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Cyclin D1: Mechanism and Consequence of Androgen Receptor Co-Repressor Activity in Prostatic Adenocarcinoma

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Cyclin D1: Mechanism and Consequence of Androgen Receptor Co-Repressor Activity in Prostatic Adenocarcinoma UNIVERSITY OF CINCINNATI Date: April 1st, 2004 I, __________________Christin E. Petre_________________________, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Cell and Molecular Biology It is entitled: Cyclin D1: Mechanism and Consequence of Androgen Receptor Co-repressor Activity in Prostatic Adenocarcinoma This work and its defense approved by: Chair: Karen E. Knudsen, Ph.D. Sohaib Khan, Ph.D. Kenji Fukasawa, Ph.D. Alvaro Puga, Ph.D. Linda Parysek, Ph.D. J. Alan Diehl, Ph.D. Robert Hennigan, Ph.D. CYCLIN D1: MECHANISM AND CONSEQUENCE OF ANDROGEN RECEPTOR CO-REPRESSOR ACTIVITY IN PROSTATIC ADENOCARCINOMA A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Cell Biology, Neurobiology, and Anatomy of the College of Medicine 2004 by Christin E. Petre B.A., Miami University, 2000 Committee Chair: Karen E. Knudsen, Ph.D. ABSTRACT It is increasingly evident that androgen receptor (AR) regulation plays a critical role in the development and progression of prostate cancer. We show that induction of cyclin D1 occurs upon ligand stimulation in androgen dependent prostatic adenocarcinoma (LNCaP) cells. Such induction results in the formation of active cyclin dependent kinase (CDK)-cyclin D1 complexes, phosphorylation of the rentinoblastoma tumor suppressor protein, and concomitant cell cycle progression. In addition, we find that cyclin D1 harbors a second cell cycle independent function responsible for restraining AR transactivation. We illustrate that cyclin D1 co-repressor activity is extremely potent, inhibiting receptor transactivation independently of cellular background, promoter context, co-activator over expression, ligand/non-ligand activators, and cancer predisposing AR mutations/polymorphisms. Cyclin D1 binds directly to the AR N-terminus, hindering its ligand dependent transactivation functions (AF-1 and AF-2). The co-repressor activity of cyclin D1 likely involves two distinct mechanisms, including the recruitment of histone deactylase (HDAC) activity and inhibition of AR N- to C- terminal interactions. These data put forth the hypothesis that cyclin D1 is a negative feedback inhibitor of the AR. Supporting this model, over expression of cyclin D1 in LNCaP (prostate cancer) cells leads to marked abrogation of cell cycle progression. Further investigation into the region(s) of cyclin D1 responsible for AR co-repression reveal a conserved central portion of the protein required for both receptor binding and inhibition. The central domain itself, elicits many of the characteristics of the wild type protein, functioning to bind HDAC3, inhibit AR transactivation, and abrogate cell cycle progression in LNCaP cells. We show that the central domain of cyclin D1 is also required for co-repression of thyroid hormone receptor beta-1 transactivation, suggesting the existence of a conserved nuclear receptor repressor motif within this region. Surprisingly, the central domain is dispensable for estrogen receptor alpha co-activation, suggesting that the co-activator and co-repressor functions of cyclin D1 are distinct and could be specifically targeted. Together, these studies provide the first in-depth analysis of AR co-repressor function, identifying the mechanism of cyclin D1 action and potentially leading to the discovery of novel therapeutic targets for the treatment of prostate cancer. ACKNOWLEDGEMENTS I must thank, and feel privileged to thank, my friends and family. Without your support and encouragement I would never have overcome the obstacles that inevitably every graduate student encounters on their journey. To my fiancé, Phil, you have been an honest and motivating presence in the toughest of times, always expressing confidence in my abilities and never allowing me to admit defeat. You have been the light at the end of my day, a constant reminder that there is life to experience outside the world of science. I only hope that I can be as much a support in your work as you have been in mine. Thank you also to my fellow graduate students, the administration, and the professors of the Cell Biology Department. Each with your own individuality, you have truly joined to create a ‘departmental family’, adding to my success through your friendship, support, and advice. In addition, the members of the Knudsen laboratories, both past and present have made these four years enjoyable. Most importantly, I thank my advisor, Dr. Karen Knudsen. I decided to come to UC because I thought I would receive the best possible training as a graduate student; I have no regrets. Karen, you have taught me more than I could ever have expected. You are truly an amazing woman, capable of successfully juggling numerous responsibilities both at home and in the office. To Karen’s husband, Dr. Erik Knudsen, I thank you for your resourcefulness and for showing me an extremely no-nonsense view of the scientific world. Together you have taught me, nurtured me, and most importantly, you have allowed me to grow scientifically and personally. You have shaped my science and have truly become a part of my life. This dissertation is dedicated to my Grandpa, Donald Beecher, who, like many other prostate cancer survivors I've met in these past few years, will never give up hope that we, as researchers, will find better ways to combat this devastating disease. It is the strength and attitude of individuals like you that drive all of us to be successful. TABLE OF CONTENTS Page List of Tables and Figures…………………..………..……………………...…….……2 Chapter I: Introduction……………………..………………………………………………….……..5 A. The evolution of prostate cancer…..……..……………..……...….…….5 B. The androgen receptor…….…..…..………………………………..…….6 C. Androgen receptor co-modulators...………...………..………….….…..8 D. AR deregulation in androgen independent prostate cancer……...….10 E. The dual actions of cyclin D1……………….……………………….…..12 F. Introductory conclusions and hypothesis……………..…………….…16 G. References……………………………………………….…….…………17 Chapter II-V: Results……………………………………………….….…………...24-124 Chapter II: Cyclin D1: Mechanism and Consequence of Androgen Receptor Co-repressor Activity………………...………...…..……….24 A. Abstract………………………………………..…….….………………….25 B. Introduction..………………..…………………..…………..……………..25 C. Experimental Procedures……………….………………………………..26 D. Results……………………………….……………………………………..27 E. Discussion.………………………………………...…..…………………..31 F. References………………………..………………………………………..33 Chapter III: Specificity of Cyclin D1 for Androgen Receptor Regulation….34 A. Abstract……………………………..………………………………...……35 B. Introduction…………………………………………………………………35 C. Materials and Methods...………………………………………………….36 D. Results……………………………………..……………….……………....37 E. Discussion…………………………………………………….…………....41 F. References…………………………………………………….…………...44 Chapter IV: Cyclin D1 Binding to the Androgen Receptor NH2-terminal Domain Inhibits AF2 Association and Reveals Dual Roles for AR Co-repression……………………………………………………………….46 A. Abstract……………...……………………………………………………..47 B. Introduction……………………………..……………………..….……….48 C. Materials and Methods………………………………………...…………52 D. Results………………………………..……………………………………54 E. Discussion……………………………...………………………………….62 F. References……………………………………………………..………….68 G. Figures & Legends………………………………………………………..72 1 Chapter V: The Central Domain of Cyclin D1 Elicits Nuclear Receptor Co-repressor Activity..………………………… ……….………. ……..84 A. Abstract………………………………………..……….……………..….85 B. Introduction…………………………………..………….……………….86 C. Experimental Procedures………………..……………….…..………..89 D. Results…………………………………..…….…………………….…...94 E. Discussion……………..……………..……………….....……….……105 F. References…………………………..………….……………….……..111 H. Figures & Legends………………..……..……………………………113 Chapter VI: Summary and Conclusions……………….….…………………....125 A. Characterization of cyclin D1 action………………………….....….125 B. Mechanism of cyclin D1 co-repressor activity………….………….130 C. Cyclin D1 as a transcriptional regulator………………………..…..133 D. Summary………...…………………………………………………….135 E. References…………………………………………………………….136 Chapter VII: Ongoing/Future Directions…………………………………….….141 A. Cyclin D1 transcript b…………………………………………….…..141 B. Identification of AR-cyclin D1 complex members………………….142 C. Therpuetic Development and Testing………………………………143 D. References……………………………………………….…………....145 LIST OF TABLES AND FIGURES Figure Page Chapter I: Introduction Fig.1 The androgen receptor…………………………………………………..…..6 Fig. 2 Cyclin D1 is a key regulatory component of the cell cycle.…………….12 Fig. 3 Cyclin D1 is induced by androgen in LNCaP cells……………………...12 Fig. 4 The structural organization of cyclin D1……………………………….…13 Fig. 5 Cyclin D1 is a CDK independent AR co-repressor…………………..….15 Fig. 6 Cyclin D1 is a negative feedback inhibitor of the AR…………………....16 Chapter II: Cyclin D1: mechanism and consequence of androgen receptor co-repressor activity Fig. 1 The LxxLL motif is dispensable for AR inhibition…………………….….27 Fig. 2 Nuclear cyclin D1 inhibits androgen receptor transactivation………….28 2 Fig. 3 Cyclin D1 binds the N-terminus of the androgen receptor ……..……29 Fig. 4 Cyclin D1 binds AR5 but fails to inhibit the constitutively active Androgen receptor…………………………………………………..…...29 Fig. 5 Cyclin D1 is dominant to androgen receptor co-activator function …30 Fig. 6 Cyclin D1 action is partially abrogated through HDAC inhibition……31 Fig. 7 Ectopic cyclin D1 abrogates cell cycle progression in androgen-dependent
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