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A Dissertation Entitled Exploring the Roles of TM4SF3 and CSN4 In A Dissertation entitled Exploring the Roles of TM4SF3 and CSN4 in Prostate Cancer by Meenakshi Bhansali Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology __________________________________________ Dr. Lirim Shemshedini, Committee Chair __________________________________________ Dr. Scott Leisner, Committee Member __________________________________________ Dr. Malathi Krishnamurthy, Committee Member __________________________________________ Dr. Paul W. Erhardt, Committee Member __________________________________________ Dr. Jeffrey G. Sarver, Committee Member __________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo December 2014 Copyright 2014, Meenakshi Bhansali 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 Exploring the Roles of TM4SF3 and CSN4 in Prostate Cancer by Meenakshi Bhansali Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo December 2014 Prostate cancer (PCa) is the most common non-skin cancer in males in the United States. Androgen Receptor (AR) is believed to remain active during progression of castration resistant PCa (CRPC). We performed gene microarray analysis and identified sGCα1 as an androgen-induced gene while TM4SF3 and BARD1 were repressed genes. Earlier we published that sGCα1 is involved in PCa cell growth and survival, independent of the classical nitric oxide (NO) signaling pathway and its association with sGCβ1. We identified by mass spectrometric analysis CSN4 as a novel binding partner for sGCα1. We observed that sGCα1 and CSN4 interact and co-localize in PCa cells. CSN4 positively regulates the sGCα1 protein stability by preventing sGCα1 proteasome- dependent degradation. Furthermore, disruption of CSN4 led to reduced PCa cell growth and these cells can be rescued significantly but not entirely by overexpression of sGCα1. This observation opened the possibility of another target for CSN4, which is p53. We observed that CSN4 negatively regulates p53 protein stability by promoting its proteasome-dependent degradation. Most importantly, CSN4 knock-down cells were rescued almost completely when we overexpressed sGCα1 and p53 knock-down. CSN5 acts downstream of CSN4 and is involved in mediating the CSN4-dependent effect on iii sGCα1 and p53 proteins. Further, we found that CK2 kinase exists in sGCα1-p53-CSN4- CSN5 protein complex and is involved in influencing the stability of sGCα1 and p53. TM4SF3 is unique in its androgen regulation as the mRNA is androgen-repressed and protein is androgen up-regulated. We focused on androgen up-regulation of TM4SF3 in different PCa cell lines. Androgen positively regulates TM4SF3 protein stability by preventing the proteasomal-degradation of TM4SF3. Our findings demonstrate that TM4SF3 is required for migration/invasiveness and epithelial mesenchymal transition of PCa cells. In addition to being localized in the membrane, TM4SF3 exhibits a novel androgen-dependent nuclear localization in AR-positive PCa cells. Interestingly endogenous TM4SF3 interacts with nuclear AR in an androgen-dependent manner in PCa cells and in vitro. Most importantly, the association of TM4SF3 with AR is required for AR protein stability, transactivation and androgen-induced proliferation of PCa cells. TM4SF3 and AR are overexpressed in prostate tumors, consistent with their mutual stabilization in PCa cells. Like TM4SF3, BARD1 is androgen-repressed gene, down- regulated at the mRNA and protein levels in PCa cells. Most importantly, overexpression of BARD1 affects endogenous AR protein levels, its transcriptional activity and endogenous gene prostate specific antigen. iv Acknowledgements I would like to thank my mentor, Dr. LIRIM SHEMSHEDINI for his encouragement, valuable time, guidance and patience while working on my projects. He provided me freedom and the opportunity to work on two novel projects. I learned art of scientific and manuscripts writings from him that helped me immensely while working on the dissertation. A special thank to my committee members for their precious time and suggestions. My sincere thanks to Dr. Richard Komuniecki and Dr. Malathi Krishnamurthy for their unconditional help and emotional support during my stay here. I take this moment to express my gratitude to the University of Toledo for funding and the opportunity to gain teaching experience. A deep sense of gratitude to the Molecular Endocrinology Journal for highlighting my publication in June 2014. I like to thank all my old lab members, especially, Dr. Shuai Gao and current lab member Jun Zhou for their support. My heartfelt thanks to all my friends in the department. I cannot forget to thank my family and my husband Pravin who was with me in my every odd and even moments. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents .............................................................................................................. vii List of Figures .................................................................................................................. viii List of Abbreviations ...........................................................................................................x 1 Introduction…. ....................................................................................................….1 2 Manuscript 1 …. ...............................................................................................….21 3 Manuscript 2…. ................................................................................................….75 4 Additional Results…. ......................................................................................….126 5 Discussion…….…. .........................................................................................….159 References ........................................................................................................................178 vi List of Figures Figure A. Structure of different functional domains of human AR…….. .........................2 Figure B. Diagram of Tetraspanin structure ....................................................................12 Figure 1-I: CSN4 is a novel binding partner for sGCα1 in prostate cancer cells. ............ 50 Figure 2-I: CSN4 positively regulates sGCα1 stability and is involved in sGCα1- dependent prostate cancer cell growth. ............................................................ 52 Figure 3-I: CSN4 negatively regulates p53 stability and provides prostate cancer cells enhanced growth. ............................................................................................. 54 Figure 4-I: CSN4 affects p53 transcriptional activity and disrupts p53-dependent apoptosis. ......................................................................................................... 56 Figure 5-I: CSN4 protein is over-expressed in prostate tumors and correlates directly with sGCα1 and inversely with p53 proteins. ......................................................... 58 Figure 6-I: CSN4 regulates CSN5 protein levels in prostate cancer cell lines. ................ 60 Figure 7-I: CSN5 antagonistically regulates p53 and sGCα1 proteins in prostate cancer.62 Figure 8-I: CK2 associates with and regulates p53 and sGCα1 proteins in prostate cancer cells ................................................................................................................... 64 Figure S1-I: CSN4 does not affect mRNA expression of sGCα1, p53, or CSN5.. .......... 66 Figure S2-I: CSN4 affects the growth of AR-positive prostate cancer cells, but not AR- negative cells. ............................................................................................... 68 vii Figure S3-I: Cyclase-deficient sGCα1 rescues the growth of prostate cancer cells depleted for CSN4. ..................................................................................................... 70 Figure S4-I: p53 does not affect sGCα1 protein levels nor the growth of prostate cancer cells. ............................................................................................................. 72 Figure S5-I: CSN4 mRNA is over-expressed in prostate tumors. .................................... 74 Figure 1-II: Androgen down-regulates TM4SF3 mRNA and up-regulates the protein.. 105 Figure 2-II: AR is required for androgen up-regulation of TM4SF3 protein.. ............... 107 Figure 3-II: TM4SF3 induces invasion, migration, and EMT of prostate cancer cells.. 109 Figure 4-II: TM4SF3 co-localizes and interacts with AR in prostate cancer cells.. ....... 111 Figure 5-II: TM4SF3 co-localizes and interacts with AR in AR-expressing PC-3 cells. 113 Figure 6-II: TM4SF3 up-regulates the AR protein and androgen signaling.. ................. 115 Figure 7-II: TM4SF3 and AR are over-expressed in prostate cancer.. ........................... 117 Figure S1-II: Androgen down-regulates TM4SF3 mRNA.. ........................................... 119 Figure S2-II: TM4SF3 induces invasion and migration of prostate cancer cells.. ......... 121
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