Histone deacetylase inhibitors affect androgen receptor activity through corepressors

Background: Histone acetylases (HATs) modulate the activity of diverse proteins including the androgen receptor which is a member of the nuclear receptor family. Deacetylation is mediated by class I histone deacetylases (HDACs) which are primarily nuclear while class II HDACs are shuttled between the nucleus and cytoplasm. The histone deacetylase inhibitors (HDACI) sodium butyrate (NaB) and trichostatin A (TSA) have shown significant antiproliferative and apoptotic effects on various cancer cells, including prostate cancer. For this reason, they are potential therapeutic agents in the treatment of prostate cancer even if the mechanism of their action remains undefined. On the other hand, the transcriptional activity of the androgen receptor (AR) is also effected by coregulatory proteins known as coactivators and corepressors. The silencing mediator for retinoid and thyroid hormone receptors (SMRT) and the nuclear corepressor (N-Cor) are linked to HDAC3 and they interact with the AR in a transcription repression manner. Overall, new approaches to silence the AR, may have therapeutic importance in the treatment of prostate cancer: repressive pathways appear to be important regulators of cancerogenesis.

Experimental procedures: We treated androgen-dependent prostate cancer cell line (LNCaP) and androgen-independent prostate cancer cell lines (C4-2, DU145 and PC3) with sodium butyrate (NaB) and trichostatin A (TSA). The effects of NaB and TSA on AR, HDAC2 and HDAC3 (class I HDACs) were assessed in whole cell extracts of these cell lines by Western blot analyses. The AR immunoprecipitates were tested with anti-SMRT monoclonal antibody by immunoblotting. The chromatin extracts incubated with SMRT (SMRTe - 1542/H7) were used for chromatin immunoprecipitation assay (ChIP). Immunoprecipitated DNA was analysed by PCRs using primers specific for AR and prostate specific antigen (PSA). Analysis of siRNAs was performed by antibodies against HDAC3, N-Cor and SMRT.

Results and conclusions: NaB and TSA suppressed AR expression in LNCaP and C4-2 cell lines containing functional AR. Immunoprecipitation analysis revealed in C4-2 cells a higher formation of the AR - SMRT complex in samples affected by NaB compared to control samples. On the other hand, the NaB effect was not relevant in the LNCaP cell line. Further, we investigated the influence of co-repressors SMRT, N-Cor and HDAC3 on AR expression in LNCaP and PC3 cell lines affected by NaB. The results suggest that the co-repressor SMRT significantly contributes to the NaB-mediated AR suppression. In addition, all four cancer cell lines were tested for expression of HDAC2 and HDAC3. After NaB and TSA treatment we detected their decreased expression only in the androgen-independent DU145 cell line.

This study was supported in part by IGA NR9475-3/2007 and MSM 6198959216.