study. To all the wonderful friends I have made on the 10th floor of TMDT Building and 4th floor of CBS Building, I want to thank you all for making my research experience memorable. I want to give special thanks to my parents, who supported me unconditionally, kept me out of trouble and taught me how to treasure and appreciate life. Last but not least, I want to thank my husband- to-be, whose patience and understanding surpassed any expectation imaginable. I thank him for being my best friend, and for always being the voice of reason when I needed it. Successful completion of this project would not be made possible without his love and support.

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Table of Contents

ABSTRACT ...... II ACKNOWLEDGEMENTS ...... IV TABLE OF CONTENTS ...... V LIST OF FIGURES ...... VIII LIST OF ABBREVIATIONS ...... XII LIST OF PUBLICATIONS ...... XVI CHAPTER 1: INTRODUCTION ...... 1

1.1 INSULIN SIGNALING...... 2 1.1.1 Overview of Insulin - Discovery, Synthesis, Secretion and Physiological Roles ...... 2 1.1.1.1 Insulin Synthesis in Pancreatic beta-cells (-cell) ...... 3 1.1.1.2 Insulin Secretion ...... 6 1.1.1.3 Physiological Roles of Insulin ...... 8 1.1.2 Initiation of Insulin Signaling ...... 9 1.1.2.1 Insulin (IR) Activation and Regulation ...... 9 1.1.2.2 Substrate (IRS) – Platform of Insulin Signaling Pathways ...... 10 1.1.3 Insulin Action via phosphatidylinosital 3- (PI3K)-Akt/ (PKB) ...... 13 1.1.3.1 Activation of PI3K ...... 17 1.1.3.2 Activation of Akt/PKB ...... 17 1.1.3.3 Akt/PKB mediated Insulin Effects ...... 19 1.1.3.4 p-21 Activated Protein Kinase 1 (PAK-1) ...... 21 1.1.4 Insulin Action via Ras-mitogen-activated protein kinase (MAPK) ...... 27 1.1.4.1 Activation of Ras GTPases ...... 27 1.1.4.2 Activation of Raf Family Serine/Threonine ...... 29 1.1.4.3 Activation of MEK/Erk ...... 30 1.1.4.4 Regulation of MAPK Pathway ...... 31 1.1.5 Implication of Insulin Signaling in Cancer ...... 32 1.1.5.1 Ras/MAPK Signaling-Mediated Cell Transformation ...... 33 1.1.5.2 PI3K/Akt Signaling-Mediated Cell Transformation ...... 34 1.1.5.3 Implication of Hyperinsulinemia in Colorectal Cancer – Evidence from Epidemiological and Animal Studies ...... 36 1.2 WNT SIGNALING ...... 40 1.2.1 Initiation of Wnt Signaling...... 40 1.2.1.1 Wnt Ligand Processing and Secretion ...... 40 1.2.1.2 Extracellular Transport of Wnt ...... 42 1.2.1.3 Serpentine Receptor (Fzd) ...... 43 1.2.2 Non-canonical Wnt Signaling Pathways...... 44 1.2.2.1 Planar Cell Polarity (PCP) – Convergent Extension Pathway ...... 44 1.2.2.2 Wnt/Ca2+ Pathway ...... 46 1.2.3 Canonical Wnt Signaling Pathway ...... 46 1.2.3.1 Transducing Wnt Signals from Receptor to cat ...... 48 1.2.3.2 Regulation of Cytoplasmic cat ...... 49 1.2.3.3 cat – Nuclear Entry and Function ...... 52 1.2.3.4 Secreted Antagonists of Canonical Wnt Signaling ...... 55

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1.2.3.5 Alternative Mechanisms for Wnt/-cat Activation and Function ...... 55 1.2.3.5.1 Growth Factors ...... 56 1.2.3.5.2 G Proteins and G-protein Coupled Receptors (GPCRs) ...... 58 1.2.3.5.3 FOXO Proteins ...... 59 1.2.3.5.4 Wnt and mammalian Target of Rapamycin (mTOR) Signaling Crosstalk...... 60 1.2.4 Non-conventional Ligands and Receptors ...... 61 1.2.5 Aberrant Regulation of Canonical Wnt Signaling ...... 63 1.2.5.1 Canonical Wnt Signals in the Intestine: Endoderm to Cancer – Mouse Models Evidence ...... 64 1.2.5.2 Canonical Wnt Signaling in Human Colorectal Cancer ...... 66 1.3 SYNOPSIS, RATIONALE AND HYPOTHESES ...... 70 CHAPTER 2: GENERAL MATERIALS AND METHODS ...... 73

2.1 CHEMICALS AND REAGENTS ...... 74 2.2 PLASMIDS UTILIZED ...... 74 2.3 CELL CULTURE AND TRANSFECTION ...... 74 2.4 LUCIFERASE (LUC) REPORTER GENE ANALYSIS ...... 75 2.5 WESTERN BLOT ANALYSIS ...... 75 2.6 QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION (QCHIP) ...... 76 2.7 SHRNAMIR SELECTIVE GENE KNOCK-DOWN ...... 77 2.8 FETAL RAT INTESTINAL CELL ISOLATION ...... 78 2.9 STATISTICS ...... 78 CHAPTER 3: BOTH WNT AND MTOR SIGNALING PATHWAYS ARE INVOLVED IN INSULIN-STIMULATED PROTO- EXPRESSION IN INTESTINAL CELLS...... 79

3.1 ABSTRACT ...... 80 3.2 INTRODUCTION ...... 80 3.3 MATERIALS AND METHODS ...... 83 3.4 RESULTS ...... 86 3.4.1 Insulin-stimulated cell proliferation involves both Akt/PKB-dependent and independent mechanisms ...... 86 3.4.2 Insulin-stimulated c-Myc expression also involves both Akt/PKB-dependent and independent mechanims ...... 92 3.4.3 Insulin stimulates -cat content and nuclear -cat translocation ...... 98 3.4.4 Insulin stimulates binding of cat to two TCF-binding sites of the c-Myc gene promoter 101 3.5 DISCUSSION ...... 102 CHAPTER 4: P-21 ACTIVATED PROTEIN KINASE 1 (PAK-1) FUNCTIONS AS A LINKER BETWEEN INSULIN AND WNT SIGNALING PATHWAYS IN THE INTESTINE ...... 110

4.1 ABSTRACT ...... 111 4.2 INTRODUCTION ...... 111 4.3 MATERIALS AND METHODS ...... 115 4.4 RESULTS ...... 117 4.4.1 Insulin stimulates PAK-1 phosphorylation in intestinal cells ...... 117 4.4.2 Insulin-stimulated PAK-1 phosphorylation is independent of Akt/PKB status both in vitro and in vivo ...... 121

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4.4.3 Functional knockdown of PAK-1 reduces insulin stimulated oncogene expression and insulin induced -cat binding to human c-Myc gene promoter ...... 126 4.4.4 Knockdown of the expression of PAK-1 blocks insulin stimulated c-Myc and cyclin D1 expression, and -cat nuclear content ...... 131 4.4.5 MEK/ERK signaling pathway is involved in insulin-stimulated c-Myc expression and nuclear -cat expression ...... 143 4.5 DISCUSSION ...... 150 CHAPTER 5: INSULIN ALTERS THE EXPRESSION OF COMPONENTS OF THE WNT SIGNALING PATHWAY INCLUDING TCF7L2 (TCF-4) IN INTESTINAL CELLS ...... 156

5.1 ABSTRACT ...... 157 5.2 INTRODUCTION ...... 157 5.3 MATERIALS AND METHODS ...... 160 5.4 RESULTS ...... 163 5.4.1 Insulin treatment alters profiles in rat intestinal cell line IEC-6 ...... 163 5.4.2 Quantitative assessment of the effect of insulin on Fzd-4 and TCF7L2/TCF-4 expression by real time RT-PCR ...... 167 5.4.3 Insulin induces TCF7L2/TCF-4 protein expression ...... 169 5.4.4 Insulin activates TCF7L2/TCF-4 promoter activity ...... 171 5.5 DISCUSSION ...... 171 CHAPTER 6: GENERAL DISCUSSION AND CONCLUSIONS ...... 178

6.1 INSULIN AND NEOPLASIA...... 179 6.2 INSULIN SIGNALING CROSSTALKS WITH WNT/-CAT SIGNALING ...... 180 6.3 WNT SIGNALING AND TYPE 2 DIABETES ...... 188 6.4 PAK-1 IN WNT SIGNALING CROSSTALK ...... 189 6.5 OVERALL IMPORTANCE OF STUDY AND CONCLUSION ...... 190 6.6 FUTURE DIRECTION...... 190 CHAPTER 7: REFERENCES...... 193 APPENDIX: LIST OF ALTERED BY INSULIN TREATMENT IN THE IEC-6 CELL LINE .. 230

A.1 UP-REGULATED GENES (>2-FOLD) IN TNE IEC-6 CELL LINE POST 4 HOURS OF INSULIN TREATMENT ...... 231 A.2 UP-REGULATED GENES (>2-FOLD) IN THE IEC-6 CELL LINE POST 24 HOURS OF INSULIN TREATMENT ...... 234 A.3 DOWN-REGULATED GENES (>2-FOLD) IN THE IEC-6 CELL LINE POST 4 HOURS OF INSULIN TREATMENT...... 237 A.4 DOWN-REGULATED GENES (>2-FOLD) IN THE IEC-6 CELL LINE POST 24 HOURS OF INSULIN TREATMENT...... 242

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List of Figures Chapter One

FIGURE 1. 1 - LOCATION OF THE PANCREATIC ISLET AND HORMONE PRODUCING CELLS...... 4 FIGURE 1. 2 - INSULIN PEPTIDE AND INSULIN RECEPTOR (IR)...... 5 FIGURE 1. 3 - MECHANISMS UNDERLYING INSULIN SECRETION BY  CELLS...... 7 FIGURE 1. 4 - STRUCTURE AND BINDING PARTNERS OF INSULIN RECEPTOR SUBSTRATES (IRSS)...... 11 FIGURE 1. 5 - SUMMARY OF CURRENT KNOWLEDGE OF INSULIN SIGNALING PATHWAYS...... 14 FIGURE 1. 6 - STRUCTURE OF CLASS IA PHOSPHATIDYLINOSITAL 3-KINASE (PI3K)...... 15 FIGURE 1. 7 - DOMAIN STRUCTURES OF AKT/PKB ISOFORMS...... 18 FIGURE 1. 8 - PAK-1 STRUCTURE AND ACTIVATION...... 23 FIGURE 1. 9 - PAK-1 REGULATED SIGNALING PATHWAYS...... 26 FIGURE 1. 10 - HIERARCHICAL ORGANIZATION OF THE MAPK PATHWAY...... 28 FIGURE 1. 11 - WNT SIGNALING PATHWAYS...... 45 FIGURE 1. 12 - CANONICAL WNT SIGNALING PATHWAY...... 47

Chapter Three

FIGURE 3. 1 - INSULIN STIMULATES THE GROWTH OF TWO HUMAN COLON CANCER CELL LINES...... 87 FIGURE 3. 2 - INSULIN STIMULATES THE GROWTH OF PRIMARY FRIC CULTURES AND A NON-CANCEROUS INTESTINAL CELL LINE IEC-6...... 88 FIGURE 3. 3 - THE STIMULATORY EFFECT OF INSULIN ON C-MYC EXPRESSION CANNOT BE COMPLETELY BLOCKED BY PKB INHIBITION...... 89 FIGURE 3. 4 - THE STIMULATORY EFFECT OF INSULIN ON CANNOT BE COMPLETELY BLOCKED BY PKB INHIBITION...... 90 FIGURE 3. 5 - INSULIN STIMULATES C-MYC AND CYCLIN D1 EXPRESSION IN A TIME-DEPENDENT MANNER...... 91 FIGURE 3. 6 - INSULIN STIMULATES C-MYC EXPRESSION IN INTESTINAL CANCER AND NON-CANCER CELL LINES, AS WELL AS THE FRIC CULTURE...... 93 FIGURE 3. 7 - INSULIN DOES NOT ACTIVATE C-MYC EXPRESSION VIA INCREASING PROTEIN STABILITY IN THE HT29 CELL LINE...... 95 FIGURE 3. 8 - ACTIVATION OF C-MYC EXPRESSION BY INSULIN INVOLVES BOTH PKB-DEPENDENT AND PKB- INDEPENDENT MECHANISMS...... 96 FIGURE 3. 9 - MTOR PATHWAY IS INVOLVED IN INSULIN ACTIVATED C-MYC EXPRESSION...... 97 FIGURE 3. 10 - INSULIN TREATMENT LEADS TO INCREASED -CAT EXPRESSION IN FRIC (WHOLE CELL LYSATE) AND HT29 CELLS (NUCLEAR FRACTION)...... 99 FIGURE 3. 11 - INSULIN STIMULATES -CAT NUCLEAR TRANSLOCATION...... 100 FIGURE 3. 12 - INSULIN STIMULATES IN VIVO BINDING OF -CAT TO THE HUMAN C-MYC GENE PROMOTER. 103 FIGURE 3. 13- INSULIN STIMULATED IN VIVO BINDING OF -CAT TO THE HUMAN C-MYC GENE PROMOTER INVOLVES PI3K BUT NOT PKB OR MTOR...... 104 FIGURE 3. 14 - INSULIN STIMULATED IN VIVO BINDING OF -CAT TO THE HUMAN C-MYC GENE PROMOTER INVOLVES PI3K BUT NOT PKB...... 105

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Chapter Four

FIGURE 4. 1 - PROPOSED MECHANISM FOR INSULIN ACTIVATED C-MYC EXPRESSION AND ASSOCIATED CELL GROWTH...... 114 FIGURE 4. 2 - INSULIN STIMULATES PAK-1 PHOSPHORYLATION ...... 119 FIGURE 4. 3 - IGFR MAY NOT BE A MAJOR PLAYER IN THE STIMULATORY EFFECT OF INSULIN ON PAK-1 PHOSPHORYLATION...... 120 FIGURE 4. 4 - INSULIN STIMULATES PAK-1 PHOSPHORYLATION IN A NUMBER OF TISSUE SAMPLES IN VIVO. . 122 FIGURE 4. 5 - INSULIN STIMULATED PAK-1 PHOSPHORYLATION IS INDEPENDENT OF AKT STATUS: EVIDENCE FROM UTILIZING AKT CHEMICAL INHIBITION...... 123 FIGURE 4. 6 - STIMULATION OF PAK-1 PHOSPHORYLATION BY INSULIN IS INDEPENDENT OF AKT STATUS: IN VITRO EVIDENCE...... 124 FIGURE 4. 7 - STIMULATION OF PAK-1 PHOSPHORYLATION BY INSULIN IS INDEPENDENT OF AKT STATUS: IN VIVO EVIDENCE...... 125 FIGURE 4. 8 - EXPRESSION OF DOMINANT NEGATIVE PAK-1 ATTENUATES INSULIN STIMULATED C-MYC AND CYCLIN D1 EXPRESSION...... 128 FIGURE 4. 9 - EXPRESSION OF CONSTITUTIVELY ACTIVE PAK-1 INCREASES BASAL C-MYC AND CYCLIN D1 EXPRESSION...... 129 FIGURE 4. 10 - EXPRESSION OF DOMINANT PAK-1 ATTENTUATES INSULIN ACTIVATED -CAT BINDING TO THE C-MYC GENE PROMOTER...... 130 FIGURE 4. 11 - KNOCK DOWN OF PAK-1 EXPRESSION USING PAK-1 SHRNA...... 132 FIGURE 4. 12 - KNOCKDOWN OF PAK-1 EXPRESSION ATTENUATES THE STIMULATORY EFFECTS OF INSULIN. 133 FIGURE 4. 13 - KNOCKDOWN OF PAK-1 EXPRESSION ATTENUATES THE STIMULATORY EFFECTS OF INSULIN – DENSITOMETRIC ANALYSIS...... 134 FIGURE 4. 14 - PKA INHIBITION HAS NO APPRECIABLE EFFECT ON INSULIN STIMULATED -CAT (SER675) PHOSPHORYLATION...... 136 FIGURE 4. 15 - KNOCKDOWN OF PAK-1 EXPRESSION ATTENUATES INSULIN INDUCED PAK-1 BUT NOT PAK-2 PHOSPHORYLATION...... 137 FIGURE 4. 16 - KNOCKDOWN OF PAK-1 EXPRESSION ATTENUATES THE STIMULATORY EFFECTS OF INSULIN ON NUCLEAR -CAT CONTENT...... 139 FIGURE 4. 17 - KNOCKDOWN OF PAK-1 EXPRESSION ATTENUATES THE STIMULATORY EFFECT OF INSULIN ON THE ASSOCIATION OF -CAT WITH THE C-MYC GENE PROMOTER...... 140 FIGURE 4. 18 - KNOCKDOWN OF PAK-1 EXPRESSION ATTENUATES THE STIMULATORY EFFECT OF INSULIN ON CELL GROWTH...... 141 FIGURE 4. 19 - PAK-1 KNOCKDOWN ATTENUATED CELL GROWTH IS ASSOCIATED WITH REDUCED PROTO- ONCOGENE (C-MYC AND CYCLIN D1) EXPRESSION...... 142 FIGURE 4. 20 - KNOCKDOWN OF PAK-1 EXPRESSION ATTENUATES THE STIMULATORY EFFECT OF INSULIN ON KI67 EXPRESSION...... 144 FIGURE 4. 21 - MEK INHIBITION BLOCKS INSULIN STIMULATED C-MYC EXPRESSION...... 146 FIGURE 4. 22 - MEK INHIBITION BLOCKS INSULIN STIMULATED C-MYC EXPRESSION – DENSITOMETRIC ANALYSIS...... 147 FIGURE 4. 23 - MEK INHIBITION BLOCKS THE STIMULATORY EFFECT OF INSULIN ON NUCLEAR -CAT EXPRESSION...... 148 FIGURE 4. 24 - MEK INHIBITION BLOCKS INSULIN STIMULATED -CAT BINDING TO THE C-MYC GENE PROMOTER...... 149

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Chapter Five

FIGURE 5. 1 - GENE EXPRESSION CHANGES AFTER 4 AND 24 H OF INSULIN TREATMENT IN IEC-6 CELLS...... 165 FIGURE 5. 2 - INSULIN STIMULATES GENE EXPRESSION OF WNT SIGNALING COMPONENTS...... 168 FIGURE 5. 3 - INSULIN STIMULATES TCF7L2/TCF-4 PROTEIN EXPRESSION IN VARIOUS CANCER AND NON- CANCER CELL LINES...... 170 FIGURE 5. 4 - INSULIN STIMULATES TCF-4-LUC PROMOTER ACTIVITY...... 172

Chapter Six

FIGURE 6. 1 - CURRENT UNDERSTANDING OF INSULIN ACTIVATED ONCOGENE EXPRESSION AND ASSOCIATED CELL GROWTH...... 187

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List of Tables

Chapter Three

TABLE 3. 1 – EXPERIMENTAL AND CONTROL PRIMERS FOR QUANTITATIVE CHIP ASSAY ...... 85

Chapter Five

TABLE 5. 1 - NUMBER OF UP- AND DOWN-REGULATED GENES INSULIN TREATED IEC-6 CELLS ...... 164 TABLE 5. 2 - THE UP- AND DOWN-REGULATED GENES IN INSULIN TREATED IEC-6 CELLS ...... 166

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List of Abbreviations

°C Degree(s) Celsius 4EBP1 eIF4E-binding protein 1 a.a Abl-1 Abelson murine leukemia viral oncogene homolog 1 AC Adenylate cyclase ADP Adenosine diphosphate Akt/PKB Protein Kinase B ALL Acute lymphocytic leukemia AML Acute myeloid leukemia AMPK AMP activated kianse AP-1 Activator protein-1 APC Adenomatous polyposis coli APC Adenomatous polyposis coli AS160 Akt substrate of 160 kDa ASV Avian sarcoma virus ATP Adenosine triphosphate Bad B=cell lymphoma/leukemia 2 (Bcl-2)/Basal cell lymphoma- extra large (Bcl-xl)-associated death promoter Bcl-2 B-cell lymphoma 2 Bcl-xl Basal cell lymphoma-extra large Bim Bcl-2 interacting mediator of cell death BMP Bone morphogenetic protein bp BSA Bovine Serum Albumin CamK Calcium/-dependent kinase cAMP Cyclic adenosine monophosphate CBP CREB binding protein Cdk Cyclin-dependent kinase ChIP Chromatin Immunoprecipitation CIP Cdk inhibitory proteins CKI Casein kinasey I Co-IP Co-immunoprecipitation CR Conserved region CRC Colorectal Cancer CRC Colorectal cancer CRD Cysteine rich domain DAG Diacylglycerol DEPC Diethylpyrocarbonate DKK Dickkopf DMSO Dimethyl sulfoxide DN Dominant Negative DNA Deoxyribonucleic acid DTT Dithiothreitol

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Dvl Disheveled E1 -activating E2 Ubiquitin-conjugating enzyme E3 Ubiquitin ECM Extra cellular matrix EDTA Ethylene diamine tetra acetic acid EGF Epidermal growth factor ENU Mutagen ethylnitrosourea ER Endoplasmic reticulum ERK Extracellular Signal Regulated Kinase FAP Familial adenomatous polyposis FBS Fetal bovine serum FEVR Familial exudative vitreoretinopathy FOXO Forkhead class of factors FZD Frizzled Gab Grb2-associated binder GAG Glycosaminoglycan GAP GTPase-activating protein GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GFP Green Fluorescent Protein GIP Gastric inhibitory polypeptide GLP-1 Glucagon-like peptide 1 GLUT2 Glucose transporter 2 GM-CSF Granulocyte macrophage-colony stimulating factor Gpi Glycophosphatidylinositol Grb Growth-factor-receptor bound protein GSK-3 Glycogen Synthase Kinase-3 beta GTP Guanosine triphosphate HBP1 HMG-box 1 HDL High-density lipoprotein HFD High Fat Diet HMG High-mobility group IGF Insulin-like Growth Factor IGF Insulin-like growth factor IGFR Insulin-like Growth Factor Receptor IMP Impedes mitogenic signal propagation IP Intra Peritoneal IP3 Inositol 1,4,5-triphosphate IR Insulin receptor IR Insulin receptor IRS Insulin receptor substrate IV Intravenous JNK c-Jun amino-terminal kinase Krm Kremen KSR Kinase suppressor of ras

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LFD Low Fat Diet Lgs Legless LPA LRP Low-density-lipoprotein receptor related protein LUC Luciferase MAPK Mitogen-Activated Protein Kinase MEK MAPK/ERK Kinase MEK Mitogen/extracellular-signal regulated kinase kinase MMP Matrix metalloproteinase MOPS 4-Morholinepropanesulfonic Acid mTOR Mammalian target of rapamycin NES Nuclear export signal NFAT Nuclear factor of activated T cells NLK NEMO-like kinase OPPG Osteoporosis-pseudogliome p70S6K p70 PAK p-21 activated kinase PAK p21-activated protein kinase PAK-1 p-21 activated kinase 1 PBS Phosphate Buffered Saline PC-1 Plasma-cell-membrane glycoprotein-1 PCP Planar cell polarity PCR Polymerase Chain Reaction PDGF Platelet-derived growth factor PDK1 Pyruvate dehydrogenase kinase, isozyme 1 PH Pleckstrin homology PHLPP PH domain and leucine rich repeat protein phosphatase PI Phosphoinositide PI3K Phosphatidylinositol 3-kinase PIAS Protein inhibitor of activated STAT PIP2 Phosphatidyl inositol 4,5-biphosphate PKA PKA Protein kinase A PKC PLC Phospholipase C PMSF Phenylmethylsulfonyl fluoride PP1 Protein phosphatase 1 PP2A Protein phosphatase 2A PPAR Peroxisome proliferator-activated receptor PTB Phosphotyrosine-binding PTEN Phosphatase and tensin homolog PTK Protein tyrosine kinase PTP1B Protein tyrosine phosphatase 1B Pygo Pygopus Q-PCR Quantitative PCR RKIP Raf kinase inhibitor protein

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ROS Reactive oxygen species RSK Ribosomal S6 kinase RSpo R=Spondin RTK Receptor tyrosine kinase SDS Sodium Dodecyl Sulphate SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SERCA Sarco/Endoplasmic reticulum Ca2+ -ATPase sFRP Secreted frizzled-related protein SGK Serum and glucocorticoid-regulated protein kianse SH Src-homology SHC Src-homology containing protein SHP SH2 domain-containing inositol 5’-phosphatase shRNA Short Hairpin RNA SOCS1 Suppressor of signaling-1 SOS Son-of-sevenless SPRED Sprouty and sprouty-related protein with EVH1 domains STAT Signal transducers and activators of transcription protein SUMO Small ubiquitin-related modifier Sur Suppressor of ras SV 40 Large T-Antigen Simian Vacuolating Virus 40 T Antigen T2DM Type 2 Diabetes Melitus TBE Tcf/Lef-binding elements TCF T-Cell Specific Factor TGF- Transforming growth factor beta TRB Tribbles TSC Tuberous sclerosis complex VEGF Vascular endothelial growth factor WIF Wnt inhibitory factor Wnt Wingless (Drosophila) and Int (MMTV integration site ) Wrch Wnt-regulated Cdc42 homolog Xtwn Xenopus twin gene -cat Beta-catenin TrCP Beta- repeat-containing protein g Microgram l Microliter M Micromolar

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List of Publications

1. Sun J, Khalid S, Rozakis-Adcock M, Fantus IG, Jin T (2009). P-21 activated protein kinase-1 functions as a Linker between Insulin and Wnt Signaling Pathways in Intestinal Cells. Oncogene. Epub ahead of print.

2. Sun J, Jin T (2008). Both Wnt and mTOR signaling pathways are involved in insulin-stimulated protooncogene expression in intestinal cells. Cell Signal 20: 219-29.

3. Sun J, Wang D, Jin T (2009). Insulin alters the expression of components of the Wnt signaling pathway including TCF7L2 (TCF-4) in the intestinal cells. Submitted to BBA.

4. Yi F*, Sun J*, Lim GE, Fantus IG, Brubaker PL, Jin T (2008). Cross talk between the insulin and Wnt signaling pathways: evidence from intestinal endocrine L cells. Endocrinology 149: 2341-51. *Co-first author

5. Jin T, Fantus IG, Sun J (2008). Wnt and beyond Wnt: Multiple mechanisms control the transcriptional property of beta-catenin. Cell Signal 20: 1697-704.

6. Wang P, Wang Q, Sun J, Wu J, Li H, Zhang N et al (2008). POU Homeodomain Protein Oct-1 Functions as a Sensor of Cyclic AMP. Journal of Biological Chemistry. Epub ahead of print.

7. Lim GE, Xu M, Sun J, Jin T, Brubaker PL (2008). The Rho GTPase, Cdc42, is required for insulin-induced GLP-1 secretion and remodeling in the intestinal endocrine L cell. Under revision for Endocrinology.

8. Lotfi S, Li Z, Sun J, Zuo Y, Lam PP, Kang Y et al (2006). Role of the exchange protein directly activated by cyclic adenosine 5'-monophosphate (Epac) pathway in regulating proglucagon gene expression in intestinal endocrine L cells. Endocrinology 147: 3727-36.

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Chapter 1: Introduction

1

1.1 Insulin Signaling

1.1.1 Overview of Insulin - Discovery, Synthesis, Secretion and Physiological Roles

In 1869, in the process of completing his doctorate work at the Berlin

Pathological Institute, Paul Langerhans observed previously un-identified clusters of cells scattered throughout the pancreas. These clusters are now known as the Islets of Langerhans [1]. In 1889, two Polish-German physicians, Oscar Minkowski and

Joseph von Mering observed a healthy dog developing glycosurea post the surgical removal of its pancreas, thereby providing one of the earliest lines of evidence linking the pancreas to diabetes [2]. Two years later, Eugene Opie suggested that it is specifically the destruction of the Islets of Langerhans that is the cause of diabetes mellitus [3, 4]. Working in J.J. R. Macleod’s lab at the University of Toronto, Fredrick

Banting and his assistant Charles Best isolated a crude extract from pancreatic islets, and found that injections of this extract reversed hyperglycemia in pancreatectomized dogs. Biochemist James Collip was subsequently invited to the

Macleod lab to purify insulin extract from fetal calf for clinical testing. In 1922, a terminal 14-year-old diabetic patient named Leonard Thompson at the Toronto

General Hospital received the first injection of insulin extract, and suffered severe allergic reaction due to extract impurity. However, less than two weeks later, James

Collip was able to improve his purification process and produced an ox-insulin extract that successfully treated Leonard Thompson [5]. Since then, extensive studies have been executed to understand the structure, synthesis and function of insulin.

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1.1.1.1 Insulin Synthesis in Pancreatic beta-cells (-cell)

The pancreas is primarily an exocrine gland, responsible for producing and secreting a cocktail of digestive including trypsin, chymotrypsin, pancreatic lipase and pancreatic amylase in an alkaline fluid that is delivered to the small intestine for digesting carbohydrates, fat and proteins [6]. Approximately 2% of the pancreas is composed of the Islets of Langerhans, which constitutes as the endocrine part of the pancreas. Each islet contains three different hormone- secreting cell types, ,  and , producing glucagon [7], insulin [8] and somatostatin

[9] respectively (Fig. 1.1). The human insulin gene INS is located on the short arm of 11, and encodes the proinsulin precursor [10]. This precursor molecule undergoes proteolytic modification carried out by prohormone convertases

(PC1 and PC2) and exoprotease carboxypeptidase E to remove the central portion of the molecule known as the C-peptide [11, 12] (Fig. 1.2A). The remaining two peptides are referred to as the A-chain (21 a.a.) and B-chain (30 a.a.). The A-chain contains an internal disulphide bond and is connected to the B-chain via two additional disulphide bonds, forming the ~5.8kDa di-peptide insulin molecule (Fig.

1.2A). Insulin plays a central role in various metabolic processes, and this is reflected in its high sequence conservation especially in vertebrates. Porcine insulin differs from human insulin in three a.a. and bovine insulin differs from human insulin in merely one a.a. This is precisely why that bovine insulin was widely utilized with success in treating diabetic patients before the breakthrough in recombinant DNA

3

Pancreas

Pancreatic Islet

 cell  cell  cell

glucagon insulin C-peptide somatostatin

Figure 1. 1 - Location of the pancreatic islet and hormone producing cells.

4

A proinsulin insulin

S S A chain S S B chain S S S S PC1 S S C chain S S PC2 Carboxypeptidase E

B alpha-subunit beta-subunit

disulphide bond  

extracellular

cell membrane   intracellular

Figure 1. 2 - Insulin peptide and insulin receptor (IR).

Diagrammatic representation of (A) proinsulin and insulin peptided, and (B) structure of the insulin receptor (IR), a heterotetramer consisting of two extracellular insulin-binding subunits (alpha) linked by a disulfide bonds to two transmembrane beta subunits. The beta subunits contain an intrinsic tyrosine kinase activity that is activated upon insulin binding to the alpha subunit.

5

technology, which allowed human insulin to be produced in bacteria in the early

1980’s.

1.1.1.2 Insulin Secretion

The pancreatic  cells synthesize insulin and store it in readily releasable granules. Insulin is usually released postprandially. Specific stimuli that trigger the secretion of insulin from  cells are primarily glucose, amino acids and incretins such as gastric inhibitory peptide (GIP) and glucagon-like peptide 1 (GLP-1). Glucose enters the  cells via glucose transporter 2 (GLUT2), and goes through glycolysis and the respiratory cycle to produce ATP molecules. This increase in ATP causes the ATP-dependent potassium (KATP) channels to close and depolarizes the cell from

-60mV to ~-40mV. Depolarization of the  cell opens voltage-dependent calcium

(Ca2+) channels and creates an influx of Ca2+. This initial increase in Ca2+ activates phospholipase C (PLC) and cleaves the membrane phospholipid, phosphatidyl inositol 4,5-biphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and

2+ diacylglycerol (DAG). IP3 then triggers the release of Ca from the endoplasmic

2+ reticulum (ER) via IP3-gated channels and further raises the intracellular Ca concentration, which leads to the exocytosis of previously synthesized insulin stored in the secretory vesicles [13] (Fig. 1.3). Amino acids alanine, glycine and arginine can also trigger insulin release via  cell depolarization. Alanine and glycine depolarize the  cell by creating an influx of sodium ions (Na+) because they share a symport that also transports Na+ [14-16]. On the other hand, arginine directly depolarizes the  cell by nature of its being a cation at physiological pH [17].

6

Insulin secretion

IP3 IP3

IP3

2+ IP3 Ca PIP2

PLC

Ca2+

ATP PKA cAMP ATP Epac

K+ AC

Na+

Figure 1. 3 - Mechanisms underlying insulin secretion by  cells.

Insulin secretion in  cells can be triggered by (1) uptake of glucose, (2) amino acids as well as (3) incretin hormones. GLUT-2: glucose transporter 2; IP3: inositol 1,4,5-triphosphate; PIP2: phospholipid phosphatidyl inositol 4,5-biphosphate; PLC: phospholipase C; PKA: protein kinase A; cAMP: cyclic adenosine monophosphate; AC: adenylate cyclase; GEF: guanine nucleotide exchange factor; GLP-1: glucagon-like peptide 1; GIP: gastrin induced peptide; ADP: adenosine diphosphate; ATP: adenosine triphosphate;

7

Incretins are gastrointestinal hormones that trigger insulin secretion upon oral glucose administration. Glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) are incretins produced by the intestinal endocrine L cells and intestinal epithelial K cells respectively [18]. These hormones are regulated primarily by nutrients and fatty acids but are also controlled by both the sympathetic and parasympathetic nervous systems. GLP-1 and GIP induce  cell insulin secretion via a distinct mechanism activating adenylate cyclase (AC), which in turn elevates intracellular cAMP levels that leads to the activation of PKA and cAMP-regulated guanine nucleotide exchange factor II (cAMP-GEFII, also known as Epac 2), and subsequently potentiates an influx of Ca2+ ions that ultimately causes  cell depolarization and insulin release [19].

1.1.1.3 Physiological Roles of Insulin

Insulin is a critical player in glucose, fat and protein metabolisms. It increases glucose uptake in muscle and adipose tissues to reduce blood glucose levels, and increases glycogen synthesis while decreasing gluconeogenesis in the liver. In fat cells, insulin increases blood lipid uptake, stimulates production of triglycerides and prevents lipolysis. In addition, insulin reduces and increases amino acid uptake in liver and muscle. The following sections will describe in detail the major players in insulin signaling, and the cellular mechanisms underlying the various physiological actions of insulin.

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1.1.2 Initiation of Insulin Signaling

1.1.2.1 Insulin Receptor (IR) Activation and Regulation

Insulin signaling is initiated the moment that the insulin molecule binds to its receptor. The insulin receptor (IR) is a 350-kDa tetrameric transmembrane glycoprotein, composed of two  subunits (135-kDa each) and two  subunits (95- kDa each) (Fig. 1.2B). The human IR gene INSR is located on the short arm of chromosome 19, and gives rise to a single-chain precursor that undergoes post translational modifications and surmounts to two subunits,  and connected by a disulphide bond. Then, the  complexes dimerize by forming a disulphide bond between their -subunits to form intact tetrameric IRs [20] (Fig. 1.2B). The IR belongs to the family of receptor tyrosine kinases, enzymes that transfer phosphate groups onto tyrosine residues of other proteins. IR behaves like a typical allosteric enzyme, having the -subunits hindering the intrinsic catalytic activity of the - subunits. Insulin binds to the extracellular -subunits of IR leading to the activation and autophosphorylation of the -subunits, which fully activates its catalytic function

[21, 22].

The activity of IR is tightly regulated by several mechanisms. Tyrosine phosphatases such as protein tyrosine phosphatase 1B (PTP1B) directly interacts with and dephosphorylates critical residues on IR and reduces its activity. It has been shown that PTP1B knockout mice exhibit significant improvements in insulin sensitivity [23]. The kinase activity of IR can also be affected when proteins such as suppressor of cytokine signaling-1 (SOCS1) and SOCS3, growth-factor-receptor bound protein 10 (Grb10) and plasma-cell-membrane glycoprotein-1 (PC-1)

9

physically block the interaction between IR and insulin receptor substrate (IRS) [24].

Indeed, elevated SOCS proteins have been observed particularly in insulin resistant states [25]. Ligand-stimulated IR internalization and degradation is another way to downregulate IR activity, which is a prevalent feature of hyperinsulinemic states and

Type II Diabetes [26]. Lastly, in general, phosphorylation of IR on its serine residues also negatively regulates IR action [27].

1.1.2.2 Insulin Receptor Substrate (IRS) – Platform of Insulin Signaling Pathways

A number of IR substrates have been identified, including insulin-receptor substrates 1-6 (IRS1-6), Grb2-associated binder-1 (Gab1), Cas-Br-M (murine) ecotropic retroviral transforming sequence homologue (Cbl) and various Src- homology-2 (SH2) containing proteins. IRS proteins contain a pleckstrin-homology

(PH) and phosphotyrosine-binding (PTB) domain in their N-termini (Fig. 1.4). These domains have high affinity for IR and are responsible for binding to IR [27]. The central and C-terminus part of the IRS proteins contain multiple potential tyrosine phosphorylation sites that can be phosphorylated by the IR, which then can serve as docking platforms for downstream effectors containing SH2 domains (Fig. 1.4) [27].

IRS proteins differ in their expression profiles, having IRS1 and IRS2 the only widely distributed isoforms. IRS3 is expressed primarily in adipocytes and the brain, and

IRS4 is expressed largely in embryonic tissues or cell lines, while IRS5 and IRS6 have limited tissue expression and function in insulin signaling [28]. General defects in body growth and insulin action in muscles tissues have been observed in IRS1 knockout animals [29]. However, in IRS2 knockout animals, only region-specific

10

PI3K Grb2 PI3K SHP2

608 628 891 935 1179 1222 IRS1 Y Y Y Y Y Y N’ PH PTB C’

S S S S S S S S 265 267 307 332 612 661 731 789

AKT/PKB JNK PKCq S6K GSK-3 ERK mTOR AMPK

PI3K Grb2 SHP2

IRS2 Y Y Y Y Y N’ PH PTB C’

Grb2 PI3K SHP2

IRS3 Y Y Y Y N’ PH PTB C’

PI3K Grb2 SHP2

IRS4 Y Y Y Y N’ PH PTB C’

Figure 1. 4 - Structure and binding partners of insulin receptor substrates (IRSs).

Insulin receptor substrate (IRS) isoforms, IRS1, IRS2, IRS3 and IRS4 share a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain at the amino terminus. IRS also contains multiple sites of phosphorylation on tyrosine and serine residues. The positions of the tyrosine residues (Y) phosphorylated by the IR and the downstream signaling proteins that bind to these sites are shown. The positions of the serine residues (S) and the kinases responsible for phosphorylating these residues are also shown. PI3K: phosphatidylinositol 3-kinase; Grb2: growth-factor-receptor- bound protein-2; SHP2: Src-homology-2 (SH2) domain-containing tyrosine phosphatase-2; AKT/PKB: AKT/protein kinase B; JNK: c-Jun-N-terminal kinase; PKC: protein kinase C; S6K: p70 ribosomal protein S6 kinase; GSK-3: glycogen synthase kinase-3; ERK: extracellular signal-regulated kinase; mTOR: mammalian target of rapamycin; AMPK: AMP-activated protein kinase;

11

(brain and pancreatic  cells) growth defects were observed, and insulin action is defective primarily in liver tissues [30]. IRS proteins may exert specific effects by targeting different SH2-containing binding partners. In vitro studies showed in L6 myotube cells that IRS1 closely regulates glucose uptake while IRS2 is closely linked with cell growth and differentiation [31]. Studies performed in hepatic-specific knockout mice showed that IRS1 and IRS2 have different roles in gene expression regulation as well [32]. Differences between the functional behaviors of IRS1 and

IRS2 can be explained by their distinct preferences and abilities to bind downstream effectors. For example, while IRS1 can bind to the Abl tyrosine kinase and SHP2,

IRS2 does not [33]. IRS1 also exhibits higher affinity for several SH2 proteins than

IRS2, such as Grb2, the Crk adaptor protein and phospholipase C.

IRS action is tightly regulated similarly to IR, via tyrosine phosphatases and serine phosphorylation. There are at least 70 potential serine phosphorylation sites in IRS1, and increased serine phosphorylation of IRS1 has been frequently observed during insulin resistant states. In a yeast-tri-hybrid assay, it was shown that phosphorylation of IRS1 at the Ser307 position located in its phosphotyrosine binding (PTB) domain disrupts its interaction with IR [34]. Other serine residues in the PTB domain of IRS1 have been implicated in IRS1 regulation by enhancing the interaction between IRS1 and 14-3-3 proteins, thereby interfering with the function of the IRS1 PTB domain and its ability to bind to IR [35].

IRS mediates insulin signaling by binding to downstream effectors that are

SH2-containing or non-SH2-containing proteins (Fig. 1.4). The SH2-containing proteins can be categorized into adaptors such as the regulatory subunit of PI3K,

12

and Grb1/SOS (leads to Ras-MAPK activation), and cytoplasmic enzymes such as

SHP2 and Fyn. IRS also binds to non-SH2-containing proteins such as SERCA1 and SERCA2, and SV40 large T antigen. Two major signaling pathways initiated by

IR activation are the PI3K-Akt/PKB pathway that is responsible for most of the metabolic actions of insulin, and the Ras-MAPK pathway that regulates gene expression and cell growth and differentiation [27, 36] (Fig. 1.5). The following sections will describe in detail these two major signaling pathways.

1.1.3 Insulin Action via phosphatidylinosital 3-kinase (PI3K)-Akt/protein kinase B (PKB)

PI3K is a crucial component of the insulin signaling pathway that carries out downstream signaling cascades that are responsible for various insulin actions, including the stimulation of glucose uptake, glycogen synthesis and the suppression of lipolysis and gluconeogenesis [37]. PI3K is divided into three classes, with Class I and Class II PI3K expressed in metazoa and class III expressed even in unicellular eukaryotes. Class I PI3K is by far the best characterized type of PI3K to date.

Class I PI3Ks are large heterodimeric proteins consisting of a catalytic subunit and a regulatory subunit (Fig. 1.6), known for their ability to catalyze the phosphorylation of phosphoinositides (PIs) on their 3’ hydroxyl group. Even though PI3K has the ability to catalyze the conversion of both PIP and PIP2 into PIP3, it has been suggested that

PIP2 is the main substrate in vivo [38]. The catalytic subunits of Class I PI3K have a molecular mass of ~110kDa and are collectively referred to as p110 subunits. There are four p110 catalytic subunits in mammals, encoded by four genes, namely

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Insulin Glucose

GLUT4 IR

P P PTP1B P P

PIP2 Shc SOS PTEN PI3K IRS Grb SHIP1/2 PIP3 PP2A Akt PDK1 Ras

PDK1 TSC1 TSC2 GLUT4 Bad GSK-3 PAK-1 Raf vesicle mTOR AS160 GS MEK GLU4 Exocytosis p70S6K 4EBP1 eIF4E Akt Glycogen Protein Erk FOXO Synthesis Synthesis Cytoplasm

Nucleus

FOXO

Gluconeogenesis  Transcription  Growth Adipogenesis Proliferation Apoptosis Figure 1. 5 - Summary of current knowledge of Insulin Signaling Pathways.

A diagrammatic representation of major pathways of insulin signaling. IR: insulin receptor; IRS: insulin receptor substrate; GLUT4: glucose transporter 4; Grb: growth factor receptor-bound protein; Shc: Src-homology containing protein; SOS: son-of-sevenless; PTP1B: protein tyrosine phosphatase-1B;

PTEN: phosphatase and tensin homolog; SHIP: SH2 domain-containing inositol 5’-phosphatase PIP2: phosphatidylinositol biphosphate; PIP3: Phosphatidylinositol (3,4,5)-trisphosphate PP2A: protein phosphatase 2A; PDK1: pyruvate dehydrogenase kinase, isozyme 1; AS160: Akt substrate of 160kDa; GS: glycogen synthase; Erk: extracellular signal-regulated kinase; FOXO: forkhead class of transcription factors; Bad:B-cell lymphoma/leukemia 2 (Bcl-2)/Basal cell lymphoma-extra large (Bcl- xl)-associated death promoter ; PI3K: phoshoinositide 3-kinase; AKT/PKB: protein kinase B; mTOR: mammalian target of rapamycin; 4EBP1: eIF4E-binding protein 1; p70S6K: p70 ribosomal S6 kinase; PAK-1: p21-activated protein kinase 1;

14

A Regulatory Catalytic subunits subunits p85 p110

PIP B 3 PIP Class IA PI3K 2 Regulatory subunits

SH3 P P SH2 p110 binding SH2 p85

P SH2 p110 binding SH2 p55 PIK3R1

P SH2 p110 binding SH2 p50

SH3 P SH2 p110 binding SH2 P p85 PIK3R2

P SH2 p110 binding SH2 p55 PIK3R3

Catalytic subunits

p85 binding Ras binding C2 Kinase domain p110 PIK3CA

p85 binding Ras binding C2 Kinase domain p110 PIK3CB

p85 binding Ras binding C2 Kinase domain p110 PIK3CD

Figure 1. 6 - Structure of Class IA phosphatidylinosital 3-kinase (PI3K).

(A) Schematic representation of PI3K, comprising of the p85 regulatory subunit and the p110 catalytic subunit.. (B) Schematic domain structures for the regulatory and catalytic subunits comprising the Class IA PI3K. PI3K: phosphatidylinositol 3-kinase; SH: Src homology domain; P: proline rich domain; C2: lipid binding domain;

15

Pik3ca, Pik3cb, Pik3cg and Pik3cd (Fig. 1.6B). However, they are usually referred to as p110, ,  and  respectively. The p110 subunits are highly homologous to each other and contain an N-terminal domain that binds to the regulatory subunits of

Class I PI3K, a Ras-binding domain, a lipid binding C2 domain and a kinase domain

(Fig. 1.6B). While p110 and p110 are expressed ubiquitously, p110 and p110 are preferentially found in leukocytes [39]. The p110, p110 and p110 catalytic subunits bind to what is referred to as the p85 family regulatory subunit, and constitute the Class IA of PI3K (Fig. 1.6B). On the other hand, Class IB is composed of the p110 catalytic subunit and p84/87 regulatory subunits. Three genes encode the regulatory subunits of Class IA PI3K, namely Pik3r1, Pik3r2 and

Pik3r3. Three splice variants are derived from Pik3r1 to produce the p85, p55 and p50, and Pik3r2 and Pik3r3 each gives rise to one regulatory subunit, p85and p55Fig. 1.6B). While p85 and p85 are expressed in all tissues [40], p50 and p55 are expressed in insulin sensitive tissues such as fat, liver and muscle [41, 42], and p50 is expressed in the brain [43]. The p85 family regulatory subunits all contain a p110-binding domain in the N-terminus, which allows for the interaction with the Class IA catalytic subunits. This p110-binding domain is flanked by two SH2 domains responsible for its interaction with phosphorylated tyrosine residues in the YXXM motif of tyrosine-phosphorylated receptors or receptor- associated adaptor signaling components such as IRS.

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1.1.3.1 Activation of PI3K

The basal activity of the p110 subunit of Class IA PI3K is relatively low because it is constitutively bound to its regulatory domain. However, once insulin stimulates IR activation and subsequent tyrosine phosphorylation of IRS, the regulatory p85 subunit is recruited to the tyrosine-phosphorylated motifs of IRS. The outcome of this event is twofold. First, the catalytic p110 subunit is brought in close proximity to its inositol lipid substrates embedded in the cell membrane [44]. Second, the binding of p85 to tyrosine phosphorylated IRS relieves its constraints on the p110 subunit and enhances the catalytic activity of p110, thereby increasing the conversion of PIP2 to PIP3 [45]. PIP3 acts as a second messenger and mediates a plethora of biological effects by recruiting and directly binding to signaling molecules of the protein kinases A, G and C (AGC) superfamily or guanine nucleotide exchange factors (GEFs) or the Rho family GTPases and TEC family of tyrosine kinases [27].

1.1.3.2 Activation of Akt/PKB

One of the most well studied signaling molecules targeted by PIP3 is Akt or protein kinase B (PKB). There are three mammalian isoforms of Akt/PKB (Akt1, 2 and 3 or PKB,  and ), encoded by three different genes. All isoforms of Akt/PKB share an N-terminal PH domain and a C-terminal catalytic domain (Fig. 1.7). Akt1 and Akt2 are both widely expressed in all tissues, and Akt3 is mainly expressed in the brain and testis. Insulin can activate Akt/PKB within minutes, and activated Akt1 is phosphorylated on Thr308 (by PDK-1) and on Ser473 (by mTOR, in complex with

17

P P PH Kinase domain HM Akt1/PKB Thr308 Ser473

P P PH Kinase domain HM Akt2/PKB Thr309 Ser474

P PH Kinase domain Akt3/PKB Thr305

Figure 1. 7 - Domain structures of Akt/PKB isoforms.

Schematic representation of domain structure of Akt/PKB isoforms. All the Akt/PKB isoforms contain a central kinase domain, a PH domain at the amino terminus and a hydrophobic domain at the carboxyl terminus. Phosphorylation sites required for Akt/PKB activation are indicated. Akt/PKB: Akt/Protein Kinase B; PH: pleckstrin homology domain; HM: hydrophobic motif;

18

rictor and Sin-1) residues, which are the equivalent of Thr309 and Ser474 on Akt2.

The activation of Akt3, on the other hand is indicated solely by the phosphorylation of Thr305 (because it terminates at residue 454), equivalent to Thr308 in Akt1.

Akt/PKB activation can be mediated by PIP3 via several mechanisms. Firstly, PIP3 interacts with PDK-1 via its PH domain and increases its enzymatic activity.

Simultaneously, Akt/PKB is being recruited to proximal regions of the plasma membrane where PDK-1 now resides, allowing more available Akt/PKB proteins to be phosphorylated/activated by PDK-1. In addition, the interaction between PIP3 and Akt/PKB triggers a conformational change that exposes its phosphorylation sites, and in turn further increases the efficiency of Akt/PKB activation [37].

1.1.3.3 Akt/PKB mediated Insulin Effects

Activated Akt/PKB has four major roles in insulin mediated signaling. A normally constitutively active enzyme glycogen synthase kinase 3 (GSK-3) is phosphorylated at serine residue 9 and 21 of the  and  isoforms, and inactivated by Akt/PKB [37]. Since GSK-3 activity promotes the inhibition of glycogen synthase

(GS) and associated inhibition of glycogen synthesis, insulin is able to induce glycogen production [38, 39, 46]. On the other hand, Akt/PKB also phosphorylates and inactivates Akt/PKB substrate of 160kDa (AS160), a GTPase-activating protein and initiates a cascade of events leading to cytoskeletal re-organization leading to the translocation of the glucose transporter GLUT4 to the plasma membrane, resulting in an increase in glucose uptake [47]. Increased protein synthesis is another outcome of insulin stimulation. Protein synthesis is positively regulated by the mammalian target of rapamycin (mTOR) pathway. The protein

19

complex formed between mTOR and raptor (regulatory associated protein of mTOR) is normally sequestered by the tuberous sclerosis complex 1 and 2 (TSC1/2).

Akt/PKB phosphorylates and inhibits TSC2 thereby relieving its constraint on mTOR/raptor, allowing it to phosphorylate/activate p70 ribosomal protein S6 kinase

(p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP-1).

Phosphorylation of 4EBP-1 triggers the release of its binding partner eIF4E, which leads to the initiation of 5’ cap-dependent translation processes. Activated p70S6K recruits and translates molecules that facilitate ribosomal biogenesis and translation elongation [48]. Interestingly, PDK-1 is also capable of phosphorylating/activating p70S6K, which further enhances insulin’s ability to induce protein translation. Last but not least, insulin stimulates gluconeogenesis and lipolysis through Akt/PKB. The expression of genes that regulate gluconeogenesis and adipogenesis is under the control of winged-helix/forkhead (Fox) class of transcription factors including FOXO1 and FOXO2 [49]. Akt/PKB induces the interaction of FOXO1 with 14-3-3 proteins by

FOXO1 Ser256 phosphorylation, preventing FOXO1 nuclear localization thereby inhibiting its transcriptional regulation of gluconeogenic and adipogenic genes [50].

Similarly, the important regulator of fasting lipid metabolism, FOXA2 is shunted from the nucleus post Thr156 phosphorylation by Akt/PKB [51].

Insulin induced PI3K/Akt pathway leads to various events important for the maintenance of metabolic and mitogenic homeostasis; insulin signaling and function is constantly regulated by other signaling molecules. The production of PIP3 by

PI3K is regulated by SH2-containing phosphatases 1 and 2 (SHIP1 and 2), and phosphatase and tensin homolog (PTEN). These are all phosphatases that

20

dephosphorylate PIP3 back to PIP2, reversing the effect of insulin. SHIP1 and SHIP2 dephosphorylate PIP3 at the 5’ position of the inositol ring [52] whereas PTEN dephosphorylates the 3’ inositol ring of PIP3 [53]. SHIP1 and 2 are important regulators of insulin signaling as the loss of SHIP2 for example, is associated with a significant increase in insulin sensitivity [54]. On the other hand, loss of PTEN expression or function is frequently observed in advanced human , suggesting that aberrantly regulated insulin signaling also contributes to metastatic cancers [55, 56]. Insulin mediated signaling can also be regulated at the level of

Akt/PKB [52]. Phosphatases including protein phosphatase 2A (PP2A) and PH domain and leucine rich repeat protein phosphatase (PHLPP) directly dephosphorylate and inactivate Akt/PKB [57, 58]. Conversely, tribbles-3 (TRB3) binds to unphosphorylated Akt/PKB and prevents its phosphorylation and activation

[59].

1.1.3.4 p-21 Activated Protein Kinase 1 (PAK-1)

p21-activated kinases (PAKs) are a family of serine threonine kinases initially identified as Rho-family GTPase binding partners [60, 61]. GTP-bound Cdc42 and

Rac were found to interact with three proteins of ~68, 65 and 62 kDa [62]. These proteins were subsequently identified as three isoforms of the p21-activated kinases,

PAK-1 (-PAK), PAK-2 (-PAK) and PAK-3 (-PAK). These Group I PAK isoforms were later confirmed by a number of studies as bonafide targets and effectors of

Cdc42 and Rac [60, 61, 63]. Three other PAK isoforms have since been identified,

PAK-4, PAK-5 and PAK-6. These PAK isoforms share less homology in their

21

catalytic domain with the Group I PAKs, therefore are categorized as the Group II

PAKs [64].

For the Group I PAKs, highest expression of PAK-1 is observed in brain, muscle and spleen; PAK-2 is expressed ubiquitously while PAK-3 is mostly expressed in the brain [62, 65]. For the less well studied Group II PAKs, the expression of PAK-4 is predominantly in prostate, testis and colon [66, 67]; PAK-5 is expressed highly in the brain [68], whereas PAK-6 was found to be expressed in testis, prostate, brain, kidney and placenta [64, 69]. Knockout animal models suggest members of the PAK family have unique biological functions during development. Pak-2-/- mice experience embryonic lethality due to multiple developmental abnormalities [70]. Pak-4-/- mice also die during embryonic development at day 11.5 due to heart defect, and exhibit neuronal abnormalities [71].

On the other hand, PAK-3 has been implicated in the regulation of neuronal gene expression and learning and memory; Pak-3 knockout mice exhibit severe impairment of late-phase hippocampal long-term potentiation, a process involving new gene expression [72]. In contrast, Pak-5-/- mice appear to develop normally and are fertile, and Pak-6 knockout mice have yet to be reported. Recently, more insights of the biological role of PAK-1 were provided by Allen et al., who showed that while Pak-1-/- mice are viable, healthy and fertile, they carry defects in MAPK signaling that causes abnormal responses in immune cells [73].

Among the six identified isoforms, PAK-1 is the most extensively studied member of PAK family. PAK-1 contains an N-terminal regulatory domain and a C- terminal catalytic domain (a.a 255-529), which is highly conserved among Group I

22

A Rac Nck Grb2 Cdc42 Pix/Cool G

PAK-1 P P P Ser21 Thr212 Thr423

Akt/PKB P35-Cdk5 PDK-1 Cdc2 Regulatory Domain Catalytic Domain (255-529 a.a) PXXP putative SH3-binding motifs Proline-rich Pix/Cool SH3-binding motif

p21 (Rac/Cdc42)-binding domain Serine/Threonine kinase domain

Autoinhibitory domain

B

Cdc42/Rac

GTP

domain domain

domain

Catalytic Catalytic Catalytic P

Figure 1. 8 - PAK-1 structure and activation.

A diagrammatic representation of PAK-1 domain structures. (A) PAK-1 coding sequence contains four SH3-binding PXXP motifs (where X is any amino acid), the Cdc42/Rac-binding domain known as the p21-binding domain (PBD), an autoinhibitory (AI) domain that overlaps with the PBD, the Cool/Pix-binding region and the serine/threonine kinase domain. Sites of phosphorylation by various kinases are shown. Sites where known binding partners that interact with Pak-1 are also indicated. (B) Depiction of Cdc42/Rac-stimulated activation of Pak-1. The binding of Cdc42/Rac relieves PAK-1 from its transinhibited homodimeric conformation. Activated Pak-1 monomer has an open conformation and is stabilized by auto-phosphorylation.

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PAKs (Fig. 1.8A) [74]. At least four known PXXP SH3-binding motifs were identified in the regulatory region of PAK-1. The first motif is responsible for PAK-1 interaction with adapter protein Nck [75], and the second motif interacts with adapter protein

Grb2 (Fig. 1.8A) [76]. Interaction with these adaptor proteins suggest that upon growth factor stimulation, PAK-1 may be recruited to the plasma membrane and become active. Indeed, it has been reported that blocking the interaction between

PAK-1 and Nck prevented PAK-1 activation [77], whereas targeting PAK-1 to the plasma membrane is sufficient to activate this kinase [78]. Adjacent to the two proline-rich SH3-binding sites is the p-21 binding domain (PBD), containing elements responsible for interaction with activated Cdc42 and Rac (Fig. 1.8A). The

PBD overlaps with an autoinhibitory domain (AID) that controls the basal catalytic activity of PAK-1 [74, 79]. In addition, there exists a for the G subunit complex of heterotrimeric G proteins at the C terminal region (Fig. 1.8A) [80].

At the basal condition, PAK-1 exists in a trans-inhibited homodimeric conformation, where the N-terminal regulatory domain of one PAK-1 binds and inhibits the C-terminal catalytic domain of the other (Fig. 1.8B). The binding of activated GTPases such as Cdc42 and Rac interrupts PAK-1 dimerization, and triggers conformational changes that destabilize the interaction between the AID and the catalytic domain, leading to autophosphorylation events required for PAK-1 full catalytic activity (Fig. 1.8B) [81]. Seven autophosphorylation sites have been identified in activated PAK-1, Ser21, Ser57, Ser144, Ser149, Ser199, Ser204 and

Thr423 [82]. Among these phosphorylation sites, the Thr423 residue was shown to be critical in sustaining PAK-1 in the autoinhibition-free conformation [81]. The

24

activation of PAK-1 can be triggered in a GTPase-independent manner as well.

Both PDK-1 and Akt/PKB are able to phosphorylate and activate PAK-1 (Fig. 1.8A)

[83, 84]. PDK-1 was shown to phosphorylate PAK-1 within its catalytic domain, at

Thr423 [83], which is important for full catalytic activity of PAK-1. Akt/PKB, on the other hand, targets PAK-1 between its first two SH3-binding motifs, and phosphorylates PAK-1 at Ser21. This modification is associated with reduced binding of PAK-1 to adaptor protein Nck, and an increase in PAK-1 mediated cell migration [84].

PAK-1 has a host of biological functions including cytoskeletal organization and regulation, cell motility, neurogenesis, angiogenesis and has also been implicated in metastatic neoplasia [74]. More than 30 PAK-1 substrates have been identified to date. Some of the better characterized substrates include MLCK

( -chain kinase), LIMK (LIM kinase), Bad, Raf and MEK1 (Fig. 1.9).

MLCK and LIMK both play important roles in actin polymerization, and actin and mysin interaction, which are events required for lamellipodia and filopodia formation, thus implicating PAK-1 function in the regulation of cytoskeletal dynamics and cell motility. Raf and MEK are both components of the MAPK signaling pathway, regulating gene expression and promote cell growth and proliferation (described in detail in the following section, Section 1.1.4). It has been reported that PAK-1 phosphorylates Raf-1 on Ser338 and induces its catalytic activity [85, 86]. In addition, PAK-1 enhances the interaction between Raf-1 and its substrate MEK1 by phosphorylating MEK1 on Ser298 [87]. On the other hand, PAK-1 phosphorylates and inhibits proapoptotic factor Bad at Ser112 and Ser136. PAK-1 phosphorylates

25

Rho PDK1 GTPases AKT/PKB

PAK-1

LIMK Raf NFkB Bad

Cofilin MEK

Cell Motility Gene Regulation Cell Survival

Figure 1. 9 - PAK-1 regulated signaling pathways.

A diagrammatic representation of a simplified version of PAK-1 signaling pathways. PAK-1 is activated by a variety of signals include Rho family GTPases, PDK1 and AKT/PKB. Activated PAK- 1 participates in a number of signaling cascades leading to cytoskeletal reorganization, gene transcription regulation and cell survival. PAK: p21-activated kinase; AKT/PKB: AKT/Protein Kinase B; PDK1: pyruvate dehydrogenase kinase, isozyme 1; LIMK: LIM kinase; MEK: mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; NFkB: nuclear factor kappa B; Bad: Bcl- xL/Bcl-2-associated death promoter;

26

and activates NIK (NFkB-inducing kinase, leading to the stimulation of NFkB

(nuclear factor kB). Both of these events have antiapoptotic effects, suggesting that

PAK-1 has an imperative role in regulating cell growth and survival. This is further supported by the observation that the expression of kinase-deficient PAK-1 was shown to prevent Ras-mediated cell transformation [88]. Consistently, PAK-1 was able to synergize with Ras signaling to promote transformation [89, 90].

1.1.4 Insulin Action via Ras-mitogen-activated protein kinase (MAPK)

Insulin signaling has been implicated in mitosis, differentiation, cell apoptosis and survival. These insulin effects can be largely attributed to the Ras-MAPK pathway, another major signaling cascade downstream of IR/IRS. MAPK pathway is organized in a way that a G-protein works upstream of three tiers of kinases, a

MAPK kinase kinase (MAPKKK) which activates a MAPK kinase (MAPKK), that further activates MAPK (Fig. 1.10) [91, 92]. The MAPK components relevant to insulin signaling are Ras as the G-protein, Raf as the MAPKKK, mitogen/extracellular-signal regulated kinase kinase (MEK) as the MAPKK and extra cellular-signal regulated kinase (ERK) as the MAPK [27, 93].

1.1.4.1 Activation of Ras GTPases

Guanine nucleotide binding protein Ras belongs to a family of membrane- associated monomeric small GTPases, which are converted to its activated GTP- bound form from its GDP-bound form, post IR activation [93]. As it has been described in Section 1.1.2, binding of insulin molecules to IR triggers tyrosine phosphorylation of IRS and subsequent production of PIP3, which are both capable

27

Stimulus Insulin Grb/SOS G-protein Ras

MAPKKK Raf

MAPKK MEK1/2

MAPK ERK1/2

Biological Cell Growth Responses

Figure 1. 10 - Hierarchical organization of the MAPK pathway.

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of binding to SH2 domain containing signaling molecules. Growth factor receptor- bound protein 2 (Grb2) is an adaptor protein that contains an SH2 domain that allows its binding to IRS or PIP3 [94]. Grb2 is also constitutively associated with guanine nucleotide exchange factor (GEF) (SOS) via its N terminal

SH3 domain, bringing SOS in the same locale as Ras [95]. SOS then activates Ras

GTPase in a two-step manner. In quiescent cells, SOS exists at an autoinhibited state. However, its interaction with Ras-GDP induces the catalytic site of SOS to have low GEF activity and generate a small number of active Ras-GTP [96]. Ras-

GTP has much higher affinity for SOS and relieves the steric occlusion of its catalytic site and fully activates SOS [97, 98].

1.1.4.2 Activation of Raf Family Serine/Threonine Kinases

Ras-GTP interacts with and activates Raf family serine/threonine kinases, A-

Raf, B-Raf and the most extensively studied C-Raf, also known as Raf-1 [93, 99].

There are three conserved regions (CRs) in Raf kinases, CR1, CR2 and CR3 [100].

CR1 contains the Ras binding domain (RBD) and a cysteine rich domain (CRD) that are both responsible for the interaction between Ras and Raf as well as the subsequent membrane recruitment of Raf [100-102]. In unstimulated conditions, Raf proteins exist in an inactive form in the cytosol. Its N terminal domain acts as an autoinhibitor of its C terminal catalytic domain. This inhibitory conformation is then stabilized by a 14-3-3 dimer complex that binds to phosphorylation sites on both N and C terminal regions of Raf. Ras-GTP binds Raf proteins and triggers not only the recruitment of Raf to plasma membrane, but also the release of 14-3-3 from the N terminal region of Raf and induces its phosphorylation and activation [95]. For

29

example, Ras-GTP induces the activation of C-Raf by phosphorylating it on residues

S338, Y341, T491 and S494 [99].

1.1.4.3 Activation of MEK/Erk

Activated Raf proteins initiate a cascade of phosphorylation events. Raf activates MEKs through the phosphorylation of Ser217/218 on MEK1 and Ser221 on

MEK2, and MEKs subsequently activate ERKs. MEKs are 43-46 kDa proteins belonging to a superfamily of dual-specific kinases, that share the ability to phosphorylate ERKs on both is threonine and tyrosine residues. MEK1 and MEK2 activate ERK1 and ERK2 through phosphorylation of Thr183 and Tyr185, respectively [94, 103]. MEK1/2 contains a nuclear export signal (NES) sequence in its N terminal domain that tethers them to the cytoplasm. Via binding interaction with

MEK1/2, ERK1/2 is also mostly localized to the cytoplasm in quiescent state [104].

ERK1 and ERK2 are 44- and 42kDa ubiquitously expressed serine threonine kinases, and share 83% amino acid sequence identity with each other. Insulin stimulated phosphorylation and activation of ERK leads to its dissociation from MEK, and translocates to the nucleus. ERK has been shown to translocate into the nucleus by (i) simple passive diffusion [105], (ii) active transport by importin- family proteins and low molecular weight GTPase Ran [106] or (iii) directly bind with the nuclear pore complex [107]. Subsequently, activated ERK targets over 50 substrates, some of which are key transcription factors, including activator protein 1

(AP-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), Myc, kinases including ribosomal s6 kinase (RSK), B-cell lymphoma 2 (Bcl-2), and cytoskeletal scaffold proteins such as paxillin [108]. The activities of these ERK

30

substrates then achieve the effects of insulin mediated cell growth, differentiation, cytoskeletal rearrangements and etc [93].

1.1.4.4 Regulation of MAPK Pathway

The three-tiered MAPK pathway can be regulated positively and negatively.

Protein phosphatase 1 (PP1) and PP2A are both able to dephosphorylate 14-3-3 binding sites located in the Raf N terminal region, thereby promoting Ras interaction, and subsequent Raf membrane recruitment and activation [109-111]. Other regulators of the MAPK pathway are scaffold proteins that act as docking platforms to colocalize with ERK components to enhance its activation. For example, suppressor of ras-8 (Sur-8) augments Raf activation by increasing the interaction between Ras and Raf [112]. On the other hand, kinase suppressor of ras-1 (KSR1) facilitates MEK/ERK activation in a more complicated manner. Without stimulation,

KSR1 interacts with 14-3-3, impedes mitogenic signal propagation (IMP), MEK1/2 and PP2A. The interaction with 14-3-3 masks the cystein-rich C1 domain of KSR1 necessary for membrane targeting, hence sequesters KSR1 from the plasma membrane [113]. IMP also deters KSR1 from the plasma membrane by mislocalizing it to a detergent-insoluble cellular compartment [114]. Upon IR activation, not only is IMP released from KSR1, C1 domain also becomes exposed, leading to the translocation of KSR1 to the membrane [114]. Membrane-associated

Ras then catalyzes the dephosphorylation of 14-3-3 binding sites on KSR1 leading to the dissociation of 14-3-3. This not only exposes the C1 membrane targeting domain of KSR1, but also the docking sites for ERK1/2, colocalizing MEK with ERK

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as well as other upstream MAPK signaling components, including Ras and Raf [115,

116].

Negative regulators of MAPK pathway operate by interfering with critical interactions between signaling components of the MAPK pathway. For example,

Erbin sterically blocks the interaction between Ras and Raf by binding with Ras

[117]. Sprouty and Sprouty-related protein with EVH1 domains (SPRED) both interact with Raf at its catalytic domain, and interferes with the phosphorylation of

Raf activating residues [118, 119]. Alternatively, Raf kinase inhibitor protein (RKIP) interacts with the kinase domain of MEK and Raf, and prevents the interaction between Raf and MEK that is required for MAPK activation [120].

1.1.5 Implication of Insulin Signaling in Cancer

Insulin signaling transduction is imperative in various metabolic and mitogenic processes. Due to the diversity of cellular events initiated by insulin, insulin signaling is tightly controlled by either its internal negative feedback system or by other negative regulators that hinder its effects at various stages described in previous sections. However, if the delicate balance between these positive and negative regulators of insulin signaling is impaired, it can lead to the development of diabetes as well as various tumors. The link between insulin signaling and diabetes has been extensively studied and characterized, therefore this section will focus on the relationship between insulin signaling and cancer development.

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1.1.5.1 Ras/MAPK Signaling-Mediated Cell Transformation

Various components of the aforementioned major pathways of insulin signaling have been implicated in human neoplasm. For example, constitutive activation of the Ras/MAPK pathway is observed in almost all cases of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL), and elevated ERK expression in AMLs and ALLs is correlated with poor prognoses [121, 122].

Approximately 30% of all human cancers contain either amplification of the ras proto-oncogenes and/or the expression of constitutively-active Ras proteins [123,

124]. These observations are not surprising as a large number of downstream effectors of the Ras/MAPK pathway alter expression of molecules that regulate progression and apoptosis [125-127]. Ras-GTP activated Raf serine/threonine kinases are involved in processes that promote cell transformation. B-Raf was found to be frequently mutated in colorectal cancer, ovarian cancer and papillary thyroid cancer [128]. In mouse embryonic fibroblast cells NIH-3T3, over-expression of A-Raf was found to increase the expression of cyclin D1, cyclin E, Cdk1 and Cdk4 and decrease the expression of Cdk inhibitor p27 [129]. This combination of changes drives cells to shift from G1 phase to enter S phase, promoting cell cycle progression and hence cell proliferation. Ras/MAPK also targets components of apoptosis pathway, inactivating B-cell lymphoma/leukemia 2 (Bcl-2)/Basal cell lymphoma-extra large (Bcl-xl)-associated death promoter (Bad) by Ser112 phosphorylation [130]. This relieves the constraint Bad holds on Bcl-2, allowing Bcl-

2 homodimer formation to occur, generating an anti-apoptotic effect. Another pro- apoptotic factor, Bcl-2 interacting mediator of cell death (Bim) is phosphorylated on

33

the Ser69 position by ERK, resulting in Bim ubiquitination and its subsequent proteosomal degradation [131]. Caspase 9, yet another pro-apoptotic protein, is phosphorylated on residue Thr125, and inactivated by Ras/MAPK signaling [132].

Not only can the Ras/Raf/MEK/ERK pathway bias cells towards survival and proliferation by directly targeting cell cycle regulators and cell apoptosis players, it also induces an autocrine effect by upregulating the expression of growth factors, which then further enhances its oncogenic effects. The promoter region of many genes encoding growth factors contain binding sites for Ras/MAPK-downstream transcription factors, suggesting a plausible mechanism for Ras/MAPK-instigated expression of various growth factors [133]. For example, over-expression of Raf in

NIH-3T3 cells induces the secretion of heparin binding epidermal growth factor

(hbEGF) [134]. Overexpression of Raf in hematopoietic cells triggers the expression of granulocyte macrophage-colony stimulating factor (GM-CSF) [127, 135]. In addition, elevated expression of B-Raf in Kaposi’s sarcoma transformed B cells is associated with an induction in vascular endothelial growth factor (VEGF) [136].

Lastly, Ras/MAPK pathway induces membrane translocation and activation of the p110 subunit of PI3K, promoting cell survival and proliferation even further by crosstalking with the PI3K/Akt pathway [95].

1.1.5.2 PI3K/Akt Signaling-Mediated Cell Transformation

There is ample evidence that ties the PI3K/Akt pathway with oncogenesis.

This link was first observed by studying transforming viruses in animals. It was reported that the cancer-causing avian sarcoma virus 16 (ASV16) in chickens encodes an oncogene derived from the cellular gene of catalytic subunit of PI3K

34

[137]. In mice, it was found that murine lymphoma virus Akt8 induces thymic lymphomas in mice via cell-derived viral oncogene Akt/PKB [138]. PI3K/Akt signaling is implicated in human cancers as well. of PIK3CA, the gene that encodes the p110 catalytic subunit of PI3K were frequently observed in colorectal cancers (32%) [139], breast cancers (18-40%) [140-142] and ovarian cancers (4-12%) [143, 144]. These mutations are usually non-synonymous missense mutations that often occur in conserved regions of PIK3CA that encode the kinase domain of the protein, conferring PI3K constitutive kinase activity [145,

146]. Amplification of the p110 locus of PIK3CA was also observed in various primary tumors, cancer cell lines and carcinomas [147-149]. Akt/PKB gene amplification has also been reported in a number of human cancers. It has been identified recently that a somatic missense of Akt1 in its PH domain (E17K) is present in colorectal, breast and ovarian cancers [150]. Furthermore, this mutation is sufficient to transform cells in vitro by allowing Akt/PKB to constitutively associate with the plasma membrane, which enhances Akt/PKB activation. Aberrant expression of negative regulators of the PI3K/Akt pathway can have tumourogenic effects as well. The tumour suppressor PTEN, which impedes the activity of second messenger PIP3 by dephosphorylating it back to PIP2 [27], was mutated or lost in both spontaneous and heritable cancers [151]. PTEN missense mutations are commonly observed in colorectal cancers (9%), endometrial cancers (39%), prostate cancers (14%) and breast cancers (6%). While homozygous PTEN knockout in mice results in embryonic lethality, PTEN+/- animals exhibit an increased incidence for tumor development, displaying neoplasms and hyperproliferative lesions in

35

multiple tissues [152-154]. Loss of PTEN expression is also implicated in the metastatic potential of certain tumors. PTEN-deficiency has been shown to activate

Rac1 and Cdc42 and small G proteins involved in cytoskeletal organization events that results in altered cell adhesion properties, and increased cellular migration and invasive capability [155-157]. Interestingly, similar to Ras/MAPK pathway, PI3K/Akt pathway targets many key regulators of the cell cycle and cell apoptosis pathways, including Bad, Bim, and procaspase-9. In order for cells to undergo G1 progression, cyclin-dependent kinase (Cdk) must be activated; this requires cyclin D1 [158].

Under quiescent conditions, glycogen synthase kinase 3  (GSK-3) phosphorylates cyclin D1 at Thr286 and targets it for ubiquitin-mediated proteosomal degradation

[159]. Insulin activated Akt/PKB phosphorylates and inhibits GSK-3, which leads to the stabilization of cyclin D1, and thus facilitates cell cycle progression. Akt/PKB can also phosphorylate and inhibit forkhead family of transcription factors AFX, FKHR and FKHR-, which then leads to reduced expression of Cdk inhibitors such as p27kip1 and p21cip1, thus promoting cell cycle progression and cell proliferation

[160-164].

1.1.5.3 Implication of Hyperinsulinemia in Colorectal Cancer – Evidence from Epidemiological and Animal Studies

Hyperinsulinemia is a consistent risk factor for both colorectal cancer (CRC) and adenoma. The relationship between hyperinsulinemia and risks of colorectal cancer or adenoma has been examined in a number of epidemiological studies.

One of the first reports was a cohort study which showed that while fasting insulin levels were not associated with increased risk or CRC, insulin levels after 2h oral

36

glucose challenge were correlated with a 2-fold increase in CRC [165]. Another method to assess hyperinsulinemia is to measure C-peptide levels [166], since it is the byproduct of insulin secretion. In a prospective study of women in New York, subjects with higher C-peptide levels were shown to be 3 times more likely in developing CRC; and when examining colon cancer alone, the risk is almost 4 times higher [167]. These observations were consistent with a nested case-control study in the Physicians’ Health Study, which showed that an increase in plasma C-peptide concentration was associated with a 2.7-fold increase in relation to CRC development [168]. Prior to the pathological transformation of colorectal cancer

(CRC), colonic epithelial cells undergo an intermediary stage and become precursor lesions, known as colorectal adenomas. There are also studies that examined insulin or C-peptide levels in relation to colorectal adenoma risks. In a case-control study carried out in patients undergoing routine colonoscopy at the University of

North Carolina hospitals, it was found that subjects with the highest quartile of insulin levels are 2.2 times more likely of having colorectal adenoma; and this is correlated with decreased apoptosis in normal rectal mucosa [169]. These observations suggest that insulin may play a role in the early stage of adenoma-carcinoma transformation to favor growth and development of colorectal adenoma by hindering normal mucosa apoptosis.

Insulin resistance and hyperinsulinemia are associated with a number of metabolic consequences; therefore epidemiological data alone may not tease out the critical factor for CRC risks. Studies using rodent models suggest that hyperinsulinemia may be the more important factor. A study examined

37

azoxymethane-induced colonic tumours formed post 17 weeks of insulin injections in rats. It was shown that rats that received insulin injections had significantly higher incidence of colonic tumor formation, and those rats that developed colonic tumors have higher number of tumors detected than those that received control saline injections [170]. Another study examined the proliferation of colorectal epithelial cells after infusing rats with intravenous (IV) insulin during a 10 h euglycemic clamp, and found that insulin infusion induced the proliferation of colorectal epithelial cells in a dose-dependent manner [171]. More interestingly, the addition of hyperglycemia to insulin infusion did not cause a further increase, and intralipid infusion did not increase proliferation of colorectal epithelial cells at all [171]. In accordance to the previous study, in the Physicians’ Health Study, C-peptide level was most strongly correlated with risks of colon cancer compared to other components of the metabolic syndrome, including high triglycerides, low plasma HDL cholesterol and high BMI

[168]. Evidence from these studies suggests that among the different metabolic components, hyperinsulinemia, rather than hyperglycemia or hyperglyceridemia is most likely the primary risk factor for promoting carcinogenesis.

Early stage of Type 2 Diabetes Mellitus is characterized by impaired glucose tolerance and compensatory hyperinsulinemia in response to insulin resistance/insensitivity. Using data from the Second National Health and Nutrition

Examination Survey and the Second National Health and Nutrition Examination

Survey Mortality Study, it was found that individuals experiencing impaired glucose tolerance but without diabetes (hyperinsulinemic) had higher mortality from colon cancer [172]. To examine a more direct role of hyperinsulinemia in Type 2 Diabetic

38

patients in relation to CRC risks, a retrospective cohort study was carried out in patients diagnosed with Type 2 Diabetes Mellitus in the General Practice Research

Database from the United Kingdom [173]. These Type 2 Diabetic patients had a 21% increase in CRC risk for each incremental year they had undergone insulin therapy

[173].

CRC is the second most common type of cancer, next to breast cancer for women and prostate cancer for men. Extensive etiological and pathological studies have led to the recognition of the effect of aberrant expression and function of the

Wnt signaling pathway in CRC formation and development. The following sections will introduce the Wnt signaling pathway and its pivotal role in colorectal neoplasia.

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1.2 Wnt Signaling

Wnt proteins were originally known as Wingless (wg) in Drosophila [174, 175] and the MMTV proto-oncogene (int-1) in mice [176, 177]. After discovering that these are homologous proteins, they were then named Wnts. Wnt signaling has a pivotal role during embryogenesis, adult tissue homeostasis and tumorigenesis [178].

The following sections will introduce and explore the mechanisms of Wnt action as well as various cellular players that are involved.

1.2.1 Initiation of Wnt Signaling

1.2.1.1 Wnt Ligand Processing and Secretion

Wnts are secreted glycoproteins encoded by a family of evolutionarily conserved genes. There are currently 19 Wnt homologs that have been identified in , and membership in this family is defined by amino acid sequence rather than functional properties [178]. Wnt signaling pathways generally categorized into the canonical or non-canonical pathways. Canonical Wnt signaling involves beta- catenin (-cat) as its mediator, and other Wnt signaling pathways not involving -cat are categorized as non-canonical Wnt pathways. Wnt glycoproteins involved in the

Wnt/-cat pathway consist of Wnt 1, 3A, 8 and 8B. Non-canonical Wnts consist of

Wnt 4, 5A and 11 and transduces signals via cat independent pathways [179]. All

Wnts share a signal sequence for secretion, many potential glycosylation sites and several highly charged amino acid residues. There is a characteristic distribution of

22 cysteine residues in all Wnt proteins with highly conserved spacing, suggesting multiple intramolecular disulfide bonds are involved in proper folding of Wnt proteins

[180].

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Examination of the primary sequence of Wnts would indicate these proteins would be adequately soluble because it contains several charged residues, suggesting hydrophilic properties. Surprisingly, secreted Wnts are rather hydrophobic and are often associated with the cell membrane and extra cellular matrix (ECM) [181]. When over-expressed in cells, N-linked glycosylated intermediate Wnt protein products were observed in cells, suggesting that Wnt proteins undergo modification after being synthesized [180-182]. It was later revealed that Wnt proteins undergo lipid modification by the addition of a palmitate moiety on a conserved cysteine residue [183]. Furthermore, this lipid modification is necessary for their functions, as enzymatic removal of the palmitate moiety renders

Wnt signaling inactive [183, 184]. Palmitoylation of Wnt proteins may facilitate Wnt signaling in several ways. One possible mechanism is by targeting Wnts to areas of the plasma membrane comprised of molecules that serve as platforms for and cell activation, such as sphigolipids and cholesterol [185]. Lipid modification has also been shown to be necessary for Wnt protein N-linked glycosylation, by anchoring Wnts to the endoplasmic reticulum (ER) where oligosaccharyl complex resides [182-184]. Glycosylation encourages the transportation of Wnt molecules between cells by enhancing its interaction with heparin sulfate proteoglycans (HSPGs) expressed on the surface of Wnt responding cells. Lastly, studies have shown that mutation of the residue required for lipid modification significantly reduces the interaction between Wnt and its receptor,

Frizzled, thus suggests that palmitate moiety may potentially facilitate and sustain

Wnt signaling by stabilizing ligand-receptor interaction with Frizzled CRD [186, 187].

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Multipass transmembrane ER protein Porcupine also plays a role in Wnt protein glycosylation. Drosophila cells with porcupine mutation fail to release Wg proteins from its association with the plasma membrane and results in the inability of Wg secretion [188]. Porcupine protein is similar to O-acyltransferase enzyme in sequence and is purported to lipid-modify Wnt proteins while in transit in the ER

[189]. Genetic screens have revealed the multipass transmembrane protein Wntless

(Wls)/Evenness interrupted (Evi)/Mom-3 to be involved in the process of Wnt protein secretion. Wnt proteins fail to be transported to the cell surface in cells deficient of

Wls/Evi/Mom-3, resulting in associated lack of Wnt secretion into media [190].

1.2.1.2 Extracellular Transport of Wnt Proteins

Wnts are secreted glycoproteins that have the capability of acting in distant and proximate regions [191, 192]. This characteristic entails that Wnt protein must somehow navigate through the aqueous extracellular milieu to reach its long-range target despite being hydrophobic in nature. Glycosaminoglycan (GAG)-modified proteins have been shown to facilitate the movement of Wnts through direct interactions. Treatment of cells with a sulfated GAG such as heparin results in an increase in Wnt signaling [181]. Consistently, in Drosophila, deficiency in genes required for the biosynthesis of heparin sulfate GAGs leads to defects in Wnt signaling [193, 194]. It was later shown that it is specifically the glypican family of the heparin sulfate GAG that facilitates the extracellular movements of Wnts.

Glypicans are integral membrane proteins bound to the plasma membrane via a glycophosphatidylinositol (gpi) chain. By acting as low-affinity binding receptors, glypicans docks and stabilize Wnt proteins in neighboring receptive cells [195]. On

42

the other hand, glypican’s plasma membrane linker gpi has been shown to associate with lipoprotein particles that have been implicated in lipid transport. Furthermore, in

Wg-producing cells, gpi co-localize with and spreads into surrounding receiving tissue at a comparable rate as Wg protein [196]. More importantly, Lipophorin knockdown in Drosophila reduced the range of Wg activity [197]. These observations suggest glypicans may also facilitate Wnt to reach its target cell receptors in long ranges.

1.2.1.3 Serpentine Receptor Frizzled (Fzd)

Frizzled proteins are localized to the surface of Wnt-responsive cells and are widely and dynamically expressed in tissues [198]. This Wnt receptor was first identified in Drosophila in a mutation screen for disruption in adult fly epidermal cell polarity [199, 200]. At least ten frizzleds (FZDs) have been found in humans (FZD1

–10), all of which encode receptors for Wnt proteins, thus further augments the complexity of Wnt signaling [201]. Frizzled proteins are between 500 to 700 amino acids in length and contain a cysteine-rich domain (CRD) at its N terminus that is extracellular. One of the characteristics of frizzled proteins is their seven hydrophobic domains that allow them to form transmembrane -helices [198]. FZD extracellular CRD contains ten conserved cysteine residues that form disulphide bonds, and is necessary for binding to Wnt ligands [187, 202]. The carboxyl terminus contains an intracellular KTXXXW motif that is highly conserved in

Frizzleds. This motif has been postulated to mediate the interaction between

Frizzled and the intracellular Wnt signaling transducer Dishevelled (Dsh/Dvl)

(discussed in Section 1.2.3.1) [203]. Frizzled also interacts with Lrp5/6, a co-

43

receptor required for the successful activation of the canonical Wnt signaling [204]

(details in Section 1.2.3).

1.2.2 Non-canonical Wnt Signaling Pathways

Wnt ligand-receptor interaction may lead to the commencement of three distinct signaling pathways, namely the planar cell polarity (PCP)-convergent extension pathway, the Wnt/calcium pathway, and the Wnt/-catenin pathway. The

Wnt/catenin pathway is known as the canonical Wnt pathway, and PCP- convergent extension pathway and Wnt/calcium pathway are known as the non- canonical Wnt pathways. Wnt signaling initiates after Wnts bind with Fzd via its

CRD region, and Dishevelled (Dvl) is suggested to act as an intracellular transducer for both canonical and non-canonical Wnt signaling (Fig. 1.11) [179]. However the exact mechanism by which Frizzled proteins transduce this signal to downstream targets remains unclear.

1.2.2.1 Planar Cell Polarity (PCP) – Convergent Extension Pathway

Non-canonical or -catenin-independent Wnt pathways were first discovered by the observation that frizzled signaling is required for the proper placement and orientation of the bristle in Drosophila, a highly regulated process known as planar cell polarity (PCP) [199]. Mammalian cells utilize a parallel process to establish a common axis of orientation in a column of cells during development, known as convergent extension [205, 206]. Dsh/Dvl becomes activated in the PCP-

Convergent extension pathway, leading to the activation of small G proteins (Rho or

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Wnt Signaling Pathways

Canonical Non-Canonical

Wnt/-cat PCP Wnt/Calcium

Fzd LRP Fzd Fzd

Dvl Dvl Dvl

GSK-3 Rho Rac Ca2+ PKC

-cat ROCK JNK CamKII

Gene Transcription Cell polarity, migration, Cell adhesion, movements; structural rearrangement inhibition of Wnt/-cat pathway

Figure 1. 11 - Wnt Signaling Pathways.

A diagrammatic representation of the canonical and non-canonical Wnt signaling pathways. PCP: planar cell polarity; Fzd: frizzled; LRP: low density lipoprotein receptor-related protein; Dvl: disheveled; GSK-3: glycogen synthase kinase 3; ROCK: Rho kinase; JNK: c-Jun N-terminal kinase; PKC: protein kinase C; CamKII: calcium/calmodulin-dependent protein kinase II;

45

Rac), and Rho kinase or c-Jun amino-terminal kinase (JNK), resulting changes in cell polarity and morphogenetic movements (Fig. 1.11).

1.2.2.2 Wnt/Ca2+ Pathway

The Wnt/Ca2+ pathway was first recognized by observing that over-expression of Wnt5a or Wnt11 in Xenopus embryos triggered intracellular calcium release, and activated the calcium-sensitive kinase protein kinase C (PKC) [207] and calcium/calmodulin-dependent kinase II (CamKII) (Fig. 1.11) [208]. Elevated intracellular calcium concentration leads to the activation of phosphatase , which dephosphorylates transcription factor nuclear factor of activated T cells (NF-

AT). This triggers NF-AT to translocate into the nucleus and activates target genes

[209, 210]. These changes ultimately lead to modification of cell adhesion and cell motility, and interestingly may also result in the inhibition of Wnt/-catenin signaling

[211, 212]. Dvl has been implicated in the activation of Wnt/Ca2+ pathway because expression of full-length Dvl exerted a modest stimulatory effect on calcium flux,

PKC and CamKII activation [213]. Studies have also implicated G proteins in Wnt stimulated Ca2+ release, even though there is still a lack of evidence showing direct interaction between G proteins and Frizzleds [214-216].

1.2.3 Canonical Wnt Signaling Pathway

Similar to the aforementioned non-canonical Wnt signaling, initiation of the canonical Wnt pathway also involves the interaction between Wnt proteins and Fzd receptors. However, unlike the non-canonical Wnt pathways, successful activation of the canonical Wnt signaling requires single-span transmembrane proteins low-

46

- cat -cat

sFRP

Wnt WIF Dkk

Cadherin Frz Frz

Krm

LPR5/6

 -

-cat cat P P P P -cat CKIe P GSK-3 P P P E3 Ub Ub Ub Dvl P E2 Ub APC Axin -cat E1 P P -cat CKI -cat -cat Degradation

Cytoplasm -cat

Nucleus CBP HBP1 -cat Pygo Tcf/Lef Tcf/Lef Groucho

Transcription Transcription Myc cyclin D1 Fra-1 MMP-7

Figure 1. 12 - Canonical Wnt signaling pathway.

A diagrammatic representation of the mechanisms involved in the regulation of -cat in the presence and absence of Wnt ligands. Frz: frizzled receptor; LRP5/6: low density lipoprotein receptor-related protein 5/6; Krm: Kremen; Dkk: Dickkopf; -cat: beta-catenin; -cat: alpha-catenin; E1: ubiquitin-activating enzyme; E2: ubiquitin-conjugating enzyme; E3: ubiqutin ligase; GSK-3: glycogen synthase kinase 3; APC: adenomatous polyposis coli; Dvl: disheveled; CKIa: casein kinase I alpha; CKIe: casein kinase I epsilon; Tcf/Lef: T-cell factor/lymphoid enhancer factor; HBP1: HMG-box transcription factor 1; CBP: CREB binding protein; Pygo: pygopus;

47

density-lipoprotein receptor related proteins 5 and 6 (LRP5/6) in vertebrates or their

Drosophila ortholog Arrow [217-219] (Fig. 1.11 and 1.12). The instigation of canonical Wnt signaling ultimately leads to the stabilization and accumulation of - catenin (-cat) by inactivating the “destruction complex” composed of Axin-APC-

GSK-3, which is responsible for phosphorylation-mediated cat proteosomal degradation. However, the exact mechanism for transducing extracellular Wnt signals from the receptor level to eventually the inactivation of the “destruction complex” is still being debated. The currently accepted dogma is that Dvl serves as an intermediary carrier for transducing signals from the receptor to the intracellular players of the pathway [220, 221].

1.2.3.1 Transducing Wnt Signals from Receptor to cat

The implication of LRP5/6 in Wnt signaling was first recognized through genetic studies. Loss of arrow in Drosophila gives rise to a phenotype similar to the wg mutant [219]. Consistently, mice harboring Lrp6 deletion display a combination of phenotypes analogous to mutations of several Wnt genes [217]. A genetic mouse study implicated Lrp5 in Wnt signaling as well [222]. Lrp6 and Arrow appear to be specifically required for canonical Wnt signaling. For example, while Fzd1 plays a critical role in Drosophila PCP determination, Drosophila arrow mutants exhibit normal PCP. This was also confirmed in Xenopus, where inhibition of Lrp6 had inconsequential effect on gastrulation movements which are orchestrated by signaling events comparable to Drosophila PCP signaling [223].

The possibility of Lrp5/6 serving as a Wnt co-receptor came from two lines of evidence. Firstly, co-immunopricipitation (co-IP) experiments showed that Lrp5/6

48

interacts with a number of Wnt proteins, including Wnt1, Wnt3a, Wnt4 and Xenopus

Wnt8 [218, 224-227]. In addition, expressing a secreted form of Lrp5 (containing only the extracellular domain) inhibited Wnt signaling, possibly by acting as Wnt ligand binding competitors [224, 228]. The second line of evidence lies in the ability of Lrp5/6 in binding Fzds. In vitro studies have shown that the extracellular domains of Lrp5/6 are capable of associating with the CRD of mouse Fzd8 in a Wnt- dependent manner [218, 223], suggesting that Wnt may trigger a complex formation between Fzd and Lrp5/6. In fact, Lrp6 mutants lacking the intracellular portion blocks Wnt signaling, whereas mutants lacking the extracellular domain activates

Wnt/cat signaling constitutively [224, 227, 229].

Wnt activation leads to the phosphorylation of Lrp6 and promotes its interaction with a member of the “destruction complex” Axin, and its subsequent recruitment to the plasma membrane [218, 230]. Lrp6-Axin interaction promotes

Axin degradation and thus the activity of the “destruction complex”, preventing cat degradation and promoting Wnt signaling transduction [224]. On the other hand, scaffolding protein Dsh/Dvl also binds with and inhibits Axin [231, 232], and is recruited to the plasma membrane by associating with the intracellular region of Fzd receptor [233].

1.2.3.2 Regulation of Cytoplasmic cat

cat was originally discovered as a segment polarity protein in Drosophila known as armadillo [234]. The cat protein consists of a phosphorylation site-rich domain at its N terminus, followed by 13 repeat units called armadillo repeats, with each repeat consisting of 42 amino acids organized in a dense alpha helical array

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[235]. The function of cat is largely dependent on its subcellular localization.

Normally, cat can be found in three intracellular locations, mostly at the plasma membrane, some in the cytoplasm and in small amounts in the nucleus. In fact, early descriptions of cat and its homologue (-catenin) focused on its role as part of the cell [236, 237]. A large pool of cat is localized to the plasma membrane in association with cadherins, -catenin and the actin cytoskeleton, thus it participates in the establishment of cell-cell adhesions and tight junctions [238, 239] (Fig. 1.12). Cytoplasmic cat is constantly subjected to regulation via the ubiquitin-mediated proteosomal pathway. Ubiquitination involves concerted actions of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein . E3 ubiquitin ligases can exist as multi- protein complexes and are responsible for recognizing and bringing together specific substrate proteins and E2 ubiquitin-conjugating enzymes [240]. The first step of ubiquitination involves the transfer of ubiquitin from E1 to an E2 enzyme in an ATP dependent manner. The E2 enzyme subsequently transfers ubiquitin onto substrate protein recognized specifically by the E3 complex [240]. In the case of cat, it is targeted for ubiquitination post a series of phosphorylation events catalyzed by the

“destruction complex”, consisting of GSK-3, Axin and adenomatosis polyposis coli

(APC) [241]. In quiescent condition, cat binds to the destruction complex by interacting with Axin and APC via its armadillo repeats domain, and is first phosphorylated by casein kinase I  (CKI) at its Ser45 position [242-244]. This primes cat to become subsequently phosphorylated by GSK-3 at Thr41, Ser37 and Ser33 [242, 245]. In humans, phosphorylated cat is recognized by a specific

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multimeric E3 complex consisting of the F-box protein -transducin repeat- containing protein (h-TrCP), Skp1 and Cul1, resulting in ubiquitination and proteosomal degradation of cat [246].

In the presence of Wnt ligand-receptor interaction, CKIe is activated and phosphorylates Dvl and enhances the binding of Dvl to Frat-1 (frequently rearranged in advanced T-cell lymphomas 1) [247, 248]. Since Dvl is able to interact with Axin via its DIX domain, this brings Frat-1 to the “destruction complex” as well [249, 250].

Frat-1 then binds to GSK-3 and interferes with its ability to phosphorylate cat, thereby reducing its degradation [247-249]. However, Axin acts also as a negative regulator of cat in the context of the “destruction complex”. When in complex with

GSK-3 and APC, Axin is phosphorylated and stabilized by GSK-3 which promotes

cat degradation [251].

Axin and APC may facilitate cat degradation by acting as scaffold proteins.

Evident in colon cancers, mutation in the APC gene is correlated with high levels of

cat and elevated transcriptional activity of cat/Tcf [252]. However, evidence suggested that this effect appears to be dependent on the interaction between APC and Axin, as APC with mutations in its Axin-binding site fails to induce cat degradation [253]. GSK-3 is able to phosphorylate APC more efficiently when in complex with Axin, which further enhances the binding of cat to both APC and

Axin, thus facilitating cat phosphorylation by GSK-3[254].

Protein kinase A (PKA) is capable of phosphorylating cat and may act as both a positive and negative regulator of cat stabilization. In the presence of the

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protein product of Alzheimer’s disease-linked gene presenilin1, a complex comprising of presenilin1, GSK-3, cat and the catalytic subunit of PKA is formed

[255, 256]. This complex stimulates cat Ser45 phosphorylation by PKA and enhances GSK-3dependent phosphorylation of cat. Consistently, presenilin deficiency in Drosophila is associated with cytoplasmic accumulation of Drosophila

-cat homolog Armadillo [257]. Interestingly, PKA may act as a positive regulator of

cat as well. Activation of PKA by prostaglandin E2 stimulation resulted in an induction in cat/Tcf transcriptional activity in the HEK293 cell line [258]. Another study observed an increase in cytoplasmic and nuclear -cat level as a result of PKA activation [259]. Recently, PKA has been shown to inhibit cat ubiquitination by directly phosphorylating its Ser675 residue [259, 260].

The previous sections have emphasized phosphorylation-dependent ubiquitin-mediated proteasomal degradation of cat; it should be noted that cat proteasomal degradation can be initiated in a phosphorylation-independent manner as well [261, 262]. The ubiquitination machineries can be brought to cat by Siah.

Siah binds to an E3 complex composed of Ebi, Skp1 and SIP, and at the same time interacts with APC with its C-terminal domain, thus bringing cat in proximity to ubiquitination enzymes and facilitates its degradation [261, 262].

1.2.3.3 cat – Nuclear Entry and Function

There are two models for -cat nuclear entry. One model suggests that by forming a complex with -cat, T-cell factor (Tcf) facilitates the nuclear entry of cat.

This is supported by the observation that when -cat is over-expressed in

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combination of Tcf over-expression, -cat primarily resides in the nucleus; cat is evenly distributed between the nucleus and cytosol in the absence of ectopic Tcf

[263]. A more recent study using live-cell microscopy and fluorescence recovery after photo-bleaching (FRAP) showed that Tcf-4 recruits and retains cat in the nucleus [264]. However, a study by Orsulic and colleagues showed that a mutant

Armadillo protein unable to interact with TCF was still able to localize to the nucleus, suggesting that -cat nuclear entry does not require Tcf binding [265]. Therefore, a second mode of cat nuclear entry has been proposed to depend on its armadillo repeats. These repeats are present in the importin protein, a major component of the nuclear import machinery [266]. Therefore, cat may directly bind to the nuclear envelope via its armadillo domain and translocates into the nucleus in an energy-dependent manner, similar to that of import [267].

Once inside the nucleus, cat binds to co-transcription factors of the Tcf/Lef family that contain the high-mobility group (HMG) box DNA-binding domain. Tcf/Lef family proteins were originally discovered as enhancer binding factors for T cell- specific genes [268-270]. Four human homologues have been identified; these are

Lef1, Tcf1, Tcf3 and Tcf4 [271]. HMG box allows Tcf/Lef to bind DNA directly, and

Tcf/Lef proteins have high affinity for the following DNA sequence,

(A/T)(A/T)CAA(A/T)GG [272]. This Tcf/Lef consensus sequence is present in the promoter region of many genes, some of which have been shown to be targets of the Wnt/cat signaling pathway. Known canonical Wnt target genes include c-myc

[273], cyclin D1 [274, 275], fra-1 [276], c-jun [276], CD44 [277], vascular endothelial growth factor (VEGF) [278], Wrch-1 [279] and many more.

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Tcf/Lef alone is not sufficient to activate gene transcription, and requires -cat as a . In the absence of cat, Tcf/Lef proteins usually interact with transcriptional repressors such as Groucho and histone deacetylases [280, 281].

cat binds to the N terminal region of Tcf/Lef and displaces the repressors, forming a bipartite transcriptional activator complex known as cat/Tcf [282]. Additional co- activators are also required for successful Wnt/-cat-mediated gene transcription.

TATA-binding protein pontin52 has been shown to interact with the N terminal region of cat, suggesting that basic transcriptional machinery may be recruited to cat/Tcf

[283]. It has been shown that cat/Tcf-mediated transcription also requires Legless

(Lgs) and Pygopus (Pygo), both serving as nuclear co-factors in complex with cat/Tcf [284-286]. Other co-activators involved in cat/Tcf-mediated transcription include acetyltransferases, p300 and CREB-binding protein (CBP) which facilitates cat/Tcf function by remodeling its surrounding chromatin and influence RNA polymerase II activity [287, 288].

The activation of cat/Tcf-mediated transcription can also be downregulated by a number of factors. ICAT and Duplin have been shown to bind -cat directly, and interfere with its interaction with Tcf/Lef, inhibiting -cat-dependent gene expression

[289-291]. On the other hand, NEMO-like kinase (NLK) phosphorylates Tcf/Lef and inhibits cat/Tcf binding to the DNA [292]. Protein inhibitor of activated STAT (PIAS) is an E3 ligase of the small ubiquitin-related modifier (SUMO) modification

(sumoylation) pathway and has been shown to interact with Lef-1 and its subsequent sumoylation, which inhibits its transcriptional activity [293].

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1.2.3.4 Secreted Antagonists of Canonical Wnt Signaling

Canonical Wnt signaling is regulated by two functional classes of secreted antagonists, the secreted frizzled-related protein (sFRP) class and the Dickkopf (Dkk) class. The sFRP class includes sFRP and Wnt inhibitory factor 1 (WIF-1). The N terminal CRD of sFRP shares high with Fzd proteins, and may block Wnt signaling by interacting with either Wnts or form non-functional complexes with the Fzd receptors [294]. WIF-1, on the other hand, does not share any sequence homology with the CRD domain of Fzd or sFRPS, but is still able to bind to Wnts and inhibit Wnt-Fzd interaction and subsequent cat stabilization [295].

Dkks are secreted cysteine-rich proteins that do not bind Wnts, and instead interact with Wnt LRP5/6 co-receptors [223, 296]. Dkk-1 also interacts with single-pass transmembrane proteins Kremen1 (Krm1) and Kremen2 (Krm2) [297]. The ternary complex of Krm, Dkk-1 and LRP6 triggers the internalization of Wnt receptor from the plasma membrane [297]. Removal of LRP5/6 from the plasma membrane also inhibits the recruitment of Axin, which facilitates the stabilization of “destruction complex”, thus further restrains Wnt signaling by promoting cat phosphorylation and degradation.

1.2.3.5 Alternative Mechanisms for Wnt/-cat Activation and Function

Canonical Wnt glycoproteins initiate the activation of bipartite transcription factor cat/Tcf. However, a number of studies suggest that cat/TCF may be activated by alternative mechanisms. In addition, Wnt glycoproteins have been shown to participate in the activation of non-Wnt related signaling pathways as well.

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1.2.3.5.1 Growth Factors

Several growth factors have been implicated in cat/TCF regulation. Desbois-

Mounthon and colleagues showed that insulin and IGF-1 treatment induce the cytoplasmic expression of -cat in HepG2 cells [298]. This effect is also associated with a 3-4 fold increase in the transcriptional activation of a Wnt-responsive reporter gene [298]. Consistently, another study reported a stimulatory effect of IGF-1 on both cytoplasmic and nuclear -cat levels in prostate cancer cells [299]. In addition,

IGF-1 was shown to enhance the stability of -cat [299, 300]. Platelet-derived growth factor (PDGF) has been recently implicated in stimulating -cat nuclear translocation [301]. Yang et al. showed that PDGF mediated phosphorylation of nuclear p68 RNA helicase (p68) at Tyr593 is associated with enhanced -cat nuclear translocation [301]. It was shown that p68 Tyr593 displaces Axin from -cat and also hinders GSK-3 instigated -cat phosphorylation, thus stabilizing -cat

[301]. A subsequent study by the same group further demonstrated that p68 Tyr593 promoted cell proliferation by activating the transcription of cyclin D1 and c-Myc

[302], two known downstream targets of the canonical Wnt signaling pathway.

These observations are supported by Shin et al., who showed that p68 and its closely-related homologue p72 form complexes with -cat and enhances its transcriptional activity [303]. Conversely, knockdown of p68 and p72 results in reduced expression of cat/Tcf target genes, including c-Myc, cyclin D2, c-jun and

Fra-1 [303].

Emerging evidence also suggests the existence of cross-talk between transforming growth factor  (TGF-) and Wnt signaling pathways [304]. TGF-

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signaling pathway regulates cell proliferation and differentiation, and has been implicated in various diseases including cancer and atherosclerosis. TGF- ligands trigger the formation of the type I and type II serine/threonine kinase receptor complex, resulting type I receptor phosphorylation and activation catalyzed by the type II receptor. Subsequently, the type I receptor recruits and phosphorylates receptor-regulated Smad (R-Smad) proteins, Smad2 and Smad3 [305].

Phosphorylated R-Smad forms a heterodimeric complex with the common Smad,

Smad4. This complex translocates to the cell nucleus and acts as a transcription factor, by recruiting coactivators as well as corepressors to the promoter region of various genes [305]. Similar to the Wnt signaling pathway, TGF- regulates a number of processes during development. It has been reported that -cat-deficient mouse embryos exhibit defective anterior-posterior axis formation [306].

Interestingly, deficiency of components of the TGF- signaling, Smad2 [307], Smad

4 [308] and receptor ActRIB [309] also failed to form normal anterior-posterior axis, suggesting cooperative efforts of TGF- and Wnt signaling in regulating developmental processes. TGF- and Wnt signaling pathways cooperate on multiple levels. Bone morphogenetic protein (BMP) is a member of the TGF- superfamily proteins. It has been reported that over-expression of Wnt3A enhanced

BMP-2 mediated chondrogenesis in murine mesenchymal cells [310]. A study by

Kim et al. showed that -cat is required for BMP-4 expression and secretion in human colon cancer cells [311]. On the other hand, TGF-1 has been shown to induce -cat nuclear translocation in a Smad3-dependent fashion in adult human mesenchymal stem cells [312]. Other studies indicate direct interactions between

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the components of Wnt and TGF- pathways. For example, Labbé et al. showed that Smad3 interacts with a member of the Tcf/Lef family Lef-1, allowing TGF- and

Wnt pathways to synergistically activate the transcription of the xenopus homeobox gene twin (Xtwn) [313]. A study by Nishita et al. showed that maximal activation of the Xtwn gene requires a transcription-activating complex comprising Smad4, -cat and Lef-1 [314], which also indicates the existence of TGF- and Wnt signaling cross-talk.

1.2.3.5.2 G Proteins and G-protein Coupled Receptors (GPCRs)

As it was described in Section 1.2.3.2, a large pool of -cat resides in the cytoplasm in association with cadherins to form adhesion structures of cells.

Heterotrimeric G proteins are comprised of a guanine nucleotide-binding -subunit and a -subunit/-subunit dimer, and are classified into four subfamilies according to their -subunit, Gs, Gi, Gq and G12 [315]. There is considerable evidence suggesting the involvement of G12 proteins in cytoskeletal shape changes, cell growth and even tumorigenesis [316-318]. Meigs and colleagues reported the interaction between G12 proteins with the cytoplasmic region of cadherins, which results in the dissociation of -cat from caderins, promoting -cat nuclear translocation [319]. Furthermore, expression of constitutively active mutant G12 proteins caused an increase in cat/Tcf-mediated transcriptional activation in a functional-APC-lacking colon cancer cell line [319].

Both prostaglandin and lipid metabolite lysophosphatidic acid (LPA) function via G-protein coupled receptors (GPCR). Activation of the FP prostanoid receptor,

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which couples to Gq, was shown to reduce phosphorylation of cytoplasmic -cat and enhance cat/Tcf-mediated transcriptional activation [320]. It was then revealed in a later study that EP(2) and EP(4) prostanoid receptors, coupling to Gs also activated cat/Tcf-mediated gene transcription in response to prostaglandin E2 (PGE2) [258].

The activation of LPA receptors, which also couple to Gs were shown to activate major signaling events in the -cat pathway, leading to increased cell proliferation in colon cancer cells [321]. These observations suggest that steroid-driven GPCR activation also participate in the activation of Wnt signaling pathways.

1.2.3.5.3 FOXO Proteins

FOXO proteins belong to a subfamily of the forkhead transcription factors and, their functions are negatively regulated by insulin signaling (described in Section

1.1.3.3) (Fig. 1.5). In the absence of insulin, the majority of FOXOs reside within the nuclei and stimulates the transcription of target genes promoting cell cycle arrest, stress resistance and apoptosis [322]. Insulin stimulation leads to exportation of

FOXOs to the cytosol, mediated via the phosphorylation of FOXOs by Akt/PKB and serum-and glucocorticoid-regulated protein kinase (SGK) [322]. Conversely, oxidative stress such as an increase in reactive oxygen species (ROS) enhances

FOXO activity [323]. FOXO was shown to interact with -cat in an evolutionary conserved fashion [324]. In mammalian cells, -cat binds directly to FOXO1 and

FOXO3, and enhanced FOXO transcriptional activity. In addition, oxidative stress stimulated the association of -cat and FOXOs [324]. Bar-1 and Daf-16 are the

Caenorhabditis elegans homolog of -cat and FOXO, respectively. Co-IP

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experiments showed that Bar-1 interacts with Daf-16, and that the loss of Bar-1 reduced the activity of Daf-16 in dauer formation and life span [324].

An antagonizing relationship between FOXO – and cat/TCF-mediated transcription activity has been observed. Almeida et al. reported that H2O2 induced oxidative stress stimulated FOXO-mediated transcriptional activation while attenuating both basal and Wnt3a-stimulated expression of Axin2 and other Wnt target genes [325]. The same group also showed that FOXO-mediated transcription requires -cat, and that over-expression of -cat attenuated the repressive effect of

H2O2 on cat/Tcf-mediated transcription [325]. Consistently, Hoogeboom and colleagues showed that FOXO knock-down, using siRNA reverted oxidative stress- induced loss of -cat binding to TCF [326]. These observations suggest that FOXO proteins may negatively regulate cat/Tcf activity by competing with TCF for a limited pool of free -cat, especially in response to cellular oxidative stress.

1.2.3.5.4 Wnt and mammalian Target of Rapamycin (mTOR) Signaling Crosstalk

In complex with Raptor and mLST8, the serine/threonine kinase mTOR plays a pivotal role in the regulation of ribosome biogenesis and protein synthesis [327].

The function of mTOR is regulated by a small and Ras homolog, Rheb.

On the other hand, Rheb function is controlled by TSC1/2 complex, which in turn is regulated by its phosphorylation status. Mutations in TSC1 or TSC2 genes result in an autosomal-dominant disease known as tumberous sclerosis, which is characterized by the formation of benign tumors in various tissues [328]. TSC2 is a

GTPase-activing protein (GAP), and it converts Rheb to its GDP-bound inactive form, thus inhibiting mTOR function. It has been well-established that insulin activated

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Akt/PKB inhibits TSC2 activity by phosphorylating it at Ser939, Ser981 and Thr1462, ultimately facilitating protein synthesis via mTOR activation [327]. Conversely, during energy stress and the increase of AMP/ATP ratio, the tumor suppressor LKB1 phosphorylates AMP activated kinase (AMPK), which in turn phosphorylates TSC2 at Ser1345, promoting its GAP activity thus inhibiting mTOR signaling [329].

Recent evidence suggests that Wnt stimulation leads to mTOR activation, via a mechanism involving GSK-3 but not -cat, and inhibition of mTOR blocked Wnt- induced cell growth and tumor development [330]. Inoki et al. showed that AMPK- mediated Ser1345 phosphorylation of TSC2 serves as a priming residue for its subsequent phosphorylation by GSK-3 at Ser1337 and Ser1341 [330]. Over- expression of constitutively active GSK-3 was sufficient to block mTOR-mediated

S6K activation. Conversely, inhibition of GSK-3 activity was sufficient to stimulate mTOR/S6K [330]. These observations suggest that Wnt function is not limited to its role in the activation of gene transcription via the bipartite transcription factor cat/Tcf, but also includes stimulation of protein synthesis via mTOR activation.

1.2.4 Non-conventional Ligands and Receptors

For many years, the dogma held that Wnt signaling activation is initiated by the binding of Wnts and Fzd receptors [331]. However, this view has been challenged by the identification of Derailed (Drl) in Drosophila and its mammalian homologue (Ryk) acting as an alternative Wnt receptors. This has been compounded by the recent discovery of Norrin [332] and R-Spondins family proteins as regulators of Wnt activity serving as potential alternative Fzd ligands [333].

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Several other proteins have been identified as alternative Wnt receptors such as Ror [334, 335] and Crypto [336]; Ryk remains most well documented and characterized. Ryk was discovered in a screen for protein tyrosine kinase (PTK) because it contains an aberrant catalytic intracellular domain. It was also determined that Ryk has a transmembrane domain and an extracellular domain with potential sites for glycosylation [337]. The Drl gene is the Drosophila homologue of

Ryk, and is expressed in interneurons, and embryonic muscles and adjacent epidermal cells. Mutations of Drl result in erroneous neuronal pathway recognition and muscle attachment. More importantly, it was shown that the catalytic domain of

Ryk was not necessary for its function in muscle attachment [338]. Drl’s human homologue Ryk was first isolated from an epithelial ovarian cancer cell line SKOV-3

[339]. However, only a low level of expression can be detected in normal ovarian epithelium, suggesting that Ryk may play a role in tumor formation [339]. This idea was supported by the study by Katso et al., which showed that over-expression of H-

Ryk in the mouse fibroblast cell line NIH3T3 induced anchorage-independent growth and tumorigenicity in nude mice [340]. Evidence of Ryk as an alternative receptor for Wnt ligands came from a study which showed that Ryk is crucial in Wnt-mediated signaling [341]. Not only was Ryk able to directly bind Wnt1 and Wnt3A, it was also able to co-operate with Wnt1 in activating the Wnt-responsive reporter system,

TOPFLASH. In addition, Ryk was shown to be necessary for the neurite outgrowth induced by Wnt-3a [341]. Lastly, co-IP analysis showed that Ryk interacts with intracellular Wnt signaling protein Dvl, which further confirms the imperative role of

Ryk in Wnt signaling [341].

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There is also evidence to suggest the existence of alterative ligands for Fzd receptors, such as Norrin and R-Spondin. Norrin is the product of the gene that is disrupted in Norrie disease in human, which is also known as Norrie disease protein

(NDP). It was coined as a potential Fzd ligand based on the similarity in its vascular phenotype compared to Fzd4 mouse mutant [332, 342]. Xu et al. demonstrated that

Norrin binds Fzd4 with high specificity and affinity, and induced Frz4- and LRP- dependent activation of the canonical Wnt signaling assessed by TOPLASH [332].

The mouse gene R-Spondin (RSpo) was hypothesized to participate in the regulation of Wnt signals because its reduced expression in Wnt1-/- and Wnt3a-/- mouse embryos [343]. Subsequently, RSpo was shown to stimulate cat stabilization using both in vitro and in vivo models [344, 345]; and this activation was attenuated by endogenous inhibitors of the canonical Wnt signaling pathway [344].

It was then demonstrated that conditioned medium from mouse R-Spondin (mRSp)- or xenopus R-Spondin (xRSp)-transfected cells could induce Tcf-luciferase reporter activation, thus suggesting that RSp proteins act in the extracellular environment

[346]. Yoon and coworkers also observed that stimulation of cells with RSp is associated with cat stabilization and Wnt target gene activation, and that RSp physically interacted with LRP6 and Fzd8 [346].

1.2.5 Aberrant Regulation of Canonical Wnt Signaling

Canonical Wnt signaling is involved in various important ceullular processes such as cell proliferation, cell survival and cell differentiation. It is critical that these processes are tightly regulated to ensure normal tissue development and homeostasis. However, if the signaling pathways escape from this fine-tuned

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balance, cancer and other diseases may be initiated. Enhanced Wnt signaling has been observed in degenerative disorder of the joints, osteoarthritis. Specifically, Wnt signaling regulates the homeostatic balance of osteoblasts and osteoclasts, and loss-of-function of Wnt co-receptor LRP5 leads to the osteoporosis-pseudogliome syndrome (OPPG) and associated low bone mass [347, 348]. Loss-of-function mutation in LRP5 is also linked to eye defects such as familial exudative vitreoretinopathy (FEVR) [347, 348]. Gain-of-function mutations in WNT4 are associated with acute renal failure and polycystic kidneys [349-351]. In addition, canonical Wnt signaling pathway is involved in cardiovascular diseases [352, 353], as well as neurodegenerative diseases such as Alzheimer’s disease [256, 354] and schizophrenia [355].

1.2.5.1 Canonical Wnt Signals in the Intestine: Endoderm to Cancer – Mouse Models Evidence

The activity of the canonical Wnt signaling is pivotal during the development of the gastrointestinal tract. It is involved in the process of endoderm specification, gut tube patterning and even maintains intestinal stem cells [356]. The endoderm embryonic gut tube undergoes a series of changes and gives rise to the gastrointestinal tract as well as the esophagus, stomach and associated organs such as liver and pancreas [357, 358]. While the small intestinal epithelium develops into alternating villi and crypts of Lieberkuehn, colonic epithelium is composed of only crypts. The self-renewal capacity of intestine resides in the crypts, as it is the niche of the intestinal stem cells. The stem cell compartment is located at the base of the epithelium, from which cells migrate upward toward the villus as they

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proliferate and differentiate into all lineages of intestinal cells, including enterocytes, goblet cells, enteroendocrine cells and Paneth cells. Wnt signaling is necessary for the lineage specification of these cell types [356]. In adults, activity of canonical Wnt signaling has only been observed in the proliferative compartment of intestinal crypts, and it plays a crucial role in maintaining crypt stem cells in the small intestine, because the Tcf4-/- mouse exhibits a complete loss of intestinal crypt progenitors

[359].

Alterations in Wnt signaling in the intestine may also lead to the development of cancers. Certainly, the discovery of silencing APC mutations in tumors of Familial

Adenomatous Polyposis (FAP) patients has implicated canonical Wnt signaling in human CRC. This is further verified after generating mouse models resembling human colonic cancer by manipulating the expression of the canonical Wnt “breaker”

APC or “activator” cat [360]. The expression of truncated APC was able to cause the development of multiple polyps throughout the intestinal tract in mice [361, 362].

ENU (mutagen ethylnitrosourea)-treated mice produce a non-sense mutation of APC and develop multiple intestinal neoplasia [363]. On the other hand, over-expression of wild type APC in mice resulted in disordered intestinal cell migration and reduced cell adhesiveness [364]. Mouse models of conditional gain-of-function and loss-of- function cat mutation have also been generated and examined in relation to intestinal epithelium formation. Mice with conditional loss-of-function cat mutation

[365, 366] display similar phenotypes to those observed in Tcf4-/- mice, with loss of crypt structure and increased differentiation of stem cells [359]. Conversely, Taketo and colleagues created a mouse model that expressed a constitutively active cat

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mutant in the intestine, and observed up to 3000 adenomatous intestinal polyps per mouse, similar to what has been observed in the APC knockout mice [367]. A recent study further pinpointed the proto-oncogene c-Myc as a downstream target of the canonical Wnt pathway essential in the development of intestinal neoplasia.

Samson et al. created a mouse model with intestine-specific Apc and Myc double knockout, and found that this mouse did not exhibit any of the phenotypes observed in mice with the Apc deletion, suggesting that c-Myc is a critical mediator in the development of intestinal neoplasia [368].

1.2.5.2 Canonical Wnt Signaling in Human Colorectal Cancer

The Wnt pathway involves many proteins that function transiently during development, some of which have also been determined to act as oncoproteins or tumor suppressors in human cancers. In the canonical Wnt signaling pathway, gain- of-function mutation of its signaling components and loss-of-function mutation of its inhibitors have both been linked to cancer development. An important piece of evidence that eluded a plausible role of Wnt signaling in human cancers was the discovery of the biochemical interaction between tumor suppressor APC and cat

[369]. Adenomatous polyposis is a type of human colon cancer and is characterized by the formation of numerous polyps in the large intestinal epithelium. It was recognized that adenomatous polyposis was a hereditary condition as early as 1900, and that Familial Adenomatous Polyposis (FAP) is correlated with deletions of a specific chromosome region that encodes APC. Truncating mutations in APC have been found in both FAP and over 85% of all sporadic colorectal cancer patients

[370]. Among the colonic carcinoma cases that retained wild-type APC, gain-of-

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function mutations of the cat gene CTNNB1, and loss-of-function mutations in

AXIN2 have been observed [371, 372]. Closer examination reveals that a large portion of these CTNNB1 mutations were localized to the N terminus. These N terminus mutations resulted in either the deletion of an N-terminal fragment, or modification of N-terminal phosphorylation sites important for ubiquitin-mediated

cat degradation.

There is compelling evidence to suggest the canonical Wnt signaling pathway contributes to the initiation of intestinal turmorigenesis. The evidence for both APC and cat in initiating intestinal tumors is strong. The majority of human small colorectal adenomas contain inactivating mutations of the APC gene; and as it was described in Section 1.2.4.1, various mouse models have also shown the development of intestinal polyps with the loss of APC function or cat stabilization.

However, it should be noted that an increase in the rate of proliferation was not observed at these early stages of polyp formation. Intestinal adenoma or polyps are precedential to the transition to malignancy, but progression from benign tumors to malignant and invasive tumors require further genetic alterations to occur [370].

One well documented example is TGF-. TGF- signaling hinders the growth rate of epithelial cells, and the loss of response to TGF- is frequently reported in cancers

[373]. Mutations in the TGF- receptor are commonly observed in colorectal tumor cell lines [374]. Significantly, a portion of the human colorectal tumors contain loss of the tumor suppressor DPC4, which encodes Smad4 that transduces all TGF-

like signals [375]. Furthermore, mice harboring double mutations of Apc and

Smad4 develop large intestinal tumors that display both hyperproliferation and

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invasiveness [376]. Supporting evidence was also found in humans that showed

TGF- mutations appearing earliest at the late adenoma stage, which is when the transitioning from benign adenoma to malignant neoplasia takes place [377].

Mutation in has also been reported in colorectal tumors, but this alone does not seem to be sufficient in initiating tumorigenesis [370]. However, In Apc mutant mice, p53 deficiency was shown to enhance the multiplicity and invasive behavior of intestinal adenomas [378].

The function of APC is not limited to its role in cat destabilization, it is also involved in cell migration and adhesion. It has been well established that cadherin- mediated cell adhesion is frequently lost in invasive front of tumors, where cells acquire migratory abilities and progress from benign to malignant tumors [379]. In aforementioned Section 1.2.3.1, a pool of cytoplasmic cat is associated with E- cadherin at adhesion cellular junctions. It is possible that APC may affect cell adhesion and migration by its interaction with cytoskeleton-associated cat.

Indeed, using GFP-tagged APC, it has been shown that APC is capable of tracking along to peripheral sites of the cell [380]. More direct evidence came from the discovery of the interaction between APC and Asef [381]. Asef is a guanine nucleotide exchange factor (GEF) that remodels actin cytoskeleton by activating the small G protein Rac. Over-expressing APC augmented the GEF activity of Asef and induced Asef-mediated signs of migratory activity in MDCK cells, including cell flattening, membrane ruffling and lamellipodia formation [381]. This is consistent with the observation that while over-expression of wild type APC in mice was associated with disordered intestinal cell migration and reduced cell adhesiveness,

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one of the prominent manifestations of APC loss is abnormal crypt structure, presumably as a result of defects in cell migration or adhesion [361, 366]. This evidence suggests that the role of APC as a tumor suppressor appears to be two fold; it restricts cat activity by promoting cat proteosomal degradation mediated by GSK-3 and CK-I; concurrently, it affects cell adhesion and migratory processes.

Mutations of cat and APC may lead to early events required for the formation of intestinal adenoma, and APC itself may advance benign tumors into invasive types. In addition, cat/Tcf targeted genes may participate in the progression toward intestinal neoplasia. Targets of the canonical Wnt pathway, such as matrix metalloproteinase 7 (MMP-7, also known as matrilysin) have been shown to contribute to tumor progression in mice and humans [382, 383]. The loss of matrilysin function in Apc mutant mice was able to markedly suppress both incidences and sizes of intestinal tumors [382]. TCF target genes also include cell cycle promoting genes such as c-Myc [273] and cyclin D1 [274], which have intuitive tumor promoting effects, though genetic evidence has only come forward to indicate functional relevance of c-Myc in CRC so far (discussed in Section 1.2.4.1). It has been observed that certain non-steroidal anti-inflammatory drugs (NSAIDs) may have beneficiary effects in preventing CRC mediated by PPAR (peroxisome proliferator-activated receptor gamma) Interestingly, another member of the PPAR family, PPAR is a known target of the canonical Wnt signaling pathway, further complicating the role of Wnt pathway in CRC development [384].

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1.3 Synopsis, Rationale and Hypotheses

Insulin insensitivity and hyperinsulinemia are hallmarks for the early stage of

Type 2 Diabetes Mellitus. Known risk factors for Type 2 diabetes include high fat diet and sedentary life style. These are also risk factors for colorectal cancer (CRC), and epidemiological and other studies have shown that subjects with Type 2 diabetes have increased risks for the development of CRC. In addition, in vivo studies have shown that colorectal tumors were formed in Type 2 diabetic animal models. These findings culminate in the hypothesis that hyperglycemia and hyperinsulinemia contribute to CRC formation.

A number of recent studies provide evidence that components of the Wnt signaling pathway can be regulated by signaling cascades that are triggered by insulin or insulin-like growth factor-1 (IGF-1). Both insulin and IGF-1 have been shown to stimulate -cat nuclear localization and Lef/Tcf-dependent transcriptional activity [298, 385]. These observations suggest that insulin/IGF-1 cross-talks with the Wnt signaling pathway. Molecular mechanisms underlying this crosstalk, however, remain largely unknown, especially in primary intestinal cells.

To elucidate the molecular mechanisms behind the observed increased risks of CRC in hyperinsulinemic or Type 2 diabetic individuals, I have examined in intestinal cells, the molecular crosstalk between insulin and Wnt signaling pathways as well as the intracellular mediators that may be involved in the crosstalk. Proto- oncogenes c-Myc and cyclin D1 were previously determined as targets of the canonical Wnt pathway and have both been implicated in promoting cell cycle progression and proliferation. Therefore, my first aim was to test the hypothesis

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that insulin stimulation leads to c-Myc and cyclin D1 induction and cell proliferation by stabilizing the mediator of canonical Wnt pathway, cat. Through the use of

Western blot and MTT analyses, I found that in colon cancer cells, insulin treatment surmounted an increase in c-Myc and cyclin D1 protein expression and cell proliferation. I demonstrated further that this effect is partially attributed to increased nuclear cat content and its interaction with transcriptional-activating sites of the c-

Myc promoter, in an Akt/PKB-independent manner.

The second aim of my study was to identify the signaling transducer responsible for transmitting insulin signals to activate the canonical Wnt pathway by affecting the nuclear content and function of cat. We hypothesized that p-21 activated protein kinase 1 (PAK-1) acts in a PI3K-dependent and Akt/PKB- independent manner to stabilize nuclear cat and activates c-Myc and cyclin D1 expression. I showed that insulin induced PAK-1 phosphorylation/activation regardless of the Akt/PKB activation status in vitro and in vivo using Western blotting analyses. Furthermore, transfecting cells with PAK-1 dominant negative (DN)- expressing plasmid reduced insulin effects on c-Myc and cyclin D1 expression, and

cat binding to the c-Myc promoter. Cells transfected with constitutively active (CA)

PAK-1 expressing plasmid exhibited elevated basal c-Myc and cyclin D1 protein levels. These results were then further verified by knocking down PAK-1 expression using lentivirus-delivered PAK-1 shRNA.

The third aim of my study was to identify other components of the Wnt signaling pathway that are under the regulation of insulin. To this end, I performed gene microarray analysis in rat intestinal epithelial cells IEC-6 after 4 and 24 h of

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insulin treatment. I was able to identify a number of genes that have been implicated in the Wnt pathway, that are potentially under the regulation of insulin signaling. Significantly, I was able to confirm that insulin increases themRNA expression of a member of the Wnt receptor family, Fzd-4. In addition, insulin has stimulatory effects on the mRNA expression of Wnt transcription factor TCF-4, and it also induced the protein expression of TCF-4 in various cancer and non-cancer cell lines.

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Chapter 2: General Materials and Methods

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2.1 Chemicals and Reagents

Cell culture medium, fetal bovine serum (FBS) and oligonucleotides were purchased from Invitrogen Life Technology Inc (Burlington, ON, Canada). Insulin was provided by Novo Nordisk (Novo Nordisk, Copenhagen, Denmark). The phosphoinositide-3 kinase (PI3K) inhibitor LY294002, Protein Kinase B (PKB)/Akt inhibitor Akti1/2,

Mitogen Activated Protein Kinase (MAPK) kinase (MEK) inhibitor PD98059 and the

Protein Kinase A (PKA) inhibitor H-89, cyclohexamide and rapamycin were all purchased from Calbiochem (EMD Biosciences Inc., San Diego, CA, USA).

Lentiviral shRNAmir gene knockdown system was that of Thermo Fisher Scientific

(Huntsville, AL, USA). The three PAK-1 shRNAmir sequences are shown in Fig. 4.11.

2.2 Plasmids Utilized

The original S33Y mutant -cat expression plasmid was a gift from Dr. Eric

Fearon[386]. The cat (S33Y)-EGFP plasmid was constructed by inserting S33Y

cat fragment into the pmaxFP-Green-C vector (Amaxa Inc., Gaithersburg, MD).

The TopFlash LUC fusion gene plasmid was a gift from Dr. Bert Vogelstein[387].

The plasmids expressing dominant negative PAK-1 (K299R) and constitutively active PAK-1 (T423E) mutants, and the vector control, were gifts from Dr. Jefferey

Field [88, 388].

2.3 Cell Culture and Transfection

All cell culture media, serum and reagents were purchased from Invitrogen Life

Technology Inc. (Burlington, ON, Canada). Cell lines were maintained in Dulbecco’s 74

Modified Eagles Medium containing 10% FBS. Lipofectamine 2000 from Invitrogen

Life Technology Inc. was used as the transfection reagent, with a protocol suggested from the manufacturer. Briefly, either 3g (for approximately 4 x 105 cells) or 9g

(for approximately 2 x 107 cells) of DNA were used for a 24 h transfection, followed for serum starvation for 24 h prior to subsequent drug treatments. Transfection efficiency was determined by side-by-side transfection of expression vectors of

EGFP, followed by visualization using fluorescent microscopy.

2.4 Luciferase (LUC) Reporter Gene Analysis

The LUC reporter gene assay was carried out by a method described previously with minor modifications [389]. Briefly, cells were washed with PBS and scraped off the plate with a rubber policeman after incubating with 0.3ml of harvesting buffer (50mM

Tris/MES, 1mM DTT and 0.1% Triton X-100). The collected samples were vortex mixed for 40 seconds and centrifuged at 4°C for 10 minutes at 16,110 x g.

Supernatant was collected for further analysis. Fifteen l of a cocktail solution

(50mM Tris/MES, 0.18M MgOAc and 40mM ATP) and 100l of the harvesting buffer were added to 100l of the collected supernatant and measured for LUC activity using a Luminometer (Lumat LB 9507 from Fisher Scientific).

2.5 Western Blot Analysis

Cells were cultured in 6cm dishes and harvested with 0.2ml radioimmune precipitation assay (RIPA) buffer. Extracted protein was quantified and resolved by

SDS-PAGE with 10% polyacrylamide gels before transfer to a nitrocellulose (Pierce,

Rockford, Ill) membrane. Blots were incubated at room temperature in 5% non-fat

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milk in 1xTBST for 1 hour, followed by overnight incubation with primary antibody in

5% BSA in 1XTBST at 4°C and 1 hour incubation at room temperature with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology,

Inc). The blot was then developed by enhanced chemiluminescence (ECL) (Pierce,

Rockford, Ill) and exposed to Maximum Sensitivity (MS) double emulsion imaging film (Kodak).

2.6 Quantitative Chromatin Immunoprecipitation (qChIP)

ChIP assay was used to examine the binding affinity of cat to the endogenous c-

Myc gene and analyzed by real time PCR with primer sets targeting the two TCF elements (TBE), as well as an region (as the control) of the c-Myc promoter region. Approximately 2 x 107 cells were used for each experiment. Following treatment with insulin or pharmacological agents, cells were fixed with 1% formaldehyde at room temperature for 10-15 minutes and neutralized with 0.125M

Glycine for 10 minutes. Cells were pelleted after collection in 1XPBS using a rubber scraper. Cells were then subjected to two sequential lysing processes with cell lysis buffer (5mM HEPES, 80mM KCl, 1% NP40, supplemented with protease inhibitor cocktail) and the nuclei lysis buffer (50mM Tris-Cl, 10mM EDTA, 1% SDS, supplemented with protease inhibitor cocktail). Nuclear contents were sonicated to shear chromatin to an average length of approximately 600bp. To reduce non- specific background binding of the subsequent immunoprocipitation (IP) step, sheared chromatin was incubated with salmon sperm DNA/protein A agarose at 4 degrees for 30 min. Polyclonal -cat antibody (Santa Cruz Biotechnology, Inc.,

Santa Cruz, CA) was used to IP DNA fragments bound with -cat overnight at 4

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degree. IP product was collected using protein A agarose beads followed by gentle centrifugation (1000rpm), and washed with low salt wash buffer, high salt wash buffer, LiCl wash buffer and TE buffer. DNA was then recovered after reversal cross-linking of protein and DNA by heating samples at 65 degrees for 6 hrs. Qiagen

PCR purification was used to purify the final DNA product, before samples were quantified using real time PCR.

2.7 shRNAmir Selective Gene Knock-Down

The GIPZ-PAK-1 shRNAmir and the GIPZ-scrambled shRNA bacterial plasmids were purchased form Thermo Fisher Scientific (Huntsville, AL). The lentivirus enveloping and packaging plasmids psPAX2 and pmD2.G4.5 were gifts from Dr.

Maria Rozakis-Adcock. Bacterial plasmids were amplified using DH5 Escherichia

Coli and plasmid DNA was purified using QIAGEN Plasmid Maxi Kit (Hilden,

Germany). Approximately 5.5x106 HEK293T cells were seeded per 10cm plate.

Twenty-four hours later, these cells were transfected with GIPZ-PAK-1 shRNA (9g) or GIPZ-scrambled shRNA (9g), psPAX2 (9g) and pmD2.G (4.5g) using

Lipofectamine 2000 per the manufacturer’s instructions (Invitrogen Life Technology

Inc.). Viruses were harvested 72 h post transfection according to manufacturer recommended instructions (Thermo Fisher Scientific, Huntsville, AL). To create cells stably expressing either PAK-1 shRNA or scrambled shRNA, cells were infected with lentiviruses expressing PAK-1 shRNA or scrambled shRNA for 72 h. Successfully infected cells were selected and maintained using 100g/ml puromycin.

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2.8 Fetal Rat Intestinal Cell Isolation

Primary intestinal cells from fetal rats were collected via a method developed by Dr.

Patricia Brubaker [390]. Briefly, intestines from a litter of 19 to 21 day gestation fetal

Wistar rats were collected and pooled followed by two sequential 30-min incubations with collagenase, hyaluronidase, and deoxyribonuclease I. The dispersed cells were washed and placed into monolayer cultures with Dulbecco’s Modified Eagles

Medium supplemented with 10% fetal bovine serum for 48 h at 37° and 5% CO2 before the indicated treatment.

2.9 Statistics

All data are presented as mean+/-SD. Statistical analysis was done by either

Student’s t test or one way ANOVA when appropriate. Significance was assumed at a p value of less than 0.05.

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CHAPTER 3: Both Wnt and mTOR Signaling Pathways are Involved in Insulin-Stimulated Proto- Oncogene Expression in Intestinal Cells

Data presented in this chapter have been published in Cell Signaling, Sun J. and Jin T. (2008) [391].

(All data presented in the figures of this chatper were contributed by Sun J.)

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3.1 Abstract

Subjects with Type II diabetes mellitus are more vulnerable to the development of colorectal tumors, suggesting that hyperinsulinemia may stimulate proto-oncogene expression, and the existence of crosstalk between insulin signaling and pathways that are involved in colorectal tumor formation. We show here that insulin stimulates cell proliferation and c-Myc expression in colon cancer cell lines

HT29 and Caco-2, the intestinal non-cancer cell line IEC-6, and primary fetal rat intestinal cell (FRIC) cultures. The effect of insulin was blocked by phosphoinositide-

3 kinase (PI3K) inhibition, but only partially attenuated by inhibition of Protein kinase

B (PKB), indicating the existence of both PKB-dependent and -independent mechanisms. The PKB-dependent mechanism of insulin-stimulated c-Myc expression in HT29 cells was shown to involve the activation of mTOR in c-Myc translation. In the investigation of the PKB-independent mechanism, we found that insulin induced nuclear translocation of -catenin (-cat), an effector of Wnt signaling.

Furthermore, insulin stimulated the expression of TopFlash, a Wnt-responsive reporter gene. Finally, chromatin immunoprecipitation (ChIP) detected significant increases in the binding of -cat to two TCF binding-sites of the human c-Myc promoter following insulin treatment. Our findings support the existence of crosstalk between insulin and Wnt signaling pathways, and suggest that the crosstalk involves an Akt/PKB-independent mechanism.

3.2 Introduction

Type II Diabetes mellitus (DM) is one of the leading causes of death in developed countries and onset of the disease is characterized by the development

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of insulin insensitivity and hyperinsulinemia. There are numerous known risk factors for the development of Type II DM, including high fat diet and sedentary life style.

Colorectal cancer (CRC) shares many of the risk factors with Type II DM, and epidemiological and other studies have shown that subjects with Type II DM have increased risks for the development of CRC [171, 392, 393]. In addition, in vivo studies have shown that colorectal tumors were formed in Type II DM animal models

[170, 394, 395]. These findings culminated in the hypothesis that hyperglycemia and hyperinsulinemia contribute to CRC formation.

CRC is the second most common type of cancer, next to breast cancer for women and prostate cancer for men [396]. Extensive etiological and pathological studies led to the discovery of the adenomatous polyposis coli (APC) tumor suppressor, and subsequent recognition of the effect of aberrant expression and function of the Wnt signaling pathway in CRC formation and development [397-399].

Wnt glycoproteins exert their effect via the transmembrane frizzled receptor and

LRP5/6 co-receptors. Following receptor binding, Wnt signals are transmitted by the association between Wnt receptors and Dishevelled (Dvl), an event that triggers the disruption of the complex containing APC, Axin, glycogen synthase kinase 3 (GSK-3) and -cat, preventing phosphorylation-dependent degradation of -cat. Thus, free - cat molecules will accumulate and form a bipartite transcription factor with a member of the T cell factor (TCF) family, namely cat/TCF, to stimulate the expression of Wnt or cat/TCF downstream target genes [400, 401]. A number of proto-oncogenes, including c-Myc and cyclin D1, have been identified as Wnt target genes [273, 274].

Recently, several studies have provided evidence that components of the Wnt

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pathway can be regulated by signaling cascades that are triggered by insulin or insulin-like growth factor-1 (IGF-1). For example, working with a hepatocarcinoma cell line HepG2, Desbois-Mounthon et al. showed that both insulin and IGF-1 stimulated Lef/Tcf-dependent transcriptional activity, and this stimulation is dependent on phosphoinositide-3 kinase (PI3K). Another study demonstrated that

IGF-1 enhances the stability and transcriptional activity of cat in the C10 human colorectal cancer cell line [385]. These observations suggest that insulin/IGF-1 crosstalks with the Wnt pathway. However, no studies have explored the molecular mechanisms underlying this potential crosstalk, particularly in primary intestinal cells.

We show herein that at pathological levels, insulin stimulates c-Myc and cyclin D1 expression and the growth of intestinal cancer and non-cancer cell lines, as well as primary fetal rat intestinal cell (FRIC) cultures. Of interest, stimulation of cell growth and c-Myc expression by insulin can only be partially attenuated by inhibition of Akt/PKB, while PI3K inhibition completely abrogated the stimulatory effect of insulin. These findings suggest that insulin stimulates the expression of proto-oncogene genes via both Akt/PKB-dependent and Akt/PKB-independent mechanisms. We confirmed that insulin stimulates c-Myc translation via activation of mTOR signaling, while PI3K-dependent and Akt/PKB-independent activation of c-

Myc expression by insulin is due to crosstalk between insulin and Wnt signaling pathways.

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3.3 Materials and Methods

Materials

Tissue culture medium, fetal bovine serum and oligonucleotides were purchased from Invitrogen Life Technology Inc. (Burlington, ON, Canada). Insulin was provided by Novo Nordisk (Novo Nordisk, Copenhagen, Denmark). The PI3K inhibitor

LY294002, and the Akt/PKB inhibitor, Akti-1/2, cyclohexamide and rapamycin were all purchased from Calbiochem (EMD Biosciences, Inc., San Diego, CA).

Plasmids

The original S33Y mutant -cat expression plasmid was a gift from Dr. Eric Fearon

[386]. The cat (S33Y)-EGFP plasmid was constructed by inserting S33Y cat fragment into the pmaxFP-Green-C vector (Amaxa Inc., Gaithersburg, MD). The

TopFlash LUC fusion gene plasmid was a gift from Dr. Bert Vogelstein [387].

Cell culture, MTT assay, DNA transfection, and LUC reporter gene analysis

The human colon cancer cell lines HT29 and Caco2, and the rat intestinal cell line

IEC-6 were purchased from American Type Culture Collection (ATCC). Method for generating FRIC cultures has been described previously [390]. Briefly, intestines from a litter of 19 to 21 day gestation fetal Wistar rats were pooled followed by two sequential 30-min incubations with collagenase, hyaluronidase, and deoxyribonuclease I. The dispersed cells were washed and placed into monolayer cultures. Method for MTT assay was described by Wang et al. [402], and method for assessing the effect of chemical treatment on TopFlash LUC reporter expression was described by Yi et al. [389].

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Antibodies, Cell Fractionation and confocal microscopy

Antibodies for Akt/PKB, phospho- Akt/PKB (Ser473), phospho-GSK-3/ (Ser21/9), phospho-4E-BP1 (Thr37/46), phospho-S6K (T389), histoneH3 and -actin were purchased from Cell Signaling Technology (Cedarlane, ON, Canada). Antibody for

GSK-3 (clone 4G-1E) was purchased from Upstate Biotechnology (Lake Placid, NY).

Antibodies for -catenin and c-Myc, and the horseradish peroxidase (HRP)- conjugated secondary antibodies were from Santa Cruz Biotechnology, Inc. (Santa

Cruz, CA). Nuclear and cytosolic protein fractions were prepared as described by

Olnes et al. [403]. Fluorescence signal in EGFP-S33Y cat transfected HT29 cells was viewed with an LSM510 confocal microscope.

Chromatin immunoprecipitation (ChIP)

Approximately 2x107 cells were used for each of the ChIP assays [404]. After sonication, the anti--cat antibody was added to precipitate sheared chromatin. In each assay, precipitated chromatin was dissolved in 60 l of TE buffer, and 2 l was taken for real time PCR. Experimental and control primers are shown in Table 3.1.

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Table 3.1 – Primer Sequences for Quantitative ChIP Assay

Primer Name Nucleotide Sequence Size of Product

TBE1 Forward 5′-GGTCCACAAGCTCTCCACTT-3′ 135 bp

TBE1 Reverse 5′-CGGTTTGCAACAGTCTCG-3′

TBE2 Forward 5′-CTTCTTTCCTCCACTCTCCCT-3′ 139 bp

TBE2 Reverse 5′-AACAGCTGCCCTCCACAC-3′

Intron Forward 5′-TGGAATCGTTGACTTGGAAA-3′ 150 bp

Intron Reverse 5′-AAGGGTAGCAGCTGTTCTGG-3′

Table 3. 1 – Experimental and control primers for quantitative ChIP Assay

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3.4 Results

3.4.1 Insulin-stimulated cell proliferation involves both Akt/PKB-dependent and independent mechanisms

To mimic hyperinsulinemic conditions, we treated HT29, Caco-2 and IEC-6 cell lines, and FRIC cultures, with pathological dosages of insulin (100, 400 and 800 nM) for 4, 8, 24 and 48 h. Using MTT assays, we found that all three concentrations of insulin elicited a significant stimulatory effect on the growth of these cell cultures.

Representative results in Fig. 3.1A and 3.1B show the stimulatory effect of insulin after 48 h in two colon cancer cell lines. In HT29 cells, insulin treatment resulted in a

2.5– 3.5-fold increase in cell number. In CaCo2 cells, however, insulin treatment resulted in only a 1.6-fold increase in cell number. This difference is consistent with a higher basal level of c-Myc expression in the Caco-2 cells and stronger stimulation of c-Myc expression after insulin treatment in HT29 cells (Fig. 3.6A, C). A significant effect of insulin on cell growth was also observed in the primary FRIC cultures (Fig.

3.2A), and in the non-cancer cell line IEC-6 (Fig. 3.2B).

To investigate signaling cascades that mediate the stimulatory effect of insulin on cell growth or proliferation, we assessed the effect of PI3K and Akt/PKB inhibition.

Fig. 3.3 shows that the PI3K inhibitor LY294002 effectively blocked insulin- stimulated Akt/PKB and GSK-3 phosphorylation in HT29 cells, detected by Western blotting with specific antibodies against phosphorylated Akt/PKB and GSK-3.

Akt/PKB inhibition with Akti-1/2 also effectively blocked insulin stimulated Akt/PKB phosphorylation and GSK-3 phosphorylation in this cell line (Fig. 3.3). Interestingly, while PI3K inhibition completely blocked insulin-stimulated cell growth (Fig. 3.4A),

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A HT29 (48h)

18 *** 16 *** 14 *** 12 10 8 6 4 Cell NumberCell(x1000) 8 2 0 - 100 400 800 Ins (nM)

B Caco2 (48h)

5 *** *** 4 **

3

2

Cell NumberCell(x1000) 1

0 - 100 400 800 Ins (nM)

Figure 3. 1 - Insulin stimulates the growth of two human colon cancer cell lines.

HT29 (A), CaCo2 (B) were serum starved for 24 h before treated with indicated concentrations of insulin for 48 h. Cell growth was monitored by MTT assay. Absorbance values were measured at 590 nm and converted into cell numbers. The data are expressed as mean relative cell numbers (n=6) +/- S.D. (*, p<0.05, **, p<0.01, and ***, p<0.001).

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A FRIC (48h)

8 *** *** 6 **

4

2 CellNumber(x1000)

0 - 100 400 800 Ins (nM)

B IEC-6 (48h)

10

8 *** ** ***

6

4

Cell NumberCell(x1000) 2

0 - 100 400 800 Ins (nM)

Figure 3. 2 - Insulin stimulates the growth of primary FRIC cultures and a non-cancerous intestinal cell line IEC-6.

(A) FRIC and (B) IEC-6 cells were serum starved for 24 h before treated with indicated concentrations of insulin for 48 h. Cell growth was monitored by MTT assay. Absorbance values were measured at 590 nm and converted into cell numbers. The data are expressed as mean relative cell numbers (n=6) +/- S.D. (*, p<0.05, **, p<0.01, and ***, p<0.001).

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HT29

p-Akt (Ser473)

AKT

p-GSK-3 (Ser21)

GSK-3 

c-Myc

-actin

- + - + - + + Ins (100nM) - - + + - - - Akti1/2 (100nM) - - - - + + - LY (50M) 9 * * 8 7 # ¥ 6 # ¥

5 * * p-Akt 4 p-GSK-3a ¥

3 c-Myc RelativeIntensity 2 ¥ 1 0 - + - + - + + Ins (100nM) - - + + - - - Akt1/2 (100nM) - - - - + + - LY (50M)

Figure 3. 3 - The stimulatory effect of insulin on c-Myc expression cannot be completely blocked by PKB inhibition.

HT29 cells were serum starved for 24 h. After a 1 h pretreatment with LY294002 (LY) or Akt inhibitor (Akti-1/2), cells were incubated with indicated concentrations of insulin for 4 h. The expression of p- AKT (Thr473), total AKT, p-GSK--3 (Ser21), total GSK-3, c-Myc and -actin (loading control) were detected by Western blotting. Bottom panel shows the densitometric analysis of the top panel. The data are expressed as mean relative intensity (n=3) +/- S.D. (#,*,¥ p<0.05).

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A HT29 (4h)

12 ** *** **

10

8 NS NS NS

6

4 Cell NumberCell(x1000) 2

0 - 100 400 800 - 100 400 800 - - Ins (nM) - - - - + + + + - - LY (50M) ------+ - RA (10M) ------+ DMSO

B HT29 (4h) 12 ** ** **

10 ** ** *** 8

6

4 CellNumber(x1000) 2

0 - 100 400 800 - 100 400 800 - - Ins (nM) - - - - + + + + - - Akti1/2 (100nM) ------+ - RA (10M) ------+ DMSO Figure 3. 4 - The stimulatory effect of insulin on cell growth cannot be completely blocked by PKB inhibition.

HT29 cells were serum starved for 24 h. After a 1 h pretreatment with LY294002 (LY, A) or Akt inhibitor (Akti- 1/2, B), cells were incubated with indicated concentrations of insulin for 4 h. Rapamycin (RA) was used as negative control. DMSO was used as vehicle control. Cell growth was monitored by MTT assay. Absorbance values were measured at 590 nm and converted into cell numbers. The data are expressed as mean relative cell numbers (n=6) +/- S.D. (*, p<0.05, **, p<0.01, and ***, p<0.001).

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HT29

c-Myc

cyclinD1

-actin

- 20’ 30’ 1h 2h 3h 4h Ins (100nM)

5 4.5 ¥ ¥ * 4 * 3.5 * ¥ 3 * * ¥ 2.5 * ¥ c-Myc ¥ 2 * cylinD1 Relative IntensityRelative 1.5 1 ¥ 0.5 0 - 20' 30' 1h 2h 3h 4h Ins (100nM)

Figure 3. 5 - Insulin stimulates c-Myc and cyclin D1 expression in a time-dependent manner.

HT29 cells were serum starved for 24 h prior to insulin (100nM) treatment for indicated time periods. The expression of c-Myc, cyclin D1, and -actin (loading control) were detected by Western blotting. Bottom panel shows the densitomeric analysis of the top panel. The data are expressed as mean relative cell numbers (n=3) +/- S.D. (*, ¥, p<0.05).

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the application of Akti-1/2 only partially attenuated the stimulatory effect of insulin on cell growth (Fig. 3.4B). These observations indicate that insulin stimulates intestinal cell growth via at least two different mechanisms, one of which depends on Akt/PKB while the other does not. Moreover, inhibition of PI3K and Akt/PKB also resulted in different degrees of mitigation of insulin- stimulated c-Myc expression (discussed below).

3.4.2 Insulin-stimulated c-Myc expression also involves both Akt/PKB- dependent and independent mechanims

We next examined the effect of insulin on the expression of c-Myc and cyclin

D1, which are known to play a role in cell proliferation. These two proto-oncogenes are also well characterized downstream targets of the Wnt signaling pathway [273,

275]. Because insulin at 100 nM was sufficient to stimulate cell growth in all the cell cultures we tested, further analyses were carried out at this concentration. First of all, we performed a time-course experiment to assess the effect of insulin on c-Myc and cyclin D1 expression in the HT29 cell line, with a representative Western blot shown in Fig. 3.5. The activation of both c-Myc and cyclin D1 expression was observed as early as 20 min reaching a maximum by 4 h after insulin exposure. We next examined the effect of insulin on c-Myc expression in CaCo2 (Fig. 3.6C), IEC-6 and

FRIC cultures (Fig. 3.6B, D). The basal level of c-Myc expression in HT29 cells (Fig.

3.6A) is lower than that in the Caco-2 cells (Fig. 3.6C). However, insulin treatment for 4 and 8 h results in an expression level comparable with that observed in Caco-2 cells after insulin treatment. As expected, for the IEC-6 cell line and FRIC cultures, the basal c-Myc expression levels were very low. However, insulin treatment was

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A HT29 B IEC-6 c-Myc c-Myc

-actin -actin - 4h 8h 24h Ins (100nM) - 4h 8h 24h Ins (100nM) * 4 c-Myc 3 c-Myc * * 2.5 3 2 * * 2 1.5 * 1 1

0.5 Relative IntensityRelative Relative IntensityRelative 0 0 - 4h 8h 24h Ins (100nM) - 4h 8h 24h Ins (100nM)

C CaCo2 D FRIC c-Myc c-Myc

-actin -actin - 4h 8h 24h Ins (100nM) - 4h 8h 24h Ins (100nM)

4 * * c-Myc 3 * c-Myc * * * 2.5 3 2 1.5 2 1 1

0.5 Relative IntensityRelative Relative IntensityRelative 0 0 - 4h 8h 24h Ins (100nM) - 4h 8h 24h Ins (100nM)

Figure 3. 6 - Insulin stimulates c-Myc expression in intestinal cancer and non-cancer cell lines, as well as the FRIC culture.

HT29 (A), CaCo-2 (B), IEC-6 (C) and FRIC (C) were serum starved for 24 h prior to insulin (100nM) treatment for indicated time periods. The expression of c-Myc, cyclin D1, and -actin (loading control) were detected by Western blotting. A representative blot from each experiment was shown, and densitometric graphs were generated from three independent experiments from each group. The data are expressed as mean relative cell numbers (n=4) +/- S.D. (*, p<0.05).

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able to elicit a notable and significant increase in c-Myc expression in both cultures

(Fig. 3.6A, C).

Previous studies have shown that ubiquitin-proteasome-mediated degradation participates in the regulation of c-Myc expression, involving factors such as Ras-activated kinases, type-2 protein serine/threonine phosphatase (PP2A), and

Pin1-prolyl- [405-408]. We therefore investigated whether insulin affects c-

Myc protein stability. Fig. 3.7A shows that after 4 h of cycloheximide treatment, c-

Myc expression in HT29 cells was barely detectable; and Fig. 3.7B shows that insulin was not able to block the inhibitory effect of cycloheximide. We therefore suggest that insulin signaling is not involved in preventing c-Myc degradation.

To further explore mechanisms underlying insulin-stimulated c-Myc expression, we investigated the role of known components of the insulin signaling pathway. First, we assessed the effect of PI3K inhibition and observed that in HT29 cells, pretreatment with LY294002 significantly reduced both the basal and insulin- stimulated c-Myc expression (Fig. 3.8A). We next examined the effect of Akt/PKB inhibition and observed that when HT29 cells were pretreated with Akti-1/2, insulin- stimulated c-Myc expression was also mitigated, but not to the same degree as with

LY294002 treatment (Fig. 3.8B). Attenuated c-Myc expression in cells treated with

LY294002 was correlated with reduced expression of phosphorylated 4EBP-1

(Thr47/36) and phosphorylated S6K (Ser386), two known targets of mTOR signaling that are essential for cap-dependent initiation and elongation of protein synthesis

(Fig. 3.8A). We therefore examined the contribution of mTOR in insulin-stimulated c-

Myc expression. Using an mTOR-specific blocker rapamycin (50 and 100 nM), we

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A c-Myc

actin

- + + + + + - CHX (100g/mL) 1.4 - 0.5 1 2 4 6 - Time (h) 1.2 1 0.8 * 0.6 c-Myc 0.4 * * * *

Relative IntensityRelative 0.2 0 - + + + + + - CHX (100g/mL) - 0.5 1 2 4 6 - Time (h)

B c-Myc

-actin

- + - + + - 4 h Ins (100nM) - - + + + - CHX (100mg/mL) 3.5 * 3 2.5 c-Myc 2 1.5 1 Relative IntensityRelative 0.5 * * * 0 - + - + + - 4 h Ins (100nM) - - + + + - CHX (100g/mL) Figure 3. 7 - Insulin does not activate c-Myc expression via increasing protein stability in the HT29 cell line.

(A) HT29 cells were treated with cycloheximide (CHX, 100 mg/ml) for indicated time periods. The expression of c-Myc and b-actin (loading control) were detected by Western blotting. Bottom panel shows the densitomeric analysis for top panel (n=3) (B) HT29 cells were treated with cycloheximide (CHX, 100mg/ml) and 100nM of insulin for 4 h. The expression of c-Myc and b-actin (loading control) were detected by Western blotting. Bottom panel shows the densitomeric analysis for top panel. The data are expressed as mean relative cell numbers (n=3) +/- S.D. (*, p<0.05).

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A c-Myc

p-4E-BP1 (Thr47/36)

p-p70 S6K (S389)

-actin

- + - + Ins (100nM) - - + + LY (50M) 4 * c-Myc 3

2

1 * Relative IntensityRelative 0 - + - + Ins (100nM) - - + + LY (50mM)

B c-Myc

-actin

- + - + - + Ins (100nM) - - + + - - Akti1/2 (100nM) 5 - - - - + + LY (50M)

4 *

3 c-Myc 2 *

1 * Relative IntensityRelative 0 - + - + - + Ins (100nM) - - + + - - Akti1/2 (100nM) - - - - + + LY (50M) Figure 3. 8 - Activation of c-Myc expression by insulin involves both PKB-dependent and PKB- independent mechanisms.

HT-29 cells were serum starved for 24 h prior to pretreatment with LY294002 (LY) (A) or Akti-1/2 (100 nM) (A, B) for 1 h before insulin (100nM) treatment for an additional 4 h. The expression of c-Myc, p-4E-BP1 (Thr47/36), p-p70 S6K (Ser389) and -actin (loading control) were detected by Western blotting. A representative blot from each experiment was shown, and densitometric graphs were generated from three independent experiments from each group. The data are expressed as mean relative cell numbers (n=3) +/- S.D. (*, p<0.05).

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c-Myc

p-4E-BP1 (Thr47/36)

p-p70 S6K (S389)

-actin

- + - + - + - Ins (100nM) 6 - - 50 50 100 100 - Rap (nM)

£ £ £ 5 *

4 #

* * c-Myc 3 ¥ * * p-4E-BP1 p-p70 S6K

2 # # Relative IntensityRelative

1

0 - + - + - + - Ins (100nM) - - 50 50 100 100 - Rap (nM)

Figure 3. 9 - mTOR pathway is involved in insulin activated c-Myc expression.

HT29 cells were starved for 24 h and then pretreated with rapamycin (Rap) (50 or 100 nM) for 1 h before an additional 4 h of insulin (Ins) treatment. The expression of c-Myc, p-4E-BP1 and p-70 S6K, and -actin (loading control) were detected by Western blotting. Bottom panel shows the densitometric analysis for top panel. The data are expressed as mean relative cell numbers (n=3) +/- S.D. (#,*, ¥, £, p<0.05).

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were able to reduce the stimulatory effect of insulin on 4E-BP-1 and p70-S6K phosphorylation (Fig. 3.9), indicating that at these two dosages, rapamycin is able to inhibit mTOR activity. Rapamycin was also shown to significantly attenuate but not completely abolish insulin-stimulated c-Myc expression (Fig. 3.9). The above observations collectively suggest that the Akt/PKB-dependent pathway involved in insulin-induced c-Myc expression is mainly mediated by mTOR activation while the residual stimulatory effect of insulin is likely due to a yet-to-be identified signaling cascade that does not involve Akt/PKB.

3.4.3 Insulin stimulates -cat content and nuclear -cat translocation

The c-Myc gene is a downstream target of the canonical Wnt pathway and the bipartite transcription factor cat/TCF is involved in c-Myc gene transcriptional activation [273]. We therefore examined whether insulin affects the expression and function of -cat. As shown in Fig. 3.10A, insulin treatment for 4, 8 and 24 h stimulated total -cat protein expression in the primary FRIC cultures. In the colon cancer cell lines, although insulin did not significantly increase total -cat expression, it was able to increase nuclear -cat content. A representative Western blotting result with the HT29 cell line is shown in Fig. 3.10B. We then transfected TopFlash, a cat/TCF responsive reporter gene into HT29 cells, and assessed the effect of insulin on the expression of this reporter, using lithium treatment as a positive control.

Fig. 3.11A shows that insulin significantly increased TopFlash expression approximately 1.5 fold. Next, we assessed the effect of insulin on subcellular localization of an EGFP-tagged -cat molecule. When HT29 cells were transfected with the control EGFP plasmid, green fluorescence signals were evenly distributed

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FRIC A -cat

-actin - 4h 8h 24h Ins (100nM) 3 * 2.5 * 2 * 1.5 b-cat 1

0.5 Relative IntensityRelative 0 - 4h 8h 24h Ins (100nM)

HT29 B -cat (nuclear)

Histone H3

-cat (cytosolic)

-actin

- 3h 8h - Ins (100nM) 2.5 * 2 * 1.5 Nuclear 1 * Cytosolic

0.5 Relative IntensityRelative 0 - 3h 8h - Ins (100nM) Figure 3. 10 - Insulin treatment leads to increased -cat expression in FRIC (whole cell lysate) and HT29 cells (nuclear fraction).

(A) FRIC cultures were serum starved for 24 h prior to insulin (100nM) treatment for the indicated time periods. The expression of -cat and -actin (loading control) was detected by Western blotting. (B) HT29 cells were serum starved for 24 h prior to insulin (100nM) treatment for indicated time periods. Expression of -cat, histone H3 (nuclear protein marker as the loading control) in the nuclear fraction and -actin (cytosolic loading control) in the cytosolic fractions were detected by Western blotting. Densitometric data are expressed as mean relative cell numbers (n=3) +/- S.D. (*,p<0.05).

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A 2.5 * 2.0 * 1.5

1.0

0.5 Relative LUC Activity (Fold)Activity LUCRelative 0 - + - LiCl (20mM) - - + Ins (100nM)

EGFP -cat(S33Y)-EGFP B - Ins + Ins - Ins + Ins

Figure 3. 11 - Insulin stimulates -cat nuclear translocation.

(A) HT29 cells were transfected with 3g TopFlash for 12 h. Transfected cells were then treated with 20 mM lithium chloride (LiCl) or 100nM insulin (Ins) for an additional 6 h before harvested for LUC reporter gene analysis. Relative LUC activity was calculated as fold change with the activity in cells received no treatment as 1 fold (mean ± SD, n=3, *, p<0.05). (B) HT29 cells were transfected with 3g of EGFP-S33Y -cat or EGFP expressing plasmid for 24 h. Transfected cells were then treated with or without 100nM insulin (Ins) for an additional 3 h before EGFP signal was detected by confocal microscopy.

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throughout the entire cell, regardless of insulin treatment (Fig. 3.11B, left panel). In the absence insulin, fluorescence signals in HT29 cells transfected with EGFP-

S33Y--cat was also observed in both cytosol and nuclei. However, after insulin stimulation, most, if not all, fluorescence was observed within the cell nucleus, suggesting that insulin stimulates -cat nuclear translocation (Fig. 3.11B, right panel).

3.4.4 Insulin stimulates binding of cat to two TCF-binding sites of the c-Myc gene promoter

It has been suggested that Wnt activation leads to increased c-Myc transcription via binding of cat/Tcf to the Tcf binding elements (TBEs) within the c-

Myc gene promoter region [273]. Fig. 3.12A shows schematically the location of the two TBEs, designated here as TBE1 and TBE2, which have been previously demonstrated to mediate the activation of c-myc promoter by cat/TCF [273]. To investigate whether β-cat nuclear accumulation in response to insulin treatment leads to enhanced binding of β-cat to the c-Myc gene promoter in vivo, we have established chromatin immunoprecipitation (ChIP) assay, showing that anti-c-Myc antibody but not the control antibody precipitated chromatin DNA that contains TBE1 and TBE2 (Fig. 3.12B). We then performed quantitative chromatin immunoprecipitation (qChIP) to measure the binding of β-cat to TBE1 and TBE2 in

HT29 cells. First, we assessed the effect of insulin on β-cat binding to TBE1 from

1 min to 4 h.More than 4-fold increase in -cat and TBE1 interaction was observed after 5 min of insulin treatment (Fig. 3.12C). From 10 min to 1 h the activation was between 2.5- to 3-fold (Fig. 3.12C). During 2-4 h, the activation reached 4.5-to 5-fold

(Fig. 3.12C, 3.13A). Also, following 4 h of insulin treatment, binding of -cat to TBE2

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was shown to be increased by more than 3-fold (Fig. 3.13B). Therefore, we limited the treatment time to 4 h and assessed the effect of PI3K, mTOR or Akt/PKB inhibition on insulin-stimulated -cat binding to the TBEs. We observed that PI3K inhibition but not mTOR inhibition blocked insulin stimulated cat binding to TBE1

(Fig. 3.13A) and TBE2 (Fig. 3.13B). Akt/PKB inhibition also did not attenuate insulin- stimulated binding of cat to TBE1 (Fig. 3.14). We therefore conclude that insulin signaling enhances the binding of cat/Tcf to the human c-Myc gene promoter, and this stimulation involves PI3K but not Akt/PKB activity.

3.5 Discussion

Hyperinsulinemia is a common symptom in Type II DM patients, and at pathological concentrations, insulin may exert its functions via both insulin and IGF-1 receptors. Aberrant expression of IGF-1 and its receptors are often associated with the development of colorectal tumors [409, 410]. Using rodent models, it has been shown that acute hyperinsulinemia stimulated colorectal epithelial cell proliferation

[171], and long-term administration of insulin induced intestinal tumor formation [170,

394, 395], These observations correlate with clinical investigations, suggesting that subjects with Type II DM are more vulnerable to the development of colorectal tumors [392, 393, 411].

Aberrant expression and function of components in the Wnt signaling pathway are also associated with intestinal tumor development [370, 387, 412]. An important component of the Wnt signaling pathway is the negative modulator GSK-3.

Working with APC, Axin, and CK1, GSK-3 proteins (GSK-3 and 3) phosphorylate

-cat and render it to proteasome-mediated degradation [413-415]. GSK-3

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A TBE1 TBE2 -1157  -1149 -584  -576 +397  +2022 +1 Intron 1

TATA

TBE1 TBE2 B

PCR product

Primers - - + + - - + + 4h 100nM Ins Flag -cat Flag -cat Flag -cat Flag -cat IP Antibody

C 6 * * TBE1 5 Intron 1 * * * 4 * * * 3

2

1

0

Relative DNA ConcentrationDNAChange)Relative (Fold - 1 5 10 20 30 60 120 180 240 T(min) Figure 3. 12 - Insulin stimulates in vivo binding of -cat to the human c-Myc gene promoter.

(A) A schematic representation of the human c-Myc gene promoter and its Intron 1 region, as well as the locations of PCR primers in the ChIP assay (GenBank accession number: AC103819). TBE1 and TBE2, two cat/TCF binding elements. TATA, TATA box. These Tcf/Lef consensus sequences are located between -1157 to -1149, -584 to -576. Intron 1 sequence is located between +397 to +2022. (B) A representative ChIP result shows that the anti-β-cat antibody but not a control (anti-FLAG tag) antibody precipitated TBE1 and TBE2 containing chromatin DNA. (C) Insulin treatment enhances binding of b-cat to TBE1. HT29 cells were serum starved for 24 h before treated with insulin (100 nM) for 1 min to 4 h, followed by ChIP assay with a polyclonal antibody against -cat. The amount of TBE1 in the chromatin DNA precipitated in each assay was then quantitatively assessed by real time PCR, and the results are presented as fold of relative copy numbers (with untreated samples defined as 1 fold, mean ± SD, n=/>3, *, p<0.05).

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A

5 * * TBE1 4 Intron 1 3

2

1

0

Relative DNA ConcentrationDNAChange)Relative (Fold - + - + - + Ins (100nM) - - + + - - LY (50M) - - - - + + Rap (100nM) B 4 TBE2 * Intron 1 * 3

2

1

0

Relative DNA ConcentrationChange) DNA (FoldRelative - + - + - + Ins (100nM) - - + + - - LY (50M) - - - - + + Rap (100nM)

Figure 3. 13- Insulin stimulated in vivo binding of -cat to the human c-Myc gene promoter involves PI3K but not PKB or mTOR.

HT29 cells were serum starved for 24 h prior to pretreatment with LY294002 (50 mM) (A, B), or rapamycin (100 nM) (A, B) for 1 h, followed by the additional 4 h of incubation with insulin (100 nM). ChIP assay is then performed with a polyclonal antibody against -cat. The amount of TBE1 (A) and TBE2 (B) in the chromatin DNA precipitated in each assay was then quantitatively assessed by real time PCR, and the results are presented as fold of relative copy numbers (with untreated samples defined as 1 fold, mean ± SD, n=/>3, *, p<0.05).

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6 * 5 TBE1 * Intron 1 4

3

2

1

Relative DNA ConcentrationDNAChange)Relative (Fold 0 - + - + - + Ins (100nM) - - + + - - LY (50M) - - - - + + Akti1/2 (100nM)

Figure 3. 14 - Insulin stimulated in vivo binding of -cat to the human c-Myc gene promoter involves PI3K but not PKB.

HT29 cells were serum starved for 24 h prior to pretreatment with LY294002 (50 M), or Akti-1/2 (100 nM) for 1 h, followed by the additional 4 h of incubation with insulin (100 nM). ChIP assay is then performed with a polyclonal antibody against -cat. The amount of TBE1 in the chromatin DNA precipitated in each assay was then quantitatively assessed by real time PCR, and the results are presented as fold of relative copy numbers (with untreated samples defined as 1 fold, mean ± SD, n=/>3, *, p<0.05).

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molecules are substrates of other protein kinases, including Akt/PKB, another major effector of insulin signaling [416]. Therefore both Wnt and insulin may inactivate their common target, GSK-3, to exert overlapping biological functions. However, examinations of the crosstalk (defined here as one pathway that uses the effector/(s) of another pathway to exert its function) between these two signaling pathways have led to a great debate. Ding et al. demonstrated in cancer cell lines and fibroblasts that 2 h of insulin treatment led to GSK-3 phosphorylation and inactivated GSK-3 enzymatic activity, but that free cytoplasmic -cat levels were not altered. On the other hand, treating cells with either Wnt conditioned medium or lithium induced free cytoplasmic -cat accumulation but did not affect the phosphorylation status of GSK-

3. Based on these observations, Ding et al. suggested that insulin and Wnt signals regulate GSK-3 via different mechanisms, leading to distinct downstream events, and that phosphorylation of GSK-3/ on Ser21/ 9 is not sufficient to induce free - cat accumulation. However, studies conducted by other groups have shown that insulin or IGF-1 could stimulate target genes of the Wnt pathway or -cat mediated reporter gene transactivation [298-300, 417-419].

Observations made in this study support the existence of crosstalk between insulin and Wnt signaling pathways, and indicate this crosstalk may not involve Akt-

GSK-3. We found that insulin stimulates cell proliferation and c-Myc expression via both Akt/PKB-dependent and independent mechanisms. In addition to c-Myc, we observed that insulin and IGF-1 stimulated the expression of cyclin D1, another proto-oncogene that falls into the category of Wnt responsive genes (Fig. 3.5 and data not shown). The activation by insulin cannot be effectively blocked by mTOR

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inhibition although PI3K inhibition was effective (data not shown). These results, combined with our observations that insulin stimulates TopFlash, -cat nuclear translocation, and the in vivo binding of -cat to the human c-Myc gene promoter, suggest that insulin indeed crosstalks with Wnt signaling, and that this crosstalk involves a PI3K-dependent but Akt/PKB-independent signaling event. A recent report shows that in response to PI3K activation, -cat interacts with a member of the 14-3-3 , 14-3-3which allows a high level of free -cat to be maintained and therefore increases -cat dependent transcriptional activation [417].

Further investigation is required to determine whether  plays a role in insulin stimulated -cat nuclear translocation in the intestinal cancer and non-cancer cells.

PI3K-mediated but Akt/PKB-independent signaling has not been recognized until recently. P21-activated protein kinase 1 (PAK-1) is an important mediator of mitogenic factors [420]. Zhang et al. have shown that in the human bronchial epithelial cells, cigarette smoke-stimulated EGF receptor activates the expression of

FRA-1 through the PI3K-(PAK-1)-(Raf)-MEK-ERK signaling cascade, without the participation of Akt/PKB [421]. The proto-oncogene PAK-1 is also a known downstream target of cat-TCF [276]. Therefore, PAK-1 or other members of the PAK family may play a role in mediating the crosstalk between the insulin and Wnt signaling pathways.

The mTOR pathway controls protein synthesis in response to signals from mitogenic factors, nutrients, cellular energy levels and stress. In quiescent cells, mTOR activity is under the control of the tumor suppressor complex TSC1/2. Insulin and other growth factors activate PI3K-Akt/PKB which mitigates TSC1/2 activity, and

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causes mTOR activation [422]. A recent study discovered that Wnt signaling also activates mTOR, involving upstream components of the Wnt signaling but not cat

[330]. It is known that low cellular energy levels lead to the activation of AMP- activated protein kinase (AMPK), which reduces protein synthesis by inhibiting mTOR via TSC1/2 activation [423]. Inoki et al. discovered that GSK-3 functions as a priming kinase in assisting AMPK to phosphorylate and activate TSC2. Essentially,

Wnt activation blocks the function of GSK-3 and hence disables the phosphorylation and activation of TSC2, therefore releasing the inhibitory effect of TSC2 on mTOR.

Since mTOR is a known effector of insulin signaling, this study provides evidence for the existence of crosstalk between the Wnt and insulin pathways from another perspective.

In summary, our study shows that at pathological concentrations, insulin stimulates growth and proto-oncogene expression in intestinal cells, and that the stimulatory effects of insulin involve both Akt/PKB-dependent and Akt/PKB- independent signaling events. In addition to demonstrating that insulin activates c-

Myc expression via Akt/PKB-dependent mTOR signaling, we have also shown that insulin stimulates -cat nuclear translocation and in vivo binding of -cat to the c-

Myc promoter in a Akt/PKB-independent manner. With the c-Myc gene as an example, Fig. 4.1 shows a summary of our current understanding of the crosstalk between insulin and Wnt signaling pathways. The expression of c-Myc at the translational level can be stimulated by both insulin and Wnt through mTOR activation. Insulin activates mTOR via Akt/PKB mediated inhibition of the TSC1/2 complex, while Wnt stimulates mTOR via inactivating GSK-3 and the TSC1/2

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complex. The expression of c-Myc at the transcription level can be activated by cat/TCF in response to either Wnt activation or insulin/IGF-1 stimulation. Therefore, insulin, and possibly IGF-1, may stimulate cat/TCF activity in intestinal epithelia via an unknown signaling cascade that involves PI3K but not Akt/PKB.

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CHAPTER 4: p-21 Activated Protein Kinase 1 (PAK-1) Functions as a Linker between Insulin and Wnt Signaling Pathways in the Intestine

Data presented in this chapter (except for Fig. 4.3, 4.5, 4.14 and 4.20) is published in Oncogene. Sun J, Khalid S, Rozakis-Adcock M, Fantus IG and Jin T (2009) [424].

(All data presented in figures of this chapter were contributed by Sun J. Animals used and materials required for generating shRNA-expressing lentiviruses in this study, except for the PAK-1 shRNA expressing plasmids were contributed by Khalid S, Rozakis-Adcock M and Fantus IG.)

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4.1 Abstract

Hyperinsulinemia and Type 2 diabetes are associated with an increased risk of developing colorectal tumors. We found previously that in intestinal cells, insulin or IGF-1 stimulates c-Myc and cyclin D1 protein expression via both Akt/PKB- dependent and Akt/PKB-independent mechanisms. The effect of Akt/PKB is attributed to the stimulation of c-Myc translation via mTOR. Akt/PKB-independent stimulation was, however, associated with an elevation of -catenin (-cat) in the nucleus as well as an increased association between -cat and TCF binding sites on the c-Myc promoter, detected by chromatin immunoprecipitation (ChIP). Here we show insulin stimulated phosphorylation/activation of p-21 activated protein kinase-1

(PAK-1) in an Akt/PKB-independent manner in vitro and in an in vivo hyperinsulinemic mouse model. Significantly, shRNA-mediated PAK-1 knockdown attenuated both basal and insulin stimulated c-Myc and cyclin D1 expression, associated with a marked reduction in ERK activation and -cat phosphorylation at

Ser675. Furthermore, PAK-1 silencing led to a complete blockade of insulin stimulated -cat binding to the c-Myc promoter and cellular growth. Finally, inhibition of MEK, a downstream target of PAK-1, blocked insulin stimulated nuclear -cat accumulation and c-Myc expression. Our observations suggest that PAK-1 serves as an important linker between insulin and Wnt signaling pathways.

4.2 Introduction

Type 2 diabetes mellitus, previously known as non-insulin dependent diabetes mellitus is classically characterized by insulin resistance, hyperinsulinemia

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and hyperglycemia. More than 90% of all diabetes cases in North America belong to this category and it affects over 150 million people worldwide [425]. Epidemiological studies have shown a positive correlation between the metabolic syndrome, Type 2 diabetes, hyperinsulinemia and the development of colorectal cancer (CRC) [426].

To explore mechanisms underlying this correlation, studies have been performed using rodent models, showing an effect of insulin on the formation of colorectal adenomas [170, 392, 393].

Although most colorectal tumors are sporadic, a proportion of them are heritable. One of these syndromes is known as familial adenomatous polyposis

(FAP). The FAP locus encodes the tumor suppressor gene Adenomatous Polyposis

Coli (APC), which is mutated and found to be inactivated in both FAP patients as well as in many sporadic cases of CRC [370, 397-399, 427]. Under quiescent conditions, Axin, Glycogen Synthase Kinase 3 (GSK-) and APC form a complex with -catenin (-cat) that facilitates -cat phosphorylation by GSK-3 and its subsequent degradation by the ubiquitin/proteasome pathway [242, 428, 429].

-cat is the key effector of the canonical Wnt signaling pathway. In the presence of Wnt ligands, activated Wnt receptors associate with Disheveled (Dvl), leading to the destabilization of the Axin/GSK-3/APC complex and the stabilization of -cat. Free -cat is then translocated into the nucleus to form a bipartite transcription factor with a member of the T cell factor (TCF) family, known as cat/TCF [400, 401] that stimulates the transcription of Wnt or cat/TCF downstream target genes. A number of proto-oncogenes, such as c-Myc and cyclin D1, have been shown to serve as the Wnt downstream targets [273, 274].

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Extensive examinations have demonstrated that the activity of cat/TCF can be stimulated by mechanisms other than the Wnt glycoproteins [430]. For example, there are a number of reports that point to the insulin and IGF-1 signaling pathway as stimulators of cat/TCF activity [298, 385]. In a previous study of ours, we were able to demonstrate that insulin stimulates the expression of the proto-oncogene, c-

Myc, via a PI3K and protein kinase B (PKB)/Akt-dependent pathway involving mTOR, as well as a PI3K-dependent but Akt/PKB-independent mechanism. The latter pathway was associated with an increase of nuclear -cat content and the stimulation of the association between -cat and the TCF binding sites within the c-

Myc gene promoter region [391]. We have therefore concluded that insulin utilizes the effector of Wnt signaling, at least in part, in exerting its stimulatory effect on gene transcription (Sun and Jin, 2008). Mediators for this crosstalk, however, remain to be identified (Fig. 4.1).

The serine/threonine kinase P-21 activated protein kinase 1 (PAK-1) is known to be involved in the development and metastasis of tumors from various tissues including the intestine [431-433]. PAK-1 was initially recognized as a downstream target of the Rho family GTPases, Rac1 and Cdc42, in regulating actin remodeling

[70, 79, 434]. PAK-1 can also be activated by Akt/PKB [84], or become directly phosphorylated/activated by PI3K [435] or phosphoinositide-dependent kinase isozyme 1 (PDK-1) on Thr 423 [83], without requiring Akt/PKB. We show here that insulin stimulates the phosphorylation/activation of PAK-1 in both intestinal cancer and non-cancer cell lines, and in several mouse tissues, including the intestines. It appears

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Wnt Insulin AMPK

PI3K Dvl TSC-1/2

PAK-1? GSK-3 Akt/PKB

mTOR -cat Tcf/Lef

-cat Nuclear Levels 4EBP1 S6K -cat/TCF binding to p p c-Myc gene promoter

Translation Transcription

c-Myc Expression/Cell Growth

Figure 4. 1 - Proposed mechanism for insulin activated c-Myc expression and associated cell growth.

A diagrammatic representation of Akt-dependent and Akt-independent mechanisms that mediate the stimulatory effect of c-Myc protein translation and gene transcription. The current study investigates whether the Akt independent stimulation is mediated by PAK-1. PI3K: phoshoinositide 3-kinase; Akt/PKB: protein kinase B; mTOR: mammalian target of rapamycin; 4EBP1: eIF4E-binding protein 1; S6K: p70 ribosomal S6 kinase; AMPK: AMP-activated protein kinase; TSC-1/2: Tumberous sclerosis protein 1/2; PAK-1: p21 activated protein kinase 1; Dvl: disheveled; GSK-3: glycogen synthase kinase-3; Tcf/Lef: T- cell factor/lymphoid enhancer factor.

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that the activation is independent of Akt/PKB. By utilizing a combination of chemical inhibitors of various signaling molecules, as well as a Lentivirus based small hairpin

RNA (shRNA) knockdown approach, we identified that PAK-1 serves as an important mediator of the crosstalk between insulin and cat/TCF in the regulation of c-Myc gene expression and cell proliferation.

4.3 Materials and Methods

Materials

Tissue culture medium, FBS, and oligonucleotides were purchased from Invitrogen

Life Technology Inc. (Burlington, ON, Canada). Insulin was provided by Novo

Nordisk (Novo Nordisk, Copenhagen, Denmark). The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 were purchased from Calbiochem (EMD Biosciences,

Inc., San Diego, CA). Lentiviral shRNAmir gene knockdown system was that of

Thermo Fisher Scientific (Huntsville, AL). The three PAK-1 shRNAmir sequences are shown in Fig. 4.11.

Animals and diets

All procedures were conducted according to protocols and guidelines approved by the Toronto General Hospital Animal Care Committee. FVB mice at the age of 13 weeks were randomly placed on a low-fat diet (LFD) (5% kcal fat), or on a high-fat content diet (HFD) (45% kcal fat, 15% fructose). The mice were housed in the

Animal Care Facility of the Toronto General Hospital with a 12h light/dark cycle.

Body weights and intra-peritoneal glucose tolerance test – Body weights were monitored and recorded weekly. Intra-peritoneal glucose tolerance tests (IPGTT)

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were performed on the mice after 15 weeks on the different diets. Following overnight fast (1.5g/kg body weight) glucose was administered by IP injection. A blood sample was drawn from a tail vein at 0, 10, 20, 30, 60, 90, and 120 minutes following glucose administration, and blood glucose levels were measured using a

Sure Step, One Touch Glucometer (Lifescan Inc., Johnson & Johnson, Milpitas, CA,

USA). For plasma insulin determinations, a blood sample (100 μl) was removed from the tail vein at the 0 min time period. Plasma was separated by centrifugation at 4°C and stored at –80°C until assayed. Plasma was assayed for insulin using a mouse insulin ELISA kit (Linco Research Inc, St. Charles, MO, USA).

Plasmids, DNA transfection, cell culture, and MTT assay

The dominant negative PAK-1 plasmid, the K299R mutant, and the vector control, were gifts from Dr. Jefferey Field [388, 436]. DNA transfection was performed using

Lipofectamine 2000 per the manufacturer’s instructions (Invitrogen Life Technology

Inc.). The human colon cancer cell lines HT29 and Caco2, the human breast cancer cell line MCF-7, and the rat intestinal cell line IEC-6 were purchased from American

Type Culture Collection (ATCC). They were maintained per ATCC instructions. The

MTT assay was carried out as described previously by Wang et al. [402].

Antibodies, cell fractionation and fluorescence microscopy

Antibodies for Akt/PKB, phospho- Akt/PKB (Ser473), phospho-GSK-3/ (Ser21/9), phospho--cat (Ser674), phospho-PAK-1/2 (Thr423/Thr402), PAK-1, histone H3 and

-actin were purchased from Cell Signaling Technology (Cedarlane, ON, Canada).

Antibody for GSK-3 (clone 4G-1E) and Myc-Tag were purchased from Millipore Corp.

(Billerica, MA). Antibodies for -cat, c-Myc, cyclin D1, and the horseradish 116

peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz

Biotechnology, Inc. (Santa Cruz, CA). Nuclear and cytosolic protein fractions were prepared as described by Olnes and Kurl (1994) (Jin and Sun, 2008). Fluorescence signal in HT29 cells expressing lentiviral delivered GFP-shRNA was viewed with an

Olympus IX71 microscope. qChromatin immunoprecipitation (ChIP)

Approximately 2 x 107 cells were used for each qChIP assay [389]. After sonication of control or insulin treated cells, anti--cat antibody was applied to precipitate sheared chromatin DNA that interacts with -cat. In each assay, precipitated chromatin DNA was dissolved in 60 l of TE buffer, and 2 l of sample was taken each time for real time PCR [389]. Experimental and control primers for ChIP assay are presented in Table 3.1.

Statistical analysis- All data are presented as mean +/- SD. Statistical analysis was done by either Student’s t test or one way ANOVA when appropriate. Significance was assumed at a p value of less than 0.05.

4.4 Results

4.4.1 Insulin stimulates PAK-1 phosphorylation in intestinal cells

As illustrated schematically in Fig. 4.1, our previous observations [391] prompted us to search for a PI3K-dependent but Akt/PKB-independent signaling pathway that mediates the crosstalk between insulin/IGF-1 and the Wnt signaling effector, cat/TCF. To assess whether PAK-1 serves as such a mediator, we first examined the effect of insulin on PAK-1 phosphorylation at its activation site, Thr423.

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The anti-PAK-1 antibody utilized in this study specifically recognizes PAK-1 but not

PAK-2, while the anti-phospho-PAK-1 (pPKA-1) antibody recognizes both pPAK-1

(at Thr423) and pPAK-2 (at Thr402). In colon cancer cell lines HT29 (Fig. 4.2A) and

Caco-2 (data not shown), pPAK-2 was constitutively expressed, regardless of insulin treatment. In the rat intestinal non-cancer cell line IEC-6 and the human breast cancer cell line MCF-7, pPAK-2 was not detected in samples without insulin treatment (Fig. 4.2A). Fig. 4.2A also shows that in IEC-6, HT29 and MCF-7 cell lines, insulin treatment led to increased PAK-1 phosphorylation, detected 1 min after insulin treatment in the MCF-7 cell line and 5 min after insulin treatment in HT29 and

IEC-6 cell lines. We then examined the effect of IGF-1, and observed that at either

50 or 100 nM, IGF-1 elicited PAK-1 phosphorylation in the HT29 cell line (Fig. 4.2B).

A representative Western blot in the HT29 cell line indicated that the stimulatory effect of IGF-1 on PAK-1 phosphorylation was sustained for up to 4 h (Fig. 4.2C).

To investigate whether the stimulation of PAK-1 phosphorylation by insulin is mainly mediated by the IGF-1 receptor (IGF-1R), we utilized a specific antibody to block the function of IGF-1R. As shown in Fig. 4.3, after HT-29 cells were pre-treated with the

IGF-1R-specific antibody, we still observed an activation of pPAK-1 expression after insulin treatment, while expression of phospho-IGF-1R is no longer detectable (Fig.

4.3).

To examine the effect of insulin on PAK-1 phosphorylation in vivo, female adult FVB mice were injected with insulin intraperitoneally (IP) (0.2 U/kg). Ten min after the injection, mice were sacrificed and various tissues were taken for Western

118

A p-PAK-1 p-PAK-2 IEC-6 PAK-1

p-PAK-1 p-PAK-2 HT29 PAK-1

p-PAK-1 MCF-7

PAK-1

C 1 5 10 20 30 60 100nM Ins (min)

B p-PAK-1 p-PAK-2 HT29 PAK-1

50 100 C nM IGF-I (4h)

p-PAK-1 C p-PAK-2 HT29 PAK-1

C 1 5 10 20 30 1 2 3 4 100nM IGF-I min h

Figure 4. 2 - Insulin stimulates PAK-1 phosphorylation

Detection of total PAK-1 as well as phosphorylated PAK-1 (Thr423) and PAK-2 (Thr402) in cell lysates. Indicated cell lines were starved overnight and treated with indicated concentrations of insulin or IGF-1 for indicated time. Cell lystates were then prepared for Western blotting. (Representative blot, n=3).

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p-IGF-1R (Tyr980)

IGF-1R

p-PAK-1 p-PAK-2

PAK-1

-actin

- + - + 100nM Ins (4h) - - + + IGF-1R Ab (11g/ml)

Figure 4. 3 - IGFR may not be a major player in the stimulatory effect of insulin on PAK-1 phosphorylation.

HT29 cells were serum starved for 24 h prior to 1 h pretreatment of IGFR antibody 11mg/ml or PBS (vehicle control) followed by 4 h of insulin (Ins) treatment (in the absence of serum). Expression of p-IGF-1R (Tyr980), IGF-1R, p-PAK-1, PAK-1 and -actin (loading control) were detected by Western blotting. (Representative blot, n=3).

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blotting analysis. A representative blot shows that acute insulin treatment stimulated the phosphorylation of both Akt/PKB (Ser473) and PAK-1 in liver, fat tissue,heart, and small as well as large intestines (Fig. 4.4).

4.4.2 Insulin-stimulated PAK-1 phosphorylation is independent of Akt/PKB status both in vitro and in vivo

We then investigated the involvement of Akt/PKB in insulin stimulated PAK-1 phosphorylation. Akt/PKB activity was blocked using a pharmacological inhibitor

Akti1/2, resulting a complete knockdown p-Akt/PKB expression both basally and post insulin stimulation. However, Akt/PKB inhibition did not affect the stimulatory effect of insulin on pPAK-1 expression (Fig. 4.5). We then examined PAK-1 phosphorylation in insulin resistant conditions where insulin induced Akt/PKB activation would be compromised. To this end, HT29 cells were exposed to chronic insulin stimulation, a condition that mimics hyperinsulinemia-induced insulin resistance. As anticipated, following overnight pretreatment of cells, we observed significantly attenuated Akt/PKB phosphorylation in response to further acute insulin treatment, indicative of these cells being in an insulin-resistant state (Fig. 4.6).

Interestingly, insulin was still able to stimulate PAK-1 phosphorylation in insulin pre- treated cells, indicating that p-Akt/PKB may not be involved in PAK-1 phosphorylation/activation. To verify this observation in a more physiological setting, we examined PAK-1 phosphorylation in the large intestinal tissue collected from Low

Fat Diet (LFD) and High Fat Diet (HFD) fed FVB mice. The mice on HFD had higher body weight and exhibited significantly reduced glucose tolerance compared to the

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LIVER FAT L.INT S.INT HEART

p-PAK-1

PAK1

p-Akt (Ser473)

Akt

-actin

- + - + - + - + - + 0.2U Ins/kg Body Wt

Figure 4. 4 - Insulin stimulates PAK-1 phosphorylation in a number of tissue samples in vivo.

Female adult FVB mice were injected with insulin intraperitoneally (IP, 2U/kg). Mouse tissues were taken 10 min after insulin injection and subject to Western blotting for examining Akt and PAK-1 phosphorylation. L.INT, large intestine; S.INT, small intestine (Representative blot, n=3).

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p-Akt

Akt

p-PAK-1 p-PAK-2

PAK-1

-actin

- + - + 100nM Ins (4h) - - + + Akti1/2 (100nM)

Figure 4. 5 - Insulin stimulated PAK-1 phosphorylation is independent of Akt status: Evidence from utilizing Akt chemical inhibition.

HT29 cells were serum starved for 24 h prior to 1 h pretreatment of DMSO (vehicle control) or AKti1/2 (100nM), followed by 4 h of insulin (Ins) treatment (in the absence of serum). Expression of p-Akt (Ser473), Akt, p-PAK-1, PAK-1 and -actin (loading control) were detected by Western blotting (Representative blot, n=3).

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WT Insulin Resistant

p-Akt (Ser473)

Akt

p-Erk1/2

Erk1/2

p-PAK-1 p-PAK-2

PAK1

- + - + 100nM Ins

Figure 4. 6 - Stimulation of PAK-1 phosphorylation by insulin is independent of AKT status: In vitro evidence.

In vitro insulin resistance was created by pre-treating HT29 cells with insulin (1nM) for 24h. After washing with serum free DMEM, both control wild type (WT) and insulin pre- treated HT29 cells were further treated with 100nM insulin (Ins) for 4 h before harvesting for Western blot. The expression of Akt, p-AKT (Ser473), PAK-1, pPAK-1 (Thr423) and - actin (loading control) were detected by Western blotting (Representative blot, n=4).

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LFD HFD

p-PAK-1

PAK-1

p-Akt

Akt

-actin

- + - + 0.2U Ins/kg Body Wt

Figure 4. 7 - Stimulation of PAK-1 phosphorylation by insulin is independent of AKT status: In vivo evidence.

Examination of the effects of insulin resistance on PAK-1 phosphorylation post insulin treatment in vivo. Large intestinal tissues were collected from LFD and HFD fed mice 10 min after IP injection of insulin. The expression of AKT, p-AKT (Ser473), PAK-1, pPAK-1 (Thr423) and -actin (loading control) were detected by Western blotting (Representative blot, n=4).

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LFD controls (data not shown). In addition, the HFD mice also had higher fasting plasma insulin levels compared with the LFD mice (0.52 +/- 0.02 ng/ml in HFD mice versus 0.44 +/- 0.006 ng/ml in LFD mice). Ten min after intraperitoneal (IP) injection of insulin (0.2 U/Kg), mice were sacrificed and the cell lysates from large intestines were prepared for the detection of Akt/PKB and PAK-1 phosphorylation by Western blotting. As shown in Fig. 4.7, in the HFD fed mice, acute insulin treatment elicited a much smaller effect on Akt/PKB phosphorylation, compared with that in the LFD fed mice. In contrast, acute insulin treatment generated a more profound effect on PAK-

1 phosphorylation in the HFD fed mice compared with that in the LFD mice. These observations further suggest that insulin activation of PAK-1 in intestinal cells is not affected by Akt/PKB activation.

4.4.3 Functional knockdown of PAK-1 reduces insulin stimulated oncogene expression and insulin induced -cat binding to human c-Myc gene promoter

To examine whether PAK-1 plays a role in insulin stimulated proto-oncogene expression, we first utilized a plasmid construct that expresses a Myc-tagged dominant negative (DN) form of human PAK-1 (K299R) [88, 437] to inhibit PAK-1 function in the HT29 cell line. We also utilized and assessed the expression of a constitutively active (CA) form of PAK-1 (T423E) [438]. The DN PAK-1 mutant was initially shown to inhibit Ras mediated transformation of Rat-1 fibroblasts and

Schwann cells [88, 437], and has been used by several other studies that required functional knockdown of PAK-1 [439, 440]. The CA PAK-1 mutant was shown to stimulate Raf-1 phosphorylation at Ser338, thereby enhancing its mitochondrial localization [438].

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Western blot analysis showed that when HT29 cells were transfected with mutant PAK-1 (K299R), we were able to detect the expression of PAK-1 (K299R) with the Myc-tag antibody (Fig. 4.8). PAK-1 (K299R) expression in HT29 cells did not alter basal cyclin D1 or c-Myc expression, but was associated with ~35% and

~25% reduction in cyclin D1 and c-Myc protein expression in response to insulin treatment, respectively (Fig. 4.8). In contrast, PAK-1 (T423E) expression led to an increase in cyclin D1 and c-Myc expression, only in the absence of insulin stimulation (Fig. 4.9).

We then examined whether the association of -cat to the two transcriptional activating sites of the c-Myc gene promoter, Tcf Binding Element 1 and 2 (TBE1 and

2), would be affected by PAK-1 (K299R) expression, using our previously described quantitative chromatin immunoprecipitation (qChIP) approach [391]. The positions of TBE1 and TBE2, as well as the positions of control primers utilized in our qChIP analysis are shown in Fig. 3.12A. While insulin treatment stimulated a nearly 4-fold increase in -cat binding to TBE1 in both un-transfected and vector transfected controls, only a 2.8-fold increase was detected in cells that were transfected with DN

PAK-1 (K299R) (Fig. 4.10A). Similarly, DN PAK-1 (K299R) expression resulted in an approximately 20% reduction in insulin stimulated -cat binding to TBE2, compared with the controls (Fig. 4.10B).

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Vector DN PAK-1

cyclin D1

c-Myc

Myc Tag

PAK-1 (K299R) PAK-1 (WT)

-actin

- + - + 100nM Ins (4h)

* * 8 3 cyclin D1 c-Myc 7 2.5 6 5 2 4 1.5 3 1

2 Relative Intensity Relative 1 Intensity Relative 0.5 0 0 - + - + - + - + 100nM Ins (4h) Vector DN PAK-1 Vector DN PAK-1

Figure 4. 8 - Expression of dominant negative PAK-1 attenuates insulin stimulated c-Myc and cyclin D1 expression.

Approximately 4 x 105 HT29 cells were transfected with 3g of Myc-tagged dominant negative (DN) PAK-1 (K299R) or pcDNA3.1 vector plasmids for 24 h. The cells were then starved for 24 h prior to a 4 h insulin (100nM) treatment. The expression of Cyclin D1, c-Myc, Myc-tag, PAK-1 and -actin were (loading control) detected by Western blotting (Top panel). Bottom panels show the densitometrical analyses of the top panel. The data are expressed as mean relative intensity +/- S.D. (*, p<0.05, (n=3)).

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Vector CAPAK-1

cyclin D1

c-Myc

Myc Tag

PAK-1 (T423E) PAK-1 (WT)

-actin

- + - + 100nM Ins (4h)

* *

14 cyclin D1 7 c-Myc 12 6 10 5 8 4 6 3

4 2 Relative Intensity Relative 2 Intensity Relative 1 0 0 - + - + - + - + 100nM Ins (4h)

Vector CA PAK-1 Vector CA PAK-1

Figure 4. 9 - Expression of constitutively active PAK-1 increases basal c-Myc and cyclin D1 expression.

Approximately 4 x 105 HT29 cells were transfected with 3g of Myc-tagged constitutively active (CA) PAK-1 (T423E) or pcDNA3.1 vector plasmids for 24 h. The cells were then starved for 24 h prior to a 4 h insulin (100nM) treatment. The expression of Cyclin D1, c-Myc, Myc-tag, PAK-1 and -actin were (loading control) detected by Western blotting (Top panel). Bottom panels show the densitometrical analyses of the top panel. The data are expressed as mean relative intensity +/- S.D. (*, p<0.05, (n=3)).

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#

A 5 * *

) 4.5

4 Intron 3.5 * 3

2.5 Control 2 100nM Insulin

Fold ChangeFold (TBE1/ 1.5

1

Relative 0.5

0 WT Vector DN PAK-1 B 5

) 4.5 #

4 Intron * * 3.5

3 * 2.5 Control

Change(TBE2/ 2 100nM Insulin

1.5

1

Relative FoldRelative 0.5

0 WT Vector DN PAK-1 Figure 4. 10 - Expression of dominant PAK-1 attentuates insulin activated -cat binding to the c- Myc gene promoter.

(A, B) Non-transfected wild type (WT), pcDNA3.1 (vector) plasmid transfected and Myc-tagged dominant negative (DN) PAK-1 (K299A) transfected HT29 cells were treated with insulin (100nM) for 4h, followed by qChIP assay with a polyclonal antibody against -cat. The amount of TBE1 (A) and TBE2 (B) in the chromatin DNA precipitated in each assay were then quantitatively accessed by real time PCR, and the results are presented as fold of relative copy numbers post adjustments of the intron copy number (as the negative control). The readings in insulin untreated and plasmid untransfected samples were defined as 1 fold (mean ± SD, n=3, *, or #, p<0.05).

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4.4.4 Knockdown of the expression of PAK-1 blocks insulin stimulated c-Myc and cyclin D1 expression, and -cat nuclear content

Although the attenuating effect of PAK-1 (K299R) on insulin stimulated c-Myc and cyclin D1 expression was statistically significant, the failure to elicit a more robust inhibition was possibly due to the low transfection efficiency of PAK-1 (K299R) in the HT29 cell line. Based on the data obtained from transfecting HT29 cells with an enhanced green fluorescent protein (EGFP) expressing control plasmid, we estimated that the transfection efficiency in this cell line by our method was about 12

– 15% (Data not shown). As an alternative approach to confirm the role of PAK-1, we utilized a Lentivirus-based delivery system to express a PAK-1 small hairpin RNA

(shRNA) to knockdown the expression of PAK-1 in the HT29 cell line (Fig. 4.11A).

Infected cells were selected by puromycin treatment and detected by the expression of GFP (Fig. 4.11B), which is driven by the same promoter that drives the expression of the shRNA. As shown in Fig. 4.12, the expression of PAK-1 shRNA led to more than 70% reduction in PAK-1 protein expression, compared to cells expressing scrambled shRNA. Knockdown of PAK-1 expression was associated with ~50% and

~65% reduction in c-Myc and cyclin D1 expression (Fig. 4.12). Furthermore, PAK-1 knockdown led to significantly reduced p-ERK1/2 expression in the presence and absence of insulin treatment. In addition, we show here for the first time that insulin stimulates -cat phosphorylation at Ser675. The Ser675 residue on -cat has recently been implicated in an increase in -cat nuclear accumulation and transcriptional activity [260, 441]. More importantly, PAK-1 knockdown in the HT29 cell line abolished the response of -cat Ser675 phosphorylation to insulin treatment

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A.

Sense Loop Antisense

1 ACCCAAGAAAGAGCTGATTATT TAGTGAAGCC AATAATCAGCTCTTTCTTGGGC ACAGATGTA 2 AGGCCTAGACATTCAAGACAAA TAGTGAAGCC TTTGTCTTGAATGTCTAGGCCG ACAGATGTA 3 CGGGCATCATGGCCATCGAAAT TAGTGAAGCC ATTTCGATGGCCATGATGCCCA ACAGATGTA

B. DIC GFP

GIPZ-Scrambled

GIPZ-PAK1shRNAmir

Figure 4. 11 - Knock down of PAK-1 expression using PAK-1 shRNA.

(A) DNA sequences of the three PAK-1 shRNAmir (provided by Thermo Fisher Scientific) utilized in this study. (B) HT29 cells were infected with a pool of three Lentivirus preparations expressing three different GIPZ-PAK-1 shRNAmir or GIPZ-Scrambled for 72h, followed by puromycin selection. GFP signal was visualized by fluorescent microscopy.

132

GIPZ-Scrambled GIPZ-PAK-1shRNAmir

PAK-1

c-Myc

cyclin D1

-cat (Ser675) (Short Exposure) -cat (Ser675) (Long Exposure)

-cat

p-Erk1/2

Erk1/2

-actin

- + - + - + - + 100nM Ins (4h) - - + + - - + + 50M PD

Figure 4. 12 - Knockdown of PAK-1 expression attenuates the stimulatory effects of insulin.

Both control (GIPZ-Scrambled) and GIPZ-PAK-1 shRNAmir expressing cells were pre-treated with or without the MEK inhibitor PD98059 (PD) for 1h prior to insulin treatment. Amounts of phosphorylated PAK-1 (Thr423), PAK-1, c-Myc, cyclin D1, Ser675 phosphorylated -cat, total - cat, p-ERK, ERK and -actin (as the loading control) were detected by Western blotting (Representative blot, n=3).

133

A. B.

1.5 PAK-1 3.5 * c-Myc 3 1 2.5 * 2 1.5 0.5 * * 1 * * * * *

* * Relative DensityRelative Relative DensityRelative 0.5 0 0 - + - + - + - + - + - + - + - + 100nM Ins (4h) - - + + - - + + - - + + - - + + 50M PD C. D. * * 6 * cyclin D1 2 -cat (Ser675) 5 1.6 4 1.2 3 * * 0.8 * 2 * *

1 0.4 Relative DensityRelative Relative DensityRelative * * * * 0 0 - + - + - + - + - + - + - + - + 100nM Ins (4h) - - + + - - + + - - + + - - + + 50M PD

E. 1.6 * p-ERK1/2 1.2 * 0.8 *

0.4 * * *

* Relative DensityRelative 0 - + - + - + - + 100nM Ins (4h) - - + + - - + + 50M PD

Figure 4. 13 - Knockdown of PAK-1 expression attenuates the stimulatory effects of insulin – Densitometric analysis.

Densitometric analysis of PAK-1 (A), c-Myc (B), -cat (675) (C) and p-Erk1/2 (D) in Fig. 30. The data are expressed as mean relative intensity +/- S.D. (*, p<0.05, (n=3)).

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(Fig. 4.12). Furthermore, the basal levels of phospho--cat (Ser675) in the PAK-1 shRNA-expressing HT29 cells was also significantly reduced (Fig. 4.12). Finally,

MEK inhibiton with PD98059 in PAK-1 shRNA expressign cells completely inhibited

ERK phosphorylation and -cat phosphorylation at Ser675. However, in scrambled shRNA expressing cells, MEK inhibition did not completely block the stimulatory effect of insulin on -cat phosphorylation at Ser675, c-Myc or cyclin protein expression (Fig. 4.12). Interestingly, while inhibtion of PKA abolished the effect of insulin on CREB phosphoryalation, -cat phosphorylation at Ser675 in response to insulin treatment was not affected, suggesting that insulin stimulates this phosphorylation event in a PKA-independent manner (Fig. 4.14). Desitometrical analysis of Fig. 4.12 is presented in Fig. 4.13.

We then assessed whether PAK-1 knockdown affects its own phosphorylation in response to insulin treatment and MEK inhibition. Fig. 4.15 shows that while the expression level of p-PAK-2 in the HT29 cell line was not affected by PAK-1 shRNA, insulin treatment, nor MEK inhibition, insulin stimulated PAK-1 phosphorylation was attenuated by PAK-1 knockdown and was blocked by MEK inhibition.

We have suggested that insulin crosstalks with the Wnt pathway by increasing nuclear-cat content [391, 404]. To further determine the role of PAK-1 in this crosstalk, we examined nuclear cat levels post PAK-1 knockdown. A significant increase in nuclear -cat content was detected in HT29 cells expressing the scrambled shRNA post insulin treatment (Fig. 4.16). The basal level of nuclear -cat in cells that express PAK-1 shRNA was reduced compared to that of the scrambled control. Even though cells expressing PAK-1 shRNA responded to insulin, the effect

135

p-CREB

CREB

p--catenin (Ser675)

-catenin

-actin

- + - + 100nM Ins (4h) - - + + H89 (10M)

Figure 4. 14 - PKA inhibition has no appreciable effect on insulin stimulated -cat (Ser675) phosphorylation.

HT29 cells were serum starved for 24 h prior to 1 h pretreatment of H89 or 2l DMSO (vehicle control), followed by 4 h of insulin (Ins) treatment (in the absence of serum). Expression of p- CREB, CREB, p--cat, -cat and -actin (loading control) were detected by Western blotting (Representative blot, n=3).

136

GIPZ-Scrambled GIPZ-PAK-1shRNAmir

p-PAK-1 p-PAK-2

PAK-1

-actin

- + - + - + - + 100nM Ins (4h) - - + + - - + + 50M PD

Figure 4. 15 - Knockdown of PAK-1 expression attenuates insulin induced PAK-1 but not PAK-2 phosphorylation.

Both control (GIPZ-Scrambled) and GIPZ-PAK-1 shRNAmir expressing cells were pre-treated with or without the MEK inhibitor PD98059 (PD) for 1h prior to insulin treatment. Amounts of PAK-1 (Thr423), PAK-1, and -actin (as the loading control) were detected by Western blotting (Representative blot, n=3).

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of insulin on -cat nuclear accumulation is significantly lower than that in the control cells (1.96 fold vs. 1.3 fold, Fig. 4.16). We then applied qChIP to assess the effect of

PAK-1 knockdown on the association of -cat with the c-Myc gene promoter in the

HT29 cell line. As presented in Fig. 4.17, insulin treatment significantly stimulated the association between -cat and TBE1 (Fig. 4.17A), or between -cat and TBE-2

(Fig. 4.17B), in both mock infected or the control shRNA expressing HT29 cells; the stimulation was not observed in the PAK-1 shRNA expressing HT29 cells.

MTT assays were then performed to assess the effect of PAK-1 knockdown on cell growth. Consistent with our previous study [391], 4, 12, 24, and 48 h insulin treatment resulted in ~1.5-5 fold increase in cell numbers of both untreated (WT) and scrambled shRNA expressing cells (Fig.4.18). However, cells expressing PAK-1 shRNA were unresponsive to 12-48 h insulin treatment, as the number of cells post insulin stimulation were comparable to those that received no insulin treatment (Fig.

4.18). Interestingly, the response to 4 h insulin treatment in the PAK-1 shRNA expressing cells was still significant (Fig. 4.18). This is consistent with the observation that 4 h of insulin treatment still stimulated c-Myc and cyclin D1 protein expression following PAK-1 knockdown (Fig. 4.19). However, PAK-1 shRNA expressing cells were no longer responsive to insulin treatment after 24-48 h, because the expression of c-Myc and cyclin D1 is no longer detectable (Fig. 4.19).

We then assessed the effect of PAK-1 knockdown on cell proliferation as well as apoptosis markers, including Ki67, PCNA, and caspase 3. In PAK-1 shRNA expressing cells, the stimulatory effect of insulin on Ki67 expression was only

138

GIPZ-Scrambled GIPZ-PAK-1shRNAmir

Nuclear -cat

Histone H3

- + - + 100nM Ins

*

* 2.5 Nuclear -cat 2

1.5

1

Relative Intensity Relative 0.5

0 - + - + 100nM Ins

Figure 4. 16 - Knockdown of PAK-1 expression attenuates the stimulatory effects of insulin on nuclear -cat content.

HT29 cells were infected with the lentivirus expressing GIPZ-PAK-1 shRNAmir or GIPZ- Scrambled for 72h, followed by puromycin selection. After 24 h serum starvation and subsequent 4 h insulin (Ins) stimulation, expression of -cat, histone H3 (nuclear protein marker as the loading control) in the nuclear fraction were detected by Western blotting (n=4). Bottom panel shows the densitometric analysis of top panel. Data is expressed as mean ± SD relative intensity (*, p<0.05).

139

# TBE1 A 9 * 8 * 7 6 5 Control 4 100nM Insulin 3 NS 2

1 Relative Fold ChangeFoldRelative(TBE1/Intron) 0 WT Scrambled KD

TBE2

B ) 5 #

Intron 4

* * 3 Control NS

2 100nM Insulin Change(TBE2/

1

0 Relative FoldRelative WT Scrambled KD

Figure 4. 17 - Knockdown of PAK-1 expression attenuates the stimulatory effect of insulin on the association of -cat with the c-Myc gene promoter.

HT29 cells were mock infected (WT), or infected with the Lentivirus expressing GIPZ-Scrambled (Scrambled), or infected with GIPZ-PAK-1 shRNAmir (KD) for 72 h, followed by puromycin selection. (A, B) After 24 h serum starvation cells were treated with insulin (100 nM) for 3 h before harvesting for the qChIP assay to determine -cat binding to TBE1 (A) and TBE2 (B). The results are presented as fold of relative copy number post adjustment against the control intron copy number. The relative copy number of uninfected HT29 cells without insulin treatment was defined as 1-fold, (mean ± SD, n=3, *, p<0.05).

140

* 12000 * 4h 12h 10000 24h * 48h 8000 NS

6000 CellNumber 4000

2000

0 - + - + - +

WT Scrambled KD 100nM Ins

Figure 4. 18 - Knockdown of PAK-1 expression attenuates the stimulatory effect of insulin on cell growth.

Mock infected control (WT), scrambled and PAK-1 shRNA (KD) expressing HT29 cells were serum starved for 24 h prior to 4, 12, 24 or 48 h insulin (100nM) treatment (in the absence of serum). Cell growth was monitored by MTT assay. Absorbance values were measured at 590 nm and converted into cell numbers, and the data are expressed as mean ± SD relative cell numbers (n=6) (*, #, p<0.05; NS, no significance).

141

GIPZ-Scrambled GIPZ-PAK-1shRNAmir

PAK-1

cyclin D1

c-Myc

-actin

- 4 24 48 - 4 24 48 h 100nM Ins

Figure 4. 19 - PAK-1 knockdown attenuated cell growth is associated with reduced proto- oncogene (c-Myc and cyclin D1) expression.

HT29 cells were infected with the lentivirus expressing GIPZ-PAK-1 shRNAmir or GIPZ- Scrambled for 72h, followed by puromycin selection. Expression of PAK-1, cyclin D1, c-Myc and -actin (loading control) were detected by Western blotting after 4, 24 or 48 h of insulin (Ins) treatment (Representative blot, n=4).

142

observed at 4 h and was returned to basal level after 24 h, while insulin treatment elevated Ki67 expression at both 4 and 24 h in scrambled shRNA expressing cells

(Fig. 4.20). PAK-1 knockdown, however, did not affect the stimulatory effect of insulin of PCNA expression (Fig. 4.20). Next, we examined both the active (cleaved) and inactive (uncleaved) caspase 3 expression. Both the scrambled control cells and PAK-1shRNA expressing cells expressed comparable levels of active caspase 3.

However, PAK-1 knockdown resulted in a significant increase in uncleaved caspase

3 expression (Fig. 4.20). Collectively, these observations support our hypothesis that PAK-1 is among the mediators of insulin that stimulate Wnt target gene transcription. The effect of insulin on protein translation, however, is not directly affected by PAK-1 silencing.

4.4.5 MEK/ERK signaling pathway is involved in insulin-stimulated c-Myc expression and nuclear -cat expression

We have also observed that in PAK-1 shRNA expressing HT29 cells, the basal expression level of pERK was extremely low, while insulin-stimulated ERK phosphorylation was completely blocked (Fig. 4.12). Since MEK has been identified as one of the downstream targets of PAK-1 [87, 442-444], we next examined whether the MEK/ERK signaling pathway participates in insulin stimulated c-Myc expression, as well as -cat nuclear content and function. PD98059 is a widely utilized inhibitor of MEK [445, 446]. PD98059 pretreatment in HT29 cells did not affect phospho-GSK-3 or phospho-Akt/PKB levels in either the basal or insulin stimulated condition (Fig. 4.21). On the other hand, pretreatment with 20 or 50M

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GIPZ-Scrambled GIPZ-PAK1shRNAmir

Ki67

PCNA uncleaved caspase 3 (Short Exposure) cleaved caspase 3 (Short Exposure) uncleaved caspase 3 (Long Exposure) cleaved caspase 3 (Long Exposure) -actin

- 4 24 - 4 24 h 100nM Ins

Figure 4. 20 - Knockdown of PAK-1 expression attenuates the stimulatory effect of insulin on Ki67 expression.

HT29 cells were infected with the Lentivirus expressing GIPZ-Scrambled (Scrambled), or infected with GIPZ-PAK-1 shRNAmir for 72 h, followed by puromycin selection. Cells were then serum starved for 24 h prior to 4 or 24 h of insulin (Ins) treatment (in the absence of serum). Expression of Ki67, PCNA, cleaved and uncleaved caspase 3 and -actin (loading control) were detected by Western blotting (Representative blot, n=3).

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PD98059 was sufficient to inhibit insulin stimulated ERK1/2 phosphorylation. This was correlated with a marked reduction in insulin stimulated c-Myc protein expression (Fig. 4.21). Furthermore, PD98059 significantly reduced nuclear -cat expression levels in insulin treated cells (Fig. 4.23).

We then performed qChIP in HT29 cells that were pretreated with either the

PI3K or the MEK inhibitor. Similar to PAK-1 shRNA expressing cells, the increase in

-cat binding to TBE1 and TBE2 post-insulin treatment was abolished in the HT29 cells that were pretreated with either PI3K or MEK inhibitor (Fig. 4.24A, B). These observations indicate that MEK-ERK functions as a downstream effector of PAK-1 in mediating the crosstalk between insulin and the Wnt signaling effector, cat/TCF.

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c-Myc

p-ERK1/2

ERK1

p-GSK-3

GSK-3

p-Akt

Akt

-actin

- + - + - + - + - + - + 100nM Ins

DMSO 1M 5M 10M 20M 50M PD

Figure 4. 21 - MEK inhibition blocks insulin stimulated c-Myc expression.

Wild type HT29 cells were starved for 24 h before being pre-treated with indicated amount of MEK inhibitor PD98059 for 45 min. Cells were then treated with or without 100nM insulin (Ins) for 4 h before harvested for immunoblotting of c-Myc, ERK, pERK, GSK-3, pGSK-3, AKT, pAKT, and - actin (as the loading control) in whole cell lysates (Representative blot, n=3). Fig. 4.22 shows the densitometric analysis of this figure.

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4.5 ¥ 4 ¥ c-Myc ¥ 3.5 * * ¥ p-ERK1/2 * 3 * 2.5 *

2 *

1.5 Relative Intensity Relative

1 * * * * 0.5 * 0 - + - + - + - + - + - + 100nM Ins

DMSO 1M 5M 10M 20M 50M PD

Figure 4. 22 - MEK inhibition blocks insulin stimulated c-Myc expression – Densitometric analysis.

Densitometric analysis for Fig. 4.21. Data are expressed as mean (n=3) +/-S.D (*, p<0.05).

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Nuclear -cat

Histone H3 - + - + 100nM Ins - - + + 50M PD

* Nuclear -cat 2.5

2

1.5 NS 1

Relative Intensity Relative 0.5

0 - + - + 100nM Ins - - + + 50M PD

Figure 4. 23 - MEK inhibition blocks the stimulatory effect of insulin on nuclear -cat expression.

Wild type HT29 cells were starved for 24 h before being pre-treated with indicated amount of MEK inhibitor PD98059 for 45 min. Cells were then treated with or without 100nM insulin (Ins) for 4 h before harvested for immunoblotting of -cat and Histone H3 (loading control) in the nuclear fraction (n=3). Bottom panel shows the densitometric analysis of top panel. Data is expressed as mean ± SD relative intensity (*, p<0.05; NS, No significance).

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A 5 *

) 4.5

4 Intron 3.5

3

2.5 Control NS NS 2 100nM Insulin

Fold ChangeFold (TBE1/ 1.5

1

Relative 0.5

0 DMSO LY PD B 5

4.5 ) 4

* Intron 3.5

3

2.5 NS NS Control 2 100nM Insulin

Fold ChangeFold(TBE2/ 1.5

1 Relative 0.5

0 DMSO LY PD Figure 4. 24 - MEK inhibition blocks insulin stimulated -cat binding to the c-Myc gene promoter.

Wild type HT29 cells were starved for 24 h before pretreatment with the vehicle (DMSO), or the PI3K inhibitor LY294002 (LY) (50M), or the MEK inhibitor PD98059 (20 M) for 45 min. The cells were then treated with or without insulin (100 nM) for 3 h prior to harvesting for qChIP assay. The stimulatory effect of insulin on the in vivo association between -cat and TBE1 (A) or between -cat and TBE2 (B) was determined as described in methods. The results are presented as fold of relative copy number post adjustment for intron copy number. Untreated samples were defined as 1-fold, (values are mean ± SD, n=3, *, p<0.05; NS, no significance).

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4.5 Discussion

Type 2 diabetes mellitus is associated with many other metabolic abnormalities such as hyperinsulinemia, insulin resistance, dyslipidemia (elevated triglycerides and low HDL) as well as hypertension and an increased risk for atherosclerosis. Epidemiological studies have also documented an association between Type 2 diabetes and a higher risk of CRC development [168, 392, 447].

While the importance of the Wnt signaling pathway in colorectal tumor formation has been well characterized [398, 448, 449], there is increasing evidence that suggests a role for hyperinsulinemia in the development of colorectal and other types of tumors, and that this phenomenon may involve molecular crosstalk between the insulin and

Wnt signaling pathways [298, 391, 404, 450, 451]. Indeed, the major effector of the canonical Wnt signaling pathway, cat/TCF, has been shown in a number of cell lineages to be activated by factors other than the Wnt ligands, including insulin and

IGF-1 [430]. In hepatocellular carcinoma, insulin was found to trigger nuclear localization and the expression of -cat [298]. In intestinal endocrine L cells, we demonstrated that insulin utilizes the same cis- and trans-elements that are employed by the Wnt signaling pathway in stimulating proglucagon gene expression and the production of the incretin hormone glucagon-like peptide- 1 (GLP-1) [404].

In intestinal non-endocrine cells, we found that insulin stimulates the expression of the Wnt target gene, c-Myc, at both the translational and transcriptional levels [391].

The stimulatory effect of insulin on c-Myc translation was attributed to mTOR activation and involved Akt/PKB. The stimulation at the transcriptional level was

PI3K-dependent but Akt/PKB-independent, and was associated with increased -cat

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nuclear content and enhanced binding of -cat to the human c-Myc gene promoter

[391].

A report by Inoki et al. supports the concept that the Wnt signaling activation also stimulates gene expression at both the transcriptional and translation level. The latter was also associated with activation of mTOR and S6K1, mediated by inactivation of GSK-3 but not requiring -cat. In the absence of Wnt activation, GSK-

3, in concert with AMP-dependent protein kinase (AMPK) catalyzed pre- phosphorylation of Ser1345, phosphorylates Ser1337 of TSC-2, increasing the GAP

(GTPase activating-protein) activity of the TSC1/2 complex to inhibit mTOR [330].

Therefore, both Wnt and insulin/IGF1 signaling result in mTOR activation as well as stimulating cat/TCF activity to augment both translation and transcription of their common targets. However, prior to this investigation, the signaling mechanism of insulin/IGF stimulation of cat/TCF-mediated gene transcription was not elucidated.

The Rho-family small G protein activated target, PAK-1, has been implicated in both progression and metastasis of tumors. PAK-1 is over-expressed in hepatocellular carcinoma, especially in the more aggressive types, and plays a role in enhancing metastasis [433]. Constitutive activation of PAK-1 rapidly induced breast cancer cell proliferation, and mutant kinase active T423E PAK-1 transgenic mice were found to develop hyperplasia in the mammary epithelium [432]. This hyperplasia was shown to respond to estrogen receptor (ER) activation, consistent with widely up-regulated PAK-1 levels detected in human breast cancers [432].

Furthermore, PAK-1 was also shown to stimulate the expression of the Wnt target gene cyclin D1 [452].

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Previous studies provided evidence of PAK-1 mediating signaling events in a

PI3K-dependent but Akt/PKB-independent manner; however none of these studies suggested the role of PAK-1 in mediating the crosstalk between growth factors and

Wnt signaling. In human bronchial epithelial cells, cigarette smoke-induced EGF receptor-mediated activation of FRA-1 expression is carried out via the PI3K-(PAK-

1)-(Raf)-MEK-ERK signaling cascade, without the participation of Akt/PKB [421, 453].

In addition, Qiao and colleagues reported that IGF-1 stimulates the activity of transcription factor RUNX2, through a PI3K-dependent but Akt/PKB-independent signaling pathway [454]. Both Fra-1 and Runx2 are known downstream targets of the Wnt or cat/TCF signaling pathway [276, 455]. In the current study, we found that insulin stimulates PAK-1 phosphorylation/activation in multiple cancer cell lines and mouse intestinal tissues on Thr423, a target residue of PDK-1, hence bypassing

Akt/PKB [83]. We have also found that PAK-1 knockdown significantly reduced insulin stimulated c-Myc and cyclin D1 expression, as well as cell growth, and completely blocked the insulin-stimulated interaction between cat and the c-Myc gene promoter. These observations suggested that PAK-1 is a mediator of Akt/PKB- independent insulin signaling to stimulate cat/TCF-dependent gene transcription.

However, it should be pointed out that the involvement of signaling components upstream of PI3K, such as IRS-1, IRS-2, in the activation of PAK-1 by insulin, requires further investigation. It is known that insulin may bind and activate the IGF receptor (IGFR). However, blockade of IGFR using an IGFR-neutralizing antibody did not affect insulin effects on PAK-1 phosohprylation, suggesting that insulin stimulated PAK-1 activation does not involve IGFR-mediated signaling events.

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However, at this time, we still cannot fully eliminate the possibility that insulin may exert this Akt/PKB- independent function via the IGF-1 receptor.

PAK-1 has been shown to increase the association between MEK1 and Raf-1 by phosphorylation of MEK1 at Thr292 [442]. PAK-1 may also phosphorylate MEK1 at Ser298 to prime MEK1 for its subsequent activation by Raf-1 [87, 444]. We observed that PAK-1 knockdown significantly reduces both basal and insulin stimulated ERK1/2 phosphorylation. Furthermore, we found that MEK inhibition reduced insulin stimulated c-Myc expression and, similar to the effect of PAK-1 knockdown, abrogated the stimulatory effect of insulin on the association between - cat and the c-Myc gene promoter. These observations suggest that the PAK-1-MEK-

ERK axis is involved in insulin stimulated Wnt target gene transcription. By analyzing macrophages from PAK-1-/- mice, Smith et al. have demonstrated recently that PAK1 signals via ERK1/2 to regulate lamellipodial stability, indicating the in vivo physiological significance of the PAK-1-MEK-ERK axis [200]. He et al., however, have shown a physical interaction between PAK-1 and -cat. They found that dominant negative PAK-1, the K299A mutant, blocked gastrin induced -cat translocation from the cell membrane to the nucleus, and -cat association with

TCF-4 [452], suggesting the existence of other mechanisms that mediate the crosstalk between hormone/growth factors and cat/TCF activity.

In the present study, we observed that PAK-1 knockdown blocked the basal and insulin stimulated -cat phosphorylation at Ser675. Phosphorylation of -cat at

Ser675 by protein kinase A (PKA) activation has been shown to stabilize -cat by attenuating its ubiquitination and subsequent degradation [441]. Taurin et al.

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suggested that the Ser675 residue enhances TCF/LEF transactivation by promoting the binding of -cat to its transcriptional coactivator, the CREB-binding protein CBP

[260]. Recently, Liu and colleagues reported that -cat phosphorylation at Ser675 in pancreatic  cells can be activated by the incretin hormone glucagon-like pepetide-1

[456]. To our knowledge, -cat phosphorylation at Ser675 in response to insulin or

IGF-1 has not been previously documented. Since -cat phosphorylation at Ser675 is important for its stimulatory effect on Wnt downstream target genes, further study is required to examine whether it is PAK-1 itself or a downstream effector that phosphorylates -cat at Ser675.

We suggest that MEK-ERK signaling is among the downstream effectors of

PAK-1 in stimulating the transcription of cat/TCF responsive genes, and this involves

-cat phosphorylation at Ser675. This is supported by two lines of evidence. First,

PAK-1 knockdown resulted in a nearly complete loss of p-ERK1/2 expression, along with the inhibition of basal -cat (Ser675) expression and the blockade of insulin stimulated -cat (Ser675) expression (Fig. 4.12). Secondly, MEK inhibition and

PAK-1 knockdown generated comparable repressive effects on nuclear -cat content and the binding of -cat to c-Myc promoter (Fig. 4.16, 4.17, 4.23 and 4.24).

However, in the control shRNA expressing HT29 cells, MEK inhibition did not block the effect of insulin on -cat phosphorylation (Fig. 4.16), suggesting insulin may potential stimulates -cat phosphorylation at Ser 675 via other pathway/kinases.

We have suggested that insulin stimulates c-Myc oncogene expression at both the translational and transcriptional levels [391, 430]. As illustrated in Fig. 4.1, the effect of PAK-1 is independent of Akt/PKB. Therefore, PAK-1 mediates the

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stimulatory effect of insulin on nuclear content and activity of -cat, the effector of

Wnt signaling, resulting in the activation of c-Myc expression at the transcriptional level. At the same time, the stimulation of translation by insulin, when PAK-1 expression level was reduced in response to shRNA mediated knockdown, likely accounts for the residual stimulation at 4 h of insulin treatment of c-Myc (and cyclin

D1) protein expression (Fig. 4.19), and cell growth (Fig. 4.18). However, the stimulatory effect of insulin on cell growth was blocked by shRNA mediated PAK-1 knockdown after ≥ 12 h of treatment (Fig. 4.18). It is important to note that PAK-1 was phosphorylated in response to insulin in vivo, and that in both cultured cells and mice rendered insulin resistant by chronic hyperinsulinemia and high fat diet respectively, in contrast to Akt/PKB, the stimulation of PAK-1 phosphorylation was not reduced. While this phenomenon remains to be further explored, the apparent lack of resistance to some of the growth promoting effects of insulin, in contrast to its metabolic pathways, may be relevant to the documented association of hyperinsulinemia/insulin resistance with cancer development in certain tissues.

In summary, we demonstrate here that insulin stimulates the phosphorylation/activation of PAK-1 in vitro and in mice in vivo. We suggest that this increases MEK-ERK phosphorylation and enhanced -cat phosphorylation at

Ser675, associated with increased nuclear -cat accumulation and cat/TCF- mediated Wnt target gene transcription. Our study shows molecular evidence implicating a role of insulin signaling in the activation of Wnt signaling pathway, providing a potential mechanism for increased CRC risk associated with hyperinsulinemia.

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CHAPTER 5: Insulin alters the expression of components of the Wnt signaling pathway including TCF7L2 (TCF-4) in intestinal cells

Data presented in this chapter have been submitted to BBA. Sun J, Wang D and Jin T (2009) [457]

(Fig. 5.1, 5.2 and 5.3 were contributed by Sun J. Fig. 5.4 was contributed by Wang D.)

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5.1 Abstract

Evidences that support the correlation between Type II diabetes mellitus and increased risk of colorectal cancer formation have led us to hypothesize the existence of molecular crosstalk between insulin and Wnt signaling pathways.

Ample amounts of evidence have shown that insulin stimulates the expression of

Wnt downstream target genes. We have demonstrated previously that insulin utilizes the same cis- and trans-elements that are employed by the Wnt signaling pathway in stimulating proglucagon gene expression in intestinal endocrine L cells; as well as c-Myc gene transcription in colorectal cancer cells, by enhancing nuclear

-cat content, associated with an increase in -cat interaction with the human c-Myc gene promoter. Here we have assessed the gene expression profile of rat intestinal

IEC-6 cell line before and after insulin treatment. cDNA microarray analyses showed that insulin altered the expression of a dozen Wnt pathway related genes including TCF-4 (=TCF7L2), Wnt-2b and Frz-4. The stimulatory effect of Insulin on

TCF-4 expression was confirmed by real time PCR, Western blotting and luciferease reporter analyses, while the activation of Fzd-4 was confirmed by real time PCR.

Our observations suggest that insulin crosstalks with the Wnt signaling pathway in a multi-level fashion, involving insulin regulation of the expression of Wnt target genes, a Wnt receptor, as well as mediators of the Wnt signaling pathway.

5.2 Introduction

An individual’s susceptibility to Type II diabetes development is determined by many genetic variations, environmental factors and life style. Colorectal cancer shares many risk factors with Type II diabetes. Increased risks of colorectal cancer 157

development have been reported in Type II diabetes mellitus patients in a number of epidemiological studies [171, 392, 393]. The canonical Wnt signaling pathway has been well identified in its role of initiation and progression of colorectal tumors [357,

399, 412]. -catenin (-cat) forms a bipartite transcription factor with a member of the Tcf/Lef family proteins (TCF-1/TCF7, LEF-1, TCF-3/TCF7L1, and TCF-

4/TCF7L2), and acts as the mediator of the canonical Wnt pathway. In response to

Wnt ligand stimulation, the negative modulator of Wnt signaling GSK-3 becomes inactivated, leading to -cat stabilization and nuclear translocation. The -cat molecule then forms the cat/Tcf bipartite transcriptional activator to stimulate Wnt target gene expression. The current dogma suggests that, in the absence of -cat,

Tcf/Lef molecules bind to Wnt target gene promoters and recruit co-repressors like

Groucho, and therefore functions as a transcriptional repressor [280]. However, - cat interaction with Tcf/Lef displaces nuclear co-repressors with nuclear co- activators, such as CBP/p300, resulting in the stimulation of Wnt or cat/Tcf downstream target gene transcription [282, 287, 288].

It has been suggested that insulin signaling may crosstalk with Wnt signaling, due to the existence of common downstream target genes, the shared signaling mediator such as the serine threonine kinase GSK-3, and the association between

T2D and increased colon cancer risks. However, mechanisms underlying this potential crosstalk are still being debated and remain unclear. Although both insulin treatment and Wnt activation, which can be mimicked in vitro as well as in vivo by lithium treatment [458], lead to GSK-3 inactivation, Ding et al. found that in a number of cultured cell lines, in contrast to Wnt activation or lithium treatment, insulin

158

treatment did not increase free cytosolic -cat content [459]. They suggested that insulin and Wnt pathways regulate GSK-3 via different mechanisms, and therefore lead to distinct downstream signaling events [459]. We have shown that in the intestinal endocrine L cells, insulin utilized the same cis-element (TCF binding site) and trans-element (cat/TCF-4) that are employed by the Wnt signaling, in stimulating transcription of the proglucagon gene and the production of its encoded peptide hormone GLP-1 [404].

It has been shown in Chapter 3 that insulin stimulates the expression of c-

Myc proto-oncogene, a known downstream target of Wnt signaling, via a PI3K- dependent but Akt/GSK-3-independent mechanism, involving the enhancement of - cat nuclear translocation and binding of cat/TCF to the Wnt target gene promoter

[391]. Discovery of a PI3K-dependent but Akt/GSK-3-independent signaling cascade that mediates the crosstalk between insulin and Wnt signaling partially resolves the dispute in the literature [421, 424]. Indeed, several other studies have also reported the stimulatory effect of insulin or IGF-I on -cat nuclear localization and cat/TCF mediated gene transcription [298, 299, 385]. Furthermore, as it was shown in

Chapter 4, the serine threonine kinase PAK-1 may function as the PI3K-dependent but Akt/GSK-3 independent effector of insulin signaling in the stimulation of c-Myc gene transcription and cell growth [424].

To further investigate the dynamics of insulin and Wnt signaling crosstalk, we examined whether Wnt pathway components would be among the downstream targets of insulin signaling. To this end, we conducted cDNA microarray analysis, examining gene expression profiles in IEC-6 cells before and after insulin treatment.

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We found that in this cell line, insulin suppressed the expression of Wnt ligand,

Wnt2b; but insulin up-regulated the expression of Wnt receptor Fzd-4, and TCF-4, a component of the cat/TCF bipartite transcription factor.

5.3 Materials and Methods

Materials

Tissue culture medium, fetal bovine serum and oligonucleotides were purchased from Invitrogen Life Technology Inc. (Burlington, ON, Canada). Insulin was provided by Novo Nordisk (Novo Nordisk, Copenhagen, Denmark).

Isolation of total RNA and GeneChip microarray assay

The total RNA from IEC-6 cells was isolated using Trizol Reagent from Invitrogen

Life Technology Inc according to manufacturer’s directions. Subsequent RNA processing procedures followed protocols in the GeneChip Expression Analysis

Technical Manual (Affymetrix, Santa Clara, CA). The Rat230.2 Chip utilized in this assay contains 31,099 probe sets, and detect over 28,000 well-substantiated rat genes. Level of transcription of each sequence represented on the GeneChip Rat

Genome 230.2 Array is measured using eleven pairs of oligonucleotide probes. cDNA Microarray data analysis and statistical treatment cDNA microarray data analysis was performed using GeneSpring Ver. 7.2 software

(Silicongenetics, Redwood City, CA). Briefly, the array measurements for all samples were normalized using the control samples (untreated). After the normalization, the detectable expressed genes were defined using P-, M- or A-calls

(according to Affymetrix algorithm) and the intensity for the genes. 17,062 genes

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were selected as the detectable expressed genes from all genes. Treatment effects were analyzed using one-way ANOVA based on the 17, 062 genes.

Quantification of gene expression level using real-time PCR

Total (5g) RNA was reverse transcribed to cDNA using SuperScript First-Strand

Synthesis System purchased from Invitrogen Life Technology Inc (Burlington, ON,

Canada). Quantification of gene expression in IEC-6 cells was measured using the real-time PCR system (Corbett Research Roter-Gene 3000, Corbett Life Science,

Mortlake, NSW, Australia). Amplification was performed in a final 20l containing

30ng of cDNA, specific primers (Invitrogen Life Technology Inc, Burlington, ON,

Canada) and SYBR Green PCR Master Mix purchased from Applied Biosystems,

Tokyo, Japan) according to manufacturer’s instructions. Results were expressed as fold increase relative to the controls after normalization using -actin gene expression level. The following primers are utilized for TCF-4 and Fzd-4 real-time

PCR analysis.

TCF-4 forward, 5’ GCCTCTCATCACGTACAGCA 3’; and TCF-4 reverse, 5’

GGATGGGGGATTTGTCCTAC 3’. Fzd-4 forward, 5’ CAGCTGCAGTTCTTCCTTTG

3’; and Fzd-4 reverse, 5’ ACATGTGGTTGTGGTCGTTC 3’.

Cell Culture

The human colon cancer cell lines HT29 and SW480, the human breast cancer cell line MCF-7, and the rat intestinal cell line IEC-6 were purchased from American

Type Culture Collection (ATCC). They were maintained per ATCC instructions.

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Construction of human TCF-4-luciferase (LUC) fusion gene construct and LUC reporter gene analysis

The 3.2 kb human TCF-4 gene promoter fragment was obtained by PCR against the human BAC clone (RP11-652A18 and RP11-978N18) with the following two primers:

TCF-4 Forward, 5’TACTGCAGTGCCAATTCTGC 3’; and TCF-4 Reverse,

5’TTTCACCCACCAGCAGCAAT 3’. The PCR product was inserted into a TA vector

(pPGEM-T easy vector, Promega), verified by DNA sequencing. The 3.2 kb NotI and

SalI fragment was then inserted into the promoter-less PGL2 luciferase (LUC) vector

(pGL2-Basic vector, Promega), and the fusion gene construct is designated as 3.2 kbTCF-4-LUC. The 1.3 kbTCF-4-LUC was generated by a sub-cloning procedure.

Approximately 3 g reporter gene construct was transfected into the human colon cancer cell line SW-480, cultured on a six-well plate by the method of calcium precipitation [389]. Methods for LUC reporter analysis have been described previously [389].

Antibodies, cell fractionation and fluorescence microscopy

Antibody for TCF7L2was purchased from Santa Cruz Biotechnology, Inc. (Santa

Cruz, CA). Antibody for -actin was purchased from Sigma-Aldrich (St. Louis, MO).

Statistical analysis

All data are presented as mean +/- SD. Statistical analysis was done by either

Student’s t test or one way ANOVA when appropriate. Significance was assumed at a p value of less than 0.05.

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5.4 Results

5.4.1 Insulin treatment alters gene expression profiles in rat intestinal cell line IEC-6

We treated IEC-6 cells with 100 nM of insulin for 0, 4 and 24 h and performed cDNA microarray analyses. As shown in Table 5.1, we found that the expression of

97 and 92 genes were up-regulated by more than 2-fold after 4 h and 24 h of insulin treatment, respectively. In addition, the expression of 181 and 165 genes were down-regulated by more than 2-fold after 4 h and 24 h of insulin treatment, respectively (Table 5.1). Fig. 5.1 shows that among the 92 genes that were up- regulated by 24 h of insulin treatment, 62 genes were also up-regulated by 4 h of insulin stimulation. Among the 165 genes that were suppressed by 24 h of insulin treatment, 118 genes were also down-regulated by 4 h of insulin stimulation. Since the main interest of this study is the direct effects of insulin on gene transcription, we focused on the gene expression changes after 4 h of insulin treatment, as the expression changes after 24 h treatment are likely due to secondary effects of insulin.

Insulin treatment affects the expression of various genes. Nineteen genes, of which expression is consistently up- or down-regulated by 4 and 24 h insulin treatment, listed in Table 5.2 are examples of some of the potential insulin targets identified in this study. Among these genes are a Wnt ligand (Wnt2b), Wnt signaling receptors (Frizzled 4 and 5), or the effector/mediator of Wnt signaling (TCF7L2/TCF-

4). The expression of several fibroblast growth factors (Fgf4 [460], 9 [461], 18 [462],

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Table 5.1 – Number of up- and down-regulated genes in IEC-6 cells treated with insulin

Up-regulated (>2-fold) Down-regulated (<2-fold)

4 h Insulin Treatment 97 181

24 h Insulin Treatment 92 165

Table 5. 1 - Number of up- and down-regulated genes insulin treated IEC-6 cells

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A. Number of genes induced by insulin 4 h Insulin 24 h Insulin

35 30 62

B. Number of genes suppressed by insulin 4 h Insulin 24 h Insulin

63 47 118

Figure 5. 1 - Gene expression changes after 4 and 24 h of insulin treatment in IEC-6 cells.

(A) Representation of the number of genes that are up-regulated by 2-fold or greater after insulin (100nM) treatment and the number of genes consistently up-regulated after 4 and 24 h of insulin stimulation. (B) Representation of the number of genes that are down-regulated by 2-fold or greater after insulin (100nM) treatment and the number of genes consistently down-regulated after 4 and 24 h of insulin stimulation.

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Table 5.2 – Up- and down-regulated genes in insulin treated IEC-6 cells

Accession Description Δ Fold 4 h Δ Fold 24 h No.

XM_342308 Wingless-type MMTV integration site family, member 2B 1.96 down 1.74 down

NM_022623 Frizzled homolog 4 1.42 up 1.78 up

NM_053369 T-cell factor 4 1.24 up 1.56 up

XP_215757 Frizzled-related protein 2.27 down 1.23 down

NM_133523 Matrix metallopeptidase 3 1.81 down 2.30 down

XP_344435 1 1.49 down 1.33 down

NM_053635 Suppression of tumorigenicity 14 1.67 down 1.24 down

NM_021867 Fibroblast growth factor 16 1.91 down 1.62 down

NM_053896 Aldehyde dehydrogenase family 1, subfamily A2 2.73 down 1.80 down

NM_013122 Insulin-like growth factor binding protein 2 1.24 down 1.56 down

NM_031590 WNT1 inducible signaling pathway protein 2 3.01 down 3.46 down

AA957545 Frizzled homolog 5 2.96 down 2.06 down

XP_217435 Serine/threonine kinase 36 2.01 down 1.65 down

XP_573183 Homeo box B5 1.77down 2.30 down

XP_220567 Epidermal arachidonate lipoxygenase 12 3.5 down 2.91 down

XP_213497 Lysozyme-like 6 2.17 down 2.01 down

XP_214621 Small nuclear ribonucleoprotein D1 2.24 up 2.16 up

XP_226016 Purine rich element binding protein A 2.13 up 1.51 up

NP_001020 Proteasome (prosome, macropain) 26S subunit, non- 2.44 up 2.23 up 860 ATPase, 14

Table 5. 2 - The up- and down-regulated genes in insulin treated IEC-6 cells

List of genes that are consistently up or down regulated by 4 and 24 h insulin treatment in IEC-6 cell line

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20 [463]) and three matrix metalloproteinases (MMP7 [464, 465], 2, 9 [466], 26) have been shown to be regulated by Wnt signaling. Our microarray analyses showed that MMP3 and Fgf16 were among the targets of the insulin signaling pathway (Table 5.2). The mRNA expression of Wnt2b, a member of the Wnt family ligands, was reduced by 1.96-fold after 4 h and 1.74-fold after 24 h of insulin treatment. Known Wnt target gene, Wnt1 inducible signaling pathway protein 2

(Wisp2) [467] was found to be down-regulated by insulin treatment by approximately

3-fold after insulin treatment. Frizzled receptor 5 (Fzd-5) mRNA expression was reduced by 3- and 2-fold post 4 and 24 h of insulin treatment, respectively. However, frizzled homolog 4 (Fzd-4) expression was found to be consistently increased after 4 and 24 h insulin treatment. Furthermore, the expression of TCF-4 was consistently up-regulated by insulin. Together, these observations suggest that insulin is able to regulate not only Wnt downstream target genes, but also players of the Wnt signaling pathway.

5.4.2 Quantitative assessment of the effect of insulin on Fzd-4 and TCF7L2/TCF-4 expression by real time RT-PCR

Using the approach of quantitative real time PCR, we assessed the effect of insulin treatment on the expression of Fzd-4 and TCF7L2/TCF-4 in the IEC-6 cell line. After 4 and 24 h insulin treatment, Fzd-4 mRNA expression level was shown to be increased by approximately 1.5- and 2.5-fold, respectively (Fig. 5.2A). While 4 h insulin treatment induced a slight but significant increase in TCF7L2/TCF-4 mRNA expression, 24 h insulin treatment increased TCF-4 mRNA expression by almost 2- fold (Fig. 5.2B). These observations, therefore, confirmed that insulin signaling is

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A. 3

Ratio 2.5 *

actin -

 2 *

4/ -

Fzd 1.5

1

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100nM Insulin Treatment

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2

actin

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100nM Insulin Treatment Figure 5. 2 - Insulin stimulates gene expression of Wnt signaling components.

IEC-6 cells were serum starved for 24 h prior to 4 h or 24 h of insulin (100nM) treatment. Total RNA was harvested for subsequent reverse transcription. cDNA was subjected to quantification using real time PCR analysis. Expression level of Fzd-4 and TCF-4 are normalized to -actin expression level, and fold changes are calculated relative to the control (untreated) sample. The readings in untreated control samples were defined as 1 fold (mean ± SD, n=3, *, p<0.05)

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capable of stimulating the expression of Wnt pathway components and effectors, i.e.

Fzd-4 and TCF7L2/TCF-4. It was unexpected that the cDNA microarray analysis was not able to detect the expression of c-Myc or cyclin D1 mRNA levels in IEC-6 cells. As it was shown in Fig. 3.2 (Chapter 3), we not only showed the expression of c-Myc protein in IEC-6 and primary FRIC cells, we demonstrated the stimulatory effect of insulin on c-Myc protein expression.

5.4.3 Insulin induces TCF7L2/TCF-4 protein expression

Next, we assessed the effect of insulin on TCF7L2/TCF-4 protein expression in a battery of cultured cell lines by Western blotting. TCF7L2/TCF-4 protein expression was detected in two human colon cancer cell lines HT29 and SW480, as well as in the rat IEC-6 cell line (Fig. 5.3A). However, we were unable to detect

TCF7L2 in the breast cancer cell line MCF-7 (Fig. 5.3B). Basal expression level of

TCF7L2/TCF-4 in HT29 and IEC-6 cells were both very low; however, 4 h insulin treatment stimulated a pronounced increase in its expression in the absence of serum. In HT29 cells, TCF7L2/TCF-4 expression undergoes a transient induction post insulin treatment, maximizing between 4 and 8 h and drops by 24 h (Fig. 5.3A).

On the other hand, insulin induced TCF7L2/TCF-4 expression in IEC-6 cells maximizes after 8 h of insulin treatment, and remains elevated even after 24 h post insulin stimulation. Interestingly, in the presence of serum, insulin treatment was no longer effective in elevating TCF7L2/TCF-4 expression in both HT29 and IEC-6 cells; the basal level of TCF7L2 in both cell lines were significantly higher compared to its basal expression in the absence of serum. In contrast, insulin was able to stimulate

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A.

B.

Figure 5. 3 - Insulin stimulates TCF7L2/TCF-4 protein expression in various cancer and non-cancer cell lines.

(A) HT29, IEC-6, SW480, FRIC and (B) MCF-7 cells were cultured either in the absence of serum or the presence of serum for 24 h prior to insulin (100nM) treatment for indicated time periods. The expression of TCF-4 and -actin (loading control) were detected by Western blotting (Representative blot, n=3).

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TCF7L2/TCF-4 expression in SW480 and primary fetal rat intestinal cells (FRIC) not only in the absence of serum but also in the presence of serum (Fig. 5.3A). These observations collectively indicate that insulin treatment induces both TCF7L2/TCF-4 mRNA and protein expression in intestinal cancer and non-cancer cells.

5.4.4 Insulin activates TCF7L2/TCF-4 promoter activity

Subsequently, we constructed two human TCF-4-LUC fusion gene constructs

(Fig. 5.4A) and assessed the effect of insulin on TCF-4 promoter expression, using the approach of LUC reporter gene analysis. As shown in Fig. 5.4B, both insulin and lithium, which mimics ligand-mediated Wnt activation, moderately but significantly stimulated the expression of 3.2kb TCF-4-LUC fusion gene, but not 1.3kb TCF-4-

LUC fusion gene in SW480 human colon cancer cell line.

5.5 Discussion

Colorectal cancer is the third most common cancer, and in the Western world, it is the second leading cause of all cancer-related deaths. Studies have shown that hyperinsulinaemia and hyperglycemia, symptoms often experienced by Type II diabetes patients, are associated with higher risks of colorectal cancer development

[447, 468, 469]. Extensive recent investigations have revealed the existence of molecular crosstalk between insulin and Wnt signaling pathway [298, 299, 330, 391].

Wnt pathway components such as APC and -cat have been shown to be involved in the initiation and progression of most colorectal carcinomas [364, 371, 427, 448].

The existence of insulin and Wnt signaling crosstalk may potentially explain the vast amount of epidemiology evidence linking Type II diabetes and the risk of colorectal

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A

+90

1298

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1.3kb TCF-4-LUC LUC

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1

Relative Luciferase Activity (Fold) ActivityLuciferase Relative 0.5

0 Reporter 3.2kb TCF-4-LUC 1.3kb TCF-4-LUC Lithium - + - - + - Insulin - - + - - +

Figure 5. 4 - Insulin stimulates TCF-4-LUC promoter activity.

(A) Schematic representation of TCF-4 promoter region, and two TCF-4-Luciferase reporter constructs containing different sizes of the TCF-4 promoter (3.2kb and 1.3kb). (B) 3 g of 3.2kb TCF-4-LUC or 1.3kb TCF-4-LUC was transfected into the human colon cancer cell line SW480 using the calcium precipitation method for 24 h. Cells were than treated with lithium chloride (10mM) or insulin (100nM) for 4 h before harvested for luciferase reporter gene analysis. Relative LUC activities were calculated as fold induction relative to its corresponding control (mean ± S.D., n=3; *, p<0.05)

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cancers [392, 469, 470]. Indeed, the crosstalk between insulin and Wnt signaling pathways have been suggested in the study of colon as well as other types of cancers [298, 385]. A study in the liver carcinoma cell line HepG2 showed that both insulin and IGF-1 stimulate Lef/Tcf dependent transcriptional activity [298]. Another study showed that IGF-1 enhances the stability and transcriptional activity of β-cat in the C10 human colorectal cancer cell line [385]. Mechanisms underlying this crosstalk, however, remain to be further explored.

In our previous studies, we demonstrated that insulin stimulates the expression of proto-oncogene c-Myc via a PI3K Akt/PKB-dependent pathway involving mTOR, as well as a PI3K Akt/PKB-independent pathway that crosstalks with the Wnt signaling pathway by enabling nuclear localization of -cat and an increase in -cat binding to TCF binding sites within the human c-Myc gene promoter region (Fig. 3.12-3.13, Chapter 3). The existence of PI3K-dependent but

Akt/PKB-independent signaling cascade was also suggested by several other studies [421, 454]. In Chapter 4, we showed that insulin and Wnt signaling crosstalk is at least partially mediated by the p21-activated protein kinase 1 (PAK-1) [424].

While there is steady emergence of evidence of insulin effects on the function and stimulation of downstream effectors and target gene expression of the Wnt signaling pathway, no studies have examined whether insulin regulates Wnt pathway components in a large scale. In this study, we performed cDNA microarray analyses, attempting to identify signaling components of the Wnt pathway that are targets of insulin signaling. We found that in addition to family members of known downstream targets of Wnt signaling such as FGF16 and MMP3, several Wnt

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pathway components or mediators were also regulated by insulin signaling. Insulin was shown to repress the mRNA expression of Wnt ligand Wnt2b, Wnt1 inducible signaling pathway protein 2 (Wisp2), as well as Wnt ligand receptor Fzd-5. How these changes might affect Wnt activity requires further investigations. Our previous observations (presented in Chapter 3) showed that insulin regulates gene expression at both transcriptional as well as translation levels; therefore, it is necessary to examine the effect of insulin on the protein expression of these Wnt components. Insulin may repress the mRNA expression of these genes and negatively affect the activity of Wnt signaling. Alternatively, reduced mRNA expression by insulin treatment may be a negative feedback response to increased protein expression of these components. Nevertheless, we did observe elevated

Fzd-4 mRNA expression by insulin treatment at both 4 and 24 h. We were unable to observe any stimulatory effects of insulin on c-Myc or cyclin D1 mRNA expression in this study. In fact, according to our cDNA microarray analysis, expression of c-Myc or cyclin D1 was below detection level regardless of insulin treatment. This may be attributed to experimental error or simply sensitivity of the microarray system we have used in this study. To verify these observations, expression of c-myc and cyclin D1 may be quantitated using a high sensitivity method such as real-time PCR.

Both Wnt2b and Fzd4 are implicated specifically in Wnt/-cat signaling. The expression of Wnt2b (also known as Wnt13) has been shown to associate with cat/Tcf transcriptional activity [471]. Furthermore, Wnt2b proteins were able to activate the Wnt/-cat pathway and induce axis duplication in Xenopus embryos and are upregulated in a number of cancers [472]. Microarray analyses presented in the

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current chapter shows that insulin treatment leads to a reduction in Wnt2b gene expression, contrary to evidence presented in Chapter 3 and 4 which suggested that insulin signaling increases cat/Tcf activity. There are several possible explanations for the observed inconsistency. In fact, two polypeptides are derived from the Wnt2b gene due to , known as Wnt2b1 and Wnt2b2 [473, 474]. While these two Wnt2b splice variants are virtually identical, their expression pattern and regulation suggest otherwise. Wnt2b2, but not Wnt2b1 is expressed in human breast cancer MCF-7 cells [475]. Wnt2b2 also appears to be preferentially upregulated in primary human gastric cancer compared to Wnt2b1 [472]. In addition, only Wnt2b2 was able to induce axis duplication in xenopus embryos [472]. Further examination is required to determine which Wnt2b isoform is down-regulated post insulin stimulation. It has also been shown that Wnt2b expression pattern differs across species. For example, a recent study by Lee and colleagues detected Wnt2b expression in islets of human pancreas but not murine pancreas [476]. Using the

HT29 human colon cancer cell line, we have shown that insulin activates canonical

Wnt signaling target gene expression via -cat involving PAK-1. However, in the rat intestinal epithelial cell line IEC-6, insulin treatment reduced the expression of

Wnt/-cat signaling components, Wnt2b and Frz5. Therefore, it is also possible that the inconsistency in insulin effects on Wnt/-cat signaling is due to differences in the experimental vessel that was utilized.

As a member of the Tcf/Lef family, TCF7L2/TCF-4 has been known to play an intricate role in the canonical Wnt signaling pathway, especially in the intestinal epithelium. TCF-4-/- mice die shortly after their birth, and exhibit depleted epithelial

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stem cell compartments in the small intestine [359]. In un-stimulated conditions,

TCF-4 molecules are mostly localized to Wnt target gene promoters along with transcriptional co-repressors such as Groucho [280]. In response to Wnt ligand stimulation, free -cat ultimately translocates to the nucleus and forms a transactivator with TCF-4 by replacing co-repressors and recruiting co-activators.

TCF transcription factor has the ability to enter the nucleus via its nuclear localizing sequence (NLS). Using live-cell microscopy and fluorescence recovery after photo- bleaching (FRAP), it was shown that TCF7L2/TCF-4 also plays a more active role in

Wnt signaling [264]. In HEK293T cells, TCF7L2/TCF-4 was shown to slow down - cat nucleo-cytoplasmic shuttling, thereby retaining and enriching -cat in the nucleus

[264]. Our study showed that insulin treatment stimulates TCF-4 mRNA expression in the IEC-6 cell line (Fig. 5.2). Insulin treatment was also shown to stimulate TCF-4 protein expression in IEC-6 and two colon cancer cell lines, as well as primary fetal rat intestinal cells (FRIC) (Fig. 5.3A). Furthermore, in the SW480 colon cancer cell line, we show that insulin as well as lithium chloride treatment stimulated TCF-4 promoter activity (Fig. 5.4B). This raises the possibility that stimulation of TCF-4 expression at both transcriptional and translational levels is yet another novel mechanism underlying the crosstalk between insulin and Wnt signaling. Although

TCF-4 functions as a transcriptional repressor in resting cells in the absence of -cat, increased TCF-4 production in response to insulin stimulation, along with increased nuclear -cat may lead to activation of Wnt target gene transcription. We found that insulin and lithium exclusively stimulated the expression of the 3.2 kb TCF-4-LUC promoter construct, but not the 1.3 kb construct (Fig. 5.4B). To determine whether

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there is/are cis-element/s located between the 3.2 kb and 1.3 kb region within the

TCF-4 promoter that mediate this activation require further investigation.

TCF7L2/TCF-4 has drawn substantial attention globally in the last few years, because genome-wide association studies revealed a few single nucleotide polymorphisms (SNPs) in TCF-4 to be strongly associated with the risk of type 2 diabetes development [477-479]. TCF-4 (rs7903146) SNP was associated with impaired insulin secretion, impaired incretin effect and hepatic insulin resistance

[480]. rs12255372 is another TCF7L2/TCF-4 SNP found to be strongly associated with risk of type 2 diabetes [478]. More recently, study by Hazra et al. suggest that the same TCF-4 SNP is also associated with colon cancer risks, further consolidating the importance of TCF7L2/TCF-4 not only in type 2 diabetes, but also in the interplay between type 2 diabetes and colon carcinogenesis [481]. Whether increased TCF7L2/TCF-4 expression serves as the linker between hyperinsulinemia and the risk of colon cancer development deserves further investigation.

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CHAPTER 6: GENERAL DISCUSSION AND CONCLUSIONS

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Insulin is a peptide hormone produced by the pancreatic beta-cells; it is important in mediating various metabolic processes. In response to elevated nutrients in the blood, insulin triggers cellular uptake of glucose, fatty acids and amino acids, and promotes glycogen synthesis and lipogenesis. Failure to elicit these responses results in the development of diabetes. Inability to produce insulin responses can arise from either deficiency in insulin secretion as a result of the loss of pancreatic beta cells (Type 1), or insulin resistance associated with the loss of insulin sensitivity (Type 2). Type 2 diabetes comprises the majority of the world’s diabetic population, as only less than 10% of diabetes patients belong to the Type 1 category. Onset symptoms of Type 2 diabetes include hyperinsulinemia, a compensatory body mechanism for reduced insulin sensitivity. However, as the disease progresses, patients develop impairment of insulin secretion and require medical intervention.

6.1 Insulin and Neoplasia

Over the last ten years, there has been an accumulation of evidence, from both epidemiological studies and laboratory experiments, which would suggest a relationship between both insulin and IGF-1 signaling with neoplasia. Laboratory studies have demonstrated a role of insulin in neoplasia dating back to as early as the 1970’s. Osborne and colleagues showed that at physiological relevant concentrations, insulin increased DNA synthesis in a human breast cancer cell line

[482]. Another group showed that insulin deficiency is correlated with reduced cancer proliferation in mice [483]. Numerous epidemiological studies observed a correlation between hyperinsulinemia (indicated by C-peptide levels, fasting insulin

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levels, as well as postptrandial insulin levels) with increased risks of a number of cancers. Moreover, one study showed that Type 2 diabetic patients whom received insulin therapy had increased risk of cancer-related mortality compared with those who used insulin-sensitizing drugs [484]. Of particular interest, a retrospective cohort study in UK observed significant increases in the risk of colorectal cancer among Type 2 diabetes patients with a history of chronic insulin therapy [173].

Consistently, other studies have shown hyperinsulinemia to be often associated with increased risks of developing colonic tumors. Canonical Wnt signaling and its participating cellular mediators have been well-established in their roles not only in colonic cancers, but also prostate, breast and liver cancers. Thus, we have hypothesized the existence of molecular crosstalk between insulin and Wnt signaling pathways, and observations presented in Chapter 3, 4, and 5 support our hypothesis.

6.2 Insulin signaling crosstalks with Wnt/-cat signaling

There is ample evidence suggesting that both insulin and IGF-1 crosstalks with Wnt signaling, particularly with the canonical Wnt signaling pathway. For example, working with a hepatocarcinoma cell line HepG2, Desbois-Mouthon et al. showed that both insulin and IGF-1 stimulated Lef/Tcf dependent transcriptional activity, and this stimulation is dependent on phosphoinositide-3 kinase (PI3K) [298].

Another study demonstrated that IGF-1 enhances the stability and transcriptional activity of -cat in the human C10 colorectal cancer cell line [385]. Consistently,

IGF-1 was shown to increase -cat stability in prostate cancer cells as well [299]. In

HepG2 cells, insulin and IGF-1 were demonstrated to activate the Wnt/-cat

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pathway, involving both Akt/PKB-dependent and Akt/PKB-independent mechanisms

[298].

I present here that insulin stimulation leads to the activation of c-Myc and cyclin D1, two known downstream target genes of the canonical Wnt signaling pathway. The observed stimulation is attributed to the ability of insulin to increase protein translation via the mTOR signaling pathway, as well as an Akt/PKB- independent pathway that leads to transcriptional activation by increased nuclear - cat content and binding of -cat to the c-Myc gene promoter. A recent study implicates the crosstalk between Wnt and mTOR signaling pathways [329]. mTOR is a serine/threonine kinase that forms a complex with Raptor and mLS8, and acts as a regulator of protein synthesis and ribosome biogenesis. The function of mTOR is regulated by a small G protein and Ras homolog Rheb, which is controlled by the

TSC1/2 tumor suppressor complex. Insulin activated Akt/PKB has been shown to inhibit TSC2 by phosphorylating it at Ser939, Ser981 and Thr1462. Phosphorylated

Ser939 and Ser981 residues in TSC2 then facilitate its interaction with the 14-3-3 protein. 14-3-3-bound TSC2 is then able to interact with Dvl, thereby obstructs its interaction with TSC1, which ultimately leads to subsequent mTOR activation. A recent report by Inoki et al. showed that over-expression of Wnt-1 or treating cells with Wnt-1 conditioned medium activated phosphorylation of mTOR substrates, S6K and 4EBP1; this activation is also dependent on GSK-3. The authors then were able to show that AMPK phosphorylates TSC2 at Ser1345 and primes TSC2 for subsequent phosphorylation by GSK-3. In this case, phosphorylated TSC2 leads to the inhibition of mTOR complex. In the same study, Inoki et al. showed that over-

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expression of a constitutively active form of GSK-3 blocked Wnt1-mediated mTOR activation. These observations suggest that Wnt signaling may relate target gene expression not only via the formation of the bipartite transcription factor cat/Tcf, but also protein synthesis via mTOR activation (Fig. 4.1, Chapter 4).

Insulin signaling leads to the activation of Akt/PKB, which in turn phosphorylates GSK-3 at their inhibitory residues Ser21/9. Inactivation of GSK-3 alleviates its inhibitory function on glycogen synthase (GS) and triggers the synthesis of glycogen. As it was described in the previous section (Section 1.2.3.2),

GSK-3 serves as a key negative modulator for the canonical Wnt pathway by destabilizing -cat. Dual roles of GSK-3 in insulin and Wnt signaling have logically led to the speculation that it acts as a point of molecular signaling convergence for insulin and Wnt/-cat pathways. However, Ding et al. reported that in various cancer cell lines and fibroblasts that 2 h of insulin treatment led to GSK-3 phosphorylation and inactivated GSK-3 enzymatic activity, but free cytoplasmic -cat levels were not altered [459]. On the other hand, treating cells with either Wnt conditioned medium or lithium induced free cytoplasmic -cat accumulation but did not affect the phosphorylation status of GSK-3. Based on these observations, Ding et al. suggested that insulin and Wnt signals regulate GSK-3 via different mechanisms, leading to distinct downstream events, and that phosphorylation of GSK-3/ on

Ser21/ 9 is not sufficient to induce free -cat accumulation. In contrast, studies conducted by other groups show that insulin or IGF-1 could stimulate target genes of the Wnt signaling pathway or -cat mediated reporter gene transactivation [298-300,

417-419].

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In line with the observations made by Ding et al., data obtained from my study suggest that insulin may crosstalk with Wnt signaling independent of Akt-GSK-3. My study demonstrated that insulin stimulation led to an increased expression of -cat in the nucleus in colon cancer cell line HT29, as well as increased binding of -cat on

TBE1 and TBE2 of the human c-Myc gene promoter, detected by qChIP (Fig. 3.12-

3.14, Chapter 3). More importantly, while pharmacological inhibition of PI3K completely abolished these insulin effects, inhibition of Akt/PKB did not (Fig. 3.3, 3.4,

3.13, Chapter 3), suggesting the involvement of a PI3K-dependent but Akt/PKB- independent mechanism. PI3K-mediated but Akt/PKB-independent signaling has been noted recently in several other studies. In human bronchial epithelial cells, cigarette smoke-stimulated EGF receptor activates the expression of FRA-1 through the PI3K-(PAK-1)-(Raf)-MEK-ERK signaling cascade, without the participation of

Akt/PKB [421]. A recent report also shows that in response to PI3K activation, -cat interacts with a member of the 14-3-3 protein family, 14-3-3which allows a high level of free -cat to be maintained and therefore increases -cat dependent transcriptional activation [417]. Further investigation is required to determine whether

 or other members of the 14-3-3 family play a role in insulin stimulated -cat nuclear translocation in the intestinal cancer and non-cancer cells.

The serine/threonine kinase p21-activated protein kinase 1 (PAK-1) has been suggested as a downstream target of cat-TCF [276]. In my study, we revealed that

PAK-1 functions as a novel signaling mediator for insulin and Wnt crosstalk in PI3K- dependent but Akt/PKB-independent manner. PAK-1 knockdown using dominant negative PAK-1 (K299R) or PAK-1 shRNA resulted in significantly reduced effects of

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insulin on proto-oncogene expression, nuclear -cat content and binding of -cat to the c-Myc gene promoter (Fig. 3.12-3.13, Chapter 3). PAK-1 was initially recognized as a downstream target of the Rho family GTPases, Rac1 and Cdc42, in regulating actin remodeling [70, 79, 434]. It is also known that PAK-1 is involved in the development and metastasis of tumors from various tissues including the intestine

[431-433]. Elevated PAK-1 expression is observed in a number of cancers and has been coined as a potential malignancy marker. Recently, PAK-1 over-expression was shown to be correlated with intestinal tumor metastasis [431]. The role of PAK-

1 in insulin-mediated -cat signaling may provide a possible explanation for observed correlation between PAK-1 expression levels in neoplastic tissues.

However, further studies are needed to clarify the potential tumor promoting effects of PAK-1 and possible involvements of Wnt/-cat pathway components in this process.

PAK-1 can also be activated by Akt/PKB [84], or become directly phosphorylated/activated by PI3K [435] or phosphoinositide-dependent kinase isozyme 1 (PDK-1) on Thr 423 [83], without the involvement of Akt/PKB. In my study, we found that insulin treatment led to Thr423 phosphorylation of PAK-1, independent of Akt/PKB activation status (Fig. 4.5-4.7, Chapter 4). As expected, during both in vitro and in vivo insulin resistance, reduced basal and insulin activated

Akt/PKB phosphorylation was observed. However, what should be noted is that

PAK-1 phosphorylation remains responsive to insulin stimulation under insulin resistant conditions. These observations suggest that insulin mediated PAK-1 activation involves a mechanism that is not subjected to hyperinsulinemia-induced

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insulin insensitivity. This is consistent with the observation that despite insulin resistance, hyperinsulinemia is still a risk factor for the development of colorectal neoplasia [171, 426]. Furthermore, a study by Bowker and colleagues showed that

Type 2 diabetic patients who had undergone treatment with insulin or sulfonylureas showed significantly higher risks for cancer-related mortality compared to those who received the insulin-sensitizing drug metformin [484]. It was postulated that hyperinsulinemia confers increased cancer risks because tumor tissues remain insulin sensitive during carcinogenesis in insulin resistant individuals. My study has not only revealed that PAK-1 functions as a downstream mediator of insulin for crosstalking with the canonical Wnt signaling, it also alludes to the involvement of an insulin activated pathway that escapes “metabolic” insulin resistance. Further studies are required to clarify the exact mechanism by which insulin phosphorylates and activates PAK-1.

CK1 and GSK-3 trigger the phosphorylation of -cat on the following residues, Ser33, Ser37, Thr41 and Ser45, which targets -cat for degradation via the ubiquitin-proteasome pathway. However, several investigations also revealed phosphorylation residues on -cat that promote its stability and transcriptional activity. Src kinase induced Tyr654 phosphorylation of -cat results its dissociation from E-cadherin [485], and is correlated with increased association of -cat to TATA- binding protein [486]. Taurin et al. showed that -cat can also be phosphorylated at

Ser675 in response to PKA activation. Phosphorylation of -cat at Ser675 facilitates its interaction with nuclear transcriptional coactivator, CBP and enhances Tcf/Lef transactivation [260]. In addition, PKA-mediated -cat phosphorylation at Ser675

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was shown to exert an inhibitory effect on -cat ubiquitination, thus favoring -cat stabilization [259]. In my study, we observed for the first time that insulin stimulated

-cat phosphorylation at Ser675 in the colon cancer cell line HT29 (Fig. 4.12,

Chapter 4). Moreover, in contrast to the observations made in studies described previously, PKA inhibition did not affect the stimulatory effect of insulin on -cat phosphorylation (Fig. 4.14, Chapter 4). However, knock down of PAK-1 expression or pharmacological inhibition of MEK1/2 dramatically reduced insulin induced -cat

Ser675 levels. These observations suggest insulin signaling utilizes a PKA- independent mechanism to mediate -cat phosphorylation at Ser675, involving PAK-

1 and MEK1/2. Whether PAK-1 or MAPK directly catalyzes the phosphorylation of

-cat requires further investigation. Interestingly, Akt/PKB which is activated by insulin was shown to phosphorylate -cat at Ser553, resulting in its dissociation from cell-cell cadherin junctions and accumulation in the cytosol and nucleus [487].

Furthermore, -cat Ser552 phosphorylation enhances its interaction with 14-3-3, and is correlated with increased cat/Tcf transcriptional activity and tumor cell invasion [487]. Collectively, these findings suggest that insulin may utilized both

Akt/PKB-dependent and Akt/PKB-independent mechanisms to manipulate Wnt signaling via -cat phosphorylation at various serine and tyrosine residues (Fig. 6.1).

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Insulin Wnt Ras Fzd

PI3K PDK-1 Raf AMPK Dvl

MEK TSC-1/2 GSK-3 Akt/PKB Akt/PKB PAK-1

Erk Src PAK-4 -cat Tcf/Lef mTOR -cat

E-cadherin Co- repressors PAK-1 4EBP1 S6K -cat Ser675 -cat Nuclear Levels phosphorylation -cat PAK-1 -cat/TCF binding to c-Myc gene promoter Translation Transcription

Oncogene Expression/ Cell Growth Figure 6. 1 - Current understanding of insulin activated oncogene expression and associated cell growth.

A diagrammatic summary of Akt-dependent and Akt-independent mechanisms for insulin stimulated oncogene (such as c-Myc) expression. This study reveals PAK-1 as a novel mediator for Akt-independent stimulation of insulin on proto-oncogene expression. PI3K: phoshoinositide 3-kinase; Akt/PKB: protein kinase B; PDK1: pyruvate dehydrogenase kinase, isozyme 1; mTOR: mammalian target of rapamycin; 4EBP1: eIF4E-binding protein 1; S6K: p70 ribosomal S6 kinase; AMPK: AMP-activated protein kinase; TSC-1/2: Tumberous sclerosis protein 1/2; PAK: p21 activated protein kinase; Dvl: disheveled; GSK-3: glycogen synthase kinase-3; Tcf/Lef: T-cell factor/lymphoid enhancer factor; Fzd: frizzled.

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6.3 Wnt signaling and Type 2 diabetes

Recent reports suggest that Wnt/-cat signaling has a role in glucose and lipid metabolism [488-490]. Mutations in Wnt/-cat signaling co-receptor LRP6 has been shown to correlate with hyperlipidemia, hypertension and diabetes [488]. Two obesity murine models demonstrated that over-expression of Wnt10b results in significantly improved insulin sensitivity and reduction in adipose tissue mass [489,

490]. In addition, Wnt3a and -cat over-expression were shown to be necessary and sufficient to drive beta cell cycle progression [491]. Lastly, LRP5 deficiency led to an increase in plasma cholesterol levels and impaired glucose tolerance in HFD fed mice; Wnt3A-induced insulin secretion was shown to be impaired in the LRP5- deficient pancreatic islets [488]. A recent report showed that Wnt2b, TCF7L2 as well as canonical Wnt targets c-Myc and cyclin D1 are all upregulated in the pancreas of type II diabetic patients [476]. In addition, HFD alone was able to induce c-Myc in murine pancreatic islet, suggesting that obesity may activate Wnt/-cat signaling to serve as an early event in the pathogenesis of type II diabetes [476]. An increasing number of reports have also suggested that a particular SNP of a member of the

Tcf/Lef family, TCF7L2 (commonly known as TCF-4) is strongly associated with the increased susceptibility of type 2 diabetes. Supporting evidence also came from the observation that TCF-4 and -cat regulate the expression of the proglucagon gene, which encodes glucogon-like peptide-1 (GLP-1) in the enteroendocrine L cells [389].

On the other hand, GLP-1 was reported to activate Wnt signaling in pancreatic beta cells, thus stimulating beta-cell proliferation [456]. In my study, we showed that insulin stimulation led to increased TCF-4 mRNA expression as well as protein

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expression in both colon cancer cells as well as primary intestinal cells from fetal rats (Fig. 5.2-5.3, Chapter 5). This finding, along with previously described evidence from other groups suggest that under hyperinsulinemic conditions, insulin may utilize components of the canonical Wnt pathway to activate GLP-1 synthesis and secretion as a compensatory mechanism for insulin resistance and loss of insulin sensitivity.

6.4 PAK-1 in Wnt signaling crosstalk

The present study identified the serine/threonine kinase PAK-1 as a novel signaling inducer downstream of the insulin pathway, that triggers cat/Tcf transcriptional activity. PAK-1 was initially recognized as a downstream target of the Rho family GTPases, Rac1 and Cdc42, in regulating actin remodeling. Wrch-1

(Wnt-responsive Cdc42 homolog) encodes a homologue of the Rho-family GTPase and shares 57 amino acid sequence with Cdc42. Supportive of observations of the present study, not only does Wrch-1 activate PAK-1, it recapitulates Wnt-1 mediated cellular changes [279]. Specifically, over-expression of a constitutively active mutant of Wrch-1 (Q107L) mimicked Wnt-1 mediated cell proliferation, cytoskeletal reorganization and cell morphology changes in mouse mammary C57MG epithelial cells. The non-canonical Wnt pathway, namely the PCP-convergent extension pathway involves small G proteins Rho and Rac. Therefore, while the currently study implicates PAK-1 in insulin and Wnt/-cat crosstalk, PAK-1 may also be postulated as a mediator for potential crosstalk between non-canonical and canonical Wnt signaling.

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6.5 Overall importance of study and conclusion

Insulin signaling is a critical player in metabolic homeostasis. This project reveals that the function of insulin is not limited to its effects in metabolic processes; it also acts as a potent mitogen by regulating components of the canonical Wnt signaling pathway which has been shown to have tumorigenic effects in numerous tissues. Significantly, this study identifies a novel mediator, PAK-1, for the crosstalk between insulin and Wnt signaling pathways. During the investigation of insulin and

Wnt signaling crosstalk, insulin was found to affect the expression of the newly coined diabetes susceptibility gene, TCF7L2. This finding may provide new insights for the relationship between TCF7L2 and diabetes, especially in Type 2 diabetes mellitus. Since TCF7L2 is a critical transcriptional mediator of the canonical Wnt signaling pathway, this study also demonstrates the potential for a non-metabolic pathway to participate in metabolic functions. Understanding the intricacies and balances between insulin and Wnt/-cat crosstalk will elucidate not only the physiological and pathological significance of insulin in tumorigenesis but also a potential role of Wnt/-cat signaling in maintaining metabolic homeostasis as well as the development of diabetes.

6.6 Future Direction

In this study, new insights of the mechanism for insulin and Wnt molecular crosstalk have been identified and characterized. Specifically, we identified PAK-1 as a novel mediator of insulin signaling that leads to the activation of the canonical

Wnt pathway via the regulation of -cat expression and function. This is reflected in the role of PAK-1 in insulin induced cell growth. We showed that PAK-1 knockdown 190

completely abrogated the stimulatory effect of insulin on colon cancer cell growth in vitro; this is associated with significant attenuation in instulin stimulated c-Myc and cyclin D1 proto-oncogene expression. These observations suggest that targeting

PAK-1 may have potential therapeutic effects on tumor growths. One may use in vivo experimental models to further verify the role of PAK-1 in cancer cell growth and proliferation. For example, colon cancer cells expressing PAK-1 shRNA or scrambled RNA can be introduced to immune-compromised NUDE mice by subcutaneous injection. The function of PAK-1 in cancer cell growth may then be evaluated by monitoring the tumors formed in these NUDE mice.

Our study shows that insulin treatment leads to an increase in -cat nuclear content and nuclear translocation; this is associated with increased -cat Ser675 phosphorylation level after insulin stimulation in the colon cancer cell line HT29.

While reports by other studies suggest that Ser675 phosphorylated -cat has enhanced ability to translocate into the nucleus as well as enhanced transcriptional activity, whether this is recapitulated in intestinal cancer cells still needs to be addressed. One approach to answer this question is to create a plasmid construct expressing GFP-tagged -cat fusion protein with mutation at the Ser675 residue

(using site-directed mutagenesis). We will then be able to discern whether this phosphorylation site is critical for insulin mediated -cat nuclear accumulation by tracking the movements of this -cat mutant after insulin treatment, using live-cell fluorescent microscopy.

Additionally, while we were able to demonstrate that insulin crosstalks with the Wnt pathway by regulating its downstream effector -cat, whether this crosstalk

191

involves upstream components of the Wnt pathway still needs to be further investigated. For example, shRNA technology may be adpoted to knock down the expression of Dvl to determine whether it plays a role in insulin mediated effects on

-cat.

Finally, there is a question that concerns the existence of cis-elements potentially located between the 3.2kb and 1.3kb TCF-4-LUC promoter constructs that are required for insulin stimulated TCF-4 promoter activity. To address this question, bioinformatics may be used first to locate consensus sequences for known transcription factors. Then, ChIP technique can be used determine whether insulin treatment enhances the interaction of the putative transcription factor/s to these specific promoter regions. We can also create a set of TCF-4-LUC promoter constructs ranging between 3.2kb and 1.3kb in sizes to pinpoint the exact region that is responsible for TCF-4 promoter activation upon insulin stimulation.

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460. Kratochwil, K., et al., FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1(-/-) mice. Genes Dev, 2002. 16(24): p. 3173-85.

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486. Piedra, J., et al., Regulation of beta-catenin structure and activity by tyrosine phosphorylation. J Biol Chem, 2001. 276(23): p. 20436-43.

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489. Longo, K.A., et al., Wnt10b inhibits development of white and brown adipose tissues. J Biol Chem, 2004. 279(34): p. 35503-9.

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229

APPENDIX: List of Genes Altered by Insulin Treatment in the IEC-6 Cell Line

230

A.1 Up-regulated Genes (>2-fold) in tne IEC-6 Cell Line post 4 hours of Insulin Treatment

Accession No. Gene Title/Description Gene Symbol Fold Change NM_031315 acyl-CoA thioesterase 1 Acot1 3.26 NM_133424 alpha 3 Actn3 2.77 NM_020302 ADAM metallopeptidase domain 3A (cyritestin 1) Adam3a 2.81 NM_030656 alanine-glyoxylate aminotransferase Agxt 4.00 AI072144 A kinase (PRKA) anchor protein 2 Akap2 4.27 NM_053781 aldo-keto reductase family 1, member B7 Akr1b7 3.20 NM_022665 alkaline phosphatase, intestinal Alpi 2.49 NM_012825 aquaporin 4 Aqp4 2.73 AI145850 armadillo repeat containing 9 Armc9 2.07 NM_130750 repeat, SAM and basic leucine zipper domain Asz1 2.65 containing 1 NM_053381 ATPase, (Na+)/K+ transporting, beta 4 polypeptide Atp1b4 2.44 NM_021850 Bcl2-like 2 Bcl2l2 2.54 NM_017178 bone morphogenetic protein 2 Bmp2 2.41 AF290214 calcium channel, voltage-dependent, T type, alpha 1I Cacna1i 2.05 subunit BF400810 calcium channel, voltage-dependent, beta 1 subunit Cacnb1 2.07 AF474979 cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) Cdkn2b 3.02 NM_133586 carboxylesterase 2 (intestine, liver) Ces2 3.38 AI230238 collagen, type X, alpha 1 Col10a1 5.55 AI715235 collagen, type XVII, alpha 1 Col17a1 2.75 NM_134465 cytokine receptor-like factor 2 Crlf2 2.79 BE102449 DENN/MADD domain containing 2A Dennd2a 2.54 NM_012698 , muscular dystrophy Dmd 2.02 NM_133606 enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme Ehhadh 2.14 A dehydrogenase NM_031826 2 Fbn2 2.05 NM_130816 fibroblast growth factor 11 Fgf11 2.51 NM_131908 fibroblast growth factor 6 Fgf6 2.12 NM_053433 flavin containing monooxygenase 3 Fmo3 2.64 BF415939 FBJ osteosarcoma oncogene Fos 14.83 BG377504 forkhead box I1 Foxi1 2.08 BE112403 fibroblast growth factor receptor substrate 2 Frs2 2.47 NM_053465 fucosyltransferase 9 (alpha (1,3) fucosyltransferase) Fut9 3.57 NM_022623 frizzled homolog 4 (Drosophila) Fzd4 2.28 NM_012849 gastrin Gast 2.88 AI170771 growth hormone receptor Ghr 2.50 BF388060 G protein-coupled receptor 64 Gpr64 3.12 NM_019309 , ionotropic, kainate 2 Grik2 2.19 NM_017010 glutamate receptor, ionotropic, N-methyl D-aspartate 1 Grin1 2.03

231

Accession No. Gene Title/Description Gene Symbol Fold Change Y18810 glutamate receptor, metabotropic 1 Grm1 3.17 AA819731 Hyaluronan and proteoglycan link protein 4 Hapln4 2.41 NM_013074 hypocretin (orexin) receptor 2 Hcrtr2 4.54 BI284294 hypermethylated in cancer 2 Hic2 2.53 BF389738 homeo box B4 Hoxb4 2.06 NM_021862 5-hydroxytryptamine (serotonin) receptor 2A Htr2a 2.72 BI276370 insulin-like growth factor 2 mRNA binding protein 1 Igf2bp1 2.66 NM_012589 interleukin 6 Il6 3.35 BI303923 , beta-like 1 Itgbl1 5.47 BF415575 Janus kinase and interacting protein 2 Jakmip2 3.00 AI145378 kaptin (actin binding protein) Kptn 2.16 BF406693 laminin, alpha 4 Lama4 2.57 AF304191 Lepr 2.21 NM_031050 lumican Lum 5.69 NM_023095 mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl- Mgat5 2.76 glucosaminyltransferase NM_022393 macrophage galactose N-acetyl-galactosamine specific Mgl1 5.51 lectin 1 NM_019188 microseminoprotein, beta Msmb 2.79 AA799423 nexilin (F actin binding protein) Nexn 2.13 NM_134375 NLR family, pyrin domain containing 6 Nlrp6 2.32 NM_023100 1 Nmur1 2.12 U16684 defensin NP-4 precursor /// defensin RatNP-3 precursor Np4 /RatNP-3b 5.32 NM_012621 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 Pfkfb1 2.23 X63434 plasminogen activator, urokinase Plau 2.15 NM_013008 POU class 1 homeobox 1 Pou1f1 2.82 BI287330 protein phosphatase 1, regulatory (inhibitor) subunit 3A Ppp1r3a 2.21 NM_017363 Prolactin family 3, subfamily d, member 1 Prl3d1 2.29 NM_021580 prolactin family 8, subfamily a, member 4 Prl8a4 6.56 AI043800 Proteasome (prosome, macropain) 26S subunit, non- Psmd14 2.44 ATPase, 14 AA923975 Protein tyrosine phosphatase, non-receptor type 4 Ptpn4 2.04 U28356 protein tyrosine phosphatase, non-receptor type 7 Ptpn7 3.04 BG372143 purine rich element binding protein A Pura 2.17 NM_012733 retinol binding protein 1, cellular Rbp1 2.06 NM_031001 ubiquitin-conjugating enzyme RGD69425 2.72 NM_019266 sodium channel, voltage-gated, type 8, alpha subunit Scn8a 2.55 BE118406 secernin 2 Scrn2 2.45 NM_031115 Sctr 5.09 NM_023951 SEBOX homeobox Sebox 2.59 BG380732 src homology 2 domain-containing transforming protein D Shd 2.55 NM_030834 solute carrier family 16, member 3 (monocarboxylic acid Slc16a3 2.17 transporter 4) NM_019230 solute carrier family 22 (extraneuronal monoamine Slc22a3 2.34 transporter), member 3

232

Accession No. Gene Title/Description Gene Symbol Fold Change NM_022287 solute carrier family 26 (sulfate transporter), member 1 Slc26a1 2.38 NM_031728 synaptosomal-associated protein 91 Snap91 2.65 AI102591 Small nuclear ribonucleoprotein D1 Snrpd1 2.24 NM_134404 SV2 related protein Svop 2.20 NM_017053 3 Tacr3 2.21 AI639401 Trichohyalin Tchh 3.24 NM_023020 transmembrane protein with EGF-like and two follistatin- Tmeff1 2.18 like domains 1 NM_031613 2 Tmod2 2.98 NM_012870 tumor necrosis factor receptor superfamily, member 11b Tnfrsf11b 2.04 NM_057149 tumor necrosis factor (ligand) superfamily, member 11 Tnfsf11 2.93 AI231999 tumor protein D52-like 1 Tpd52l1 3.63 AI059620 TRAF3 interacting protein 3 Traf3ip3 3.01 NM_080898 transient receptor potential cation channel, subfamily C, Trpc5 2.37 member 5 BE106848 tyrosine ligase-like family, member 3 Ttll3 2.96 BE096453 ubiquitin-like modifier activating enzyme 7 Uba7 5.57 AW530193 Utrn 2.96 BF391286 wingless-type MMTV integration site family, member 16 Wnt16 2.06 AW524041 zinc finger and BTB domain containing 9 Zbtb9 3.48 BE103689 zinc finger homeobox 4 Zfhx4 5.55 AW529624 zinc finger protein 91 Zfp91 2.69

233

A.2 Up-regulated Genes (>2-fold) in the IEC-6 Cell Line post 24 hours of Insulin Treatment

Accession No. Gene Title/Description Gene Symbol Fold Change NM_013084 acyl-Coenzyme A dehydrogenase, short/branched Acadsb 2.34 chain NM_133424 Actn3 3.00 AI072144 A kinase (PRKA) anchor protein 2 Akap2 2.05 NM_053781 aldo-keto reductase family 1, member B7 Akr1b7 9.52 NM_053381 ATPase, (Na+)/K+ transporting, beta 4 polypeptide Atp1b4 4.28 NM_017178 bone morphogenetic protein 2 Bmp2 2.48 NM_031542 breast cancer 2 Brca2 2.40 BF400810 calcium channel, voltage-dependent, beta 1 subunit Cacnb1 2.34 NM_031518 Cd200 molecule Cd200 2.24 NM_031333 cadherin 2 Cdh2 3.10 AF474979 cyclin-dependent kinase inhibitor 2B (p15, inhibits Cdkn2b 3.02 CDK4) NM_133586 carboxylesterase 2 (intestine, liver) Ces2 2.53 AI230238 collagen, type X, alpha 1 Col10a1 5.22 AI715235 collagen, type XVII, alpha 1 Col17a1 3.67 NM_031560 cathepsin K Ctsk 2.37 NM_012730 cytochrome P450, family 2, subfamily d, Cyp2d2 3.06 polypeptide 2 NM_133606 enoyl-Coenzyme A, hydratase/3-hydroxyacyl Ehhadh 3.64 Coenzyme A dehydrogenase NM_022294 EGF, and seven transmembrane domain Eltd1 2.46 containing 1 NM_130816 fibroblast growth factor 11 Fgf11 2.32 AJ312745 fibroblast growth factor receptor 2 Fgfr2 2.28 NM_053433 flavin containing monooxygenase 3 Fmo3 2.53 BF415939 FBJ osteosarcoma oncogene Fos 13.92 NM_012742 forkhead box A1 Foxa1 2.70 BE112403 fibroblast growth factor receptor substrate 2 Frs2 2.01 NM_053465 fucosyltransferase 9 (alpha (1,3) Fut9 5.52 fucosyltransferase) AI170771 growth hormone receptor Ghr 1.60 NM_012774 glypican 3 Gpc3 3.73 NM_133573 G protein-coupled estrogen receptor 1 Gper 4.49 BF388060 G protein-coupled receptor 64 Gpr64 2.32 NM_019309 glutamate receptor, ionotropic, kainate 2 Grik2 4.28 NM_017010 glutamate receptor, ionotropic, N-methyl D- Grin1 1.23 aspartate 1 Y18810 glutamate receptor, metabotropic 1 Grm1 2.22 AA819731 Hyaluronan and proteoglycan link protein 4 Hapln4 2.03 NM_013074 hypocretin (orexin) receptor 2 Hcrtr2 3.42 BI284294 hypermethylated in cancer 2 Hic2 3.52

234

Accession No. Gene Title/Description Gene Symbol Fold Change NM_133285 histone cluster 1, H1d Hist1h1d 2.07 BI276370 insulin-like growth factor 2 mRNA binding protein 1 Igf2bp1 2.00 BF399783 insulin-like growth factor binding protein 5 Igfbp5 2.01 BF396448 insulin-like growth factor binding protein-like 1 Igfbpl1 2.68 NM_013037 interleukin 1 receptor-like 1 Il1rl1 2.01 NM_031691 integrin, alpha D Itgad 2.12 BF415575 Janus kinase and microtubule interacting protein 2 Jakmip2 4.11 NM_022235 potassium voltage-gated channel, Isk-related Kcne3 2.03 subfamily, gene 3 AI145378 kaptin (actin binding protein) Kptn 2.24 BF406693 laminin, alpha 4 Lama4 2.00 AF304191 leptin receptor Lepr 3.51 NM_031050 lumican Lum 3.19 NM_023095 mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N- Mgat5 2.09 acetyl-glucosaminyltransferase AA799423 nexilin (F actin binding protein) Nexn 2.39 NM_023100 Nmur1 2.12 U16684 defensin NP-4 precursor /// defensin RatNP-3 Np4 /// RatNP- 3.49 precursor 3b NM_013001 paired box 6 Pax6 2.13 NM_012621 6-phosphofructo-2-kinase/fructose-2,6- Pfkfb1 2.17 biphosphatase 1 X63434 plasminogen activator, urokinase Plau 3.72 NM_012625 pro-melanin-concentrating hormone Pmch 2.35 BI287330 protein phosphatase 1, regulatory (inhibitor) subunit Ppp1r3a 2.50 3A NM_022627 protein kinase, AMP-activated, beta 2 non-catalytic Prkab2 3.17 subunit NM_017363 Prolactin family 3, subfamily d, member 1 Prl3d1 3.44 NM_021580 prolactin family 8, subfamily a, member 4 Prl8a4 6.56 NM_138851 prokineticin 1 Prok1 2.02 AI043800 Proteasome (prosome, macropain) 26S subunit, Psmd14 2.23 non-ATPase, 14 AA923975 Protein tyrosine phosphatase, non-receptor type 4 Ptpn4 2.80 U28356 protein tyrosine phosphatase, non-receptor type 7 Ptpn7 3.12 NM_080901 recoverin Rcvrn 3.35 NM_031001 ubiquitin-conjugating enzyme RGD69425 2.03 NM_022669 secretogranin II (chromogranin C) Scg2 2.44 NM_019266 sodium channel, voltage-gated, type 8, alpha Scn8a 3.42 subunit BE118406 secernin 2 Scrn2 2.17 NM_023951 SEBOX homeobox Sebox 2.43 NM_022957 serine (or cysteine) peptidase inhibitor, clade A, Serpina5 2.95 member 5 BF396545 secreted frizzled-related protein 2 Sfrp2 2.01 NM_031672 solute carrier family 15 (H+/peptide transporter), Slc15a2 2.63 member 2

235

Accession No. Gene Title/Description Gene Symbol Fold Change NM_030834 solute carrier family 16, member 3 (monocarboxylic Slc16a3 2.09 acid transporter 4) NM_019230 solute carrier family 22 (extraneuronal monoamine Slc22a3 2.18 transporter), member 3 NM_022287 solute carrier family 26 (sulfate transporter), Slc26a1 2.29 member 1 NM_022632 slit homolog 2 (Drosophila) Slit2 2.44 AI102591 Small nuclear ribonucleoprotein D1 Snrpd1 2.16 NM_053730 stromal antigen 3 Stag3 3.08 NM_017053 Tacr3 2.56 AI639401 Trichohyalin Tchh 3.37 NM_031613 Tmod2 2.24 NM_013049 tumor necrosis factor receptor superfamily, member Tnfrsf4 2.03 4 AI059620 TRAF3 interacting protein 3 Traf3ip3 2.89 NM_080898 transient receptor potential cation channel, Trpc5 2.15 subfamily C, member 5 BE106848 tubulin tyrosine ligase-like family, member 3 Ttll3 4.16 BE096453 ubiquitin-like modifier activating enzyme 7 Uba7 2.58 AW530193 utrophin Utrn 3.09 BF391286 wingless-type MMTV integration site family, Wnt16 2.65 member 16 BF397665 wingless-type MMTV integration site family, Wnt8b 2.31 member 8B AW524041 zinc finger and BTB domain containing 9 Zbtb9 3.42 BE103689 zinc finger homeobox 4 Zfhx4 2.75 AW529624 zinc finger protein 91 Zfp91 2.46

236

A.3 Down-regulated Genes (>2-fold) in the IEC-6 Cell Line post 4 hours of Insulin Treatment

Accession No. Gene Title/Description Gene Symbol Fold Change BI291076 Septin 3 3-Sep 2.77 AA818804 ataxin 2 binding protein 1 A2bp1 2.51 AI102811 AP2 associated kinase 1 Aak1 3.52 AF257746 ATP-binding cassette, sub-family B (MDR/TAP), member 1 Abcb1 3.00 AI105070 actin binding LIM protein family, member 3 Ablim3 2.40 a disintegrin-like and metalloprotease (reprolysin type) with BF416285 thrombospondin type 1 motif, 9 Adamts9 3.10 AI170431 actin filament associated protein 1-like 1 Afap1l1 2.12 BI277442 arylformamidase Afmid 3.86 AW520369 ATP/GTP binding protein 1 Agtpbp1 2.82 NM_017196 allograft inflammatory factor 1 Aif1 3.38 BF392600 ankyrin 1, erythrocytic Ank1 2.92 BF390984 ankyrin repeat domain 6 Ankrd6 2.01 U14007 aquaporin 4 Aqp4 3.40 BG672534 Cdc42 guanine nucleotide exchange factor (GEF) 9 Arhgef9 2.88 AI227963 cyclic AMP-regulated phosphoprotein Arpp-21 4.05 AI146251 aristaless related homeobox Arx 2.00 U45946 ATPase, Na+/K+ transporting, beta 2 polypeptide Atp1b2 3.62 AI407473 Bromodomain adjacent to zinc finger domain, 1B Baz1b 2.72 BF398531 B-cell CLL/lymphoma 11B (zinc finger protein) Bcl11b 4.00 BF406639 Bone morphogenetic protein 6 Bmp6 2.52 Bone morphogenetic protein receptor, type II AI137474 (serine/threonine kinase) Bmpr2 2.23 BF284922 complement component 7 /// tubulin, beta 2c C7 /// Tubb2c 2.90 calcium channel, voltage-dependent, L type, alpha 1D NM_017298 subunit Cacna1d 3.06 NM_017346 calcium channel, voltage-dependent, beta 1 subunit Cacnb1 2.96 NM_012920 calcium/calmodulin-dependent protein kinase II alpha Camk2a 2.06 NM_031338 calcium/calmodulin-dependent protein kinase kinase 2, beta Camkk2 3.54 NM_019292 carbonic anhydrase 3 Car3 3.00 NM_131914 caveolin 2 Cav2 2.74 AW919314 cerebellin 1 precursor Cbln1 10.42 NM_031538 CD8a molecule Cd8a 2.26 BG057557 cadherin 1 Cdh1 4.44 BE111632 cadherin 11 Cdh11 3.22 BI282750 cadherin 13 Cdh13 2.23 AI412693 (chimaerin) 1 Chn1 2.12

237

Accession No. Gene Title/Description Gene Symbol Fold Change AI454607 cholinergic receptor, nicotinic, beta 4 Chrnb4 2.79 cytidine monophosphate (UMP-CMP) kinase 2, BI276216 mitochondrial Cmpk2 2.30 U12425 cyclic nucleotide gated channel alpha 4 Cnga4 3.49 BI290821 collagen, type XIV, alpha 1 Col14a1 2.07 AI556056 Cr2 3.89 NM_017334 cAMP responsive element modulator Crem 5.00 AF187323 cathepsin Q Ctsq 2.76 AI237598 CUG triplet repeat, RNA binding protein 2 Cugbp2 1.98 NM_012840 cytochrome c, testis /// phosphodiesterase 11A Cyct /// Pde11a 9.94 BF407867 dual specificity phosphatase 13 Dusp13 2.05 AI136886 early B-cell factor 3 Ebf3 2.11 AW534202 ER degradation enhancer, mannosidase alpha-like 1 Edem1 2.65 BE097384 eukaryotic translation initiation factor 4 gamma, 3 Eif4g3 2.50 BF408426 family with sequence similarity 65, member A Fam65a 2.62 NM_139194 Fas (TNF receptor superfamily, member 6) Fas 4.13 AI172585 fibulin 1 Fbln1 2.36 AW520855 F-box and leucine-rich repeat protein 7 Fbxl7 3.40 AW528693 Ferredoxin 1-like Fdx1l 2.23 AI072045 fibrinogen alpha chain Fga 8.21 U05675 fibrinogen beta chain Fgb 3.93 AA963213 fibroblast growth factor receptor 2 Fgfr2 2.52 NM_019306 fms-related tyrosine kinase 1 Flt1 2.61 BI289361 forkhead box K1 Foxk1 2.86 AW525701 Forkhead box K2 Foxk2 2.24 BE106518 Forkhead box O1 Foxo1 2.47 fucosyltransferase 4 (alpha (1,3) fucosyltransferase, NM_022219 myeloid-specific) Fut4 3.81 NM_023091 gamma-aminobutyric acid (GABA) A receptor, epsilon Gabre 2.34 M83092 gap junction protein, alpha 5 Gja5 4.03 NM_031803 glucocorticoid modulatory element binding protein 2 Gmeb2 3.15 BF394843 Golgi integral membrane protein 4 Golim4 2.68 BF560816 G protein-coupled receptor 125 Gpr125 4.33 BF403075 G protein-coupled receptor 176 Gpr176 5.89 BF416118 G protein-coupled receptor 68 Gpr68 2.23 BF283627 growth factor receptor bound protein 10 Grb10 8.22 NM_017263 glutamate receptor, ionotropic, AMPA4 Gria4 2.33 BE108608 Glutamate receptor, ionotropic, N-methyl-D-aspartate 3B Grin3b 3.28 AF112182 glutamate receptor interacting protein 2 Grip2 2.40 Y18809 glutamate receptor, metabotropic 1 Grm1 2.27 AW144332 general transcription factor IIH, polypeptide 5 Gtf2h5 2.38 BI300243 H2A histone family, member Y2 H2afy2 3.31 BF399794 Hyaluronan and proteoglycan link protein 2 Hapln2 2.91

238

Accession No. Gene Title/Description Gene Symbol Fold Change BF394504 histone deacetylase 2 Hdac2 4.00 NM_017254 5-hydroxytryptamine (serotonin) receptor 2A Htr2a 15.33 AI230709 insulin-like growth factor 2 mRNA binding protein 3 Igf2bp3 2.97 NM_012588 insulin-like growth factor binding protein 3 Igfbp3 5.98 BF399783 insulin-like growth factor binding protein 5 Igfbp5 2.34 AI179741 IGF-like family member 3 Igfl3 3.83 NM_013110 interleukin 7 Il7 2.42 NM_053917 inositol polyphosphate-4-phosphatase, type II Inpp4b 2.49 BF408149 katanin p60 subunit A-like 1 Katnal1 2.25 potassium voltage gated channel, Shal-related family, NM_031730 member 2 Kcnd2 5.58 Potassium voltage-gated channel, Isk-related subfamily, AW920063 gene 2 Kcne2 4.36 potassium inwardly-rectifying channel, subfamily J, member NM_053870 4 Kcnj4 4.86 NM_130429 lymphoid enhancer binding factor 1 Lef1 3.44 U53144 leptin receptor Lepr 4.45 AW526088 plasticity related gene 1 Lppr4 3.11 BI276243 leucine rich repeat (in FLII) interacting protein 1 Lrrfip1 2.20 AI145796 microtubule associated serine/threonine kinase 3 Mast3 2.83 BF408498 Malonyl CoA:ACP acyltransferase (mitochondrial) Mcat 2.76 BF409340 MAX gene associated Mga 3.85 mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl- AI501069 glucosaminyltransferase Mgat5 2.72 AI502076 Midline 1 Mid1 5.19 NM_017027 myelin protein zero Mpz 2.20 AI501673 Myc target 1 Myct1 6.12 AI535411 Neuroguidin, EIF4E binding protein Ngdn 3.20 AI711265 nucleoside phosphorylase Np 1.98 NM_053344 nuclear receptor subfamily 5, group A, member 1 Nr5a1 6.40 AI145667 neuronal cell adhesion molecule Nrcam 13.71 U02315 neuregulin 1 Nrg1 6.44 NM_022671 one cut homeobox 1 Onecut1 1.99 NM_017167 , kappa 1 Oprk1 2.67 NM_017294 protein kinase C and casein kinase substrate in neurons 1 Pacsin1 3.35 M82845 peptidylglycine alpha-amidating monooxygenase Pam 2.50 BE109825 parvin, beta Parvb 2.84 AA901054 p300/CBP-associated factor Pcaf 1.99 AW523567 Protocadherin 8 Pcdh8 2.33 BF408410 PHD finger protein 13 Phf13 2.21 BE114779 alpha 2 Phka2 2.05 BI282311 PNMA-like 2 Pnmal2 2.84 BF409210 proline-rich nuclear receptor coactivator 1 Pnrc1 2.36 X12658 POU class 1 homeobox 1 Pou1f1 3.87 239

Accession No. Gene Title/Description Gene Symbol Fold Change AI237137 Protein phosphatase 1F (PP2C domain containing) Ppm1f 2.33 D86136 protein phosphatase 1, regulatory subunit 3D Ppp1r3d 2.08 (formerly 2A), regulatory subunit B, NM_053999 alpha isoform Ppp2r2a 4.98 AI175403 Protein phosphatase 3, catalytic subunit, gamma isoform Ppp3cc 3.13 M13706 protein kinase C, beta Prkcb 5.33 NM_022846 prolactin family 8, subfamily a, member 2 Prl8a2 3.83 BF283674 Proline rich 12 Prr12 4.51 proteasome (prosome, macropain) 26S subunit, non- AW527866 ATPase, 11 Psmd11 3.21 NM_032076 prostaglandin E receptor 4 (subtype EP4) Ptger4 2.38 protein tyrosine phosphatase, non-receptor type 22 AW915666 (lymphoid) Ptpn22 2.03 BF397707 Poliovirus receptor-related 3 Pvrl3 4.67 BF559179 Rab40b, member RAS oncogene family Rab40b 2.32 BG381176 RAB6B, member RAS oncogene family Rab6b 3.98 AI172341 RAB GTPase activating protein 1 Rabgap1 2.06 AI170661 RAS p21 protein activator 3 Rasa3 4.38 AI145237 RasGEF domain family, member 1A Rasgef1a 5.05 AI577569 RAS protein-specific guanine nucleotide-releasing factor 1 Rasgrf1 12.65 RAS guanyl releasing protein 1 (calcium and DAG- AF081196 regulated) Rasgrp1 2.49 BI286015 Ras association (RalGDS/AF-6) domain family member 4 Rassf4 6.85 NM_019219 retinoblastoma binding protein 9 Rbbp9 4.50 AI555453 Retinol dehydrogenase 10 (all-trans) Rdh10 2.11 AI556106 retinol dehydrogenase 5 Rdh5 2.54 AW920332 arginine-glutamic acid dipeptide (RE) repeats Rere 2.29 AI408117 regulatory factor X, 7 Rfx7 2.10 AI179271 regulator of G-protein signaling 17 Rgs17 2.48 BE097598 Rho-related BTB domain containing 2 Rhobtb2 5.36 BF416022 ras homolog gene family, member V Rhov 2.01 BG373273 ring finger protein 207 Rnf207 3.22 BI279526 RT1 class II, locus Db1 RT1-Db1 4.50 AW530219 ryanodine receptor 1, Ryr1 2.09 NM_017301 sphingosine-1-phosphate receptor 1 S1pr1 10.40 AF353637 sodium channel, voltage-gated, type 5, alpha subunit Scn5a 5.11 AA957500 signal peptide, CUB domain, EGF-like 1 Scube1 1.98 AI716896 secreted frizzled-related protein 1 Sfrp1 2.81 AA955140 Splicing factor, arginine/serine-rich 2, interacting protein Sfrs2ip 2.94 solute carrier family 16, member 1 (monocarboxylic acid AI556510 transporter 1) Slc16a1 3.38 solute carrier family 25 (mitochondrial carrier; phosphate BF399958 carrier), member 23 Slc25a23 2.68 solute carrier family 6 (neurotransmitter transporter), AW526310 member 17 Slc6a17 1.98

240

Accession No. Gene Title/Description Gene Symbol Fold Change AA997044 SMAD family member 6 Smad6 2.81 Smg-7 homolog, nonsense mediated mRNA decay factor H34608 (C. elegans) Smg7 2.21 AI177589 sortilin-related receptor, LDLR class A repeats-containing Sorl1 2.89 M27882 serine peptidase inhibitor, Kazal type 3 Spink3 3.93 scavenger receptor cysteine rich domain containing, group B AI556291 (4 domains) Srcrb4d 5.50 serum response factor (c-fos serum response element- BF558969 binding transcription factor) Srf 4.28 NM_031558 steroidogenic acute regulatory protein Star 2.89 BF388802 Sulfatase 2 Sulf2 2.88 AW533329 , protein Synm 2.37 AI454612 synaptotagmin IV Syt4 2.50 AW533648 Testis expressed 2 Tex2 2.30 AI229000 T-cell leukemia, homeobox 3 Tlx3 7.67 AI556940 transmembrane channel-like 4 Tmc4 2.54 NM_139254 tubulin, beta 3 Tubb3 2.36 AW520508 ubiquitin-conjugating enzyme E2 variant 1 Ube2v1 10.13 BF285820 ubiquitin-like 5 Ubl5 2.00 NM_031533 UDP glycosyltransferase 2 family, polypeptide B Ugt2b 16.15 AI385361 Vesicle-associated membrane protein 5 Vamp5 3.03 NM_012685 vasoactive intestinal peptide receptor 1 Vipr1 2.19 AI227991 visinin-like 1 Vsnl1 2.02 AA945807 wingless-type MMTV integration site family, member 11 Wnt11 3.13 BF419013 WW domain containing E3 ubiquitin protein ligase 1 Wwp1 2.84 BF545972 zeta-chain (TCR) associated protein kinase Zap70 2.30 NM_053583 zinc finger protein 423 Zfp423 2.15 AW535213 Zinc finger protein 709 Zfp709 2.65 BF408982 Zinc finger protein, multitype 2 Zfpm2 2.28 BE114251 zinc finger protein 592 Znf592 2.53 BG381715 zinc finger with UFM1-specific peptidase domain Zufsp 2.44

241

A.4 Down-regulated Genes (>2-fold) in the IEC-6 Cell Line post 24 hours of Insulin Treatment

Accession No. Gene Title/Description Gene Symbol Fold Change AI102811 AP2 associated kinase 1 Aak1 2.95 AF257746 ATP-binding cassette, sub-family B (MDR/TAP), member 1 Abcb1 2.35 AI105070 actin binding LIM protein family, member 3 Ablim3 2.89 AI170431 actin filament associated protein 1-like 1 Afap1l1 2.16 BI277442 arylformamidase Afmid 2.21 AW520369 ATP/GTP binding protein 1 Agtpbp1 2.16 NM_017196 allograft inflammatory factor 1 Aif1 4.18 BF392600 ankyrin 1, erythrocytic Ank1 2.33 BF390984 ankyrin repeat domain 6 Ankrd6 3.36 U14007 aquaporin 4 Aqp4 0.95 AI013384 Rho GTPase activating protein 10 Arhgap10 2.64 AI178168 Rho GTPase activating protein 15 Arhgap15 2.98 BM384457 Rho GTPase activating protein 22 Arhgap22 2.17 AI071719 Rho guanine nucleotide exchange factor (GEF7) Arhgef7 2.61 BG672534 Cdc42 guanine nucleotide exchange factor (GEF) 9 Arhgef9 2.09 AI227963 cyclic AMP-regulated phosphoprotein Arpp-21 3.13 AI146251 aristaless related homeobox Arx 2.20 U45946 ATPase, Na+/K+ transporting, beta 2 polypeptide Atp1b2 3.62 AI407473 Bromodomain adjacent to zinc finger domain, 1B Baz1b 2.26 BF398531 B-cell CLL/lymphoma 11B (zinc finger protein) Bcl11b 2.44 BF406639 Bone morphogenetic protein 6 Bmp6 1.10 BF284922 complement component 7 /// tubulin, beta 2c C7 /// Tubb2c 2.23 NM_017298 calcium channel, voltage-dependent, L type, alpha 1D subunit Cacna1d 1.29 NM_017346 calcium channel, voltage-dependent, beta 1 subunit Cacnb1 3.33 NM_012920 calcium/calmodulin-dependent protein kinase II alpha Camk2a 5.11 NM_131914 caveolin 2 Cav2 3.53 AW919314 cerebellin 1 precursor Cbln1 3.01 NM_031538 CD8a molecule Cd8a 0.83 BG057557 cadherin 1 Cdh1 3.66 BI282750 cadherin 13 Cdh13 1.72 BI276216 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial Cmpk2 2.29 U12425 cyclic nucleotide gated channel alpha 4 Cnga4 4.28 AI412936 procollagen, type IV, alpha 4 Col4a4 2.11 NM_017334 cAMP responsive element modulator Crem 0.73 AF187323 cathepsin Q Ctsq 3.12 AI237598 CUG triplet repeat, RNA binding protein 2 Cugbp2 3.00 NM_012840 cytochrome c, testis /// phosphodiesterase 11A Cyct / 3.02 Pde11a AI136886 early B-cell factor 3 Ebf3 2.63

242

Accession No. Gene Title/Description Gene Symbol Fold Change AW534202 ER degradation enhancer, mannosidase alpha-like 1 Edem1 3.05 NM_139194 Fas (TNF receptor superfamily, member 6) Fas 3.28 AW520855 F-box and leucine-rich repeat protein 7 Fbxl7 3.40 AW528693 Ferredoxin 1-like Fdx1l 2.57 AI072045 fibrinogen alpha chain Fga 2.47 U05675 fibrinogen beta chain Fgb 2.36 AA963213 fibroblast growth factor receptor 2 Fgfr2 2.72 NM_019306 fms-related tyrosine kinase 1 Flt1 2.00 AW525701 Forkhead box K2 Foxk2 3.51 M83092 gap junction protein, alpha 5 Gja5 3.29 NM_031803 glucocorticoid modulatory element binding protein 2 Gmeb2 2.86 BF394843 Golgi integral membrane protein 4 Golim4 3.47 BF403075 G protein-coupled receptor 176 Gpr176 3.25 BF283627 growth factor receptor bound protein 10 Grb10 7.59 NM_017263 glutamate receptor, ionotropic, AMPA4 Gria4 2.23 AF112182 glutamate receptor interacting protein 2 Grip2 2.13 Y18809 glutamate receptor, metabotropic 1 Grm1 2.04 AW144332 general transcription factor IIH, polypeptide 5 Gtf2h5 3.69 BI300243 H2A histone family, member Y2 H2afy2 3.31 BF399794 Hyaluronan and proteoglycan link protein 2 Hapln2 3.39 AI230709 insulin-like growth factor 2 mRNA binding protein 3 Igf2bp3 2.59 BF399783 insulin-like growth factor binding protein 5 Igfbp5 10.28 NM_031730 potassium voltage gated channel, Shal-related family, Kcnd2 3.88 member 2 AW920063 Potassium voltage-gated channel, Isk-related subfamily, gene Kcne2 9.08 2 NM_053870 potassium inwardly-rectifying channel, subfamily J, member 4 Kcnj4 2.92 AI412474 Potassium channel tetramerisation domain containing 13 Kctd13 2.15 U53144 leptin receptor Lepr 2.88 AW526088 plasticity related gene 1 Lppr4 14.33 NM_053847 mitogen-activated protein kinase kinase kinase 8 Map3k8 3.24 AI237423 mitogen activated protein kinase kinase kinase kinase 1 Map4k1 2.60 BF399528 mitogen-activated protein kinase 8 interacting protein 3 Mapk8ip3 2.00 BF408498 Malonyl CoA:ACP acyltransferase (mitochondrial) Mcat 6.05 BF561921 methyltransferase 11 domain containing 1 Mett11d1 2.11 BF409340 MAX gene associated Mga 4.19 AI502076 Midline 1 Mid1 5.58 AF087697 membrane protein, palmitoylated 3 (MAGUK p55 subfamily Mpp3 4.57 member 3) NM_017027 myelin protein zero Mpz 2.05 NM_019188 microseminoprotein, beta Msmb 2.80 AI501673 Myc target 1 Myct1 2.07 BF419995 myosin, light polypeptide 2, regulatory, cardiac, slow Myl2 3.05 AA891242 myosin, light chain 7, regulatory Myl7 2.90

243

Accession No. Gene Title/Description Gene Symbol Fold Change AA892404 Na+ dependent glucose transporter 1 Naglt1 3.27 AI535411 Neuroguidin, EIF4E binding protein Ngdn 2.50 AI502150 Notch homolog 1, translocation-associated (Drosophila) Notch1 2.91 BF411864 neuronal PAS domain protein 1 Npas1 4.77 AA956855 Nuclear receptor subfamily 1, group H, member 2 Nr1h2 2.38 NM_031627 nuclear receptor subfamily 1, group H, member 3 Nr1h3 2.31 NM_053344 nuclear receptor subfamily 5, group A, member 1 Nr5a1 6.40 AI145667 neuronal cell adhesion molecule Nrcam 3.56 U02315 neuregulin 1 Nrg1 1.12 AW533345 nuclear receptor interacting protein 3 Nrip3 2.06 AI575310 Neurotrophic tyrosine kinase, receptor, type 1 Ntrk1 2.09 NM_017167 opioid receptor, kappa 1 Oprk1 2.67 NM_017294 protein kinase C and casein kinase substrate in neurons 1 Pacsin1 2.07 M82845 peptidylglycine alpha-amidating monooxygenase Pam 1.88 BF402788 phosphatidic acid phosphatase type 2 Pap2d 2.33 BE113130 p53-associated parkin-like cytoplasmic protein Parc 2.24 AA901054 p300/CBP-associated factor Pcaf 2.31 NM_053572 protocadherin 21 Pcdh21 1.99 AW523567 Protocadherin 8 Pcdh8 2.33 NM_133584 phosphodiesterase 5A, cGMP-specific Pde5a 9.28 BF408410 PHD finger protein 13 Phf13 2.21 AI178193 phosphatidylinositol transfer protein, membrane-associated 2 Pitpnm2 3.09 AI556803 pleckstrin Plek 2.26 BI287470 Pleckstrin homology domain containing, family A Plekha1 4.24 (phosphoinositide binding specific) member 1 AI535522 pleckstrin homology domain containing, family F (with FYVE Plekhf2 2.01 domain) member 2 BF547200 polymerase (DNA directed) sigma Pols 3.98 X12658 POU class 1 homeobox 1 Pou1f1 3.71 AI237137 Protein phosphatase 1F (PP2C domain containing) Ppm1f 4.35 BF288039 protein phosphatase 1, regulatory (inhibitor) subunit 12B Ppp1r12b 2.40 BF418702 Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha Ppp2ca 4.32 isoform NM_053999 protein phosphatase 2 (formerly 2A), regulatory subunit B, Ppp2r2a 3.04 alpha isoform AI175403 Protein phosphatase 3, catalytic subunit, gamma isoform Ppp3cc 2.34 BM392159 PR domain containing 8 Prdm8 2.14 BF283674 Proline rich 12 Prr12 2.20 BE126475 paired related homeobox 1 Prrx1 3.92 AW527866 proteasome (prosome, macropain) 26S subunit, non-ATPase, Psmd11 5.19 11 NM_032076 prostaglandin E receptor 4 (subtype EP4) Ptger4 3.00 AI070060 Protein tyrosine phosphatase, non-receptor type 14 Ptpn14 3.14 AW915666 protein tyrosine phosphatase, non-receptor type 22 (lymphoid) Ptpn22 3.16 BF397707 Poliovirus receptor-related 3 Pvrl3 4.52

244

Accession No. Gene Title/Description Gene Symbol Fold Change BG381176 RAB6B, member RAS oncogene family Rab6b 3.75 AI172341 RAB GTPase activating protein 1 Rabgap1 2.06 BF291124 Rab geranylgeranyltransferase, alpha subunit Rabggta 2.05 AI535169 Rap2 interacting protein Rap2ip 2.44 AI577569 RAS protein-specific guanine nucleotide-releasing factor 1 Rasgrf1 2.81 AF081196 RAS guanyl releasing protein 1 (calcium and DAG-regulated) Rasgrp1 2.63 BF406688 Ras interacting protein 1 Rasip1 2.17 BI286015 Ras association (RalGDS/AF-6) domain family member 4 Rassf4 2.05 AW920332 arginine-glutamic acid dipeptide (RE) repeats Rere 2.12 AI172174 RAS-like, estrogen-regulated, growth-inhibitor Rerg 2.05 AI408117 regulatory factor X, 7 Rfx7 2.13 BF283938 RGD1562890 RGD1562890 2.21 AI179271 regulator of G-protein signaling 17 Rgs17 1.09 BE097598 Rho-related BTB domain containing 2 Rhobtb2 4.80 BF416022 ras homolog gene family, member V Rhov 2.75 BE111378 ribosomal protein L3-like Rpl3l 2.08 BI279526 RT1 class II, locus Db1 RT1-Db1 2.70 AW530219 ryanodine receptor 1, skeletal muscle Ryr1 3.21 NM_017301 sphingosine-1-phosphate receptor 1 S1pr1 2.24 AF353637 sodium channel, voltage-gated, type 5, alpha subunit Scn5a 2.49 AA957500 signal peptide, CUB domain, EGF-like 1 Scube1 2.56 AI716896 secreted frizzled-related protein 1 Sfrp1 2.22 AI556510 solute carrier family 16, member 1 (monocarboxylic acid Slc16a1 3.14 transporter 1) BF399958 solute carrier family 25 (mitochondrial carrier; phosphate Slc25a23 3.83 carrier), member 23 AW526310 solute carrier family 6 (neurotransmitter transporter), member Slc6a17 1.50 17 H34608 Smg-7 homolog, nonsense mediated mRNA decay factor (C. Smg7 2.67 elegans) NM_030991 synaptosomal-associated protein 25 Snap25 2.34 AI412208 small nuclear RNA activating complex, polypeptide 4 Snapc4 2.10 AI177589 sortilin-related receptor, LDLR class A repeats-containing Sorl1 1.89 M27882 serine peptidase inhibitor, Kazal type 3 Spink3 2.96 AI556291 scavenger receptor cysteine rich domain containing, group B Srcrb4d 2.79 (4 domains) AI703710 Sterol regulatory element binding transcription factor 2 Srebf2 2.00 BF409823 sulfatase 1 Sulf1 2.74 BF388802 Sulfatase 2 Sulf2 2.36 AW533329 Synemin, intermediate filament protein Synm 2.68 AW533648 Testis expressed 2 Tex2 2.15 NM_012775 transforming growth factor, beta receptor 1 Tgfbr1 2.00 AI229000 T-cell leukemia, homeobox 3 Tlx3 3.02 BI294972 transmembrane emp24 protein transport domain containing 6 Tmed6 2.04 NM_139254 tubulin, beta 3 Tubb3 2.56

245

Accession No. Gene Title/Description Gene Symbol Fold Change AW520508 ubiquitin-conjugating enzyme E2 variant 1 Ube2v1 4.75 NM_031533 UDP glycosyltransferase 2 family, polypeptide B Ugt2b 6.18 AI385361 Vesicle-associated membrane protein 5 Vamp5 1.63 NM_012685 vasoactive intestinal peptide receptor 1 Vipr1 4.68 NM_053583 zinc finger protein 423 Zfp423 2.15 AW535213 Zinc finger protein 709 Zfp709 2.14

246