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IDENTIFICATION OF SMALL MOLECULE INHIBITORS TARGETING TRUNCATED ADENOMATOUS POLYPOSIS COLI (APC) AS A NOVEL THERAPEUTIC STRATEGY IN COLORECTAL CANCER APPROVED BY SUPERVISORY COMMITTEE Jerry W. Shay, Ph.D., Mentor Woodring E. Wright, M.D., Ph.D., Mentor Ralph Deberardinis, M.D., Ph.D., Chair Michael Roth, Ph.D. Michael White, Ph.D. ACKNOWLEDGMENTS I would like to express my deepest gratitude to my mentors Dr. Jerry Shay and Dr. Woodring Wright, for their support, encouragement, valuable guidance and instrumental criticism throughout my graduate study. Thank you for sharing your knowledge with me and providing me a strong foundation for my future career as a research scientist. I would like to thank my thesis committee members, Dr. Ralph Deberardinis, Dr. Michael Roth and Dr. Michael White for their invaluable suggestions and constructive feedbacks. I would also like to thank all the current and former lab members of the Shay/Wright lab. In particular, I would like to thank my colleagues Dr. Sang Bum Kim, Dr. Ugur Eskiocak and Dr. Guido Stadler for their experimental guidance and assistance. Likewise, thanks to Sze Wong, Ilgen Mender, Ejun Huang and Gaoxiang Jia for their kind friendship and encouragement. Special thanks to Kevin Kennon for his administrative support. Finally, I would like to thank my parents, Jie Wang and Bin Zhang, and my husband Wenhao Chen for their unconditional support, encouragement and everlasting love. Thank you for being with me every step throughout my life. IDENTIFICATION OF SMALL MOLECULE INHIBITORS TARGETING TRUNCATED ADENOMATOUS POLYPOSIS COLI (APC) AS ANOVEL THERAPEUTIC STRATEGY IN COLORECTAL CANCER By Lu Zhang DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of Doctor Of DOCTOR OF PHILOSOPHY University Of Texas Southwestern Medical Center Dallas, Texas August, 2014 Copyright by Lu Zhang, 2014 All Rights Reserved IDENTIFICATION OF SMALL MOLECULE INHIBITORS TARGETING TRUNCATED ADENOMATOUS POLYPOSIS COLI (APC) AS A NOVEL THERAPEUTIC STRATEGY IN COLORECTAL CANCER Lu Zhang, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2014 Jerry W.Shay, Ph.D., Woodring E. Wright, M.D./Ph.D. Abstract Adenomatous polyposis coli (APC) is a tumor suppressor gene that is mutated in the vast majority of colorectal tumors. Inactivation of this gene is a key and early event in colorectal tumorigenesis. APC primarily acts as a negative regulator of Wnt pathway by aiding in degradation of β-catenin. Further studies have suggested that APC plays important roles in several other cellular processes, including cell adhesion and migration, organization of actin and microtubule networks, spindle formation and chromosome segregation. Mutations in APC gene generate truncated gene products, leading to deregulation of these processes. Accumulating evidence suggest that these C terminally truncated APC may have gain of function properties beyond their loss of tumor suppressive function. Both the gain and v vi loss of function of APC truncations contribute to CRC initiation and progression. Utilizing a series of isogenic immortalized human colonic epithelial cells (HCECs), we have screened a 200K compound library for small molecules that exhibited selective cytotoxicity towards HCECs expressing truncated APC. We identified a compound, TASIN-1(Truncated APC Selective Inhibitor-1), which showed selective cytotoxicity towards HCECs and colorectal cancer (CRC) cells with APC truncations. TASIN-1 induces apoptotic cell death in APC truncated cells and its effect is independent of canonical Wnt pathway activity. Short hairpin RNA (shRNA) mediated knockdown of truncated APC confers resistance to TASIN-1. Additionally, TASIN-1 can interact with in vitro translated APC fragments, implicating truncated APC in the mechanism of action of TASIN-1. TASIN-1 inhibits tumor growth in both xenograft and genetic CRC mouse models. TASIN-1 may represent a novel therapeutic strategy for colon cancer prevention and intervention. Table of Contents Page Abstract ......................................................................................................................................................... v Prior Publications ......................................................................................................................................... vi List of Figures ............................................................................................................................................. vii List of Tables ............................................................................................................................................... ix Chapter 1 Introduction .................................................................................................................................. 1 Colorectal Cancer (CRC) .......................................................................................................................... 1 Adenomatous polyposis coli (APC) .......................................................................................................... 6 The APC gene ....................................................................................................................................... 6 APC Protein and its Many Functions .................................................................................................... 7 APC Mutation in Sporadic CRC ......................................................................................................... 11 Loss of Tumor Suppressive Function of APC in CRC ....................................................................... 12 Dominant-negative Effects of APC .................................................................................................... 15 APC Truncation: Switch from Tumor Suppressor to Oncogene? ....................................................... 16 Lessons from genetic mouse models ...................................................................................................... 20 Overview of chemical genetics-based target identification in drug discovery ...... Error! Bookmark not defined. Target-based versus chemical genetics drug discovery approaches ................................................... 22 Chemical proteomics for protein target identification in chemical genetics drug discovery .............. 23 Additional approaches for target identification-transcriptional profiling ........................................... 26 Additional approaches for target identification-genetics screening .................................................... 27 Purpose and significance ......................................................................................................................... 28 Chapter 2 . Cell Survival Pathways in Colorectal Cancer (CRC) Cells: Targeting Mutant Adenomatous Polyposis Coli (APC) .................................................................................................................................. 36 Introduction ............................................................................................................................................. 36 Materials and Methods ............................................................................................................................ 37 Results ..................................................................................................................................................... 43 Discussion ............................................................................................................................................... 48 Chapter 3 . Identification of Novel Driver Tumor Suppressors through Functional Interrogation of Putative Passenger Mutations in Colorectal Cancer ................................................................................... 68 Introduction ............................................................................................................................................. 69 Materials and Methods ............................................................................................................................ 71 iv v Results ..................................................................................................................................................... 72 Discussion ............................................................................................................................................... 76 Chapter 4 . Exome Sequencing of Normal and Isogenic Transformed Human Colonic Epithelial Cells (HCECs) Reveals Novel Genes Potentially Involved in the Early Stages of Colorectal Tumorigenesis ... 96 Introduction ............................................................................................................................................. 96 Materials and Methods ............................................................................................................................ 98 Results ..................................................................................................................................................... 99 Discussion ............................................................................................................................................. 103 Chapter 5 . Conclusions and Future Perspectives ....................................................................................
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