The transcriptional co-activator cAMP response element-binding protein-binding protein is expressed in prostate cancer and enhances androgen- and anti-androgen-induced androgen receptor function

Abstract

Progression of human prostate cancer toward therapy resistance occurs in the presence of wild-type or mutated androgen receptors (ARs) that, in some cases, exhibit aberrant activation by various steroid hormones and anti-androgens. The AR associates with a number of co-activators that possess histone acetylase activity and act as bridging molecules to components of the transcription initiation complex. In previous reports, it was shown that the transcriptional co-activator CREB (cAMP response element-binding protein)-binding protein (CBP) enhances AR activity in a ligand-dependent manner. In the present study, we have investigated whether CBP modifies antagonist/agonist balance of the nonsteroidal anti-androgens hydroxyflutamide and bicalutamide. In prostate cancer DU-145 cells, which were transiently transfected with CBP cDNA, hydroxyflutamide enhanced AR activity to a greater extent than bicalutamide in the presence of either wild-type or the mutated AR 730 val-->met. In two sublines of LNCaP cells that contain the mutated AR 877 thr-->ala and overexpressed CBP, increase in AR activity was observed after treatment with hydroxyflutamide but not with bicalutamide. Anti-androgens did not influence AR expression in cells transfected with CBP cDNA, as judged by Western blot analysis. Endogenous CBP protein was detected by Western blot in nuclear extracts from the three prostate cancer cell lines, LNCaP, PC-3, and DU-145, all derived from therapy-resistant prostate cancer. In addition, CBP was expressed in both basal and secretory cells of benign prostate epithelium, high-grade prostate intraepithelial neoplasia, and prostate cancer clinical specimens, as evidenced by immunohistochemical staining. Taken together, our findings demonstrate the selective enhancement of agonistic action of the anti-androgen hydroxyflutamide by the transcriptional co-activator CBP, which is a new, potentially relevant mechanism contributing to the acquisition of therapy resistance in prostate cancer.

Figure 1.

AR activity in the absence (dotted bars) or presence (open bars) of overexpressed CBP in DU-145 cells transfected with the reporter gene ARE₂TATA-CAT, either wild-type (A) or mutant AR 730 val→met (B), and CBP cDNA. The cells were treated with R1881 and/or hydroxyflutamide (HFM) or bicalutamide (Bic) and CAT activity was measured. Reporter gene activity measured after treatment of CBP-transfected cells with 1 nmol/L of R1881 was set as 100% and all other activities are expressed in relation to that value.

Figure 2.

Enhancement of AR activity by CBP in LNCaP (A) and long-term steroid-deprived LNCaP-abl (B) cells. LNCaP cells were transfected with the reporter plasmid pGL3E-probasin and CBP cDNA and treated with R1881 and/or anti-androgens for 24 hours. Luciferase activities are expressed in relation to values measured in CBP-overexpressing cells treated with R1881; dotted bars, cells transfected with empty plasmid; open bars, cells transfected with CBP cDNA.

Figure 3.

Regulation of AR protein expression in DU-145 cells overexpressing CBP. DU-145 cells were transfected as described in the legend for Figure 1 ▶ and the cells were treated with androgen, hydroxyflutamide, or bicalutamide. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blots for the AR (top) and β-actin (internal control, bottom) were performed; lane 1, transfected DU-145 cells, treatment with either hydroxyflutamide (A) or bicalutamide (B); lane 2, transfected cells, treatment with R1881; lane 3, untreated transfected cells; lane 4, nontransfected cells.

Figure 4.

Expression of CBP in prostate cancer cells. Nuclear extracts were prepared from the cell lines LNCaP, PC-3, and DU-145 using nuclear and cytoplasmatic extraction reagents. Western blots for CBP (top) and β-actin (bottom) in nuclear (lanes 1 to 3) and cytoplasmatic (lanes 4 to 6) preparations were performed; lanes 1 and 4, DU-145 cells; lanes 2 and 5, PC-3 cells; lanes 3 and 6, LNCaP cells.

Figure 5.

Expression of CBP in benign (A), high-grade prostate intraepithelial neoplasia (B), and malignant [primary prostate cancer, Gleason pattern 5 (C); and lymph node metastasis (D)] prostate tissue. The sections were incubated with the polyclonal anti-CBP antibody, dilution 1:100. For visualization, the PicTure kit was used. In benign prostate tissue, basal and secretory cells are CBP-positive. Original magnifications, ×400.