BAF57 governs androgen receptor action and androgen-dependent proliferation through SWI/SNF

Abstract

Androgen receptor (AR) activity is required for prostate cancer development and progression. Thus, there is a major impetus to understand the regulation of AR action. We and others have previously shown that AR transactivation potential is dependent on the presence of an active SWI/SNF chromatin remodeling complex. However, the mechanisms underlying SWI/SNF regulation of the AR remained unsolved. We show here that the BAF57 subunit, an accessory component of the remodeling complex, is a critical regulator of AR function. We show that BAF57 is expressed in the luminal epithelia of the prostate and is required for AR-dependent transactivation in prostatic adenocarcinoma cells. Our data reveal that BAF57 can directly bind to the AR and is recruited to endogenous AR targets upon ligand activation. Loss of BAF57 or inhibition of BAF57 function severely compromised AR activity, as observed with both exogenous and endogenous AR targets. Rescue of BAF57 function restored AR activity, thus demonstrating a specific requirement of BAF57 for AR activity. This action of BAF57 proved to be dependent on SWI/SNF ATPase function. BAF57 has previously been implicated in nuclear receptor coactivator function, and we show that, although BAF57 facilitated coactivator activity, only a selected subset required BAF57 for coactivator function. Lastly, we demonstrate that both BAF57 and BRM are required for the proliferation of AR-dependent prostatic adenocarcinoma cells. In summary, these findings identify BAF57 as a critical modulator of the AR that is capable of altering AR activity, coactivator function, and AR-dependent proliferation.

FIG. 1.

BAF57 is expressed in the luminal epithelia. Mouse prostates were resected and the individual lobes (ventral lobe [VL, lane 3], dorsolateral lobe [DLL, lane 4], and anterior lobe [AL, lane 5]) were isolated and prepared for either immunoblotting or immunohistochemistry as described in the text. (A) BAF57 and AR were detected by immunoblotting. LNCaP cell lysate (lane 1) and BT549 cell lysate (lane 2) were used as positive and negative controls, respectively. (B) Tissue from mouse prostate was sectioned and stained by standard immunohistochemistry techniques with no primary antibody control (left panels) or BAF57 (right panels). Antibody reactivity was visualized with diaminobenzidene chromogen, and nuclei were counterstained with hematoxylin. Magnifications, ×20 (top panels) and ×40 (bottom panels).

FIG. 2.

BAF57 binds the AR independent of ligand. (A) Immobilized GST or GST-BAF57 was incubated with [³⁵S]methionine-labeled AR in the presence of 0.1% EtOH vehicle (lanes 2 and 3) or 10 nM DHT (lanes 4 and 5) for 3 h at 4°C, washed, and denatured. Input and bound protein was subjected to SDS-PAGE, and ³⁵S-labeled AR was detected by autoradiography. (B) Clarified protein lysates from LNCaP cells was utilized for coimmunoprecipitation with antibodies to AR (lane 1), BAF57c (specific to C terminus of BAF57) (71) (lane 2), or preimmune serum (lane 3). The resultant immunoprecipitates were subjected to SDS-PAGE, and the presence of AR was detected by immunoblotting.

FIG. 3.

BAF57 is critical for AR activity. (A) MCF7 (lane 1), BT549 (lane 2), LNCaP (lane 3), and CV1 (lane 4) cells were harvested, lysed, and subjected to SDS-PAGE. Immunoblots were performed with antibodies to BAF57 and CDK4 (loading control). (B) MCF7, CV1, and BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.5 μg of pSG5AR, and 0.25 μg of pTK-Renilla luciferase (to normalize for transfection efficiency). Posttransfection, cells were stimulated with either 0.1% EtOH vehicle or 0.1 nM DHT for 24 h. Cells were harvested, lysed, and monitored for luciferase activity by utilizing the Dual Luciferase Assay Reporter System. AR activity is presented as the fold activation by ligand (DHT) versus EtOH. Averages and standard deviations are shown. (C) BT549 cells either untransfected (lane 1) or transfected as described above with pGreenLantern (indicating transfection) and pSG5AR (lane 2) were harvested 24 h posttransfection. Immunoblot analysis demonstrates AR expression in transfected BT549 cells. (D) To elucidate BAF57 function on AR reporter activity, BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.5 μg of pSG5AR, 0.25 μg of pTK-Renilla luciferase, and either 2.25 μg of parental vector or pBabe-WTBAF57. Posttransfection, cells were treated with either 0.1% EtOH vehicle or 0.1 nM DHT for 24 h. Cells were harvested and lysed, and the luciferase activity was measured by using the dual Luciferase Assay Reporter System. Ligand-induced AR activity in the presence of vector was set to 100, and the relative luciferase activity is shown. (E) BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pGreenLantern GFP (transfection control), 0.5 μg of SG5AR, and either 2.25 μg of vector (lane 1) or BAF57 expression plasmid (lane 2). Immunoblotting was performed for AR and GFP at 24 h posttransfection.

FIG.4.

Inhibition of BAF57 results in reduced endogenous AR activity. (A) LNCaP cells (2 × 10⁵) were transfected with 1 μg of pARR2-Luc reporter, 0.25 μg of pTK-Renilla luciferase, and either 2.25 μg of pBabe-BAF57ΔN (dominant-negative BAF57) or parental vector (to 4.5 μg of total DNA). Cells were treated with either 0.1% EtOH vehicle or 1 nM DHT for 48 h (with restimulation after 24 h) and then harvested and lysed. The luciferase activity was measured as in Fig. 3D, and the AR activity in the presence of vector alone plus DHT was set to 100. The average relative luciferase activity and standard deviations are shown. ✽, P < 0.05. (B) LNCaP cells transfected with either vector (lane 1) or BAF57ΔN (lane 2) were harvested, and the expression of endogenous AR and CDK4 (loading control) was determined by immunoblotting. (C) LNCaP cells (4.5 × 10⁵) were transfected with 1 μg of pBabe-PURO, 1 μg of H2B-GFP, and 4 μg of parental vector (lane 1), BAF57ΔN (lane 2), or dominant-negative AR (dnAR) (lane 3) expression plasmids. Posttransfection, cells were selected with puromycin until >90% of cells were H2B-GFP positive (ca. 3 days). Cells were then harvested, and RNA was isolated and subjected to RT-PCR. cDNAs were amplified in the presence of [³²P]dCTP to accurately quantify the signal (upper panel). Product levels were quantified by using a PhosphorImager. Relative levels of PSA over GAPDH from two independent experiments, and standard deviations are shown (lower panel).

FIG.5.

BAF57 action is dependent upon SWI/SNF ATPase activity. (A) BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.25 μg of pTK-Renilla luciferase, 0.5 μg of pSG5AR, and 2.25 μg of pBabe-WTBAF57, 1 μg of dominant-negative BRM (dnBRM), or parental vector (to 4.5 μg of total DNA) as indicated. Cells were treated posttransfection with 0.1% EtOH or 0.1 nM DHT for 24 h. Cells were harvested and lysed, and the dual luciferase activity was measured. Ligand-activated AR activity was set to 100, and relative luciferase activity and standard deviations are shown. ✽, P < 0.05. (B) BT549 cells were transfected with 0.5 μg of SG5AR, 0.5 μg of pGreenLantern GFP, and 1 μg of vector (lane 1) or dnBRM (lane 2) expression plasmid. Cell lysates were obtained, and immunoblot analysis was performed for AR and GFP. (C) SW13 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.25 μg of pTK-Renilla luciferase, 0.5 μg of pSG5AR, and 2.25 μg of pBabe-WTBAF57, 1 μg of BRM, and parental vector (to 4.5 μg of total DNA) as indicated. Posttransfection, cells were treated with 0.1% EtOH or 0.1 nM DHT for 24 h. Cells were harvested, lysed, and dual luciferase activity was measured. Ligand-activated AR activity was set to 100, and relative luciferase activity and standard deviations are shown.

FIG. 6.

BAF57 is recruited to the AR regulatory regions in the presence of ligand. LNCaP cells (5 × 10⁶) were cultured in steroid-free medium for 4 days and then stimulated with either 0.1% EtOH vehicle (lane 1) or 10 nM DHT (lane 2) for 60 min. After treatment the cells were formaldehyde cross-linked, and chromatin was recovered for use in ChIP assays. (A and B) Antibodies to acetylated histone H4 (AcH4), AR, BAF57h, and preimmune serum were used for ChIP analyses of the PSA enhancer region, as indicated. (C) A total of 10⁵ LB1-Luc cells (with integrated pARR2-Luc) were serum starved for 3 days and then treated with the indicated concentrations of DHT for 24 h. Reporter analysis was performed and demonstrated activation of the AR in a dose-dependent manner (left panel). For the ChIP analysis (right panel), LB1-Luc cells (5 × 10⁶) were cultured in steroid-free medium for 4 days and then stimulated with either 0.1% EtOH vehicle (lane 1) or 10 nM DHT (lane 2) for 60 min. After treatment, the cells were formaldehyde cross-linked, and chromatin was recovered for use in ChIP assays. Antibodies to AR and BAF57c were used for ChIP analyses of the ARR2 promoter region.

FIG. 7.

BAF57 cooperates with p160 coactivators. BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.25 μg of pTK-Renilla luciferase, 0.5 μg of pSG5AR, and 2.25 μg of pBabe-WTBAF57, as well as 1 μg of coactivator (pRC3.1-SRC1 [A] or pCMV-TIF2 [B]) or parental vector (to 4.5 μg of total DNA) as indicated. Posttransfection, cells were treated with 0.1% EtOH or 0.1 nM DHT for 24 h. Cells were harvested and lysed, and the dual luciferase activity was measured. Ligand-activated AR activity was set to 100, and the average relative luciferase activity and standard deviations are shown.

FIG. 8.

BAF57 facilitates non-p160 AR coactivator function. (A) BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.25 μg of pTK-Renilla luciferase, 0.5 μg of pSG5AR, and 0.01 to 2.25 μg of pBabe-WTBAF57 in the absence (▪) or presence of 1 μg of pSG5ARA70 (▨). As described in Materials and Methods, all transfections were brought to a total 4.5 μg by using empty vector. Posttransfection, cells were treated with 0.1% EtOH or 0.1 nM DHT for 24 h. Cells were harvested and lysed, and the luciferase activity was measured. The ligand-activated AR activity was set to 100 (data not shown), and the average relative luciferase activity and standard deviations are shown (P < 0.05). (B to D) BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.25 μg of pTK-Renilla luciferase, 0.5 μg of pSG5AR, and 2.25 μg of pBabe-WTBAF57, vector, or the indicated coactivator (pSG5ARA55 and pFLAG-Ubc9) or mutant (pcDNA3-ARA70N2) expression plasmid (1 μg). Posttransfection, cells were treated with 0.1% EtOH or 0.1 nM DHT for 24 h. Cells were harvested and lysed, and luciferase activity was measured. The ligand-activated AR activity in the absence of BAF57 was set to 100, and the average relative luciferase activity and standard deviations are shown (P < 0.05).

FIG. 9.

Inhibition of BAF57 or BRM attenuates AR-dependent proliferation. (A) LNCaP cells (10⁵) were transfected with 0.5 μg of pEGFP-H2B-GFP and 4.5 μg of parental vector, pBabe-BAF57ΔN, or dominant-negative AR (pSG5AR-Δ46-408). At 2 days posttransfection, cells were labeled with BrdU for 16 h and fixed. The percentage of transfected cells (H2B-GFP positive) incorporating BrdU was determined (left panel, representative image). The effect of dnAR or BAF57ΔN is shown as the percent inhibition of BrdU incorporation over vector control (right panel). (B) LNCaP cells were transfected with 0.5 μg of H2B-GFP and 4.5 μg of either pHTP-vector (lane 2) or pHTP-BRMi (lane 3). After transfection, cells were selected by using puromycin. Once cells were >90% H2B-GFP positive, they were lysed and subjected to immunoblot analyses for BRM and actin (loading control). SW13 cells (lane 1) are shown as a positive control for BRM deficiency. (C) LNCaP cells were transfected with 0.5 μg of H2B-GFP and 4.5 μg of parental vector, pHTP-BRMi scrambled, pHTP-BRMi, or pCMV-p16ink4a. The percentage of transfected cells (H2B-GFP positive) incorporating BrdU was determined. The effect of the BRM attenuation is shown as the percent inhibition of BrdU incorporation over vector control.