ABERRANT GENE EXPRESSION: DIAGNOSTIC MARKERS AND THERAPEUTIC TARGETS FOR PANCREATIC CANCER Jeran Kent Stratford A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology. Chapel Hill 2014 Approved by: Jen Jen Yeh Channing J. Der Gary L. Johnson Adrienne D. Cox Timothy C. Elston © 2014 Jeran Kent Stratford ALL RIGHTS RESERVED ii ABSTRACT Jeran Kent Stratford: Aberrant gene expression: diagnostic markers and therapeutic targets for pancreatic cancer (Under the direction of Jen Jen Yeh and Channing J. Der) Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer and the fourth leading cause of cancer-related death in the United States. The overall median survival for patients diagnosed with PDAC is five to eight months. The poor outcome is due, in part, to a lack of disease-specific symptoms that can be used for early detection, and as such, most patients present with locally advanced or metastatic disease at the time of diagnosis. Therefore, the need for diagnostic tools is both great and urgent. Furthermore, current chemotherapies have low response rates and high toxicity, limiting their use, and there are currently no effective targeted therapies for PDAC. Therefore, a greater understanding of the underlying biology of pancreatic cancer is needed to identify tumor-specific vulnerabilities that can be therapeutically exploited. Pancreatic cancer development is driven by genomic changes that alter gene expression. Aberrant gene expression produces changes in protein expression, which in turn may confer growth advantages to the tumor; often the tumor then develops a dependency on continued aberrant gene and protein expression. Determining how aberrant genome-wide gene expression changes affect the biology of the tumor is of paramount importance in our continued efforts to improve patient therapy. By identifying and establishing a role for the overexpressed genes in PDAC we can discover new avenues for therapeutic intervention and iii potentially predict the most effective therapy for each individual and avoid therapies that may have little clinical efficacy. My research aimed to identify aberrantly expressed genes in primary tumor samples from PDAC patients and characterize the diagnostic and therapeutic value of the identified genes. The work outlined in this dissertation focuses first on identifying a prognostic signature of genes with the ability to stratify patients into high and low risk groups, and second on assessing the biological importance of overexpression of the dual-specificity protein kinase TTK for pancreatic cancer oncogenesis. Together these two investigations provide a basis for translating molecular changes in tumor biology into improved therapy for pancreatic cancer patients. iv To Mindy, my ever steady companion v ACKNOWLEDGEMENTS First I wish to thank my mentors, Jen Jen Yeh and Channing Der. I sincerely thank you for all of the encouragement and time you have spent on my behalf as advisors. It was only through your continued support and guidance that the work contained in this dissertation was made possible. Your combined knowledge has aided me in discovering how to apply basic research to key clinical questions, one of the main goals I wanted to accomplish in my graduate career. I appreciate the patience and guidance I have received from both of you as I have struggled through developing experimental methods and the ever-daunting process of writing. I will always appreciate the sacrifices you have made on my behalf to mold me into the scientist I have become. Secondly I wish to thank the Department of Pharmacology for all of the support I have received. I would not have been able to accomplish this work had I not been fortunate enough to collaborate with such exceptional faculty and students. This has been such a unique opportunity to learn and grow as a scientist and as a person. In addition I would like to thank the members of the Yeh and Der labs both past and present. Thank you for helping guide me through the hard times and share in the joy of the good times. Lastly, I thank my wonderful and ever-growing family. Family is forever and I appreciate your patience and interest in me as I have pursued this doctorate. I’d like to thank my father and mother for their encouragement and support even though I moved over 2000 miles away from them. Thank you for your positive outlook and well-timed packages of vi candy. Finally, a thank you to Mindy, my sweet wife. This would have been impossible had I not had you by my side. You have provided me with the strength I didn’t have on my own to stay the course through your amazing example and kind words. You’re simply the best. I love you and can never say thank you enough. vii TABLE OF CONTENTS LIST OF TABLES .................................................................................................................. X LIST OF FIGURES .............................................................................................................. XI LIST OF ABBREVIATIONS ............................................................................................ XII CHAPTERS I. INTRODUCTION ........................................................................................................ 1 PANCREAS ANATOMY AND FUNCTION ........................................................................... 2 PANCREATIC DUCTAL ADENOCARCINOMA DEVELOPMENT ............................................ 2 STAGING OF PANCREATIC CANCER ................................................................................ 5 PANCREATIC DUCTAL ADENOCARCINOMA OUTCOME AND TREATMENT ........................ 9 GENOMIC INSTABILITY ................................................................................................ 14 SPINDLE ASSEMBLY CHECKPOINT ................................................................................ 14 TTK ............................................................................................................................ 16 APC/C ........................................................................................................................ 18 GENETICS OF PANCREATIC DUCTAL ADENOCARCINOMA .............................................. 23 GENE EXPRESSION PROFILING ..................................................................................... 27 SUMMARY ................................................................................................................... 29 viii II. A SIX-GENE SIGNATURE PREDICTS SURVIVAL OF PATIENTS WITH LOCALIZED PANCREATIC DUCTAL ADENOCARCINOMA .......................... 32 OVERVIEW .................................................................................................................. 32 INTRODUCTION ............................................................................................................ 33 EXPERIMENTAL PROCEDURES ...................................................................................... 34 RESULTS ..................................................................................................................... 39 DISCUSSION ................................................................................................................ 48 III. GENETIC AND PHARMACOLOGIC INHIBITION OF TTK IMPAIRS PANCREATIC CANCER CELL LINE GROWTH BY INDUCING LETHAL LEVELS OF CHROMOSOMAL INSTABILITY .................................................... 54 OVERVIEW .................................................................................................................. 54 INTRODUCTION ............................................................................................................ 55 EXPERIMENTAL PROCEDURES ...................................................................................... 58 RESULTS ..................................................................................................................... 64 DISCUSSION ................................................................................................................ 84 IV. CONCLUSIONS AND FUTURE DIRECTIONS ..................................................... 90 DIAGNOSTIC GENE EXPRESSION FOR PANCREATIC DUCTAL ADENOCARCINOMA .......... 90 GENE EXPRESSION SIGNATURES AND SUBTYPES OF DISEASE ....................................... 91 THERAPEUTIC VALUE OF TTK FOR PANCREATIC DUCTAL ADENOCARCINOMA ............. 92 TTK PHOSPHORYLATION SUBSTRATES ........................................................................ 95 USP16 SUBSTRATES AND REGULATION ........................................................................ 96 SUMMARY ................................................................................................................... 97 REFERENCES ........................................................................................................................ 98 ix LIST OF TABLES Table 1. Pancreatic cancer TNM classification ........................................................................... 6 Table 2. AJCC stage groupings for pancreatic cancer ................................................................ 7 Table 3. ECOG Performance Status ........................................................................................... 15 Table 4. Patient, tumor, and treatment characteristics in the derivation set ................................ 40 Table 5. Patient, tumor, and treatment characteristics