Analysis of Somatic Copy Number Gains in Pancreatic Ductal Adenocarcinoma Implicates ECT2 as a Candidate Therapeutic Target by Nardin Samuel A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Nardin Samuel 2012 Analysis of Somatic Copy Number Gains in Pancreatic Ductal Adenocarcinoma Implicates ECT2 as a candidate therapeutic target Nardin Samuel Master of Science Department of Molecular Genetics University of Toronto 2012 Abstract This study presents an integrated analysis of pancreatic ductal adenocarcinoma (PDAC) for identification of putative cancer driver genes in somatic copy number gains (SCNGs). SCNG data on 60 PDAC genomes was extracted to identify 756 genes, mapping to 20 genomic loci that are recurrently gained. Through copy number and gene expression analysis on a panel of 29 human pancreatic cancer cell lines, this gene catalogue was refined to 34 PDAC high-confidence candidate genes. The performance of these genes was assessed in pooled shRNA screens and only ECT2 showed significant essentiality to cell viability in specific PDAC cell lines with genomic gains at the 3q26.3 locus that harbor this gene. Targeted shRNA-mediated interference of ECT2, as well as pharmacological inhibition, are supportive of the pooled shRNA screen findings. These results favor ECT2 as a candidate target gene for further evaluation in the subset of PDACs presenting with 3q26 somatic copy number gains. ii Acknowledgements First I would like to acknowledge my supervisor and mentor, Dr. Thomas Hudson, for giving me the opportunity to work in his lab and for his immense support, guidance and encouragement. I also thank Dr. Jason Moffat for all of his support and for welcoming me into his lab to learn new techniques and think critically about my work, as well as Azin Sayad and Dr. Kevin Brown from the Moffat Lab, for their willingness to help with this project. I also thank Dr. Fei-Fei Liu and Dr. Brenda Gallie for kindly mentoring me and serving on my supervisory committee. I would also like to thank the entire Hudson lab, especially Mathieu Lemire, for all of his help and support with statistical analyses. At OICR, I also thank Drs. David Uehling, Gennadiy Poda, Rima Al-Awar, Quang Trinh and Lakshmi Muthuswamy for helpful discussions and feedback. I am also very grateful to Dr. Troy Ketela, Kajaal Nagar, Sonali Weerawardane and Jasmyne Carnevale for support with shRNA studies and for accommodating me in the lab. Lastly, I am so grateful for the endless support of my wonderful family and friends. During this work, a championed scientist, Dr. Ralph Steinman, was awarded one of the most prestigious awards in research, a Nobel Prize in Medicine, but passed away from pancreatic cancer before he could be presented with the award. For pancreatic cancer in particular, therapeutic options are limited and it is these types of stories that remind me of the potential impact research can achieve and inspire me to be involved in cancer research. iii Table of Contents Acknowledgements……………………………………………………………………………………….……………………………iii Table of Contents…………………………………………………………………………………………….………………………….iv List of Tables……………………………………………………………………………………………………….…………………....vii List of Figures…………………………………………………………………………………………………………………………..viii List of Appendices……………………………………………………………………………………………………………………....x List of Abbreviations……………………………………………………………………………………………………………….…xi Chapter 1…………………………………………………………………………………………………………………………………….1 1 Introduction……………………………………………………………………………………………………………………………..1 1.1 Pancreatic Ductal Adenocarcinoma…………………………………………………………………………….1 1.1.1 Incidence and Mortality……………………………………………………………………………1 1.1.2 Molecular Biology of Pancreatic Ductal Adenocarcinoma…………………………...1 1.2 Current Therapeutic Options for Pancreatic Ductal Adenocarcinoma…………………………...2 1.2.1 Rationale for identifying novel molecular targets……………………………………….4 1.3 Somatic Mutations in Pancreatic Ductal Adenocarcinoma……………………………………………5 1.3.1 Driver vs. Passenger Mutations…………………………………………………………………5 1.3.2 Known Driver Mutations in Pancreatic Ductal Adenocarcinoma…………………6 1.4 Somatic Copy Number Gains in Human Caner……………………………………………………………..7 1.4.1 Methods for Genome-Wide Detection of Somatic Copy Number Gains………...7 1.4.2 Studies of Structural Mutations in Pancreatic Ductal Adenocarcinoma…..……8 1.5 Features of Ideal Therapeutic Targets……………………………………………………………………….10 1.6 Epithelial cell-transforming oncogene 2 (ECT2)………………………………………………………….10 1.6.1 ECT2 Structure and Function…………………………………………………………………..10 1.6.2 ECT2 Copy Number Gains and Over-Expression in Human Cancer…………….12 iv Chapter 2…………………………………………………………………………………………………………………………………..15 2 Identification of ECT2 as a Candidate Therapeutic Target Gene in Pancreatic Ductal Adenocarcinoma………………………………………………………………………………………………………………….15 2.1 Introduction…………………………………………………………………………………………………………….15 2.2 Hypothesis………………………………………………………………………………………………………………16 2.3 Project Aims………………….…………………………………………………………………………………………16 2.3.1 Identification of Coding Regions of Recurrent Copy Number Gain in Human Pancreatic Ductal Adenocarcinoma…………………………………………………………16 2.3.2 Analysis of Candidate Gene List in an Independent Cohort of Human Pancreatic Ductal Adenocarcinoma Cell Lines …………….…………………………...16 2.3.3 Assembling a Catalogue of Candidate Genes for Further Study………………….17 2.3.4 Modulation of Candidate Target Gene by shRNA-Mediated Interference and Pharmacological Approaches…………………………………………………………………..17 2.4 Materials and Methods……………………………………………………………………………………………18 2.4.1 Publically Available Pancreatic Ductal Adenocarcinoma Genome Datasets..18 2.4.2 Integrated Analysis of Pancreatic Cancer Genome Datasets………………………18 2.4.3 Copy Number Analysis of Candidate Genes in Human Pancreatic Ductal Adenocarcinoma Cell Lines…………………………………………………..…………………18 2.4.4 Gene Expression Analysis of Candidate Genes in Human Pancreatic Ductal Adenocarcinoma Cell Lines……………………………………………………………………..19 2.4.5 Integrated Analysis of Copy Number and Gene Expression of Candidate Genes to Refine List of Putative Target Genes…………………………………………20 2.4.6 Assembly of Pancreatic Ductal Adenocarcinoma Candidate Target Gene Database………………………………………………………………………………………………...20 2.4.7 Compilation of ‘Druggable Genome’ Database………………………………………….21 2.4.8 Integration of RNA-interference Pooled Screen Studies to Identify Candidate Target Gene for Laboratory-Based Study…………………………………………………21 2.4.9 Tissue Culture and Cell Lines…………………………………………………………………..22 2.4.10 ECT2 and Control Lentivirus Production………………………………………………….22 2.4.11 Lentivirus Titration………………………………………………………………………………...23 v 2.4.12 Cell Viability Assay in shRNA Experiment………………………………………………..24 2.4.13 Pharmacologic Modulation Assay……………………………………………………………24 2.5 Results…………………………………………………………………………………………………………………….26 2.5.1 Genomic Regions of Recurrent Somatic Copy Number Gains in Pancreatic Ductal Adenocarcinoma………………………………………………………………………….26 2.5.2 Integrated Copy Number and Expression Analysis of Candidate Genes……..33 2.5.3 Database of Top-Ranked Candidate Target Genes……………………………………36 2.5.4 Identification of ECT2 for Laboratory Study Through Integration of shRNA Pooled Screen Results……………………………………………………………………………..39 2.5.5 Targeted shRNA studies of ECT2 in Pancreatic Ductal Adenocarcinoma Cell Lines………………………………………………………………………………………………………43 2.5.6 Functional Effects of Pharmacologic Inhibition of the ECT2 Pathway on Cell Viability…………………………………………………………………………………………….……61 Chapter 3…………………………………………………………………………………………………………………………………..64 3 Discussion…………………………………………………………………………………………………………………………...64 3.1 Pooling Data from Genome-Wide Analyses………………………………………………………………..64 3.2 Analysis of Top-Ranked Candidate Genes and Identification of ECT2 as a Putative Target…………………………………………………………………………………………………………………...…65 3.3 Dependence on ECT2 for Cell Viability in Cell Lines Bearing a Genomic Gain at the 3q26 Locus.....................................……………………………………………………………………………….…………...67 3.4 Differential Sensitivity to Inhibitors of ECT2-Mediated Cellular Pathway in Cell Lines Bearing Genomic Copy Number Gains at the 3q26 Locus………………………....………..………68 3.5 Future Directions……………………………………………………………………………………………………..70 3.5.1 Rationale……………………………………..……………………………………………..…………..70 3.5.2 Specific Aims………………………………………………………………………………………….71 References……….……………………………………………………………………………………………………………………….72 Appendices…………………………………………………………………………………………………………………………….…80 vi List of Tables Table 1 Genomic loci encompassed in SCNGs identified in this study…. …………………...………………….29 Table 2 Regions of genomic gain identified in this analysis of pancreatic tumors as well as a survey of 26 histological subtypes in human cancer by Beroukhim et al, 2011………………………………………..32 Table 3 Database of top-ranked candidate PDAC genes……………………………………………………………….38 Table 4 Results of copy number measures for ECT2 in cell lines utilized for targeted shRNA analyses obtained through different computational methods……………………………………………………………………45 Table 5 Copy number analysis of pancreatic cancer cell lines in Barretina J, et al. 2012……………..…46 Table 6 Comparison of targeted shRNA analysis with results from pooled shRNA screen……………..60 vii List of Figures Figure 1 ECT2 protein structure……………………………………………….………………………………………………..11 Figure 2 ECT2 is mislocalized to the cytoplasm of primary non-small lung cancer tumors…………....13 Figure 3 Number of genes encompassed in genomic gains multiple datasets………………………………..26