A Signaling Network Controlling Androgenic Repression of c-Fos Protein in Prostate Adenocarcinoma Cells

Abstract

The transcription factor c-Fos controls many important cellular processes, including cell growth and apoptosis. c-Fos expression is rapidly elevated in the prostate upon castration-mediated androgen withdrawal through an undefined mechanism. Here we show that androgens (5α-dihydrotestosterone and R1881) suppress c-Fos protein and mRNA expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) or EGF in human prostate cancer (PCa) cell lines. Such suppression transpires through a transcriptional mechanism, predominantly at the proximal serum response element of the c-fos promoter. We show that androgen signaling suppresses TPA-induced c-Fos expression through repressing a PKC/MEK/ERK/ELK-1 signaling pathway. Moreover, our results support the hypothesis that p38(MAPK), PI3K, and PKCδ are involved in the androgenic regulation of c-Fos through controlling MEK/ERK. Stable silencing of c-Fos and PKCδ with shRNAs suggests that R1881 promotes cell death induced by low-dose TPA through a mechanism that is dependent on both PKCδ and loss of c-Fos expression. Reciprocally, loss of either PKCδ or c-Fos activates p38(MAPK) while suppressing the activation of ERK1/2. We also provide the first demonstration that R1881 permits cell death induced by low-dose TPA in the LNCaP androgen-dependent PCa cell line and that TPA-induced cell death is independent of exogenous androgen in the castration-resistant variants of LNCaP, C4-2 and C4-2B. Acquisition of androgen-independent killing by TPA correlates with activation of p38(MAPK), suppression of ERK1/2, and loss of c-Fos. These results provide new insights into androgenic control of c-Fos and use of PKC inhibitors in PCa therapy.

Keywords: Akt PKB; MAPK; PKC; androgen; androgen receptor; c-Fos; c-Jun transcription factor; p38 MAPK; phorbol ester; prostate cancer.

FIGURE 1.

Androgen down-regulates the protein level of c-Fos in prostate epithelial cells. A, LNCaP, C4-2B, MDA-PCa-2b, and VCaP cells were incubated with either 10 nm R1881 or DHT 48 h prior to a 2-h treatment with 10 ng/ml TPA, and expression of c-Fos and β-actin in whole cell lysates were analyzed by Western blotting. B, comparison of 24 h of pretreatment with R1881 versus co-treatment with R1881 on the induction of c-Fos by TPA in LNCaP cells as assessed by Western blotting. C, Western blotting of the effective dose of R1881 that suppresses c-Fos induction by TPA in LNCaP cells. D, R1881 (48 h) suppresses EGF-induced (10 ng/ml, 30 min) c-Fos expression. E, Western blotting of the expression of c-Fos in CWR22Rv1 cells following 22-h treatment with 1 nm R1881 before a 2-h treatment with 10 ng/ml TPA or a 1-h treatment with 10 ng/ml EGF. F, R1881 suppression of TPA-induced c-Fos expression in LNCaP cells is not reversed by the proteasomal inhibitor MG132, as determined by Western blotting. Cells were treated for 20 h with 10 nm R1881 or vehicle prior to 30-min pretreatment with 5 μm MG132 or vehicle, followed by 2 h of treatment with 10 ng/ml TPA or vehicle. Results are representative of two to three independent experiments.

FIGURE 2.

Suppression of c-Fos mRNA by R1881. A and B, LNCaP cells were pretreated with R1881 (10 nm) for 48 h and incubated with TPA (10 ng/ml) for the indicated times as c-Fos expression was measured by Northern blotting analysis (A) and semiquantitative RT-PCR with total RNA (10 μg) (B). Data were quantified by a PhosphorImager (A, right panel) or by ImageJ (B, right panel). The data in B are shown normalized to β-actin. C and D, real-time quantitative RT-PCR analysis of TPA-induced (2 h) or EGF-induced (1 h) c-fos expression (normalized to GAPDH) in LNCaP cells pretreated with R1881 (n = 4 replicates). Error bars represent the mean ± S.E. of triplicate to quadruplicate determinations. p Values were calculated by Student's t test (two-tailed). Data are representative of two to three different experiments/treatment.

FIGURE 3.

Androgen controls c-fos expression at the promoter level. A, LNCaP cells were co-transfected with either pGL3-basic-luc (control) or c-Fos promoter-luc (p-c-Fos-luc) along with CMV-Renilla, followed by treatment with R1881 (10 nm) or DHT (10 nm) for 48 h before TPA (10 ng/ml) treatment for 2 h. B, separately for EGF treatment, cells were preincubated with R1881 for 24 h prior to EGF treatment (10 ng/ml) for 24 h. C, cells were first transfected with p-c-Fos-luc and CMV-Renilla, and then incubated with R1881 (10 nm) for 48 h prior to TPA treatment (10 ng/ml, 2 h). U0216 (10 μm), PD98059 (5 μm), SP600125 (10 μm), SB202190 (10 μm), and GF109203X (25 μm) were added 1 h before R1881. Data shown are relative values of firefly luciferase normalized to Renilla luciferase. D, LNCaP cells were co-transfected with SRE-luc, SRF-Luc, or CRE-Luc along with CMV-Renilla, followed by treatment with R1881 (10 nm) for 48 h before TPA treatment (10 ng/ml) for 2 h. Data are relative values of firefly luciferase normalized to Renilla luciferase. Error bars represent the mean ± S.E. of triplicate determinations. p Values were calculated by Student's t test (two-tailed). Data are representative of two to three different experiments/treatment.

FIGURE 4.

Androgenic control of the PKC/MEK/ERK pathway in PCa cells. A, LNCaP and C4-2B cells were pretreated with 10 nm R1881 48 h prior to 2-h treatment with 10 ng/ml TPA, and cell lysates were analyzed by Western blotting for expression of total and phosphorylated MEK1/2, ERK1/2 p38 MAPK, and SAPK/JNK. B and C, the effect of R1881 (10 nm, 48 h) on c-Fos promoter activity (assayed as in Fig. 3) by enforced expression of wild-type (B) MEK1 and constitutively active (Act) MEK1 (C) constructs transfected in LNCaP cells. D, LNCaP cells treated as in A were analyzed for expression of total and phospho-PKCs. E, LNCaP cells treated as in A were analyzed for expression of total and phospho-PKCs following treatment with TPA and R1881 as indicated. Data are representative of two to three experiments.

FIGURE 5.

R1881 promotes TPA induced cell death through a mechanism that involves the suppression of c-Fos expression and activation of PKCδ. c-Fos was stably silenced by pLKO.1 lentivirus-mediated transduction of LNCaP cells, and silencing was assessed by Western blotting of TPA-treated (2 h, 10 ng/ml) cells (A). Silencing of c-Fos enhanced and suppressed TPA-induced P-p38^MAPK and P-ERK1/2, respectively (A), and was permissive to cell death induced by low-dose TPA treatment (B). PKCδ was stably silenced by pLKO.1 lentivirus-mediated transduction of LNCaP cells, as assessed by Western blotting of TPA-treated (2 h, 10 ng/ml) cells (C). Silencing of PKCδ suppressed and enhanced TPA-induced P-p38^MAPK and P-ERK1/2, respectively (C and D) and repressed cell death induced by low-dose TPA + R1881 treatment (E). Relative changes in cell density were assessed by crystal violet staining of adherent cells following 5 days of treatment (B and E). F, dose response of TPA on changes in the expression of c-Fos, P-p38, and P-ERK1/2 in vehicle versus R1881 pretreatment LNCaP cells (48 h, 1 nm).

FIGURE 6.

Cross-talk of MEK1/2, p38^MAPK, PI3K and AR in TPA-induced signals controlling c-Fos expression. A and B, LNCaP cells were pretreated with vehicle or an inhibitor of p38^MAPK (SB203580, 10 μm), MEK1/2 (selumetinib, 1 μm) or PI3K (ZSTK474, 1 μm) for 1 h, followed by vehicle or 1 nm R1881 (22 h) before 2-h pretreatment with 2.5 ng/ml TPA or vehicle. C, effect of selumetinib and ZSTK474 on EGF-induced (10 ng/ml, 30 min) c-Fos and P-ERK. D, effect of MK2206 versus ZSTK474 on c-Fos expression induced by TPA (2 h) with or without R1881 (24 h). E, dose dependence of R1881 on alterations in the expression of c-Fos and prostate-specific antigen (PSA) and the activation of ERK1/2 and p-38^MAPK. Cell lysates were analyzed by Western blotting for the expression of the indicated proteins. F, schematic of our model showing pathways involved in the control of c-Fos expression by AR and how AR signaling may promote TPA-induced death.

FIGURE 7.

Androgenic control of the c-Fos promoter. A, effect of R1881 on nuclear localization of P-ERK following treatment with TPA in LNCaP cells, as assessed by Western blotting of cytosolic and nuclear fractions. LNCaP cells were treated with or without 1 nm R1881 (22 h), followed by TPA (2.5 ng/ml, 2 h) or no TPA. B, effect of R1881 on expression and TPA-induced phosphorylation of ELK-1 and SRF expression in LNCaP (treated as in A), as assessed by Western blotting analysis of whole cell lysates. C, immunoprecipitation of ELK-1 by AR in TPA- and R1881-treated LNCaP cells (treated as in A). Cell lysates were immunoprecipitated with rabbit anti-AR (catalog no. sc-816) or control rabbit IgG and immunoblotted with mouse anti-AR (catalog no. sc-7305), anti-ELK-1 (catalog no. sc-56896), and anti-ERK1/2 (catalog no. 9107) antibodies. D, nuclear lysates of LNCaP cells (pretreated with or without 10 nm R1881 for 46 h, followed by 2 h with or without 10 ng/ml TPA) were subjected to EMSA using radiolabeled WT SRE and mutant SRE containing WT TCF-dimerized oligonucleotides. The DNA-protein complex (complex 1) was supershifted by treatment of the dimerized oligos with 1 μg of anti-SRF IgG (catalog no. sc-335) 30 min prior to electrophoresis. exp, exposure.

FIGURE 8.

Androgen-dependent and -independent killing of PCa cells by TPA. A—C, R1881 permits TPA-induced death of LNCaP cells (A) but not CRPC variants of LNCaP, C4-2 (B), and C4-2B (C), which are killed by low-dose TPA in the absence of androgen, as shown in Fig. 6, B and E. D, androgen-independent killing of C4-2 and C4-2B cells by low-dose TPA correlates with androgen-independent activation of p38^MAPK and reduced activation of ERK1/2 by TPA, as assessed by Western blotting and growth conditions described in Fig. 6A. E, TPA failed to kill RWPE-1 and DU145 cells but suppressed R1881-induced growth in VCaP, MDA-PCa-2b, and CWR22Rv1 cells assayed as in A. F, Western blotting analysis of the expression of AR in CWR22Rv1 cells treated with or without R1881, TPA, or EGF for 3 days. Error bars represent the mean ± S.E. of triplicate to six replicates. p Values were calculated by Student's t test (two-tailed) and two-way analysis of variance). Data are representative of at least two independent experiments. #, p < 0.05; *, p < 0.01; **, p < 0.001.