Increase of androgen-induced cell death and androgen receptor transactivation by BRCA1 in prostate cancer cells

Abstract

Although mutations of the breast cancer susceptibility gene 1 (BRCA1) may play important roles in breast and prostate cancers, the detailed mechanism linking the functions of BRCA1 to these two hormone-related tumors remains to be elucidated. Here, we report that BRCA1 interacts with androgen receptor (AR) and enhances AR target genes, such as p21((WAF1/CIP1)), that may result in the increase of androgen-induced cell death in prostate cancer cells. The BRCA1-enhanced AR transactivation can be further induced synergistically with AR coregulators, such as CBP, ARA55, and ARA70. Together, these data suggest that the BRCA1 may function as an AR coregulator and play positive roles in androgen-induced cell death in prostate cancer cells and other androgen/AR target organs.

Figure 1

Potentiation of the AR transactivation by BRCA1. (A) BRCA1, but not p53, potentiates the wild-type AR transactivation in prostate cancer cells. In each 60-mm dish of DU145 cells, 1 μg of pSG5-AR, 3 μg of MMTV-CAT, and/or 4.5 μg of pCR3-BRCA1, or 4.5 μg of p53 were transfected into cells by calcium phosphate method (14). The total plasmid amount was adjusted with pSG5, pCR3, or pCMV parent vector to 11 μg for each 60-mm transfection by calcium phosphate precipitation method. (B) BRCA1 can potentiate the AR transactivation in PC-3 cell. Cells were transfected as mentioned above. (C) BRCA1 can potentiate the AR transactivation in LNCaP cells in the presence of androgen without changing the expression of AR. 0.5 μg of PSA-LUC and 1.0 μg of pCR3 or pCR3-BRCA1 were transfected into LNCaP cells in 35-mm dish for 2 h by SuperFect. Cells were then treated with 1 nM DHT for an additional 24 h and harvested for LUC assay. The relative LUC activity was normalized against Renilla LUC activity (Promega). Data represent an average of three independent experiments. Duplicate LNCaP cells were harvested, and 60 μg of whole-cell extract was assayed with Western blotting for the detection of AR protein. The ectopically expressed BRCA1 cannot affect the expression of endogenous AR in LNCaP cells. (D) AR coregulators could cooperate with BRCA1 to synergistically enhance the AR transactivation. DU145 cells were cotransfected with 3 μg of MMTV-CAT, 1 μg of pSG5-AR, and 3 μg of alone or together with 3 μg CBP, ARA70N, ARA55, or BRCA1, in the absence or presence of 1 nM DHT. The error bars represent the mean ± SD of four independent experiments. (E) BRCA1 can potentiate the AR transactivation in MCF-7 and T47D cells. 0.5 μg of PSA-LUC reporter plasmid or 1.0 μg of pCR3-BRCA1 were transfected into T47D and MCF-7 cells. Cells were treated with 1 nM DHT after transfection as mentioned above.

Figure 2

The interaction between BRCA1 and AR. (A) Mapping the domains of BRCA1 that are responsible for AR interaction. Six recombinant GST–BRCA1 fusion proteins, fragments #1, #2, #3, #4, #5, and #6, were generated in Escherichia coli as described. BRCA1 residues are marked relative to the translation initiation site. The Coomassie blue-stained SDS polyacrylamide gel, showing the relative abundance of each fusion protein, was used in the GST pull-down assay as described. The 110-kDa protein bound to GST–BRCA1 #4 and #6 is a product of ³⁵S-methionine-labeled full-length AR. (B) Mapping the domain of AR that is required for BRCA1 interaction. 20 μl of in vitro translated ³⁵S-methionine-labeled AR N (from amino acids 36–553) and AR DBD-LBD (from amino acids 553–918) protein was used to perform the pull-down assay. The results indicated that GST–BRCA1 fragment #4 (amino acids 758-1064) can interact both with the N-terminal and DBD-LBD of AR. In contrast, GST–BRCA1 #6 (amino acids 1314–1863) can associate with only the DBD- LBD of AR. Our data indicate that there are two contact pockets between BRCA1 and AR. (C) The interaction between AR and BRCA1 by mammalian two-hybrid assay. PC-3 cells in 35-mm dishes were transiently cotransfected with 0.5 μg of reporter plasmid pG5-LUC, and 0.75 μg of Gal4DBD fused BRCA1 constructs, amino acids 1–304, amino acids 231-1314, amino acids 1560–1863, with or without 0.75 μg of VP16 fused AR (VP16-AR) construct for 2 h by SuperFect. 1 nM DHT was added for another 24 h, and then the cells were harvested for LUC assay. Arrows indicate VP16-fused SV40 large T antigen and ARA70 were applied here to assure the interaction specificity between BRCA1 and AR. The results indicate that BRCA1 amino acids 231-1314 and amino acids 1560–1863 are responsible for AR interaction; these results are consistent with the results from GST pull-down assay.

Figure 3

Mutations of BRCA1 reduce the enhancement of AR transactivation. PC-3 cells in 35-mm dishes were cotransfected with 0.5 μg of reporter plasmid MMTV-LUC, 0.5 μg of pSG5AR, and 1 μg of BRCA1 construct for 2 h. 1 nM DHT was then added for 24 h before cells were harvested for LUC assay. As compared with the wild-type BRCA1, the cotransfection of three mutant BRCA1s, one of the constructs lacks the C terminus of BRCA1 (BRCA1 amino acids 1–772), one has a point mutation at its C terminus (BRCA1-P1749R), and the other lacks exon 11 (BRCA1-Δ11), reducing the enhancement of AR activity (lane 3 vs. lanes 4–6).

Figure 4

The AR and BRCA1 could cooperatively regulate the promoter activity and protein expression of p21^(WAF1/CIP1) and stimulate the cell death in PC-3(AR2). (A) The coexpression of AR and BRCA1 cooperatively induces the −291 and −2326 p21^(WAF1/CIP1) promoter but not the basal promoter activity. In each transfection, 0.5 μg of reporter gene, 0.5 μg of AR, with or without 1 μg of BRCA1, were cotransfected into PC-3 cells. After 2 h of transfection, the medium was changed, and 10 nM DHT was added for another 30 h. (B) The endogenous p21^(WAF1/CIP1) expression was induced by DHT-AR and BRCA1 in MCF-7 cells. MCF-7 cells were seeded on 60-mm wells and cotransfected with 2 μg of AR with or without 4 μg of BRCA1 by SuperFect. After 2 h of transfection, the medium was changed, and 10 nM DHT was applied for another 30 h. In each experiment, 60 μg of whole-cell extract was applied for the Western blotting. (C) The p21^(WAF1/CIP1) protein is enhanced by DHT/AR and inhibited by hydroxyflutamide (HF) in PC-3(AR2) cells. The expression of protein p21^(WAF1/CIP1) can be induced by DHT, and this induction can be inhibited by 1 μM HF (lane 2 vs. lane 3). (D) The p21^(WAF1/CIP1) protein is enhanced by DHT/AR and BRCA1 in PC-3(AR2). PC-3(AR2) cells were cotransfected with or without 4 μg of BRCA1. After 2 h of transfection, the medium was changed, and 10 nM DHT was applied for another 30 h. In each experiment, 60 μg of whole-cell extract was applied for the Western blotting. (E) Cell growth is regulated by DHT/AR and BRCA1 in PC-3(AR2) cells. Duplicate PC-3(AR2) cells (as in D) were applied to MTT assay for the relative cell number determination. (F) Dead cells were indicated as loss of cell membrane integrity assayed by PI inclusion. PC-3(AR2) cells were transfected with 4 μg of BRCA1. After 2 h of transfection, the medium was changed, and 10 nM DHT or vehicle was applied for another 4 days. The medium was changed on day 2. Attached cells were trypsinized and collected with floating cells, stained with 20 μg/ml PI on day 4. After 10 min of staining, the PI-positive cells were then counted under fluorescent microscope.