The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa.
Multiple genetic alterations are associated with prostate carcinogenesis. Tumor-suppressor genes phosphatase and tensin homolog deleted on chromosome 10 (Pten) and androgen upregulated gene 19 (U19), which encodes ELL-associated factor 2 (EAF2), are frequently inactivated or downregulated in advanced prostate cancers. Previous studies showed that EAF2 knockout caused tumors in multiple organs and prostatic intraepithelial neoplasia (PIN) in mice. However, EAF2-knockout mice did not develop prostate cancer even at 2 years of age. To further define the roles of EAF2 in prostate carcinogenesis, we crossed the Pten +/− and EAF2 +/− mice in the C57/BL6 background to generate EAF2 −/− Pten +/−, Pten +/−, EAF2 −/− and wild-type mice. The prostates from virgin male mice with the above four genotypes were analyzed at 7 weeks, 19 weeks and 12 months of age. Concomitant loss of EAF2 function and inactivation of one Pten allele induced spontaneous prostate cancer in 33% of the mice. Prostatic tissues from intact EAF2 −/− Pten +/− mice exhibited higher levels of phospho-Akt, -p44/42 and microvessel density. Moreover, phospho-Akt remained high after castration. Consistently, there was a synergistic increase in prostate epithelial proliferation in both intact and castrated EAF2 −/−Pten +/− mice. Using laser-capture microdissection coupled with real-time reverse transcription–PCR, we confirmed that co-downregulation of EAF2 and Pten occurred in >50% clinical prostate cancer specimens with Gleason scores of 8–9 (n = 11), which is associated with poor prognosis. The above findings together demonstrated synergistic functional interactions and clinical relevance of concurrent EAF2 and Pten downregulation in prostate carcinogenesis.
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