A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes

Abstract

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.

FIGURE 1.

Comparison of AR level and activity in HeLaA6 and HeLa13 cells. A, a Western blot shows AR levels in HeLaA6 and HeLa13 cells. After at least 3 days in medium containing 5% CD-FBS, ethanol (−) or 10 nm DHT (+) was added. After 24 h, the cells were harvested, protein was extracted, and equal amounts of protein were fractionated on 10% polyacrylamide gels and analyzed by Western blotting. Tubulin served as a loading control. B, shown are the DHT dose-response curves for HeLaA6 and HeLa13 cells. HeLaA6 or HeLa13 cells were plated 1 day before treatment with the indicated concentrations of DHT or ethanol (vehicle) control. After 24 h of incubation, lysates were prepared, and PSA luciferase activity was measured in extracts from three wells of cells (mean ± S.E.). Activity in the presence of saturating DHT was set to 100%.

FIGURE 2.

CPIC inhibits androgen induction of PSA-luciferase activity. A, shown is the structure of CPIC. B, dose-response studies of CPIC inhibition of DHT-AR induced PSA-luciferase in HeLaA6 and HeLa13 cells. Cells were seeded in 24-well plates and treated with medium ± 10 nm DHT containing DMSO (Veh), the indicated concentrations of CPIC, or 10 μm bicalutamide (Bic) for 24 h and assayed for PSA luciferase activity. Activity of the reporter in the presence of 10 nm DHT and DMSO vehicle was set to 100%. Data represent the mean of three experiments ±S.E. C, LAPC-4 cells transiently transfected with 400 ng of PSA-Enh-luciferase plasmid and 100 ng of CMV-Renilla luciferase were treated with the indicated concentrations of CPIC or DMSO in the presence of 2 nm R1881 for 48 h. Cell lysates were assayed for firefly luciferase and normalized with Renilla-luciferase activity. Bars represent the mean of three experiments ± S.E.

FIGURE 3.

CPIC competes with androgens for binding to AR and reduces the AR N/C interaction. A, shown is a competitive radioligand binding assay. Relative binding affinity of CPIC for AR was determined using 5 nm [³H]R1881 and a range of CPIC concentrations as described (31). Data are the average of duplicate experiments. B, shown is the effect of DHT concentration on CPIC inhibition of AR-induced luciferase activity. HeLaA6 cells were seeded in 24-well plates and maintained for 24 h in medium containing 0.1 nm DHT (circle) or 10 nm DHT (triangle) and the indicated concentrations of CPIC. Data represent the average of triplicate experiments ± S.E. C, HeLa cells were transfected with 100 ng of PSA-Enh-Luc reporter plasmid, 50 ng of pCMV-AR-(1–503), and 50 ng of pCMV-AR-(507–919). Cells were incubated in the absence or presence of 10 nm DHT and the indicated concentrations of CPIC or Bic. Luciferase units of DHT-treated wells were set at 100%. The luciferase activity is representative of two independent experiments.

FIGURE 4.

Partial inhibition of ERα activity at high concentrations of CPIC. T47D-KBluc and T47D/(A1–2) cells were seeded in 24-well plates in medium containing 5% CD-FBS. After the cells attached, treatment medium was added with 5 nm 17β-estradiol for ER in T47D-KBluc cells and 5 nm dexamethasone for GR in T47D/(A1–2) cells and the indicated concentrations of CPIC (or DMSO vehicle). After 24 h cell lysates were analyzed for luciferase activity. Data are the average of triplicate experiments ± S.E.

FIGURE 5.

Effects of CPIC on prostate cancer cell growth. LNCaP cells (A), LAPC-4 cells (B), and CWR-R1 cells (C) were treated with the indicated concentrations of CPIC in the presence or absence of 1 nm R1881. After 4 days of treatment for LNCaP and CWR-R1 cells and 8 days for slow-growing LAPC-4, cell proliferation was measured using MTS. Cell growth in R1881+DMSO was set to 100%. D, AR-negative PC-3, MDA-MB-231, and DU145 cells were inoculated at 2000 cells/well in 96-well plates and treated with 100 μl of medium containing the indicated concentrations of CPIC or DMSO (vehicle). Growth of the cells was evaluated after 3 days. DMSO wells were set to 100%. Data points represent the mean of 8 wells ± S.E.

FIGURE 6.

CPIC inhibits anchorage-independent growth of LAPC-4 prostate cancer cells in soft agar. 5000 LAPC-4 cells were plated into top agar. Cells were treated with medium containing DMSO (vehicle), 1 nm R1881, 1 nm R1881 + 10 μm CPIC, or 1 nm R1881 + 10 μm Bic and replenished every 3–4 days. After 28 days, colonies were counted and photographed at 5× magnification. Inset, the bar graph represents the average of the total number of colonies counted in each well of the treatments. Photographs are representative of the entire well and of triplicate experiments.

FIGURE 7.

CPIC inhibits expression of endogenous AR-regulated genes. LNCaP (A) and LAPC-4 (B) cells were seeded in 6-well plates at 300,000 cells/well in medium containing 5% CD-FBS for at least 3 days. Cells were treated with or without 1 nm R1881 and the indicated concentrations of CPIC or Bic for 24 h before RNA extraction. mRNA was quantitated using quantitative RT-PCR and normalized to β-actin. Data represent the mean of three independent experiments ±S.E.

FIGURE 8.

CPIC does not decrease AR levels or alter nuclear localization of AR. A, shown is a Western blot of AR levels in LNCaP cells treated with CPIC. LNCaP cells were plated in medium containing 5% CD-FBS and maintained for at least 3 days. Treatment medium with (+) or without (−) 10 nm DHT containing DMSO (vehicle) or the indicated inhibitor was added to the cells. After 24 h, cell lysates were prepared, and equal amounts of protein were analyzed by Western blotting and tubulin used as a loading control. B, visualization of intracellular AR in LNCaP cells is shown. LNCaP cells were incubated with 10 nm R1881, 10 μm Bic, or 10 μm CPIC for 24 h, and intracellular AR was visualized by fluorescent microscopy using AR polyclonal antibody (Abcam, ab3510). Objective magnification, 40×.

FIGURE 9.

CPIC inhibits R1881-AR occupancy at AREs in LNCaP cells. Before ChIP, LNCaP cells were maintained for 3 days in medium containing 5% CD-FBS. After 1 h of incubation with DMSO, 10 μm CPIC, or 10 μm Bic, the cells were treated with 1 nm R1881 or ethanol and incubated for 4 h. Protein complexes were cross-linked, and AR (A, B) or RNA polymerase II (RNAPoIII; C, D) or control IgG antibody was used to pull down protein-bound chromatin fragments. Occupancy at the PSA enhancer, PSA promoter, and a control ARE-free region (middle region) between the two sites was determined using quantitative RT-PCR. -Fold enrichment over IgG control was plotted. Data represent the mean of three PCRs ± S.E. and are representative of other experiments. Significance of the differences between CPIC and the DMSO control was tested using Student's t test. p < 0.01 when compared with the respective controls. IP, immunoprecipitate.

FIGURE 10.

CPIC inhibits R1881-AR occupancy at AREs in LAPC-4 cells. LAPC-4 cells were maintained for 3 days in medium containing 5% CD-FBS, and ChIP was carried out as described in Fig. 9 and under “Experimental Procedures.” AR and RNA polymerase II occupancy at the PSA enhancer, PSA promoter, and a control ARE-free region (middle region) between the two sites was determined using quantitative RT-PCR. -Fold enrichment over IgG control was plotted. Data represent the mean of three PCRs ± S.E. Significance of the differences between CPIC and the DMSO control was tested using Student's t test and p < 0.05 when compared with the respective controls. For AR recruitment to the PSA promoter, R1881 + 10 μm Bic was not significantly different from R1881 + DMSO. IP, immunoprecipitate.