Mechanisms of cell death induced by histone deacetylase inhibitors in androgen receptor-positive prostate cancer cells.

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.

Inhibition of p53 expression modifies the specificity of chromatin binding by the androgen receptor.

The androgen receptor (AR) is known to play a critical role in prostate cancer (PC). p53 likely also plays a role given that p53 mutations are commonly found in advanced PC, and loss of wild-type protein function contributes to the phenotype of castration-resistant prostate cancer (CRPC). Nevertheless, the extent of the contribution of p53 dysfunction to PC remains unclear. Here we analyze the effects of p53 inhibition in PC cells and show that it has significant consequences for both the interaction between AR, and chromatin and the proliferative capacity of these cells. Inhibition of p53 expression enabled LNCaP cells to proliferate independently of androgens. Moreover, it modified the genome-wide binding pattern of AR. ChIP-sequnce analyis (ChIP-seq) revealed that fewer AR-binding sites were present in the context of p53 inhibition, suggesting that wild-type p53 is required for stable binding of AR to certain chromatin regions. Further analysis revealed that a lower AR occupancy was accompanied by a reduction in FoxA1 binding at regulatory regions of AR-dependent genes. Our study also identifies a pool of genes that may be transcriptionally regulated by AR only in the absence of p53, and that may contribute to the CRPC phenotype. Overall, our results point to p53 playing an important role in regulating AR activity across the genome.

Caspase-2-Based Regulation of the Androgen Receptor and Cell Cycle in the Prostate Cancer Cell Line LNCaP.

Caspase-2 can induce apoptosis in response to extrinsic and intrinsic signals. Unlike other caspases, this protein is not expressed solely in nonnuclear compartments; a subpopulation is constitutively localized in the nucleus. As one of the most evolutionarily conserved caspases, caspase-2 may have roles in multiple cellular processes. However, its contribution to nonapoptotic processes remains a mystery. In this study, we show that caspase-2 activity is important for proliferation by cells of the androgen-dependent prostate cancer cell line LNCaP. LNCaP cells expressing either a dominant-negative (dn) form of caspase or an siRNA against caspase-2 had lower androgen receptor (AR)-dependent proliferative responses than control cells, and application of the siRNA resulted in downregulation of the expression of both AR-dependent prostate-specific antigen (PSA) and AR-dependent reporter luciferase. Also, caspase-2 formed complexes with the cell cycle regulatory proteins cyclin D3, CDK4, and p21/Cip1, and caspase-2 regulated AR transactivation by inhibiting the repressive function of cyclin D3. Taken together, these results reveal, for the first time, that caspase-2 is involved in cell cycle promotion and AR activation. Given that prostate cancer cells depend on AR activity in order to survive, the fact that our data indicate that caspase-2 positively regulates AR activity suggests that caspase-2 has potential as a target in the treatment of prostate cancer.