The aryl hydrocarbon receptor nuclear translocator (ARNT) transcriptional co-regulator complex: Effects on estrogen and hypoxia signaling

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(Thesis) Ph.D.
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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.
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Thesis advisor: Beischlag, Timothy
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