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Role of Sam68 in Dna Damage Responses and Tumorigenesis

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Role of Sam68 in Dna Damage Responses and Tumorigenesis ROLE OF SAM68 IN DNA DAMAGE RESPONSES AND TUMORIGENESIS by Xin Sun A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland Dec 2016 © 2016 Xin Sun All Rights Reserved Abstract The ability to recognize and repair DNA damage through rapid and appropriate DNA damage responses is pivotal to safeguard genomic information, which is persistently challenged by internal and environmental offenses. DNA lesion initiated poly(ADP- ribosyl)ation (PARylation), catalyzed primarily by poly(ADP-ribose) polymerase 1 (PARP1), is one of the earliest post-translational modifications to orchestrate downstream DNA damage response (DDR) signaling. However, the precise mechanisms through which PARP1 is activated and poly(ADP-ribose) (PAR) is robustly synthesized are not fully understood. Converging evidence support the emerging role of RNA- binding proteins (RBPs) in promoting DNA damage repair at different stages of DDR. We discovered Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel- signaling molecule in DDR. In the absence of Sam68, DNA damage-triggered PARylation and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we revealed that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PARylation. Utilizing cell lines and knockout mice, we demonstrated that Sam68- deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. In addition, loss of Sam68 delays basal cell carcinoma (BCC) onset and progression in a mouse model for basal cell carcinoma (BCC) of the skin, suggesting Sam68 is required for BCC tumorigenesis, likely through promoting tumor cell survival. Together, our findings demonstrate that Sam68 plays a crucial role in DDR via ii regulating DNA damage-initiated PARylation, and that Sam68 is crucial in protecting normal cells from environmental genotoxic stress under physiological conditions and facilitating tumor cells to undergo malignant transformation in BCC. Thesis Readers Dr. Fengyi Wan Dr. Pierre Coulombe Dr. Alan Scott Dr. Luis Garza Dr. Michael Matunis Dr. Jay Bream Dr. Xuhang Li Preface My passion has always been applying new discoveries from the bench to the diagnosis and treatment of human tumors at bedside. There are six biological competencies acquired during the initiation and progression of human tumors, including ‘‘sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion iii and metastasis’‘, as summarized by Drs. Douglas Hanahan and Robert A. Weinberg in the prominent document ‘‘Hallmarks of Cancer: The Next Generation’‘ (Hanahan and Weinberg 2011). Remarkably, alteration of genomic information underlies all six hallmarks of cancer to generate mutations that promote every aspect of tumorigenesis and progression. With my thesis, I wanted to explore the underlying mechanisms of this multifaceted disease in relation to DNA damage responses (DDRs). I first examined the hypothesis that Src-associated substrate during mitosis of 68 kDa (Sam68), a previously known RNA binding protein (RBP), is a novel DNA damage-signaling molecule, governing poly(ADP-ribose) polymerase 1 (PARP1)-mediated poly(ADP-ribosyl)ation. Having made the observation that genetic ablation of Sam68 decreases tumor burden in the mouse colon (Fu, Sun et al. 2016), I then assessed whether Sam68 is also involved in skin basal cell carcinoma (BCC) tumorigenesis. In this dissertation, the main focus has been to investigate the role of Sam68 as a novel DNA damage response (DDR) protein. The resulting thesis is divided into six parts. Chapter 1 introduces the background information and our scientific questions. It first describes the concept of DDR, and then discusses the essential role of PARP1 and PARylation during DDR and the importance in its tight regulation. Lastly it introduces Sam68, our protein of interest, and our hypothesis that Sam68 may be a previously unappreciated DDR protein regulating PARylation. Chapter 2 reports on the approaches and observations in addressing our scientific questions. Chapter 3 discusses conclusions iv we drew from the study, the significance of identifying Sam68 as a regulator of PARP1 catalytic activity, the emerging of RBPs in different phases of DDR and the uniqueness of Sam68 that distinguishes it from other RBPs in the context of DDR. In addition, I reviewed what are the burning questions that are left unanswered, and proposed how we can further uncover the mechanism through which Sam68 is activated and the mechanism through which Sam68 regulate PARP1. Chapter 4 summarizes the unpublished data about the potential role of Sam68 in BCC as an appendix to further discuss the involvement of Sam68 in tumorigenesis. This dissertation represent an expanded and updated version of the recent by Sun, X., Fu, K., Hodgson, A., Wier, E. M., Wen, M. G., Kamenyeva, O., Xia, X., Koo, L.Y., and Wan, F., published in PLoS Biology in 2016. Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP- ribosyl)ation. PLoS Biol, 14(9), e1002543, with the addition of unpublished data. Figures 1.1, 1.3 and 1.5 were summaried from current literature and made by myself; Figures 1.2, 1.4 and 1.6 were adopted from the cited literature and remade by myself; Figures 2.1, 2.2, 2.3, 2.4, 2.5, 2.6B, 2.6D-E, 2.8, 2.11E-G, 2.13, 2.14, 2.15, 2.16, 2.18, 3.1, 4.1D-E and 4.3 were generated entirely by my own data; Figure 2.6A, 2.6C-D, 2.7, 2.11B, 2.11H-I, 2.12, 4.1A-B were generated entirely by Dr. Kai Fu; Figures 2.11A and 2.11C were generated in collaboration with Dr. Kai Fu; Figures 2.6F-G, 2.9, and 2.10 were generated by myself with help from Drs. Lily Y. Koo and Olena Kamenyeva; Figues 4.1C and 4.2 were generated by myself with help from Dr. Kai Fu, Mr. Matthew Wen and Ms. Xin Guo; Figure 3.3 was summarized from two of our published manuscripts and made by myself (Fu, Sun et al. 2016, Sun, Fu et al. 2016). v Acknowledgement Graduate school has been a long voyage with many obstacles along the way. But it is not a lonely journey. With the warm company and generous support from my amazing mentors, colleagues, friends and family, and with a determined mind, my thesis has become a reality, and I have been rewarded. It is all the laughers and tears that allow me to uncover my potential and keep moving forward in science and in life. Foremost, I would like to express my gratefulness to the great set of mentors that I have. I cannot thank my advisor Dr. Fengyi Wan enough for taking me into his lab and granting a first year graduate student plentiful trust to open a new field in the lab. He provided me not only the techniques, but also the intellect to tackle the scientific questions. He always gave me an opportunity to describe my plan, before offering suggestions and explaining how he would improve it. It is all the open discussions we had over the years that fostered my independent scientific thinking. Fengyi’s passion about science has driven everyone in the lab to always try our best. I feel lucky to have such an incredible advisor, and I appreciate his tremendous guidance and support during my graduate career. I also would like to thank members of my thesis committee, Drs. Pierre Coulombe, Michael Matunis, Alan Scott, and Luis Garza, who have encouraged, challenged and guided me to be faithful to science and myself. I want to thank Dr. Pierre Coulombe for mentoring me through our fruitful collaboration and our invaluable joint lab meetings. He showed me that a true scientist is one with broad knowledge, a talent who continuously ask insightful questions and a mentor who is vi always approachable for advise and help. My special thanks also goes to Dr. Michael Matunis. His great suggestions and substantial support through the whole duration of graduate school helped shape my thesis work and career. Conversations in his office or small talks in the hallway prepared me for my meetings and career development. I want to thank our collaborators Drs. Stephane Richard from McGill University, Johnny He from University of North Texas Health Science Center, Jeremy Stark from Beckman Research Institute of City of Hope, John Pascal from University of Montreal, Anthony Leung from Johns Hopkins University and Zhaoqi Wang from Fritz Lipmann Institut for sharing experimental material; Dr. Cory Brayton and Ms. Xin Guo from Johns Hopkins University for help with histological analyses; Drs. Owen Schwartz, Lily Y. Koo and Olena Kamenyeva from National Institutes of Health for help with laser micro-irradiation microscopy. Your help significantly improved my thesis project. Thank you to all my friends and colleagues inside and outside of school. I want to thank all the previous and current members of the Wan Lab, Dr. Kai Fu, Dr. Andrea Hodgson, Dr. Eric Wier, Xue (Summer) Xia, Yue (Harry) Liu, Matthew Wen, Dr. Wenxin Zheng and Jackline Lasola. You build such an incredible positive and collaborative working environment that I learned a lot from each of you. My graduate school would not be so rewarding without your friendship and support. I especially want to thank Kai for our fruitful and enjoyable collaboration on so many projects over the years. Kai is so knowledgable with hands on bench techniques that he is the MASTER of western blotting and immunoprecipitation in our lab. He is always willing to lend a helping vii hand. His contribution to my thesis project significantly enhanced our understanding of the molecular mechasim.
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