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Title: Interactions of microRNA27a and prohibitin in the androgen signalling pathway
Author: Fletcher, Claire E.
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Prohibitin (PHB) is a highly conserved, multi-functional protein that acts as a tumour suppressor and androgen receptor (AR) corepressor. We found that downregulation of PHB by androgens is required to drive androgen-dependent prostate cancer cell growth. This effect can be mimicked through the use of PHB siRNA, whereby reduction in PHB protein levels enhances cancer cell and xenograft tumour growth. Conversely, PHB overexpression results in rapid accumulation of prostate cancer cells in G0/G1. The potential importance of PHB in prostate cancer development merits investigations into possible androgen-mediated mechanisms of PHB regulation. We observed significant reduction in endogenous PHB mRNA and protein levels, and a 20% decrease in transfected PHB promoter activity, following androgen treatment. We have also demonstrated interaction between AR and the proximal PHB promoter. These results suggest that AR may directly regulate levels of its own corepressor. In addition, we have shown that mutagenesis of residues with potential importance for PHB post-translational modification alters PHB subcellular localisation and permits PHB to function as a coactivator of AR. Recently, PHB has been identified as a potential target of microRNA 27a (miR-27a). Knockdown of miR-27a in gastric cancer cells increased PHB mRNA and protein expression (Liu et al., 2009). We therefore hypothesised that miR-27a may function as an 'oncomiR' in prostate cancer through PHB suppression. It was established that androgen-mediated downregulation of PHB can be attributed largely to miR-27a, since we found that: 1) MiR-27a expression leads to reduced PHB mRNA and protein levels through association with PHB 3'UTR, resulting in enhanced expression of AR target genes, PSA and TMPRSS2, and increased prostate cancer cell growth. These effects can be abrogated through miR-27a binding site mutation, anti-androgen treatment, exogenous PHB expression, or addition of miR-27a anti-sense inhibitor oligo (ASO) 2) MiR-27a is upregulated by androgens and downregulated by anti-androgens in prostate cancer cells. This represents a novel mechanism of PHB regulation, and implies that miR-27a may function as an "oncomiR" in prostate cancer. Hence, decreasing miR-27a levels may provide a potential thereapeutic strategy for the treatment of prostate cancer. Upon investigation of mechanisms of miR-27a regulation by androgens, a complex and novel mechanism of regulation was found: AR associates with the miR-27a promoter, leading to increased polycistronic transcription of miR-27a and its cluster partners, miR-23a and miR-24-2. Thus AR exhibits an element of regulation at the level of transcription. However, all three mature miRs show accumulation upon androgen treatment in the absence of transcription, whilst pri-miR levels are increased following AR silencing. These data thus suggest that AR signalling accelerates processing of primiR-23a27a24-2 in addition to its transcriptional role, increasing levels of mature miR-27a and its cluster partners. In contrast, anti-androgen treatment blocks processing of the pri- miR, reducing mature miR-27a levels and thus increasing PHB expression. We propose that AR- mediated post-transcriptional regulation of miR-27a occurs during Drosha-mediated primiR- 23a27a24-2 processing, since activity of a primiR-23a27a24-2-specific Drosha processing reporter is reduced by androgen treatment. GSK3B inhibition led to reduced miR-27a biogenesis, suggesting that GSK3B kinase activity may be involved in androgen-enhanced Microprocessor primiR-23a27a24-2 cleavage. I have also demonstrated that Drosha is transcriptionally androgen regulated through AR association with the Drosha promoter but this occurs too late to mediate androgen-regulated miR- 27a accumulation. It was also found that miR-27a targets the receptor tyrosine kinase EGFR, which has multiple cell signalling functions in cancer. Taken together, these data demonstrate that PHB is a target of oncomiR-27a in prostate cancer and that PHB is regulated by androgen both at the levels of transcription and translation, mediated by several related pathways: 1. Androgen binding to AR facilitates association with potential AREs within the PHB promoter, repressing gene transcription 2. Androgen signalling leads to upregulation of miR-27a, through increased transcription and enhanced Drosha-mediated primiR processing, which then associates with PHB 3'UTR, resulting in PHB translational inhibition. 3. Post-translational modification of PHB by phosphorylation and/or acetylation is vital in determining PHB subcellular localisation and corepressive function. We identify a novel mechanism of androgen-mediated post-transcriptional regulation of miR maturation, generating a positive feedback loop by which AR maintains its own activity by reducing corepressor activity. Additionally, miR-27a ASO may represent a potential novel therapeutic for prostate cancer.
Supervisor: Bevan, Charlotte ; Dart, Alwyn Sponsor: Medical Research Council ; Rpsetrees Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available