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CHARACTERIZATION OF A NOVEL REGULATOR AND PREDICTORS OF SENSITIVITY TO TRAIL-INDUCED APOPTOSIS IN BREAST CANCER CELLS A Dissertation Presented to the Faculty of the Graduate School at the University of Missouri In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by JENNIFER L. DINE, RN Dr. Jane Armer, Dissertation Supervisor DECEMBER 2015 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled CHARACTERIZATION OF A NOVEL REGULATOR AND PREDICTORS OF SENSITIVITY TO TRAIL-INDUCED APOPTOSIS IN BREAST CANCER CELLS presented by Jennifer L. Dine, a candidate for the degree of Doctor of Philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. Dr. Jane Armer Dr. Tina Bloom Dr. Elizabeth Bryda Dr. Stanley Lipkowitz Dr. Deidre Wipke-Tevis DEDICATION This work is dedicated to my parents, with thanks. ACKNOWLEDGEMENTS I would like to acknowledge the first of many research mentors, my advisor and committee chair, Dr. Jane Armer. Her unsurpassed energy and support enabled my initial forays into science and helped open a multitude of opportunities in exploration and inquiry. Without Jane’s early vision, I would not now have my own. Thank you, Jane, as well as Dr. Steve Hadwiger, who co-mentored my first research project. I would also like to acknowledge Dr. Elizabeth Bryda, who allowed me the time (and an immeasurable amount of patience) to experience the basic science work environment before I transitioned to NIH. Otherwise, I would have most certainly drowned. Drs. Denise Bouvrette McKinney, Kristen Taylor, and Rui-Hong Wang were also instrumental in drowning prevention. For this, I am eternally grateful. I am not even sure how to begin to thank my dissertation supervisor, Dr. Stanley Lipkowitz, whose willingness to mentor me through thick (sometimes really thick) and thin with humor, insight, and Dolly Parton has made this dissertation possible. I know the following statement is cliché, but: this work (very literally) would not have happened without him. And I feel fortunate to have had the opportunity to help lay down a few of the puzzle pieces (of a very big puzzle) in what I hope will become a viable therapeutic strategy for breast cancer. Thanks, Stan, for letting me participate in this exciting venture. I am greatly appreciative, too, of the support from the other members of my dissertation committee, Drs. Tina Bloom and Deidre Wipke-Tevis. I thank you both for the faith you put in me by allowing me to work so very far away from Mizzou during such an important part of the PhD. Your guidance balanced with granting me autonomy ii and independence has been integral to the entire dissertation process. Thank you so much again. I am also grateful to both past and present members of the Lipkowitz lab, including Juan Crespo-Barreto, Yoshimi Greer, Steve Kales, Mariya Liyasova, Ke Ma, Marion Nau, Phil Ryan, and Donna Voeller for their support, good ideas, and great fun. I would especially like to thank Marion for her wisdom and joy as my former baymate and Donna whose cookies and kindness always made things seem a little better when they didn’t seem like they could get much worse. Special thanks, too, to Yoshimi, whose insightful feedback (and figure-making skills) have helped bring clarity to my science. I would also like to thank members of the Lipkowitz lab’s former home, the Laboratory of Cellular and Molecular Biology, particularly Christina Stuelten, Sarah Clatterbuck Soper, and Connie Sommers, who helped in ways both personal and professional. The National Institute of Nursing Research Division of Intramural Research, especially Director of Training, Dr. Mary Engler, made all of this possible. Without the Graduate Partnerships Program Fellowship, so many nursing doctoral students would not have the chance to train within the state-of-the-art facilities and with world-renowned researchers housed in the NIH intramural program. Thank you for this unequaled opportunity. Finally, my family and friends have really provided the psychological grounding to undertake this hefty endeavor. I would first like to thank all of my graduate student comrades (alphabetized because how can I rank you?) who have lived similar experiences, especially Melanie Austin, Jo-Ana Chase, Lydia Cook, Judy Gohndrone, Yao Matchim, Orawan Nukaew, Tiffany Reed, Kate Robbins, Mattie Robinson, Christina iii Slota, Erica Stein, Ausanee Wanchai, and Maria Yefimova. Donna Martin and Tova Steiner are perhaps my only non-grad school-related friends and teachers in the Bethesda area but have listened to me so much, I am certain they know this process as well as any grad student. My stalwart pre-grad school friends, Emily Haghighi, Erin Horak, Ally Johnson, Deborah Miller, Emma Rogers, Kate Stam, and Iryna Sydorenko have always been there, and I am relentlessly grateful for their friendship. And Megan Schoor… what can’t I say about Megan Schoor? Just know that it is all very, very good. At the end of the day, though, my Aaron has lived through my grad school experience with me in real time and still manages to hold down a job, do the dishes, and make spaghetti. For this, and a few other things, I love him. I love my parents, too, whom I should also thank, but since this whole thing is dedicated to them, I’m just going to end it now. They know already, anyway. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………………..ii LIST OF TABLES……………………………………………………………………..viii LIST OF FIGURES..........................................................................................................ix ABBREVIATIONS AND DEFINITIONS……………………………………………..xi ACADEMIC ABSTRACT………………………………………...…………………...xv CHAPTER ONE: GENERAL INTRODUCTION…………………………………….1 CHAPTER TWO: TRAIL PATHWAY SIGNALING IN THE TREATMENT OF CANCER…………………………………………………………………………………4 Abstract...............................................................................................................................4 TRAIL and its Receptors………………………………………………………………..6 TRAIL Signaling Pathways……………………………………………………………..9 Clinical Results with TRAIL DR Agonists……………………………………………12 Evaluating Pharmacokinetic and Pharmacodynamic Characteristics of TRAIL DR Agonists………………………………………………………………………………….15 Strategies to Improve TRAIL DR Agonist Activity…………………………………..19 Conclusions and Future Directions……………………………………………………25 v CHAPTER THREE: METHOD………………………………………………………57 Research Strategy………………………………………………………………………62 Aim 1: Select a candidate TRAIL pathway regulator for further characterization in the triple negative breast cancer cell line MB-MDA-231……………………..62 Aim 2: Elucidate the functional relationship between gp78 and TRAIL-induced apoptosis………………………………………………………………………....65 CHAPTER FOUR: GP78 NEGATIVELY REGULATES TRAIL-INDUCED APOPTOSIS IN BREAST CANCER CELLS…………………………………….….77 Abstract……………………………...…………………………………………………..77 Materials and Methods…………………………………………………………………82 Cell culture……………………………………………………………………….82 siRNA transfection……………………………………………………………….82 Plasmid and generation of MB231 stably expressing +/-shRNA targeting gp78..83 Caspases-3/7 activity…………………………………………………………….83 Viability assay……………………………………………………………………84 Caspase inhibitor experiments…………………………………………………...84 Transcript quantitation……..……………………………………………………84 Lysate preparation and immunoblotting…………………………………………85 DR5 cell surface expression……………………………………………………...85 Sub-G1 analysis………………………………………………………………….86 Results…...………………………………………………………………………………86 gp78 knockdown sensitizes MB231 to TRAIL……………………………………86 gp78 knockdown sensitizes MB231 to TRAIL in a caspase-dependent manner…90 gp78 knockdown sensitizes MB231 to TRAIL in a non UPR-dependent manner..91 Discussion……………………………………………………………………………….92 Conclusions...……………………………………………………………………………94 CHAPTER FIVE: THE TRAIL RECEPTOR AGONIST DROZITUMAB TARGETS BASAL B TRIPLE NEGATIVE BREAST CANCER CELLS THAT EXPRESS VIMENTIN AND AXL…………………………………………………..114 Abstract………………………………………………………………………………...114 Materials and Methods………………………………………………………………..119 Cell culture……………………………………………………………………...119 vi Inhibitors………………………………………………………………………..119 Viability assay…………………………………………………………………..119 Lysate preparation and immunoblotting………………………………………..120 Caspase-3/7 glo………………………………………………………………...120 Sub-G1analysis…………………………………………………………………120 Analysis of publically available microarray data………………………………121 Immunohistochemistry………………………………………………………….121 Statistical models for analysis of clinical parameters………………………….122 Results………………………………………………………………………………….124 Drozitumab induces apoptosis in breast cancer cells…………………………..124 Drozitumab preferentially kills basal B TNBC cells……………………………127 Vimentin and Axl are expressed in human TNBC……………………………....128 Discussion……………………………………………………………………………...131 CHAPTER SIX: CONCLUSIONS…………………………………………………...159 COMPREHENSIVE REFERENCE LIST…………………………………………..164 APPENDIX: IDENTIFICATION OF NOVEL MOLECULAR REGULATORS OF TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND (TRAIL)-INDUCED APOPTOSIS IN BREAST CANCER CELLS BY RNAi SCREENING………………………………………………………………………….202 VITA…………………………………………………………………………………...271 vii LIST OF TABLES Table Page 4.1 siRNA Targeting Sequences……………………………………………………..…101 5.1 Patient Characteristics………………………………………………………………145 5.2 Vimentin and Axl Correlate with DFS and OS, Respectively……………………...147 viii LIST OF FIGURES Figure Page 2.1 TRAIL and its Receptors…………………………………………………………….55 2.2 The Extrinsic and Intrinsic Apoptotic Pathways ………………………………..…..56 3.1 Negative Regulators of