A Dissertation Entitled Role of Androgen Receptor in Folate Receptor α Regulation and in Prostate Cancer by Suneethi Sivakumaran Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences ___________________________________________________________ Dr. Manohar Ratnam, Committee Chair ____________________________________________________________ Dr. Beata Lecka-Czernik, Committee Member ____________________________________________________________ Dr. Cynthia M. Smas, Committee Member ____________________________________________________________ Dr. Ivana De La Serna, Committee Member ____________________________________________________________ Dr. Lirim Shemshedini, Committee Member ____________________________________________________________ Dr. Robert J. Trumbly, Committee Member ____________________________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo December, 2012 Copyright 2012, Suneethi Sivakumaran This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author An Abstract of Role of Androgen Receptor in Folate Receptor α Regulation and in Prostate Cancer by Suneethi Sivakumaran Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo June 2012 Folic acid is an essential water soluble vitamin required for nucleic acid and protein biosynthesis. The first trimester of pregnancy needs constant folate transport from maternal circulation to support rapid fetal cell division, growth and proliferation. Folate receptor α mediates the transplacental folate transport and facilitates normal embryonic growth. Female sex hormones, estrogen and progesterone regulate the complicated process of embryogenesis. Estrogen and progesterone through their receptors regulate folate receptor α, but the role of androgens that increase during first trimester in folate receptor α regulation is unknown. We show that androgen/androgen receptor increases folate receptor α mRNA and protein expression. We utilized folate receptor α promoter deletion and mutant constructs to identify the androgen receptor binding sites. We show that androgen receptor and CAAT enhancer binding protein α bind the folate receptor α promoter regions and cause promoter activation. Co-immunoprecipitation studies show that androgen receptor and CAAT enhancer binding protein α interact in placental trophoblast cells. These results suggest that androgen receptor and CAAT enhancer binding protein α interact, bind the iii folate receptor α promoter and regulate folate receptor α gene in cancer and placental trophoblast cells. In normal prostate, androgen-bound androgen receptor activates its target genes by binding to cis androgen response elements. This is the classical mechanism of androgen receptor action. In prostate cancer, androgen receptor exerts its growth promoting effects by tethering to other transcription factors. We identified Elk-1 transcription factor that needs androgen receptor for its transcriptional activation. Co- immunoprecipitation and mammalian two-hybrid assays show that Elk-1 interacts with N-terminal domain of androgen receptor. Microarray analysis and gene validation show that Elk-1 and androgen receptor association regulates genes involved in cell growth. Growth assays show that Elk-1 and androgen receptor association is required to support prostate cancer growth. Chromatin immunoprecipitation studies identified chromosomal regions at which Elk-1 and androgen receptor are recruited. These results show that Elk-1 tethers androgen receptor to target gene promoters and suggest Elk-1 mediated androgen receptor tethering supports prostate cancer growth. iv Dedicated to my family who have supported and egged me on Acknowledgements First and foremost, I thank my major advisor Dr. Manohar Ratnam for providing me an opportunity to work in his laboratory and specifically for making decisions keeping my best interests in mind. I am thankful to the scientific committee members Dr. Cynthia Smas, Dr. Ivana De La Serna, Dr. Beata Lecka-Czernick, Dr. Robert Trumbly and Dr. Lirim Shemshedini for their support, encouragement and comments on the research project. My committee members molded me scientifically and prepared me for the competitive scientific world through challenges. They made me aware of the realities of science. I thank Dr. De La Serna for her continuous support, genuine feedback, and for all our discussions. I thank Dr. Smas for encouraging me to exert maximum efforts and be the best. I thank Dr. Lecka-Czernick for her feedback and comments during the committee meetings. I thank Dr. Trumbly for patiently answering my questions and for being helpful at times of need. I thank Dr. Shemshedini for agreeing to be on my committee at the last moment and for being helpful by sharing plasmids and reagents for the project. I like to thank the entire faculty who took time and enormous effort to provide us the basic foundations through coursework. I extend special thanks to Dr. John David Dignam with whom I have had several interesting scientific discussions. Dr. Dignam helped me in several ways professionally and encouraged me to shoot beyond the horizon. His genuine feedbacks are my best gifts. He and Dr. De La Serna played very important roles when I started applying for post-doctoral positions. They provided vi guidance and advice at every possible step. I thank Dr. Randall Ruch who supported in several ways and made sure the journey during Ph.D. is as comfortable as possible. I thank Dr. William Maltese for being very supportive, particularly during the last several months of Ph.D. I like to thank my former lab members Mesfin Gonit and Marcella D’Alincourt Salazer who made me at ease when I joined the lab and taught several technical aspects, and my present lab members Venkatesh Chari for his support and Mugdha Patki. I like to thank Mesfin Gonit for allowing me to challenge his scientific perspectives, for intellectually stimulating arguments. I thank him for his encouragement, feedback on my technical expertise and scientific knowledge, and for being my well wisher. I thank my mother, sister and brother for their patience, support, encouragement, and understanding and for challenging me with scientific questions from a common man point of view. I thank my friends for their support and suggestions. vii Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of contents .............................................................................................................. viii List of figures……………………………………………………………………………………..x Chapter 1 Introduction and Literature Review ..............................................................1 Introduction ......................................................................................................................1 Literature review ..............................................................................................................4 Chapter 2 Androgen receptor and C/EBPα interaction in folate receptor α gene activation in human trophoblasts and cancer cells……………………………….......37 Abstract ..........................................................................................................................37 Introduction ....................................................................................................................38 Results ............................................................................................................................46 Discussion ......................................................................................................................56 Materials and Methods ...................................................................................................59 References ......................................................................................................................66 Figure Legends ...............................................................................................................74 Figures ............................................................................................................................79 Chapter 3 Elk-1 directs a critical component of growth signaling by the androgen receptor in prostate cancer cells……………………….………………………………84 Abstract ..........................................................................................................................84 Introduction ....................................................................................................................85 viii Results ............................................................................................................................87 Discussion ....................................................................................................................102 Materials and Methods .................................................................................................109 References ....................................................................................................................121 Figure