A new compound targets the AF-1 of androgen receptor and decreases its activity and protein levels in prostate cancer cells

Abstract

Increased expression levels of constitutively active androgen receptor splice variants (AR-Vs) cause alterations in AR signaling, resulting in drug resistance and failed hormone therapy among patients with advanced prostate cancers. Several available compounds targeting the androgen axis and AR signaling have not demonstrated efficacy in preventing prostate cancer recurrence. Here, we investigated whether a new agent, 6-[6-ethoxy-5-ispropoxy-3,4-dihydroisoquinolin-2[1H)-yl]-N-[6-methylpyridin-2-yl]nicotinamide (EIQPN), has the potential for treating advanced prostate cancer. EIQPN interacted with the AR-activation fragment-1 (AF-1) domain and blocked its androgen-independent activity, robustly decreased the protein levels of AR and variants in prostate cancer cells by inducing AR protein degradation, and inhibited the androgen-independent proliferation of various AR-positive prostate cancer cells. In xenograft mouse models, EIQPN blocked the tumor growth of androgen-independent prostate cancer cells. Overall, these findings indicate that EIQPN could serve as a novel therapeutic agent for advanced recurrent prostate cancers.

Keywords: Prostate cancer; androgen receptor; androgen-independent activity; protein degradation; splice variant.

Figure 1

EIQPN inhibits androgen-independent AR transactivation as well as androgen-dependent. (A) Chemical structure of (6-[6-ethoxy-5-ispropoxy-3,4-dihydroisoquinolin-2[1H)-yl]-N-[6-methylpyridin-2-yl]nicotinamide (EIQPN). (B-E) EIQPN inhibits endogenous AR transactivation in prostate cancer cells. LNCaP (B-D) and CWR22rv (E) cells were transiently transfected with pARE2-TATA-luc reporter and incubated with 10 µM EIQPN and with or without 10 nM DHT (B), 50 ng/mL IL-6 (C), or 50 µM FSK (D). (F, G) EIQPN inhibits exogenous AR transactivation in PPC1 cells. Cells were transiently transfected with full-length AR (AR-FL) (F) or AR N-terminal domain (AR-NTD) (G) expression construct along with pARE2-TATA-luc, and incubated with 10 µM EIQPN, MDV-3100, or BIC, and with and without 10 nM DHT. Luciferase activity was normalized to β-galactosidase activity. Data are shown as means ± SEM of at least three independent experiments. *, P < 0.01; **, P < 0.001; one-way ANOVA with Tukey post hoc tests. ns, not significant. (H) EIQPN dose-dependently inhibits AR-NTD transactivation. HEK 293T cells overexpressing AR-NTD were incubated with various doses of EIQPN or BIC. Inhibitory concentration (IC₅₀) was obtained by nonlinear regression analysis.

Figure 2

EIQPN blocks AR activation at various stages. (A, B) EIQPN inhibits the N/C interaction of AR. PPC1 cells co-transfected with AR N-terminal (VP16/AR1-660) and C-terminal (GAL4/AR-LBD658-919) domain constructs along with 5xGAL4-luc3 reporter, were incubated with 10 µM EIQPN, MDV-3100, or BIC, and 10 nM DHT (A) or 50 µM FSK (B). (C, D) EIQPN blocks AR nuclear translocation. HEK 293T cells overexpressing GFP-AR-FL (C) and GFP-AR-NTD (D) were incubated with 10 µM EIQPN, MDV-3100, or BIC, and with or without 10 nM DHT for 2 h. Subcellular localization of ARs was detected as green fluorescent protein (GFP) signals. Nuclei were stained blue with TOPRO-3. Images were acquired using a confocal microscope. Scale bars, 25 µm. (E) EIQPN prevents AR recruitment to ARE. Recruitment of AR protein to ARE-2 and ARE-3 within PSA promoter was determined by ChIP assays using anti-AR (C-19) antibody. LNCaP cells were incubated with 10 µM EIQPN and 10 nM DHT for 2 h. Changes in AR enrichment at ARE was examined by PCR. The loading control was β-actin. (F, G) EIQPN inhibits coactivator recruitment to AR. PPC1 cells co-transfected with AR expression construct and SRC-1 or empty vector (EV) along with pARE2-TATA-luc were incubated with 10 µM EIQPN, MDV-3100, or BIC, and with or without 10 nM DHT (F) or 50 µM FSK (G). Data represent means ± SEM of at least three independent experiments. *, P < 0.01, and **, P < 0.001; one-way ANOVA with Tukey post hoc tests.

Figure 3

EIQPN interacts with AR-AF1 domain, but not with LBD. (A) EIQPN does not bind AR-LBD. HEK 293T cells overexpressing AR-FL were incubated with 5 nM [³H]5α-DHT and various concentrations of unlabeled ligand (DHT or EIQPN). K _d values were obtained by nonlinear regression analysis. (B) Schema of cloned AR-AF1 and docking region within TAU-5 domain. AR-AF1 domain (amino acids 110-485) was cloned for protein-substrate interaction assays in vitro. Docking region (H385-G410) contains 26 amino acids of TAU-5 domain. (C) Conformational change in AR-AF1 protein caused by interaction with EIQPN. Changes in intrinsic fluorescence spectrum of native or urea-denatured AR-AF1 protein caused by EIQPN were monitored using fluorescence spectroscopy. (D-F) Computational modeling of EIQPN interaction with AF-1 domain. Molecular PDB model of docking region of AR-AF1 was obtained using SWISS-MODEL. Novel interaction between EIQPN and docking region was determined by 1-Click docking. Summary of docking interaction scores (D). Images show 3D structure of H385-G410 TAU-5 docking region (orange) and EIQPN interacting in a K389/E391/P393/W400 pocket (E). EPI-001 was the positive control (F).

Figure 4

EIQPN reduces the AR protein levels. (A, B) Protein levels of AR and AR-Vs are decreased by EIQPN. LNCaP (A) and CWR22rv (B) cells were incubated with 10 µM EIQPN, MDV-3100, or BIC. Amounts of AR proteins were analyzed by western blotting, with GAPDH as loading control. (C) Western blots show decreased protein levels of endogenous AR and exogenous AR-NTD in LNCaP cells incubated with various doses of EIQPN. (D) Western blots show decreased protein levels of exogenous AR-NTD in DU145 cells incubated with 10 µM EIQPN. (E, F) Western blots show no effects of 10 µM EIQPN on protein levels of exogenous AR-NTD in PPC1 cells (E) and of different doses of EIQPN on exogenous AR-NTD (left) and AR-FL (right) in HEK 293T cells (F). (G, H) Ubiquitin-proteasome inhibitor MG-132 fully recovered AR protein levels decreased by EIQPN. CWR22rv (G) and C4-2 (H) cells cultured with or without 10 µM EIQPN for 16 h were incubated with MG-132 or chloroquine (CQ) for 8 h. (I) EIQPN significantly enhances ubiquitinated AR (ubi-AR) levels. CWR22rv cells were transfected with HA-tagged E3 ubiquitin ligase Mdm2 (HA-ubi) expression construct and incubated with 10 µM EIQPN for 2 h in the presence of 10 µM MG-132.

Figure 5

EIQPN inhibits androgen-dependent and -independent growth of AR-positive prostate cancer cells. (A-F) Proliferation of AR-positive prostate cancer cells is inhibited by EIQPN. LNCaP (A, B), C4-2 (C) and CWR22rv (D) cells were incubated for 5 days with 10 µM EIQPN, MDV-3100, or BIC, and with or without 50 ng/mL IL-6 (A) or 1 nM DHT (B). C4-2 (E) and CWR22rv (F) cells were incubated with different doses of EIQPN or MDV-3100. Half maximal inhibitory concentrations (IC₅₀) were determined by nonlinear regression analyses. Cell growth was assessed by MTS assays (A-C and E) or counting cells stained with trypan blue (D and F). (G) Proliferation of AR-negative prostate cancer cells is not affected by EIQPN. DU145 cells were incubated with 10 µM EIQPN, MDV-3100, or BIC, then growth was assessed using MTS assays. Data represent means ± SEM of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed t-test analysis. ns, not significant.

Figure 6

CWR22rv xenograft tumor growth in vivo is inhibited by EIQPN. (A-D) Tumor growth in intact (A, C) and castrated (B, D) mouse xenograft models is prevented by EIQPN. CWR22rv cells were injected into shoulders of intact and castrated 4-week-old, male NOD.CB17-PrkdcSCID/J mice. After 1 week, mice were injected i.p. with 25 mg/kg EIQPN in DMSO:PEG300 (2:8) or vehicle three times per week for 7 weeks. Two days after the final injection, tumor (left), testis (middle) and body (right) weight of intact (A) and castrated (B) mice were assessed. Intact mice, n = 9; castrated mice, n = 7. (C, D) Representative tumors dissected from intact (C) and castrated (D) mice. Scale bars, 1 cm. (E, F) Levels of cleaved caspase 3 (cleaved C3) and AR are significantly increased and decreased, respectively, by EIQPN in tumors dissected from intact (E) and castrated (F) mice. Protein band signals were scanned by Image Studio Lite, and visualized by GraphPad Prism 5.0. (G, H) EIQPN downregulated expression of AR target genes, PSA and TMPRss2, in tumors from intact (G) and castrated (H) mice. Levels of mRNA were analyzed by RT-PCR. Data represent means ± SEM of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed t-test analysis. ns, not significant.