Glucocorticoid receptor activity contributes to resistance to androgen-targeted therapy in prostate cancer

Abstract

Despite new treatments for castrate-resistant prostate cancer (CRPC), the prognosis of patients with CRPC remains bleak due to acquired resistance to androgen receptor (AR)-directed therapy. The glucocorticoid receptor (GR) and AR share several transcriptional targets, including the anti-apoptotic genes serum and glucocorticoid-regulated kinase 1 (SGK1) and Map kinase phosphatase 1 (MKP1)/dual specificity phosphatase 1 (DUSP1). Because GR expression increases in a subset of primary prostate cancer (PC) following androgen deprivation therapy, we sought to determine whether GR activation can contribute to resistance to AR-directed therapy. We studied CWR-22Rv1 and LAPC4 AR/GR-expressing PC cell lines following treatment with combinations of the androgen R1881, AR antagonist MDV3100, GR agonist dexamethasone, GR antagonists mifepristone and CORT 122928, or the SGK1 inhibitor GSK650394. Cell lines stably expressing GR (NR3C1)-targeted shRNA or ectopic SGK1-Flag were also studied in vivo. GR activation diminished the effects of the AR antagonist MDV3100 on tumor cell viability. In addition, GR activation increased prostate-specific antigen (PSA) secretion and induced SGKI and MKP1/DUSP gene expression. Glucocorticoid-mediated cell viability was diminished by a GR antagonist or by co-treatment with the SGK1 inhibitor GSK650394. In vivo, GR depletion delayed castrate-resistant tumor formation, while SGK1-Flag-overexpressing PC xenografts displayed accelerated castrate-resistant tumor initiation, supporting a role for SGK1 in GR-mediated CRPC progression. We studied several PC models before and following treatment with androgen blockade and found that increased GR expression and activity contributed to tumor-promoting PC cell viability. Increased GR-regulated SGK1 expression appears, at least in part, to mediate enhanced PC cell survival. Therefore, GR and/or SGK1 inhibition may be useful adjuncts to AR blockade for treating CRPC.

Conflict of interest statement

Conflict of Interest R.Z.S and S.D.C and The University of Chicago have a pending patent application with claims related to the use of concomitant glucocorticoid and androgen receptor antagonism in prostate cancer treatment (US patent application no. PCT/US2013/027150).

Figures

Fig. 1

GR expression in prostate cancer following androgen signaling inhibition. A Western blot of GR and AR expression for PC cell lines PC3, DU145, LNCaP, LAPC4, CWR-22Rv1, and VCaP. BNRC31 (GR) mRNA expression (above) and protein expression (below) in PC cell lines after long-term (>30 days) treatment with AR antagonist MDV3100. Error bars represent standard error of the mean, and asterisk indicates p<0.05 paired t test. For Western blot, parental cell lines (P) are compared to the MDV3100 (MDV) treated. The GRα isoforms are labeled to the left of the immunoblot. C Xenografted PC tumors from host mice with intact androgen production versus 30-day-status post-castration were analyzed using an immunofluorescent anti-GR antibody and were also stained with DAPI. D Protein immunoblots from prostate cancer xenografts analyzed grown in intact (In) non-castrated mice, 48 h (48h), 7 days (7D), and 30 days (30D) post-castration

Fig. 2

GR expression/activation levels following AR inhibitor therapy. Hormonal therapies utilized included: R1881 (1 nM), a synthetic androgen; dexamethasone (Dex, 100 nM), a GR agonist; and MDV3100 (MDV, 10 μM), an AR antagonist. A 22Rv1 and LAPC4 GR mRNA expression analysis by qRT-PCR after 3 days of treatment; normalized to GAPDH and depicted as fold change relative to vehicle. B Western blot analysis of 22Rv1 and LAPC4 GR and AR protein expression after 3 and 7 days of treatment. All conditions were treated with Dex and R1881. Asterisk denotes AR splice variants within 22Rv1 cells. C 22Rv1 and LAPC4 cells were treated for 14 days under various conditions

Fig. 3

GR expression/activation and PC cell viability. Cells were cultured for 14 days under the same conditions described in Fig. 2 that also included mifepristone (Mif, 100 nM), a GR antagonist. Total viable cells (A) and relative viable cells (B) are shown. For relative viability (b), the control denominator is listed within the figure for each data set. R = R1881, D = Dex, MDV = MDV3100, and Mif = Mifepristone. Error bars represent standard error of the mean (SEM), and asterisk indicates p<0.05 paired t test. C Kaplan–Meier analysis of time to initiation of castration-resistant tumors for LAPC4 cells stably expressing a GR-specific shRNA versus SS shRNA (p=0.038, log-rank). Western blot analysis of GR protein expression [inset]. D Progression-free survival for 22Rv1 cells stably expressing doxycycline (Dox)-inducible shRNA construct with and without Dox treatment in the setting of castration (p=0.009, log-rank). Western blot of GR showing GR depletion with Dox [inset]

Fig. 4

Ligand-bound GR regulation of AR target genes in the absence of AR signaling. A Conditioned media were collected after 3 days of agonist and/or antagonist treatment, and secreted PSA was measured by ELISA. B SGK1 protein expression by Western blot analysis after 7 days of treatment. C Protein immunoblots from PC xenografts at various time points post-castration as in Fig. 1d. D SGK1 immunoblots of GR-depleted shRNA expressing cell lines shown in Fig. 2 showing decreased SGK1 expression (normalized densitometry shown) in the setting of GR depletion. E mRNA was collected from LAPC4 and 22Rv1 cells treated for 7 days under various hormonal conditions, and MKP1 gene expression was assessed by qRT-PCR. The conditions used included: R1881 (1 nM), a synthetic androgen; dexamethasone (Dex, 100 nM), a GR agonist; MDV3100 (MDV, 10 μM), an AR antagonist; and mifepristone (Mif, 100 nM), a GR antagonist

Fig. 5

Increased regulation of PSA by the GR following AR antagonism. A mRNA was collected from LAPC4 and 22Rv1 cells treated for 2 h under various hormonal conditions, and PSA gene expression was assessed by qRT-PCR. B Both 22Rv1 and LAPC4 cells were treated for 3 days in either vehicle, R1881 (1 nM), or R1881 + MDV3100 (10 μM) containing media. Conditions specifying dexamethasone treatment were stimulated with Dex (100 nM) for 1 h prior to chromatin harvest. Targeted qPCR was performed for the PSA promoter after ChIP, and y-values represent fold chromatin enrichment (relative to IgG controls for each condition). Error bars represent standard deviation of mean cycle threshold values, and asterisk indicates p<0.05 of performed paired t tests (only included for key statistical analysis)

Fig. 6

Increased regulation of SGK1 by the GR following AR antagonism. A mRNA was collected from LAPC4 and 22Rv1 cells treated for 2 h under various hormonal conditions, and SGK1 gene expression was assessed by qRT-PCR. B Both 22Rv1 and LAPC4 cells were treated for 3 days in either vehicle, R1881 (1 nM), or R1881 + MDV3100 (10 μM)-containing media. Conditions specifying dexamethasone treatment were stimulated with Dex (100 nM) for 1 h prior to chromatin harvest. Targeted qPCR was performed for the region within the SGK1 promoter after ChIP, and y-values represent fold chromatin enrichment (relative to IgG controls for each condition). Error bars represent standard deviation of mean cycle threshold values, and asterisk indicates p<0.05 of performed paired t tests (only included for key statistical analysis)

Fig. 7

The functional role of SGK1 in androgen signaling-deprived PC A PC cells were treated with SGK1 inhibitor GSK650394 (GSK, 1 μM) for 7 days, and cell viability was assessed with trypan blue exclusion assay. Cell survival results are displayed relative to R1881 plus dexamethasone control (first bar). Error bars represent standard error of the mean, and asterisk denotes p<0.05 using a paired Student’s t test. B Median time to tumor initiation of LAPC4 cells ectopically expressing SGK1-Flag versus control within a castrate host is 76 days versus not reached (log-rank test p=0.002). C Time to 22Rv1 xenograft establishment showing median castration-resistant tumor-free survival of mice bearing 22Rv1 SGK1-Flag tumors versus control tumors 22 versus 37 days (p=0.033)