Altered SMRT levels disrupt vitamin D3 receptor signalling in prostate cancer cells

We hypothesized that key antiproliferative target genes for the vitamin D receptor (VDR) were repressed by an epigenetic mechanism in prostate cancer cells resulting in apparent hormonal insensitivity. To explore this possibility, we examined nuclear receptor corepressor expression in a panel of nonmalignant and malignant cell lines and primary cultures, and found frequently elevated SMRT corepressor mRNA expression often associated with reduced sensitivity to 1alpha,25-dihydroxyvitamin D-3 (1alpha, 25(OH)(2)D-3). For example, PC-3 and DU-145 prostate cancer cell lines had 1.8-fold and twofold increases in SMRT mRNA relative to normal PrEC cells (P<0.05). Similarly, 10/15 primary tumour cultures ( including three matched to normal cells from the same donors) had elevated SMRT mRNA levels; generally NCoR1 and Alien were not as commonly elevated. Corepressor proteins often have associated histone deacetylases (HDAC) and reflectively the antiproliferative action of 1 alpha, 25(OH)(2)D-3 can be 'restored' by cotreatment with low doses of HDAC inhibitors such as trichostatin A (TSA, 15 nM) to induce apoptosis in prostate cancer cell lines. To decipher the transcriptional events that lead to these cellular responses, we undertook gene expression studies in PC-3 cells after cotreatment of 1 alpha, 25(OH)(2)D-3 plus TSA after 6 h. Examination of known VDR target genes and cDNA microarray analyses revealed cotreatment of 1 alpha, 25(OH)(2)D-3 plus TSA cooperatively upregulated eight (outof1176) genes, including MAPK-APK2 and GADD45 alpha. MRNA and protein time courses and inhibitor studies confirmed these patterns of regulation. Subsequently, we knocked down SMRT levels in PC-3 cells using a small interfering RNA ( siRNA) approach and found that GADD45 alpha induction by 1 alpha, 25(OH)(2)D-3 alone became very significantly enhanced. The same distortion of gene responsiveness, with repressed induction of GADD45 alpha was found in primary tumour cultures compared and to matched peripheral zone ( normal) cultures from the same donor. These data demonstrate that elevated SMRT levels are common in prostate cancer cells, resulting in suppression of target genes associated with antiproliferative action and apparent 1 alpha,25(OH)(2)D-3-insensitivity. This can be targeted therapeutically by combination treatments with HDAC inhibitors.