HDACs are traditionally associated with transcriptional repression, particularly suppressing gene expression by a number of nuclear receptors (35
). Hormone receptor antagonists, such as bicalutamide for AR and tamoxifen for the estrogen receptor, promote the assembly of HDAC complexes at hormone receptor binding sites to block ligand-induced gene expression. Here, we show, using genetic and pharmacologic approaches, that HDACs are also paradoxically required for activation of a substantial fraction of AR target genes including the transcriptional driver of ETS
fusion mRNAs (TMPRSS2
) implicated in ~70% of human prostate cancers. Our data illustrate the important clinical implications for the development of HDAC inhibitors as potential prostate cancer drugs.
Using shRNA, we showed that knockdown of individual HDACs recapitulates what is seen with treatment of HDAC inhibitors on transactivation of nearly all the androgen receptor target genes and AR mRNA levels. This was specifically observed with loss of HDAC1 and, to a lesser extent, loss of HDAC3. Indeed, knockdown of these two HDACs can account for the majority of the antiandrogen effect. However, neither loss of HDAC1 nor loss of HDAC3 phenocopies the effect of HDAC inhibitors on androgen-induced TMPRSS2 expression. This suggests that androgen regulation of TMPRSS2 is not dependent on a single class I HDAC but rather a combination of them. This question can be addressed by performing combinatorial knockdown studies of the individual histone deacetylases and ChIP on individual promoters.
Further investigation is required to discern precisely how HDAC inhibition interferes with AR complex assembly on chromatin. The cyclical nature of AR (and ER) binding to target genes is well established, including the nonproductive first wave of receptor binding that terminates in the presence of HDACs at the promoter/enhancer. Correspondingly, each wave of transcription is associated with a round of acetylation and deacetylation of histone H3 and H4. HDAC inhibitors could interfere with a proposed, albeit controversial, action of HDACs in resetting the promoter for assembly of a competent transcriptional complex (32
). Our data, showing that TSA treatment shifts the phase of cyclical AR binding to the PSA enhancer, are consistent with this model. ChIP experiments indicate that HDAC inhibitor treatment rapidly and substantially enhances histone H3 acetylation at the PSA enhancer,5
indicating the constitutive presence of strong HAT activity at the loci. Therefore, one possibility is that hyperacetylated chromatin is incompatible with recruitment of coactivators and RNA Pol II.
Prior work has shown that HDAC inhibitors potentiate AR transcription, consistent with the transcriptional repression model. We reconcile our findings with these earlier reports in two ways. First, hyperactivation of AR function by HDAC inhibitors was observed using AR-dependent reporter constructs, which are unlikely to reflect the chromatinized state of endogenous genes. Second, the stimulatory effect of HDAC inhibitors on endogenous genes, such as PSA, was seen at doses (100 nmol/L TSA) below those required for achieving an increase in global histone acetylation. At this dose, we did not see a significant affect on PSA () or ARGs in expression profile (). Higher doses are required to suppress AR function and may be indicative of a biphasic response to HDAC inhibition in certain contexts that could have clinical relevance if traditional end points, such as serum PSA levels, are used to monitor response to treatment. The HDAC-dependent and HDAC-repressed AR target genes identified in our gene array studies could serve as biomarkers for selecting those that ensure sufficient levels of HDAC inhibition in future clinical trials of these agents.
The most immediate clinical implication of our data is that HDAC inhibitors may have activity in prostate cancer. Conventional antiandrogens, such as bicalutamide, are highly effective as initial therapy but inevitably fail due to the emergence of drug-resistant disease. Here, we show that HDAC inhibitors remain potent inhibitors of AR function even in the setting of bicalutamide resistance and have antitumor activity in hormone refractory xenograft models.
Collectively, these data justify a careful examination of the therapeutic potential of HDAC inhibitors in prostate cancer. However, the magnitude of HDAC inhibition required to inhibit AR activity is critical because the effects we observed are clearly dose-dependent. Pharmacokinetic studies of the HDAC inhibitor SAHA suggest that the levels required for maximal AR inhibition are unlikely to be achieved using the oral regimen currently approved for lymphoma therapy (36
). Indeed, phase I studies of SAHA, LBH589, and depsipeptide in CRPC, conducted by us and others, have been disappointing, with zero of eight PSA responders to LBH589 (37
) and 2 of 31 PSA responders to depsipeptide (38
). However, doses capable of AR inhibition should be possible with i.v. delivery or possibly through high dose of intermittent oral therapy, and the HDAC-regulated AR target genes defined here could be useful biomarkers to guide dose selection. This has led us to initiate a phase I study of intermittent i.v. LBH589.