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MED12 Mutations Promote Castration-Resistant Prostate Cancer Through Hyperactivated GLI3/SHH Signalling

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MED12 Mutations Promote Castration-Resistant Prostate Cancer Through Hyperactivated GLI3/SHH Signalling University of the Incarnate Word The Athenaeum Theses & Dissertations 5-2019 MED12 Mutations Promote Castration-Resistant Prostate Cancer Through Hyperactivated GLI3/SHH Signalling Soundharrya Muthukumar University of the Incarnate Word, [email protected] Follow this and additional works at: https://athenaeum.uiw.edu/uiw_etds Part of the Biology Commons, and the Cancer Biology Commons Recommended Citation Muthukumar, Soundharrya, "MED12 Mutations Promote Castration-Resistant Prostate Cancer Through Hyperactivated GLI3/SHH Signalling" (2019). Theses & Dissertations. 361. https://athenaeum.uiw.edu/uiw_etds/361 This Thesis is brought to you for free and open access by The Athenaeum. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of The Athenaeum. For more information, please contact [email protected]. MED12 MUTATIONS PROMOTE CASTRATION-RESISTANT PROSTATE CANCER THROUGH HYPERACTIVATED GLI3/SHH SIGNALLING by SOUNDHARRYA MUTHUKUMAR A THESIS Presented to the Faculty of the University of the Incarnate Word in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE UNIVERSITY OF THE INCARNATE WORD May 2019 ii Copyright by Soundharrya Muthukumar 2019 iii ACKNOWLEDGMENTS Firstly, I’d like to thank my mentor, Dr. Marieke Burleson for her valuable time and constant guidance throughout the project. Dr. Burleson always had a constant belief and motivation which pushed me forward towards thesis completion. Her convenient office hours and constant passion towards this thesis kept me driving forward every single day of my thesis period. I would also thank my graduate advisor Dr. Russell Raymond for his valuable advice and great support in time of need. He always had confidence in me and motivated me every single time I encountered obstacles during my grad life. I would like to make a special thanks to Thu Duong (PhD student from Dr. Boyer’s Lab) for her constant help throughout the project. I would also like to thank Dr. Thomas Boyer for letting me use his valuable lab space during the time of need. I’d also like to thank my parents and my boyfriend for being a great support during my entire graduate life and always making sure that my confidence levels are up while facing struggles. My parent’s constant moral and financial support drove me towards a progressive path without making me to lose focus. Soundharrya Muthukumar iv MED12 MUTATIONS PROMOTE CASTRATION-RESISTANT PROSTATE CANCER THROUGH HYPERACTIVATED GLI3/SHH SIGNALLING Soundharrya Muthukumar University of the Incarnate Word, 2019 When cells of the prostate gland start growing uncontrollably, it results in cancer due to genetic aberrations. The development and progression of prostate cancer depend on androgenic stimulation. Although prostate cancer is temporarily treated by depriving a tumor of androgen, the patient relapses getting a castration-resistant form of the disease called castration resistant prostate cancer (CRPC). CRPC can specifically develop through crosstalk of androgen receptor signalling pathways. One such pathway that is of specific interest in this paper is the MED12 regulated SHH signalling pathway. Mediator is a signal processor that helps in the transduction of gene-specific transcription factors to RNA Polymerase II (Pol II).The activation of SHH ligand activates a transcription factor called GLI3 which physically targets the MED12 interface within the mediator complex so as to functionally reverse mediator dependent suppression of SHH target gene transcription. In this paper, we have shown that MED12 expression is critical in regulating androgen independent prostate cancer cell and therefore progression towards CRPC. Also, GLI3- dependent SHH signalling is indeed required for the progression of MED12 mutated prostate cancer cells to CRPC, after castration. v TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... viii PROSTATE CANCER OVERVIEW ...............................................................................................1 Precancerous Conditions ......................................................................................................2 Key Statistics .......................................................................................................................3 PSA ..........................................................................................................................3 Gleason score ...........................................................................................................3 REATMENT OPTIONS FOR PROSTATE CANCER ....................................................................4 Radical Prostectomy ............................................................................................................4 Radiation Therapy ................................................................................................................5 External Beam Radiation Therapy .......................…………………………………5 Internal Beam Radiation Therapy .......................………………………………….6 Chemotherapy ...............…………………………………………………………………...6 Immunotherapy ...............………………………………………………………………….6 Cellular Immuno Therapy ...................…………………………………………….7 Antibody Therapy ............…………………………………………………………7 Hormone Therapy ...............……………………………………………………………….8 Surgical Castration .................……………………………………………………..8 LHRH Agonist .......………………………………………………………………..9 LHRH Antagonist ..........…………………………………………………………..9 Anti-Androgen ...........……………………………………………………………..9 vi Limitations of Hormone Therapy ..................……………………………………10 Castration Resistant Prostate Cancer .............................…………………………………10 SIGNALLING PATHWAYS INVOLVED IN PROSTATE CANCER ... ………………………..11 Androgen Receptor Signalling ..................... ……………………………………………..11 Androgen Receptor ............ ………………………………………………………11 Canonical Signalling Pathway ..................…………………………………….....12 Hedgehog Signalling ..............……………………………………………………………13 Mechanism of Action ............…………………………………………………….13 Hedgehog Signalling In Human................……………………………………….13 Hedgehog Signalling in Prostate Cancer .......................…………………………15 Mediator Complex and Med12 Gene ....................……………………………………….16 Mediator ......……………………………………………………………………...16 MED12 ........……………………………………………………………………...18 METHODS………………………………………………………………………………………..19 Lenti Virus Particle Production……..………………………………………………….....19 Virus Infection…..………………………………………………………………………...19 Cell Culture…….……………………………………………..…………….………….....20 Quantitative Real Time PCR……………………………………………………………...20 Proliferative Assay………………………………………………………………………..20 Colony Formation Assay…………………………...……………………………………..21 RESULTS………………………………………………….……………………………………..22 MED12 Knockdown Promotes Androgen Independent Growth…..…………………………22 GLI3 Target Genes Are Upregulated in the Absence of Androgen…………………….…….24 vii GLI3 is required for Androgen Independent Growth of Med12 Knockdown Cells…….......26 MED12 Knockdown Promotes IN-VITRO Colony Formation…….…………………..……28 SUMMARY……………….…………………………………………………………………..…29 CONCLUSIONS AND DISCUSSION……………………………………………………….....29 REFERENCES……………………………………………………………………………...…...32 viii LIST OF FIGURES Figure Page 1. Overview of Prostate Cancer Progression ...................................................................................2 2. Androgen Receptor Signalling ...................................................................................................12 3. Hedgehog Signalling Pathway ...................................................................................................15 4. Mediator Complex .....................................................................................................................17 5. MED12 promotes androgen independent growth ......................................................................23 6. (A) & (B) GLI3 target gene transcription under androgen replete and androgen independent conditions ...........................................................................................................................25 7. (C) GLI3 target genes were up regulated in the absence of androgen .......................................26 8. GLI3 is required for androgen independent MED12 knockdown cell growth ..........................27 9. In-Vitro model for androgen independent MED12 knockdown cell growth .............................28 10. Model .......................................................................................................................................31 1 Prostate Cancer Overview When cells of the prostate gland start growing uncontrollably, it results in cancer. The prostate is an endocrine gland in most mammals which is a part of the male reproductive system.1 It is a compound tubuloalveolar gland that secrets an alkaline fluid that in human constitutes 30% of the seminal fluid.3 Prostate cancer might happen due to genetic aberrations that inactivate tumor suppressor genes or activate oncogenes. Most of the mutations are acquired during the evolution of cancer, however, some of them may be inherited.4 Human prostate is a small walnut-sized organ that is located just below the bladder and surrounds the urethra. It contains a system of branching ducts comprising pseudo-stratified epithelium surrounded by a fibro-muscular stroma. The prostate is a male sex accessory gland that functions
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