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(ARNT) Transcriptional Co-Regulator Complex: Effects on Estrogen and Hypoxia Signaling

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(ARNT) Transcriptional Co-Regulator Complex: Effects on Estrogen and Hypoxia Signaling The aryl hydrocarbon receptor nuclear translocator (ARNT) transcriptional co-regulator complex: Effects on estrogen and hypoxia signaling by Mark Labrecque B.Sc., University of British Columbia, 2007 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Doctor of Philosophy Program Faculty of Health Sciences Mark Labrecque 2016 SIMON FRASER UNIVERSITY Summer 2016 Approval Name: Mark Labrecque Degree: Doctor of Philosophy Title: The aryl hydrocarbon receptor nuclear translocator (ARNT) transcriptional co-regulator complex: Effects on estrogen and hypoxia signaling Examining Committee: Chair: Dr. Tim Takaro Professor Dr. Timothy Beischlag Senior Supervisor Associate Professor Dr. Frank Lee Supervisor Associate Professor Dr. Gratien Prefontaine Supervisor Associate Professor Dr. Christopher Beh Internal Examiner Associate Professor Department of Molecular Biology and Biochemistry Dr. Judy Wong External Examiner Associate Professor Faculty of Pharmaceutical Sciences University of British Columbia Date Defended/Approved: August 11, 2016 ii Abstract The basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH-PAS) domain family of proteins mediates cellular responses to a variety of stimuli. The bHLH-PAS proteins are heterodimeric transcription factors that are further sub-classified into sensory and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins. The ARNT protein is constitutively expressed and heterodimerizes with hypoxia-inducible factors (HIFs) to mediate oxygen-sensing mechanisms and heterodimerizes with the aryl hydrocarbon receptor (AHR) to combat environmental contaminant exposure. Firstly, a reciprocal disruption relationship exists between AHR ligands, like 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) and estrogen receptor (ER) ligands, like 17β-estradiol (E2). Ligand-bound ER tethers to the AHR/ARNT transcription factor complex and represses TCDD- inducible gene transcription. However, the tethering paradigm and molecular mechanisms employed by AHR and ARNT at ER-regulated genes remains to be determined. Secondly, thyroid hormone receptor/retinoblastoma-interacting protein 230 (TRIP230) interacts with ARNT and is a coactivator required for hypoxia-regulated transcription. The retinoblastoma protein (Rb) is a negative regulator of the cell cycle and also negatively regulates TRIP230 coactivator potential. Thus, Rb may influence ARNT transcription factor functions via TRIP230. Rb-loss in many solid tumours directly precedes the activation of HIF-regulated genes and correlates with increased angiogenesis and metastasis. As most solid tumours contain regions of hypoxia and only correlative data between HIF/ARNT activity and Rb-loss has been gathered, we have identified a need to rigorously examine the role of Rb-loss in concert with hypoxia in breast and prostate cancer models. In this thesis, I used siRNA technology to knockdown ARNT and AHR expression and found that TCDD-mediated disruption of ER-signalling is AHR-dependent and that ARNT is a coactivator in MCF7 cells and a corepressor in ECC1 cells. Additionally, I used siRNAs and shRNA technology in concert with microarray analysis in LNCaP prostate cancer cells and MCF7 breast cancer cells to delineate the role of the ARNT-TRIP230-Rb transcriptional complex in hypoxia- regulated transcription. I found that Rb-depletion in conjunction with hypoxia exacerbates HIF1-mediated transcription and promotes a more invasive and late stage phenotype in both breast and prostate cancer models. The molecular mechanisms and gene pathways described herein should prove useful for developing chemotherapies for late stage breast and prostate cancers. Keywords: ARNT; AHR; ER; TRIP230, Retinoblastoma protein; HIF1 iii Dedication For my wife, Alexa, and our son, Lincoln, Thank you for all the love and support and for always bringing a smile to my face iv Acknowledgements My journey at SFU has been long, interesting and slightly unexpected and it would not have been possible without the support and guidance from several key individuals. I would like to thank my senior supervisor, Dr. Timothy Beischlag, for giving me the opportunity to develop as a scientist and for sparking a curiosity that has led to both personal and intellectual growth. I am grateful for his mentorship, leadership and encouragement; my time as a graduate student under his guidance has profoundly changed my life. I am indebted to my supervisory committee, Dr. Gratien Prefontaine and Dr. Frank Lee, for their valuable feedback over the years and for helping me stay on track. I would also like to extend my thanks to my internal examiner, Dr. Christopher Beh, and my external examiner, Dr Judy Wong, for taking the time to read my thesis and for sitting on my examining committee. To all the past and present members of the Beischlag Lab, thank you for making the lab an enjoyable and productive environment. Kevin Tam, Julienne Jagdeo, Mandeep Takhar, Lillian Wong, Alex Reers, Stephanie Santacruz and Rebecca Nason, thank you for your support and for all the hardwork that has made several of our publications a reality. To Shabnam Massah and Sam Khakshour, thank you for your friendship and for allowing me to collaborate with you on interesting research projects that have enriched my time as a graduate student at SFU. To Dr. Michael Cox and his team at the Vancouver Prostate Centre, and to Dr. Kevin Bennewith and his team at the BC Cancer Research Centre, thank you for all your research insights, for being so generous with your time and for the mentorship and learning opportunities that were provided. I will forever cherish my graduate student experiences at your world-class organizations. Lastly but most importantly, thank you to my family. My graduate student journey would not have been possible without the steadfast support of my wife, mom, dad, brother and sister. You inspire me to be a better man and to reach for the stars. This work was supported in part by graduate fellowships from Simon Fraser University and by a graduate studentship award from Prostate Cancer Canada. v Table of Contents Approval .............................................................................................................................ii Abstract ............................................................................................................................. iii Dedication .........................................................................................................................iv Acknowledgements ........................................................................................................... v Table of Contents ..............................................................................................................vi List of Tables ..................................................................................................................... x List of Figures....................................................................................................................xi List of Acronyms .............................................................................................................. xiii Chapter 1. Introduction ............................................................................................... 1 1.1. The aryl hydrocarbon receptor nuclear translocator (ARNT) family of proteins .................................................................................................................... 2 1.2. The aryl hydrocarbon receptor (AHR) ...................................................................... 5 1.2.1. Endocrine disruption and the estrogen receptor (ER) ................................. 7 1.2.2. AHR, ER and ARNT crosstalk .................................................................... 9 1.3. Carcinogenesis ...................................................................................................... 11 1.4. Tumour Microenvironment ..................................................................................... 13 1.4.1. Normoxia and Hypoxia .............................................................................. 13 1.5. Hypoxia Inducible Factors (HIFs) ........................................................................... 15 1.6. Thyroid hormone receptor/retinoblastoma-interacting protein 230 (TRIP230) ....... 16 1.6.1. TRIP230 and interactions with ARNT ....................................................... 18 1.7. The Pocket Proteins ............................................................................................... 18 1.7.1. Molecular components of Rb, p107 and p130 .......................................... 18 1.7.2. Rb and cell cycle control. .......................................................................... 19 1.7.3. Rb function in apoptosis and the DNA damage response. ....................... 22 1.7.4. Rb and cancer ........................................................................................... 24 1.8. Prostate Cancer ..................................................................................................... 25 1.8.1. The androgen receptor and roles in prostate cancer. ............................... 25 1.8.2. Neuroendocrine differentiation in prostate cancer .................................... 26 1.8.3. NED and Rb .............................................................................................. 28 1.9. Breast Cancer .......................................................................................................
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