Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6

Abstract

High consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer in epidemiological studies. There is preliminary evidence that sulforaphane, derived from glucoraphanin found in a number of crucifers, may prevent and induce regression of prostate cancer and other malignancies in preclinical models, but the mechanisms that may explain these effects are not fully defined. Recent reports show that sulforaphane may impair prostate cancer growth through inhibition of histone deacetylases, which are up-regulated in cancer. Indeed, one of these enzymes, histone deacetylase 6 (HDAC6), influences the acetylation state of a key androgen receptor (AR) chaperone, HSP90. AR is the central signaling pathway in prostate cancer, and its inhibition is used for both prevention and treatment of this disease. However, it is not known whether the effects of sulforaphane involve suppression of AR. We hypothesized that sulforaphane treatment would lead to hyperacetylation of HSP90 and that this would destabilize AR and attenuate AR signaling. We confirmed this by demonstrating that sulforaphane enhances HSP90 acetylation, thereby inhibiting its association with AR. Moreover, AR is subsequently degraded in the proteasome, which leads to reduced AR target gene expression and reduced AR occupancy at its target genes. Finally, sulforaphane inhibits HDAC6 deacetylase activity, and the effects of sulforaphane on AR protein are abrogated by overexpression of HDAC6 and mimicked by HDAC6 siRNA. The inactivation by sulforaphane of HDAC6-mediated HSP90 deacetylation and consequent attenuation of AR signaling represents a newly defined mechanism that may help explain this agent's effects in prostate cancer.

Fig. 1.

Sulforaphane treatment of prostate cancer cells increases HSP90 acetyation and dissociates it from AR. (A and B) Immunoprecipitations were carried out after treatment with sulforaphane (SFN) 20 μM or vehicle for 4 h followed by a Western blot for (A) HSP90 and (B) AR. Enrichment was quantified for each immunoprecipitation. Inputs were probed with the indicated antibodies by Western blot and were quantified.

Fig. 2.

Sulforaphane treatment of prostate cancer cells lowers AR protein levels. (A) Western blot of protein lysates from LNCaP cells treated with vehicle, increasing doses of sulforaphane, or TSA (trichostatin A) at the indicated time points. (B) Western blot of protein lysates from VCaP cells at 24 h. AR and GAPDH levels by Western blot were quantified.

Fig. 3.

Sulforaphane treatment of prostate cancer cells reduces AR target gene expression. (A) Real-time PCR of PSA expression from LNCaP cells treated with vehicle, increasing doses of sulforaphane, or TSA at the indicated time points. (B and C) Real-time PCR of PSA (B) and TMPRSS2-ERG (C) gene expression from VCaP cells at 24 h. The vehicle-treated sample was set to 1. 18S was used as an endogenous control in all assays.

Fig. 4.

Proteasome inhibitor treatment of prostate cancer cells rescues AR protein from sulforaphane treatment. LNCaP cancer cells were treated for 24 h with 20 μM sulforaphane with or without 10 μM MG132, 300 nM TSA with or without 10 μM MG132, 10 μM MG132, or vehicle. AR levels and GAPDH levels by Western blot were quantified.

Fig. 5.

Sulforaphane treatment inhibits HDAC6. Tubulin dimers were either incubated without recombinant HDAC6 or with recombinant HDAC6 in the presence of vehicle, sulforaphane, or TSA. Levels of HDAC6, acetylated tubulin, and tubulin by Western blot were quantified.

Fig. 6.

Ectopic overexpression of HDAC6 attenuates sulforaphane-mediated AR and HDAC6 protein depletion, and HDAC6 siRNA recapitulates the findings seen with sulforaphane. (A) LNCaP cells were transfected with pCDNA3.1 or FLAG-HDAC6. Cells were then treated with either vehicle or 15 μM sulforaphane. Intensity values for bands by Western blot in the respective vehicle controls were set to 1. (B) LNCaP cells were transfected with siRNA to either the luciferase gene (si LUC) or HDAC6 gene (si HDAC6). Levels of protein expression by Western blot were quantified, and the bands from the HDAC6 siRNA samples were compared to the luciferase control samples from the same time point.

Fig. 7.

Model of sulforaphane attenuation of AR signaling via HDAC6 inactivation. Normally, HSP90 is deacetylated by HDAC6, which enables it to chaperone client proteins such as AR. HDAC6 also deacetylates alpha-tubulin. With sulforaphane treatment, HDAC6 is inhibited or targeted for protein degradation. This leads to hyperacetylated alpha-tubulin; the functional sigificance of this remains unclear. The HDAC6 inactivation with sulforaphane also leads to hyperacetylated, inactive HSP90 protein, which dissociates from AR, and AR is then targeted for protein degradation. Consequently, AR binding to its target gene androgen response elements (ARE), including TMPRSS2-ERG, is diminished, which reduces their expression.