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1 the Origin of Genome Instability in Cancer

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1 the Origin of Genome Instability in Cancer The Origin of Genome Instability in Cancer: Role of the Fragile Site Gene Product FHIT Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Joshua Charles Saldivar, M.S. Biomedical Sciences Graduate Program The Ohio State University 2013 Dissertation Committee: Kay Huebner, PhD, Advisor Carlo Croce, MD Joanna Groden, PhD Denis Guttridge, PhD 1 Copyright by Joshua Charles Saldivar 2013 2 ABSTRACT The transformation of normal cells to cancer cells involves multiple steps mediated by the acquisition of mutations, selection and clonal expansion of cells with favorable mutations. Most cancers exhibit remarkable genomic instability, defined as an elevated rate of genetic mutation at the single nucleotide and chromosome levels. Genomic instability is a facilitating hallmark of cancer in that it raises the probability of generating cancer-promoting mutations. Multiple factors contribute to the genome instability phenotypes seen in cancer, but the molecular processes initiating instability in sporadic cancer are unknown. In dysplastic cells, genomic alterations are first seen at chromosome fragile sites. These fragile sites are exquisitely sensitive to agents that stress DNA replication forks, and thus, it is thought that replicative stress is a major source of genome instability in cancer. A frequent and very early genetic alteration in precancerous cells is deletion within fragile site FRA3B, which overlaps exons of the large FHIT gene, resulting in loss of FHIT protein expression. Here it is shown that loss of FHIT expression triggers endogenous replication stress hindering replication fork progression and inducing fork stalling and collapse. Consequently, FHIT-deficient cells develop spontaneous DNA breaks and chromosome instability. Mechanistically, FHIT loss-induced replication stress ii is due to an imbalance in the deoxyribonucleotide triphosphate pool and an insufficient supply of thymidine triphosphate. FHIT up-regulates the S-phase-specific expression of thymidine kinase 1, a component of the pyrimidine salvage pathway for the production of thymidine triphosphate. Balanced precursors of DNA are needed for efficient and accurate DNA replication, and notably restoration of nucleotide balance rescues DNA replication defects in FHIT-deficient cells. Under selective pressure, FHIT-deficient clones enabled by oncogenic mutations emerge with newly acquired precancerous phenotypes, suggesting that FHIT loss-induced genome instability facilitates tumorigenesis. Collectively, these findings support a model where loss of FHIT expression initiates genomic instability in dysplastic lesions, linking alterations at chromosome fragile sites to the origin of genome instability and cancer progression. iii Dedicated to Catherine Waters for her love and companionship To my mother for her love and support, for valuing my education, and for cultivating my curiosity To my father for teaching me to work hard, to be courageous, to never quit, and inspiring me to always do my best To Robert Story and Dr. Michael Johnson for giving me the opportunity as a high-school student to work in a research laboratory, and in doing so, kindling my love for science iv ACKNOWLEDGMENTS I am especially grateful to my advisor, Dr. Kay Huebner, for supporting and mentoring me during my graduate research years. I am especially thankful for the many opportunities to travel and attend prestigious meeting; these trips have given me countless ideas, molded my hypotheses and kept me up to date on the hot topics in the field. I am very grateful for Dr. Huebner’s tireless commitment to any and all of my student needs. She has challenged my thinking, encouraged my ideas, and promoted my research. It is because of her that I have been able to achieve my goals. I would like to thank my thesis committee members, Drs. Carlo Croce, Joanna Groden, and Denis Guttridge, for their helpful comments and critiques during committee meetings, for their suggestions on current and future projects, and for monitoring my timely progression through the program. I would like to thank the Biomedical Sciences Graduate program for accepting me into the program back in 2008, and special thanks to Dr. Ginny Sanders, the program director at the time, for being an inspirational director. Special thanks to the program office staff, including Ms Amy Lahmers and Ms Kelli Kaiser, for their continued dedication to the graduate program and students. v I would like to thank current and former members of Dr. Huebner’s lab, for making the lab a friendly, cheerful environment every day. I would like to thank Drs. Hidetaka Shibata and Satoshi Miuma for teaching me many research techniques, offering insightful discussions, and for their friendship in the lab. I would like to thank Dr. Jin Sun for his technical expertise, daily maintenance of our animal colony, assistance with presentations and room reservations, and for managing the lab safety protocol. I would especially like to thank Jessica Bene and Larissa Topeka who as talented undergraduate students performed many critical experiments some of which have been used in this dissertation. I would like to thank Dr. Chris Mathews and Linda Wheeler for their willingness to collaborate and offer their expertise in nucleotide metabolism. Their contributions led to significant advances in our understanding of FHIT function. I greatly appreciate financial support provided by the Ohio State University Wexner Medical Center and the National Institutes of Health. vi VITA November 24, 1982……………………...Born – Springdale, Arkansas 2000-2005……………………………….Undergraduate Laboratory Assistant P.I.: Michael G. Johnson, PhD, University of Arkansas, Fayetteville, Arkansas 2005……………………………...............B.S. in Biochemistry, University of Arkansas, Fayetteville, Arkansas 2007……………………………………...M.S. in Food Science, University of Arkansas, Fayetteville, Arkansas 2007-2008………………………………..Hourly Assistant P.I.: Steven C. Ricke, PhD University of Arkansas, Fayetteville, Arkansas 2008-present……………………………..Graduate Research Associate Dissertation Advisor: Kay Huebner, PhD The Ohio State University, Biomedical Sciences Graduate Program, Columbus, Ohio vii PUBLICATIONS Peer-Reviewed Manuscripts: 1. Saldivar JC, Bene J, Hosseini SA, Miuma S, Horton S, Heerema NA, and Huebner K. Characterization of the role of FHIT in suppression of DNA damage. Adv Biol Regul. 2013:53(1) 77-85. 2. Saldivar JC, Miuma S, Bene J, Hosseini SA, Shibata H, Sun J, Wheeler LJ, Mathews CK, and Huebner K. Initiation of genome instability and preneoplastic processes through loss of FHIT expression. PLoS Genetics. 2012; 8(11):e1003077. 3. Hu B, Nandhu MS, Sim H, Agudelo-Garcia PA, Saldivar JC, et al. Fibulin-3 Promotes glioma growth and resistance through a novel paracrine regulation of Notch signaling. Cancer Res. 2012;72(15) 3873-85. 4. Milillo SR, Friedly EC, Saldivar JC, et al. A review of the ecology, genomics, and stress response of Listeria innocua and Listeria monocytogenes. Crit Rev Food Sci Nutr. 2012:52(8) 712-25. 5. Shibata H, Miuma S, Saldivar JC, Huebner K. Response of subtype-specific human breast cancer-derived cells to PARP and Chk1 inhibition. Cancer Science. 2011; 102(10) 1882-8. 6. Huebner K, Saldivar JC, Sun J, Shibata H, Druck T. Hits, FHITs and Nits: beyond enzymatic function. Adv Enzyme Regul. 2011; 51(1) 208-217. 7. Saldivar JC, Shibata H, and Huebner K. Pathology and biology associated with the fragile FHIT gene and gene product. J Cell Biochem. 2010:109(5) 858-865. 8. Lungu B, Saldivar JC, Story R, Ricke S, Johnson MG. 2009. The combination of energy-dependent internal adaptation mechanisms and external factors enable Listeria monocytogenes to express a strong starvation survival response during multiple-nutrient starvation. Foodborne Pathog Dis. 2010;7(5):499-505. FIELDS OF STUDY Major Field: Biomedical Science Research Emphasis: Genetics viii TABLE OF CONTENTS Page Abstract…………………………………………………………….…………………….ii Dedication…………………………………………………………….………………….iv Acknowledgements……………………………………………….………...……………v Vita………………………………………………………………………...……………vii List of Tables……………………………………………………………………………xii List of Figures………………………………………………………………………….xiii Chapters: 1. FHIT, a Guardian of the Preneoplastic Genome?....................................................1 1.1 Introduction………………………………….……………...……………….1 1.2 The FHIT gene and gene product…………………………….………..…….2 1.3 Tumor suppressor function of FHIT………………………...….……………5 1.4 Loss of FHIT expression in early preneoplasias…………....…….…………..7 1.5 Genomic instability in FHIT-deficient cells………………………………….9 1.6 Conclusion………………………………………………………..…………11 2. Initiation of Genome Instability and Preneoplastic Process through Loss of FHIT Expression…………………..…………….………………..13 2.1 Introduction…………………………………………………..…….……….13 2.2 Materials and Methods………………………………………..………….…16 2.3 Results………………………………………………………….....…………20 2.3.1 FHIT-deficient cells spontaneous DNA breaks………...……...….20 ix 2.3.2 FHIT prevents endogenous replication stress and replication fork stalling…………………………….………....25 2.3.3 Genome instability induced by FHIT-deficiency does not activate the S/G2 checkpoint………………….………...30 2.3.4 Genomic alterations caused by FHIT loss expedite cell immortalization…………………………………….…………35 2.4 Discussion……………………………………………………..…..………..38 2.4.1 Model for FHIT loss-induced genome instability………………...38 2.4.2 FHIT deletion as a cancer-driving mutations……………...……...41 3. FHIT Supports DNA
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