Sirtuin 1 is required for antagonist-induced transcriptional repression of androgen-responsive genes by the androgen receptor

Abstract

Androgen antagonists or androgen deprivation is a primary therapeutic modality for the treatment of prostate cancer. Invariably, however, the disease becomes progressive and unresponsive to androgen ablation therapy (hormone refractory). The molecular mechanisms by which the androgen antagonists inhibit prostate cancer proliferation are not fully defined. In this report, we demonstrate that sirtuin 1 (SIRT1), a nicotinamide adenosine dinucleotide-dependent histone deacetylase (HDAC) linked to the regulation of longevity, is required for androgen antagonist-mediated transcriptional repression and growth suppression. Androgen antagonist-bound androgen receptor (AR) recruits SIRT1 and nuclear receptor corepressor to AR-responsive promoters and deacetylates histone H3 locally at the prostate-specific antigen promoter. Furthermore, SIRT1 down-regulation by small interfering RNA or by pharmacological means increased the sensitivity of androgen-responsive genes to androgen stimulation, enhanced the sensitivity of prostate cancer cell proliferative responses to androgens, and decreased the sensitivity of prostate cancer cells to androgen antagonists. In this study, we demonstrate the ligand-dependent recruitment of a class III HDAC into a corepressor transcriptional complex and a necessary functional role for a class III HDAC as a transcriptional corepressor in AR antagonist-induced transcriptional repression. Collectively, these findings identify SIRT1 as a corepressor of AR and elucidate a new molecular pathway relevant to prostate cancer growth and approaches to therapy.

Figure 1. SIRT1 represses AR-dependent gene transcription

A) Cell-based NAD-dependent HDAC activity assay of cells treated with 50 μM resveratrol or 10 mM NAM for 24 hr. B) Upper panel: The effects of resveratrol and nicotinamide on PSA-LUC transcription. LNCaP cells cultured in media containing charcoal-stripped serum were transfected with the PSA-LUC reporter vector plasmid. Transfected cells were then exposed to resveratrol (RES) at 50 μM or nicotinamide (NAM) at 10 mM for 24 hr, and treated with 10 nM DHT or vehicle-treated (Veh) for 24 hr before assay of luciferase activity, expressed here in arbitrary units. Insert shows the results from vehicle treatment, with an expanded y-axis. Lower panel: Immunoblot analysis of AR protein levels in cells treated with nicotinamide or resveratrol. C) Upper panel: Effects of SIRT1 over-expression or SIRT1 knockdown on PSA-LUC transcription. LNCaP cells were co-transfected with PSA-LUC and an empty control vector (pcDNA3.1), or a wt-SIRT1 expression vector (pcDNA3.1-SIRT1), or a vector expressing SIRT1 siRNA (pSUPER-SIRT1), or the empty siRNA expression vector (pSUPER), then treated with 10 nM DHT or vehicle-treated for 24 hr, and cells were harvested for luciferase assay. Insert shows the results from vehicle treatment, with an expanded y-axis. Lower panel: Immunoblot analysis of SIRT1 protein levels in cells transfected with wt-SIRT (pCDNA3.1-SIRT1), or DN-SIRT1 (pCDNA3.1-H363Y), or empty vector (pCDNA3.1), or empty siRNA vector (pSUPER), or SIRT1 siRNA expression vector (pSUPER-SIRT1). Cell extracts were normalized for protein content, separated by PAGE, transferred, probed with an anti-SIRT1 antibody or anti-AR antibody or an anti-β-actin antibody, and developed with a chemoluminescence kit. D) The effect of resveratrol or nicotinamide on endogenous AR-dependent and –independent PSA gene transcription. LNCaP cells cultured in media containing charcoal-stripped serum were exposed to 10 mM nicotinamide (NAM), 50 uM resveratrol (RES) or vehicle (control) for 2 hr, and treated with 10 nM DHT or vehicle-treated for 48 hr. Transcript levels of PSA was measured by quantitative RT-PCR analysis of RNA extracted from the cells. Transcript levels are expressed relative to β-actin transcripts. E) The effect of resveratrol or nicotinamide on endogenous AR-dependent and –independent KLK2 gene transcription. LNCaP cells were cultured in media containing charcoal-stripped serum were exposed to 10 mM nicotinamide (NAM), 50 uM resveratrol (RES) or vehicle (control) for 2 hr, and treated with 10 nM DHT or vehicle-treated for 48 hr. Transcript levels of KLK2 were measured by quantitative RT-PCR analysis of RNA extracted from the cells. Transcript levels are expressed relative to β-actin transcripts. F) Upper panel: SIRT1 knockdown increases endogenous AR-dependent PSA genes transcription. Transcript levels of PSA were measured by quantitative RT-PCR analysis of RNA extracted from empty vector-transfected LNCaP cells (Ctrl) and LNCaP cell lines in which SIRT1 expression levels had been knocked down by stable expression of siRNA (RNAi). The cells were cultured under androgen-deprivation conditions for 3 days, followed by treatment with DHT or vehicle for 48 hr. Lower panel: Immunoblot analysis of SIRT1 and AR protein levels in an empty vector-transfected LNCaP cell line (Ctrl) and LNCaP cell lines in which SIRT1 expression levels had been knocked down by stable expression of siRNA (RNAi). G) SIRT1 knockdown increases endogenous AR-dependent KLK2 gene transcription. Transcript levels of KLK2 were measured by quantitative RT-PCR analysis of RNA extracted from empty vector-transfected LNCaP cells (Ctrl) and a LNCaP cell line in which SIRT1 expression levels had been knocked down by stable expression of siRNA (RNAi). The cells were cultured under androgen-deprivation conditions for 3 days, followed by treatment with DHT or vehicle for 48 hr. H) SIRT1 deacetylase activity is required for SIRT1 effects on AR-dependent gene transcription. LNCaP cells cultured in media containing charcoal-stripped serum were co-transfected with the PSA-LUC vector plus an empty vector (Vector), or a SIRT1 expression vector (SIRT1), or a dominant-negative SIRT1 vector (H363Y), or SIRT1 expression vector plus DN-SIRT1 vectors (SIRT1+H363Y), then treated with 10 nM DHT or vehicle, and harvested for assay of luciferase activity. Insert shows the results from vehicle treatment, with an expanded y-axis. In all relevant figures, relative luciferase activities were normalized to β-gal activity to control for transfection efficiency. The error bars represent the SEM. Asterisks indicate significant differences between two groups (** p<0.01, *p<0.05).

Figure 2. ChIP analysis of the endogenous PSA gene promoter region

LNCaP cells were cultured in charcoal-stripped serum (S) for three days and certain cultures were treated for 4 hr with DHT (10 nM) (D) or bicalutamide (15 μM) (CDX), or vehicle control (S). ChIP assays were performed using primers sets which amplified the PSA promoter region, the enhancer region, or a distal region upstream of known PSA regulatory elements. Immunoprecipitations were carried out using antibodies directed against SIRT1, androgen receptor (AR), NCoR, polymerase II (Pol II) and an irrelevant protein (Rag1). The bound and input DNA were analyzed by ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, California) by the ΔΔ Ct method. The results are presented as the relative level of the protein associated with the PSA promoter or enhancer, normalized to irrelevant control antibody and input DNA. A) SIRT1 on the PSA promoter. B) SIRT1 on the PSA enhancer. C) AR on the PSA promoter. D) AR on the PSA enhancer. E) Pol II on the PSA promoter. F) Pol II on the PSA enhancer. G) NCoR on the PSA promoter. H) NCoR on the PSA enhancer. I) Immunoblot analysis of AR protein levels in DU145 cells transfected with wt-AR or empty vector. J) Bicalutamide-induced recruitment of SIRT1 to the endogenous PSA promoter requires the AR. DU145 cells were cultured in DMEM+10% charcoal-treated FBS. The cells were transfected with AR or mock-transfected, and treated with bicalutamide (CDX) or DHT (D). ChIP assays were performed using antibodies directed against SIRT1.

Figure 3. Androgen and antagonists alter histone H3 acetylation at the PSA promoter

A) Histone H3 acetylation at the PSA promoter. Cross-linked chromatin was extracted from cells cultured under androgen-deprivation conditions and then treated with 10 nM DHT (D), or DHT plus 25 μM resveratrol (D+RES), or vehicle (S) for 4 hr. Anti-Histone H3 antibody (AcH3) or an irrelevant antibody (anti-RAG) were used for immunoprecipitation. The ethidium-stained PCR products of the ChIP assay are shown. B) Quantitative PCR results from the same ChIP assays, analyzing both the PSA promoter and enhancer. C) Quantitative PCR analysis of ChIP assays for acetylated histone H3 at the PSA promoter or enhancer in the presence of DHT in control-transfected LNCaP cells (Ctrl) and in LNCaP cell lines in which SIRT1 expression levels had been knocked down by stable expression of siRNA (RNAi). Asterisks (*) indicates significant differences between two groups (p<0.05).

Figure 4. Reversal of bicalutamide-mediated transcriptional and growth suppression of cells by SIRT1 depletion

A) SIRT1 knockdown impairs bicalutamide-mediated PSA transcription repression. A SIRT1 knockdown cell line (RNAi) and control-transfected LNCaP cells (Ctrl) were cultured in charcoal-stripped serum, transfected with the PSA-LUC reporter vector, and then treated with DHT (1 nM) (D), or DHT plus bicalutamide (10 μM) (D+CDX), or vehicle (S). Cells were harvested after 24 hr and lysates were assayed for luciferase activity. The data are presented as a percent of the activity obtained in DHT alone (assigned the value of 100). B) Immunoblot analysis of SIRT1, AR and β-actin levels in control-transfected LNCaP cells (Ctrl) and a LNCaP line in which SIRT1 had been knocked down by stable expression of siRNA (RNAi). C) SIRT1 over-expression can partially rescue the defect of bicalutamide-mediated PSA transcription suppression induced by SIRT1 depletion. SIRT1 knockdown cells were cultured in charcoal-stripped serum, co-transfected with PSA-LUC and empty-vector (RNAi) or with PSA-LUC and SIRT1 expression vector (RNAi+SIRT1wt), or with PSA-LUC and SIRT1 catalytic inactive mutant (RNAi+SIRT1H363Y) and then treated with DHT (1 nM) (D), or DHT plus bicalutamide (10 μM) (D+CDX), or vehicle (S). Cells were harvested after 24 hr and lysates were assayed for luciferase activity. The data are presented as a percent of the activity obtained with exposure to DHT alone (assigned the value of 100). D) Immunoblot analysis of SIRT1 and AR protein levels in SIRT1 knockdown cell lines transfected with empty vector (RNAi), or wt-SIRT (SIRT1wt), or catalytic inactive mutant (SIRT1H363Y). E) SIRT1 is required for bicalutamide-mediated cell growth suppression. Parental empty-vector transfected LNCaP cells (Ctrl) or LNCaP cells in which SIRT1 had been stably knocked down by siRNA expression (RNAi) were cultured in charcoal-stripped serum three days, and exposed to DHT (1 nM) for another three days, then treated with addition of bicalutamide (2.5 μM) (D+CDX) or vehicle (D) for 48 hr. Viable cells were quantitated by MTS assay, and the results expressed relative to the values obtained from wells cultured without added bicalutamide (assigned as value of 100). Asterisks (*) indicates significant differences between two groups (p<0.05).

Figure 5. SIRT1 depletion increases the sensitivity of AR-dependent gene transcription and cell proliferation to DHT

A) SIRT1 knockdown increases the sensitivity of AR-dependent gene transcription to DHT. Parental empty-vector transfected LNCaP cells (Ctrl) or LNCaP cells in which SIRT1 had been stably knocked down by siRNA expression (RNAi) were cultured in charcoal-stripped serum, transfected with the PSA-LUC reporter vector, and then treated with nicotinamide (NAM) or vehicle (Control), and exposed to the indicated concentrations of DHT (0–1 nM). Cells were harvested after 48 hr and assayed for luciferase activity. B) Immunoblot analysis of SIRT1, AR and β-actin levels in control-transfected LNCaP cells (Ctrl) and three LNCaP lines in which SIRT1 had been knocked down by stable expression of siRNA (RNAi-1, RNAi-2 and RNAi-3). C) SIRT1 knockdown increases the sensitivity of the proliferative response of LNCaP cells to DHT. Control, empty-vector transfected LNCaP cells (Ctrl) or LNCaP cell lines in which SIRT1 had been stably knocked-down by siRNA expression (lines RNAi-1, RNAi-2 and RNAi-3) were made quiescent by culture in charcoal-stripped serum, and exposed to the indicated concentrations of DHT (0–10 nM). Viable cells were quantitated at 72 hr by MTT assay, and the results expressed relative to values obtained from plates cultured without added DHT (assigned an arbitrary value of 1). In all relevant figures, relative luciferase activities were normalized to β-gal activity to control for transfection efficiency. The error bars represent the SEM. Asterisks (*) indicates significant differences between two groups (p<0.05).