Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer

Abstract

The androgen receptor (AR) is a transcription factor that has a pivotal role in the occurrence and progression of prostate cancer. The AR is activated by androgens that bind to its ligand-binding domain (LBD), causing the transcription factor to enter the nucleus and interact with genes via its conserved DNA-binding domain (DBD). Treatment for prostate cancer involves reducing androgen production or using anti-androgen drugs to block the interaction of hormones with the AR-LBD. Eventually the disease changes into a castration-resistant form of PCa where LBD mutations render anti-androgens ineffective or where constitutively active AR splice variants, lacking the LBD, become overexpressed. Recently, we identified a surfaced exposed pocket on the AR-DBD as an alternative drug-target site for AR inhibition. Here, we demonstrate that small molecules designed to selectively bind the pocket effectively block transcriptional activity of full-length and splice variant AR forms at low to sub-micromolar concentrations. The inhibition is lost when residues involved in drug interactions are mutated. Furthermore, the compounds did not impede nuclear localization of the AR and blocked interactions with chromatin, indicating the interference of DNA binding with the nuclear form of the transcription factor. Finally, we demonstrate the inhibition of gene expression and tumor volume in mouse xenografts. Our results indicate that the AR-DBD has a surface site that can be targeted to inhibit all forms of the AR, including enzalutamide-resistant and constitutively active splice variants and thus may serve as a potential avenue for the treatment of recurrent and metastatic prostate cancer.

Keywords: Androgen Receptor; DNA-binding Protein; Drug Discovery; Nuclear Receptor; Prostate Cancer.

FIGURE 1.

Small molecules target the DBD of full-length hAR. A, chemical structure of VPC-14228, VPC-14449, and VPC-14337 (pyrvinium). B, multiple sequence alignment of nuclear receptor DBD domains. The amino acid sequences from human androgen, estrogen, glucocorticoid, and progesterone receptors were aligned with ClustalW, and visualized using JalView with Blossom62 color scheme. The positions that were mutated in this study, Gln-592 and Tyr-594, are highlighted in red. Numbering is according to the human AR. C–E, 50 ng of hAR_WT, hAR_Y594D, or hAR_Q592D plasmids were co-transfected with ARR₃tk-luciferase into PC3 cells. 0.1 nm R1881 and the indicated concentrations of compounds were added for 24 h. 100% refers to luminescence recorded with 0.1% DMSO only. Bottom panels in C–E present Western blots (AR-441/actin) of cell lysates incubated with DMSO or 50 μm compound. Lysates from hAR_WT were also probed with anti-PARP/cleaved PARP antibodies. Errors bars represent the mean ± S.D. from 4 to 6 replicates per point. IC₅₀ values (micromolar) were calculated by fitting curves to a sigmoidal dose-response (variable slope) equation. F and G, Y594A or Q592A mutants of the full-length human AR were tested in the luciferase reporter assay and analyzed by Western blot as described in C. enz, enzalutamide.

FIGURE 2.

DBD-interacting compounds are specific for AR. Enzalutamide (enz) (A), VPC-14228 (B), and VPC-14449 (C) were tested at the indicated concentrations in luciferase assays against transiently expressed AR, GR, and PR or against endogenous ER-α in MCF-7 cells. AR, GR, and PR activity was assessed with the ARR₃tk-luciferase reporter. MCF-7 cells include a stably transfected estrogen-response element-luciferase gene. 100% refers to luciferase activity of each receptor with 0.1% DMSO only. Errors bars represent the mean ± S.D. six replicates. enz, enzalutamide.

FIGURE 3.

Effect of DBD-interacting compounds on expression of AR target genes. A, LNCaP cells were treated with 1 nm R1881 and compounds for 2 days at the indicated concentrations. Secreted PSA was quantified by analyzing 150 μl of cell culture media from each well from two independent experiments. B, gene expression changes of AR target genes and a non-androgen-responsive gene (β-actin, ACTB) in the presence of R1881, compound 14449, and enzalutamide (Enz). * = significant reduction in gene expressions (p value < 0.05) based on two-sample t test between 14449 + R1881 and DMSO + R1881 and between enzalutamide + R1881 and DMSO + R1881. C, overlap among three gene sets: 1) down-regulated genes by compound 14449; 2) down-regulated genes by enzalutamide; and 3) previously published androgen-up-regulated genes. Individual androgen-up-regulated genes in the overlapping regions are shown.

FIGURE 4.

Effect of DBD-interacting compounds on AR splice variants. The luciferase reporter assay was performed as described in Fig. 1C but with 5 ng of pcDNA3.1 AR-V7 (A) or AR-V7_Y594D expression plasmid (B). Western blots of cell lysates containing AR-V7 were performed as in Fig. 1C. R1-AD1 cells expressing full-length AR (C) and R1-D567 (D) cells expressing the ARv567es splice variant were tested in luciferase assays as described in Fig. 1C, but with transfection of only 50 ng of ARR₃tk-luciferase reporter per well. R1-AD1 cells were stimulated with 0.1 nm R1881, whereas no androgen was administered to R1-d567 cells. E, Western blots with cell lysates before luciferase assay in C and D were performed as in Fig. 1C. F, cells were incubated with 50 μm compounds and 0.1 nm R1881 (R1-AD1 cells only; no androgen with R1-D567) for 2 days before cell lysates were probed with anti-FKBP5 antibody. enz, enzalutamide.

FIGURE 5.

Effect of compounds on AR nuclear localization. A, YFP-AR (50 ng/well) or YFP-V7 (5 ng/well) were co-transfected with ARR₃tk-luciferase reporter construct as described in Fig. 1C. Where indicated, the assay was performed in the presence of 25 μm compound (or 0.1% DMSO only) and 0.1 nm R1881 (or ethanol only). The data are presented as raw luminescence values without normalization. B, PC3 cells were transfected with YFP-AR plasmid and treated with 10 nm R1881 and 25 μm of the indicated compounds. Cells were fixed, mounted, and stained with DAPI. Confocal microscopy reveals the location the cell nucleus (DAPI, 388 nm) and YFP-AR (508 nm) in the 1st two rows of images. The 3rd row depicts the merged images of DAPI and YFP-AR visualizations. Scale bars, 20 μm. C, same as B, but with a plasmid expressing YFP-V7. enz, enzalutamide.

FIGURE 6.

Protein-DNA complexes are diminished in the presence of compounds. A, ChIP analysis of AR binding to the PSA or FKBP5 enhancers, or the GAPDH promoter, in LNCaP cells. Where indicated, nuclear translocation of the AR was stimulated with 1 nm R1881 (or ethanol only), and compounds were administered at 10 μm concentration. Sheared chromatin-protein complexes were precipitated with the AR-N20 antibody, reverse cross-linked, and analyzed by quantitative PCR. The results are normalized as fold enrichment over precipitation with a rabbit isotype control IgG antibody for each condition tested. Error bars represent the mean ± S.D. between three replicates. Fold enrichment from each tested condition (AR-N20 antibody) was statistically compared against DMSO + 1881 with a two-tailed t test (paired): *, p value < 0.05; **, p value < 0.01. B, purified AR-DBD + hinge domain at the indicated concentration was mixed with ∼2 μm ARE or scrambled 42-bp dsDNA and separated by native-PAGE. C, same as B but with 0.83 μm AR-DBD in the presence of 50 μm compound. D, biotinylated AR-DBD + hinge was loaded onto streptavidin sensors for biolayer interferometry analysis. t = 0 s. DBD was equilibrated with the indicated compound (50 μm); t = 100 s (arrow), dsDNA association was monitored with 3 μm ARE + 50 μm compound; t = 220 s, dissociation in buffer + compound. The y axis represents the nanometer shift in wavelength resulting from ligand (dsDNA) binding/dissociation. E, same as D but with the DBD bearing the Y594D mutation. enz, enzalutamide.

FIGURE 7.

VPC-14449 reduces tumor volume and abolishes PSA production in LNCaP xenograft model. Castrated mice were dosed twice daily with VPC-14449 (100 mg/kg) or enzalutamide (Enz) (10 mg/kg) for 4 weeks and assessed for LNCaP xenograft tumor volume (A) and serum PSA (B). Data are presented as mean ± S.E., n = 4. p value <0.05 was considered significant (*) compared with vehicle control; p value < 0.001 was considered extremely significant (**) compared with vehicle control.