Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 10-12-2018 Genome-Wide Screening Identifies Genes and Biological Processes Implicated in Chemoresistance and Oncogene-Induced Apoptosis Tengyu Ko Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Cancer Biology Commons, Cell Biology Commons, and the Genomics Commons Recommended Citation Ko, Tengyu, "Genome-Wide Screening Identifies Genes and Biological Processes Implicated in Chemoresistance and Oncogene- Induced Apoptosis" (2018). LSU Doctoral Dissertations. 4715. https://digitalcommons.lsu.edu/gradschool_dissertations/4715 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. GENOME-WIDE SCREENING IDENTIFIES GENES AND BIOLOGICAL PROCESSES IMPLICATED IN CHEMORESISTANCE AND ONCOGENE- INDUCED APOPTOSIS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical and Veterinary Medical Sciences through the Department of Comparative Biomedical Sciences by Tengyu Ko B.S., University of California, Santa Barbara 2010 December 2018 ACKNOWLEDGEMENTS I would like to express my sincerest gratitude to my major supervisor Dr. Shisheng Li for giving me the opportunity to join his team and the freedom to pursue projects. I appreciate all of his thoughts and efforts. Truly, none of these findings would be possible without his supervisions, supports, insightful discussions, and patience. I am deeply indebted to the members of my committee Dr. Yoshimura, Dr. Yao, Dr. Larkin, and Dr. Marzilli. Without Dr. Yoshimura’s encouragements, Dr. Yao’s useful equipment, Dr. Larkin’s constructive suggestions, and Dr. Marzilli’s willingness to help out, I would not be able to finish this dissertation. Special thanks to Dr. Penn for letting me go rampage on his sophisticated pathway analysis software, and for encouraging me, calming me down, and trying not to shoot me in the face when I make bad presentations. I would also like to thank my colleagues Dr. Wentao Li, Dr. Kathiresan Selvam, Dr. Mingyang Li, and Mr. Wenzhi Gong for their helpful discussions and unwavering friendships even though I always puzzled them in lab meetings. Last but not the least, I would like to extend my gratitude to my Father in Heaven, my family, Ms. Fuqi Sun, Mr. Shane Chang, Ms. Laura Yen, Ms. Zora Yo, Ms. Iris Chen, Dr. Zhong Li, Ms. Mary Price, Ms. Sue, Mr. Finch, Dr. John Fu, and many more that I don’t have the time to mention. Thanks for putting up with me all these years. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................................ ii LIST OF TABLES .......................................................................................................................... v LIST OF FIGURES ....................................................................................................................... vi LIST OF ABBREVIATIONS ....................................................................................................... vii ABSTRACT ................................................................................................................................... ix CHAPTER 1. TUMORIGENESIS AND MELANOMA TREATMENTS ................................... 1 1.1 Introduction ........................................................................................................................... 1 1.2 Oncogene ............................................................................................................................... 1 1.3 Oncogene-induced Senescence ............................................................................................. 2 1.4 Oncogene-induced Apoptosis ............................................................................................... 3 1.5 Melanoma treatments and resistance..................................................................................... 5 1.6 CRISPR/Cas9 ........................................................................................................................ 7 1.7 Statement of the Problem and Specific Aims........................................................................ 8 1.8 References ............................................................................................................................. 9 CHAPTER 2. GENOME-WIDE SCREEN IDENTIFIES NOVEL GENES AND BIOLOGICAL PROCESSES IMPLICATED IN CISPLATIN RESISTANCE ................................................... 14 2.1 Introduction ......................................................................................................................... 14 2.2 Material and Methods.......................................................................................................... 15 2.3 Results ................................................................................................................................. 21 2.4 Discussion ........................................................................................................................... 41 2.5 References ........................................................................................................................... 45 CHAPTER 3. GENOME-WIDE SCREEN IDENTIFIES NOVEL GENES IMPLICATED IN TUMORIGENESIS ...................................................................................................................... 50 3.1 Introduction ......................................................................................................................... 50 3.2 Materials and Methods ........................................................................................................ 51 3.3 Results ................................................................................................................................. 58 3.4 Discussion ........................................................................................................................... 70 iii 3.5 References ........................................................................................................................... 74 CHAPTER 4. CONCLUDING REMARKS................................................................................. 80 4.1 Research Summary .............................................................................................................. 80 4.2 Future Direction .................................................................................................................. 81 4.3 Reference ............................................................................................................................. 81 APPENDIX. GENES IMPLICATED IN CISPLATIN SENSITIVITY AND RESISTANCE, AND ONCOGENE-INDUCED APOPTOSIS AND SENESCENCE ......................................... 82 VITA ........................................................................................................................................... 130 iv LIST OF TABLES Table 2.1. Oligonucleotides used for creation of sequencing libraries of GeCKO gRNAs integrated in the genome of transduced cells ............................................................................ 17 Table 2.2. shRNA-encoding DNA sequences that are effective for gene knockdown ............. 18 Table 2.3. Reverse transcription PCR primers .......................................................................... 21 Table 2.4. Top 40 disrupted biological processes positively selected by cisplatin ................... 25 Table 2.5. Top 40 disrupted biological processes negatively selected by cisplatin .................. 28 Table 3.1. Primers for Genome Screening and for creating indel profiles................................ 54 Table 3.2. Common candidates in P1F/hTERT and H4C cells. ................................................ 62 Table 3.3. Distribution of GPR4 knockouts in cells survived BRAF-V600E expressiona ....... 65 Table 3.4. Distribution of DBT knockouts in cells survived BRAF-V600E expressiona ......... 65 Table 3.5. Distribution of GPR4 knockouts in cells without BRAF-V600E expressiona ......... 66 Table 3.6. Distribution of DBT knockouts in cells without BRAF-V600E expressiona .......... 67 Table A1. Top 1000 disrupted genes positively selected by cisplatin ...................................... 82 Table A2. Top 1000 disrupted genes negatively selected by cisplatin ................................... 105 Table A3. Top disrupted genes positively selected by BrafV600E in P1F/hTERT fibroblasts ................................................................................................................................................. 127 Table A4. Top disrupted genes positively selected by BrafV600E in H4C melanocytes ....... 129 v LIST OF FIGURES Figure 1.1. Oncogene-induced apoptosis and senescence through p53- and p16-mediated pathways. ..................................................................................................................................... 3 Figure 1.2. Coupling of cellular proliferation and apoptosis by oncogenic stress