Direct regulation of androgen receptor activity by potent CYP17 inhibitors in prostate cancer cells

Abstract

TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [(3)H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5' cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation.

FIGURE 1.

The effect of 17-HASs on the levels of AR protein and AR mRNA. A, AR protein from LNCaP cells cultured in charcoal-stripped serum (CSS) and treated with the indicated concentrations of TOK-001 (5–15 μm). Control (C) cells were treated with an equal volume of DMSO. A representative Western blot of whole cell extracts collected on day 1 or day 3 post-treatment. The levels of α-tubulin were also determined as a loading control. B, Western blot analysis for AR protein from LNCaP cells cultured in CSS and treated for 3 days with the indicated concentrations of abiraterone alcohol (5–15 μm). Control (C) cells were treated with an equal volume of ethanol. The levels of α-tubulin were also determined as a loading control. C, Western blot analysis for intracellular PSA protein levels in LNCaP cells cultured in FBS and treated for 3 days with the indicated concentrations of TOK-001 or abiraterone alcohol. Control (C) cells were treated with an equal volume of either DMSO or ethanol. The levels of α-tubulin were also determined as a loading control. D, recovery of AR protein expression after TOK-001 treatment. Western blot analysis of AR steady-state protein levels in LNCaP cells cultured in CSS and treated with DMSO (D) or 5 μm TOK-001 (+T) for 5 days (Days 0–5, left panel). Treated cells were then subcultured into fresh media without drug (-T) and incubated for an additional 5 days (Days 6–10, right panel). The levels of α-tubulin were also determined as a loading control. E, Western blot of AR protein levels in LAPC-4 cells cultured in CSS for 3 days with the indicated concentrations (0.25–2.5 μm) of TOK-001 and abiraterone alcohol. Control (C) cells were treated with an equal volume of either DMSO or ethanol. The levels of α-tubulin were also determined as a loading control. F, qRT-PCR of AR steady-state mRNA levels in LNCaP cells cultured in CSS and treated for 24 h with the indicated concentrations of TOK-001. AR mRNA levels were normalized to RPLPO. Data represent the mean and S.D. of independent experiments.

FIGURE 2.

Competitive binding assay of TOK-001 and abiraterone alcohol for the AR. A, competitive binding curve of TOK-001 and abiraterone alcohol for the T877A mutant AR in LNCaP cells. B–F, competitive binding curve of TOK-001 for the wild type AR (LAPC-4 (B)), mutant AR (LNCaP, T877A) (C)), (D–F) AR expressed in PC3 cells: wild type AR (PC3 AR-WT (D)), mutant W741C AR (PC3 AR-741C (E)), mutant W741L (PC3 AR-W741L (F)). In PC3 cells, the WT AR and mutant AR proteins were expressed by transient transfection, as described. K_c was determined by non-linear regression using the One-site fit K_i equation of the GraphPad Prism Software. Plotted are AR-bound counts (cpm) versus [17-HAS] (log [M]). The average k_c ± S.D. (n = 3) is shown in the lower left corner of each graph.

FIGURE 3.

Suppression of androgen receptor trans-activation by 17-HASs. A, PC3 clones that stably express the WT AR protein were transiently transfected with the reporter vector pARE4-Luciferase and pCMV-Renilla. 24 h later, cells were stimulated with R1881 (1 nm) and treated with serial dilutions of TOK-001, Casodex, or abiraterone alcohol (n = 6 ± S.E.). The amount of transcriptional activity was normalized to Renilla luciferase and is expressed as normalized relative light units (RLU). Solid lines represent the best-fit sigmoidal dose response (variable slope). IC_50[TOK-001] = 1.3 μm; IC_{50[abiraterone alcohol]} = 4.7 μm; IC_50[Casodex] = 0.89 μm. B, PC3 clones that stably express the wild type (black), W741C (white), or W741L (gray) AR proteins were transiently transfected with the reporter vector pARE-4X-Luciferase and then stimulated with R1881 (1 nm). Cells were co-treated with the indicated concentrations of TOK-001. The amount of transcriptional activity was normalized to Renilla luciferase and is expressed as normalized relative light units (RLU). C, PC3 clones that stably express the WT, W741C, or W741L AR proteins were transiently transfected with the reporter vector pARE-4-Luciferase. The cells were untreated (mock or control), treated with 1 nm R1881, or treated with 10 μm Casodex, 10 μm TOK-001, or 10 μm abiraterone in the absence of R1881. The amount of transcriptional activity was normalized to Renilla luciferase and is expressed as normalized relative light units (RLU). D, LNCaP cells were either pretreated with DMSO (TOK-) or pretreated with 20 μm TOK-001 (TOK+) for 1 h. Cells were then stimulated with either 1 nm R1881 (R1881+) or DMSO (R1881-) for an additional 2 h before fractionation of the cytoplasmic (C) and nuclear (N) proteins by differential lysis and centrifugation. A representative Western blot is depicted showing the levels of AR in the C and N fractions. The levels of PARP and α-tubulin indicate the quality of the fractionation procedure.

FIGURE 4.

Neither TOK-001 nor abiraterone increase the rate of AR degradation. A and B, representative AR degradation time-course experiment in LNCaP cells is shown. AR protein was measured at 4-h intervals from LNCaP cells cultured in androgen-free media and co-treated with cycloheximide (CHX, 100 μm) and 20 μm TOK-001 (TOK-20) or 20 μm abiraterone alcohol (abiraterone). AR protein expression was also measured from vehicle control cells treated with CHX+DMSO (for TOK-001 (A)) or CHX+ethanol (for abiraterone alcohol (B)). AR levels are expressed relative to the time at t = 0. For both compounds, compared with vehicle, p = NS (Mann-Whitney). In a separate experiment, LNCaP cells were co-treated with cycloheximide (100 μm) and MG132 (5 μm). C, Western blot analysis of AR steady-state protein levels in LNCaP cells treated for 24 h with 17-HASs (TOK, 20 μm TOK-001; ABIR, 20 μm abiraterone alcohol) in the absence or presence of MG132 (5 μm). The levels of α-tubulin were determined as a loading control. D, Western blot analysis of AR steady-state protein levels in LAPC-4 cells treated for 24 h with TOK-001 (20 μm) in the absence or presence of MG132 (5 μm). The levels of α-tubulin were determined as a loading control.

FIGURE 5.

The AR translational machinery is a target of 17-HASs. A and B, luciferase reporter assay, in which translation of the luciferase mRNA is under the control of AR 5′-UTR (test) or IRES (control). Cells were treated with 20 μm TOK-001 or an equal volume of vehicle (DMSO) in A or treated with increasing doses of TOK-001 in B. C and D, cap-binding assay with 7^mG-pull down in total lysates from LNCaP cells treated with 10 μm or 20 μm TOK-001 in C or 10 μm or 20 μm abiraterone alcohol (ABI) in D for 36 h. Western blot analysis with the indicated antibodies is depicted. E, Western blot analysis of 4EPB1 and phospho-4EBP1 (p-4EBP1) in total lysates from LNCaP cells treated with vehicle (mock), 20 μm TOK-001, or 20 μm abiraterone alcohol (ABI). F, cell proliferation with MTS; *,** p < 0.007. LNCaP cells were incubated with mock (control), TOK-001 (10 μm), or abiraterone (10 μm) in serum-fed media for 72 h. Statistical analyses were performed using unpaired Student's t test. The data represent at least three independent experiments.