NUCLEAR FUNCTIONS OF ADENOMATOUS POLYPOSIS COLI: REGULATION OF THE G2-M CELL CYCLE TRANSITION & INTERMEDIATE FILAMENT INTERACTION BY ©2009 Yang Wang B.S., Xiamen University, 2003 Submitted to the graduate degree program in Molecular Biosciences and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy Committee members: _______________________ Chairperson – Kristi L. Neufeld _______________________ Robert J. Coffey _______________________ Yoshiaki Azuma _______________________ Robert S. Cohen _______________________ Erik A. Lundquist _______________________ John D. Robertson _______________________ Robert E. Ward Date defended: 1/21/2009 The Dissertation Committee for Yang Wang certifies that this is the approved version of the following dissertation: NUCLEAR FUNCTIONS OF ADENOMATOUS POLYPOSIS COLI: REGULATION OF THE G2-M CELL CYCLE TRANSITION & INTERMEDIATE FILAMENT INTERACTION Committee members: ________________________ Chairperson – Kristi L. Neufeld _______________________ Robert J. Coffey ________________________ Yoshiaki Azuma ________________________ Robert S. Cohen ________________________ Erik A. Lundquist ________________________ John D. Robertson ________________________ Robert E. Ward Date approved: 1/21/2009 __________________ ii Abstract Mutation of tumor suppressor Adenomatous Polyposis Coli (APC) is an early, if not the first step in the majority of colorectal cancers. As a multi-functional protein, APC has been implicated in regulating cell cycle and cytoskeletal integrity. To further understand APC function in cell cycle regulation, I immunoprecipitated APC and identified novel associated proteins. I identified topoisomerase IIα (topo IIα), a critical regulator of the G2 decatenation checkpoint, as a potential binding partner of APC. The interaction of endogenous APC and topo IIα was verified by co-immunoprecipitation, co-localization, and Förster resonance energy transfer (FRET). Both the fifteen (M2-APC) and twenty (M3-APC) amino acid repeat regions of APC interacted with topo IIα when expressed in cultured cells. Cells expressing M2- or M3-APC arrested in G2, but only if the cells contain normal levels of topo IIα. This G2 cell cycle arrest likely resulted from reduction of endogenous topo IIα activity, consistent with the established model whereby inhibition of topo IIα activates the G2 decatenation checkpoint. APC has been shown to interact with microtubules and the actin cytoskeleton, inplicating APC in cell polarity and migration. Using a novel APC antibody raised against M2-APC, I co-immunoprecipitated potential APC binding proteins from HCT116βw cells. I identified 42 proteins in complex with APC by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Among these were intermediate filament (IF) proteins lamin B1 and keratin 81. Lamin B1 interacts with APC in both cultured cells and human colonic tissue. APC also associated with IF iii proteins throughout a sequential extraction procedure, which removed actin microfilaments and microtubules. Based on evidence described in this dissertation, I hypothesize that two central APC domains interact with topo IIα to regulate the G2 decatenation checkpoint. In addition, I also provide evidence supporting a role for APC in the regulation of cytoskeletal integrity. iv Dedicated to my parents and my husband 给我亲爱的爸爸妈妈 v Acknowledgements First and the foremost, I would like to thank my parents, Yongyi Wang and Xiuju Wang. They have always been the NO.1 source of motivation and courage for me in my whole life. They helped me to develop various skills that I am still benefiting from today. They helped me to make my decision to pursue science. Without their encouragement and firm belief in me, I would never gain the kind of confidence and achievement like this. I would also like to thank my husband, Zeqiang Ma. I feel so lucky to marry him during graduate school, because he has been so understaining and supportive of my dream to become a female scientist. He has been always standing by me and backing me up in life. Second, I would like to thank my mentor Dr. Kristi Neufeld. I don’t know how I can possibly express my tremendous gratitude to her. She has been one of the greatest female scientists and role models to me. I feel extremely fortunate to have met her and been guided by her in graduate school. She supports me in every way that she can to help me develop my career and at the same time manage my family and marriage. Due to her great effort and support, I got to move to Nashville to do research in Vanderbilt University and be with my husband. I have learned a lot from her not only about how to be a female scientist but also how to be a great woman. Third, I would like to thank my co-mentor Dr. Bob Coffey. He helped make my move to Nashville to follow my love, come true. He has been supportive in both life and science. He is the one who helps me to gain condidence in science and always tells me to aim high and shoot high. I feel fortunate and happy that I will be able to work with him as a postdoctoral fellow for the next few years. vi Fourth, I would also like to acknowledge members of my graduate committee, Dr. Yoshiaki Azuma, Dr. Bob Cohen, Dr. Erik Lundquist, Dr. Robert Ward and Dr. John Robertson for all their advice. Especially, I would like to thank Dr. Azuma, who together with Dr. Neufeld and I, initiated this project. He is a wonderful and patient mentor. From him, I learnt not only a lot of techniques but also experimental design when I was doing my research proposal for prelim and at later work for my thesis. Fifth, I want to thank my labmates from both the Neufeld and the Coffey labs, for all the helpful discussions and suggestions, and for making both labs the greatest places to work every day. Especially, I would like to thank Dr. Jamie Cunningham from Neufeld lab and Dr. Yina Li from Coffey Lab. They have become my best friends in school and being with them everyday have become one of the most fun parts of life. Last, I would also like to thank Kozo Kaibuchi (Nagoya University, Japan) for providing expression constructs for APC fragments fused to GFP, Bert Vogelstein (The Johns Hopkins University) for providing the HCT 116βw (mut ko, β-cat w/-) cell line, Joe Holden (University of Utah) for providing the anti-topo IIα sera, Dave Gard (University of Utah) for providing anti-(α+β)-tubulin antibodies, Ian Tomlinson (Imperial Cancer Research Fund, London) for providing the HCA46 cell line, Martha Stampfer (Lawrence Berkeley Laboratory, Berkeley, CA) for providing the 184A1 cell line, Cooperative Human Tissue Network (CHTN, NCI) for providing normal human colonic tissues, Yina Li (Vanderbilt University) for technical help with cryosectioning tissues, James Higginbotham (Vanderbilt University) for technical assistance with FACS based cell cycle analysis, and Jo Ann Byl (Vanderbilt University) for providing technical support with the topoisomerase activity assays. Chapter 2 was originally published in Mol vii Biol Cell 19, 4076-4085. Work in this thesis was funded in part by NIH RO1 CA10922 (Y.W. and K.L.N.), GM33944 (N.O.), GI Special Program of Research Excellence CA95103 (R.J.C), Mouse Models of Human Cancers Consortium 5U01 CA084239-10 (R.J.C), NCI RO1 CA46413 (R.J.C), and Higuchi Biosciences Center J.R. & Inez Jay Award (Y.W., Y.A., and K.L.N.). viii Table of Contents Page Abstract.............................................................................................................................. iii Acknowledgements............................................................................................................ vi Table of Contents...............................................................................................................ix List of Figures.................................................................................................................... xi List of Tables .................................................................................................................... xii List of Abbreviations ....................................................................................................... xiii CHAPTER 1 INTRODUCTION: COLORECTAL CANCER, ADENOMATOUS POLYPOSIS COLI & CELL CYCLE REGULATION ............................................... 1 Part I. Pathogenesis of Colorectal Cancer................................................................... 1 The colonic epithelium ....................................................................................... 1 Molecular Pathogenesis of CRC......................................................................... 4 Part II. Adenomatous Polyposis Coli.......................................................................... 8 APC gene and protein ......................................................................................... 8 APC in Wnt signaling ....................................................................................... 11 Nuclear APC ..................................................................................................... 14 APC and cytoskeletal integrity ......................................................................... 17 APC and cell cycle regulation........................................................................... 19 Part III. Topoisomerase IIα ...................................................................................... 21 Topo II enzyme and DNA topology ................................................................