Integrative genomic approaches towards nuclear receptors and their regulators in cancer
Long, Mark Damon
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Nuclear receptors (NRs) comprise a superfamily of ligand activated transcription factors which are integral to normal development and physiology in animals. As such, disruptions to NR functions are associated with the development of diseases such as cancer. While this fact is generally realized, the extent to which this statement remains true in cancer is unknown, and furthermore so are the causes and precise contributions of NR disruptions in different tissue types. Integrative analyses of publically available data available through The Cancer Genome Atlas were performed to determine that NR expression is commonly and significantly downregulated in primary prostate cancer (PCa), a phenomenon also observed across six different cancer types. No evidence of common NR disruption was observed at the levels of mutation frequency or copy number changes, suggesting the importance of epigenetic mechanisms in driving dampened expression of NRs. Specifically in PCa, retinoic acid receptor gamma (RARγ / RARG) was amongst the most commonly downregulated NRs. RARs respond to retinoic acid, the biologically active form of vitamin A, which has well established roles in prostate development and PCa biology and thus represents a potential therapeutic target in this setting. However, the mechanism of RARγ downregulation and how it contributes to retinoic acid resistance and general tumorigenic properties in prostate cells remains enigmatic. MicroRNA (miRNA) prediction algorithms were employed to identify putative NR targeting miRNA, which were commonly elevated in expression, suggesting a broader contribution of miRNA in the downregulation of NRs in PCa. This analysis revealed the highly elevated miR-96 as a putative regulator of RARγ. Of interest, miR-96 expression was found to associate with indicators of aggressive disease and stratified patients based on time to biochemical recurrence post radical prostatectomy (RP). Expression patterns in publically available data were validated by RT-qPCR in an independent cohort of 36 tumor/matched normal pairs obtained from men who had undergone RP at Roswell Park Cancer Institute. Correlative analyses found significant, inverse relationships between RARG and miR-96 across all human cohorts and along the time course of tumor development in the TRAMP murine model of PCa. Furthermore, in vitro approaches in prostate cells including miR-mimic transfection and luciferase assay validated a direct interaction between miR-96 and the RARG -3’UTR in prostate cells. RAR G knockdown cells were established to interrogate the relationship of this interaction towards retinoic acid resistance and general transcriptional control in prostate cells. Cell proliferation assays, cell-cycle analysis and transcriptome wide expression profiling were utilized in these in vitro models, identifying a limited role of RARG in mediating the classical anti-proliferative responses towards retinoic acid, but revealing novel, ligand independent functions of RARγ as a regulator of cell-cycle and androgen response. Additionally, a biotin-miRNA-pulldown approach was employed to profile the miR-96 targetome in prostate cells to address RARγ independent function. This approach revealed 400 additional mRNA targets, highly enriched for miR-96 predicted target sites and for direct downregulation by miR-96 upon overexpression in vitro, including those involved in cell-cycle control and responses to external stimuli. NRs act in concert with a variety of coregulators with both activating and repressive function. However, these functions have been characterized only in candidate studies and have yet to been examined genome wide. To address this, publically available data from The ENCODE Project was obtained to interrogate the global function of nuclear receptor corepressor 1 (NCOR1 / NCOR1) in K562, chronic myelogenous leukemia (CML) cells. Integrative analyses of ENCODE generated ChIP-seq and RNA-seq data revealed that genomic binding of NCOR1 correlated with elevated expression, even when considering additional factors associated with chromatin status. NCOR1 knockdown cells were established and subject to gene expression profiling, identifying NCOR1 as having both activating and repressing functions in certain contexts. Additionally, a pharmacogenomics approach was developed leveraging data available through the NCI-60 to assess the druggability of the NCOR1 cistrome. Experimental validation of predictions made by this approach revealed a role of NCOR1 in mediating the differentiating effects of Imatinib in CML cells.