Abstract
Prohibitin (PHB) is a tumour suppressor molecule with pleiotropic activities across several cellular compartments including mitochondria, cell membrane and the nucleus. PHB and the steroid-activated androgen receptor (AR) have an interplay where AR downregulates PHB, and PHB represses AR. Additionally, their cellular locations and chromatin interactions are in dynamic opposition. We investigated the mechanisms of cell cycle inhibition by PHB and how this is modulated by AR in prostate cancer. Using a prostate cancer cell line overexpressing PHB, we analysed the gene expression changes associated with PHB-mediated cell cycle arrest. Over 1000 gene expression changes were found to be significant and gene ontology analysis confirmed PHB-mediated repression of genes essential for DNA replication and synthesis, for example, MCMs and TK1, via an E2F1 regulated pathway—agreeing with its G1/S cell cycle arrest activity. PHB is known to inhibit E2F1-mediated transcription, and the PHB:E2F1 interaction was seen in LNCaP nuclear extracts, which was then reduced by androgen treatment. Upon two-dimensional western blot analysis, the PHB protein itself showed androgen-mediated charge differentiation (only in AR-positive cells), indicating a potential dephosphorylation event. Kinexus phosphoprotein array analysis indicated that Src kinase was the main interacting intracellular signalling hub in androgen-treated LNCaP cells, and that Src inhibition could reduce this AR-mediated charge differentiation. PHB charge change may be associated with rapid dissociation from chromatin and E2F1, allowing the cell cycle to proceed. The AR and androgens may deactivate the repressive functions of PHB upon E2F1 leading to cell cycle progression, and indicates a role for AR in DNA replication licensing.
Highlights
Prostate cancer is the most commonly diagnosed male cancer in the Western world.[1]
Several E2F-regulated genes, for example, the MCM family, DNA biosynthetic precursor genes, for example, TK1 and DHFR, showed strong reduction in the we demonstrated that doxycycline-induced PHB ectopic overexpression significantly repressed androgen receptor (AR) activity in LNCaP cells and presence of ectopic PHB cDNA expression
We set out to analyse how the PHB modulated the cell cycle and how its activity is regulated by AR in prostate cancer cells
Summary
Prostate cancer is the most commonly diagnosed male cancer in the Western world.[1]. Tumour growth is initially androgendependent; driven by the androgen receptor (AR). Hormonal therapies frequently fail and patients may relapse with ‘castrate-resistant’ prostate cancer.[2,3,4] Resistance results from clonal selection of cells that circumvent androgen requirement by mechanisms including AR mutation, amplification or changes in AR cofactor (coactivator and corepressor) levels One such corepressor is prohibitin (PHB), previously found to be downregulated by androgen treatment.[5] PHB has multiple roles in the cell, including (i) forming a part of a chaperone in the inner mitochondrial membrane;[6] (ii) an attenuator of Raf-Mek signalling[7,8] and (iii) a repressor of various transcription factors (including E2Fs and steroid receptors). It has tumour suppressor, antiproliferative and cell cycle regulation activities
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