Differential targeting of androgen and glucocorticoid receptors induces ER stress and apoptosis in prostate cancer cells: a novel therapeutic modality

Abstract

Androgen (AR) and glucocorticoid (GR) receptor signaling play opposing roles in prostate tumorigenesis: in prostate, AR acts as an oncogene, and GR is a tumor suppressor. Recently, we found that non-steroidal phyto-chemical Compound A (CpdA) is AR/GR modulator acting as anti-inflammatory anti-androgen. CpdA inhibits AR and prevents GR transactivation while enhancing GR transrepression. GR and AR are controlled by proteasomal degradation. We found that prolonged exposure of LNCaP, LNCaP-GR, DU145 and PC3 prostate carcinoma (PCa) cells to proteasome inhibitor Bortezomib (BZ) caused AR degradation and GR accumulation. BZ enhanced CpdA ability to inhibit AR and to augment GR transrepression. We also found that CpdA+BZ differentially regulated GR/AR to cooperatively suppress PCa cell growth and survival and to induce endoplasmic reticulum stress (ERS). Importantly, CpdA+BZ differentially regulated GR-responsive genes. CpdA+BZ blocked activation of glucocorticoid-responsive pro-survival genes, including SGK1, but activated BZ-induced ERS-related genes BIP/HSPA5 and CHOP /GADD153. Using ChIP, we showed that SGK1, BIP/HSPA5 and CHOP regulation was due to effects of CpdA and CpdA+BZ on GR loading on their promoters. We also found that AR and GR are abundant in advanced PCa from patients treated by androgen ablation and/or chemotherapy: 56% of carcinomas from treated patients expressed both receptors, and the other 27% expressed either GR or AR. Overall, our data validate the concept of dual AR/GR targeting in prostate cancer (PC) and suggest that BZ combination with dual-target steroid receptor modulator CpdA has high potential for PC therapy.

Figures

Figure 1

AR and GR are highly expressed in PCa from patients after androgen ablation or chemotherapy. Expression of AR (A.1–A.6) and GR (B.1–B.6) in benign prostate (A.1 and B.1), prostate carcinoma from untreated patient (A.2 and B.2) (as in ref. or Sup. 2), prostate carcinomas from patients treated with androgen ablation or chemotherapy (A.3–A.6 and B.3–B.6). (C) AR and GR expression in PCa from treated patients. The number of prostate epithelial cells with either GR or AR nuclear signal was evaluated by + to +++ scoring. Tumors with GR/AR staining intensity ++/+++ were considered GR+ or AR+ respectively. Note: (1) extensive nuclear AR and GR expression in benign prostate; absence of GR in untreated PCa and high GR and AR nuclear staining in treated PCa tissues; (2) overlapping patterns of expression of AR and GR in PCa from treated patients.

Figure 2

Differential AR and GR regulation by Bortezomib correlates with PC cell apoptosis. (A) Western blot analysis of AR/GR in whole-cell lysates of LNCaP, LNCaP-GR, PC3 and DU145 treated with 10−6–10−10 M BZ for 24 h (A.1) and 10−8 M BZ for 2–32 h (A.2). (B) Immunostaining for AR/GR in cells treated with 10−8 M BZ (24 h). (C and D) Western blot analysis of PARP cleavage. (C) LNCaP and LNCaP-GR treated with 10−6–10−9 M BZ (32 h). (D) LNCaP-GR-shNS (expressing non-silencing shRNA) and LNCaP-GR-shGR treated with 10−8 M BZ (32 h). Note: (1) GR upregulation and AR downregulation after BZ correlates with PARP cleavage; (2) decreased PARP cleavage in LNCaP-GR-shGR cells.

Figure 3

Bortezomib increases anti-PC activity of CpdA. (A.1) Proliferation of LNCaP-GR cells treated with BZ or with CpdA (10−5–10−10 M, 4 d). (A.2) Proliferation of LNCaP-GR-shGR, LNCaP-V and LNCaP-GR-shNS cells treated with 5 × 10−7 M CpdA and 10−8 M BZ (4 d); (B) Apoptosis after treatment with 10−8 M BZ and 5 × 10−7 M CpdA (24 h) in LNCaP-GR-shGR, LNCaP-GR-shNS, PC3 and DU145 as measured by (1) PARP cleavage using western blotting (B.1), (2) immunofluorescent microscopy with anti-bodies against cleaved PARP (B.2) and (3) quantitative analysis of cleaved PARP staining and propidium iodide (PI) incorporation (B.3) using Celligo cell analyzer (Cyntellect Inc.). (C) Caspase activity in LNCaP-GR treated as described in (B); data were normalized to control (0.01% DMSO). (A.1, B.3 and C) Statistically significant differences compared with vehicle control; (A.2) statistically significant differences compared with LNCaP-GR-shNS cells. Note: CpdA+BZ cooperate at low, singly ineffective doses to suppress PCa cell growth and induce apoptosis.

Figure 4

Effect of Bortezomib on AR and GR transcription activity. (A) Western blot of cytoplasmic and nuclear AR/GR in LNCaP-GR (A.1) and LNCaP (A.2) treated with CpdA (5 × 10−7 M), DHT (10−7 M), FA (10−7 M) and BZ (10−8 M) for 16 h. (B) Assessment of GR activity (B.1) and AR activity (B.2) using MMTV-Luciferase reporter in LNCaP-GR, PC3 and LNCaP cells treated as in (A) for 24 h; *p < 0.05 statistically significant difference compared with vehicle control; #p < 0.05 difference compared with BZ treated cells. (C) DNA binding by GR in LNCaP-GR (C.1) and AR in LNCaP-V (C.2) cells treated as in (A) for 16 h (EMSA). AR/GR-binding hormone responsive element (HRE) labeled with infrared dye IR-680. (C.3 and C.4) Confirmation of DNA complexes formed with GR in LNCaP-GR cells stimulated with FA (C.3) and AR in LNCaP cells stimulated with DHT (C.4) using dilutions with non-labeled HRE and mutated HRE as described in Materials and Methods. Note: CpdA+BZ strongly inhibit AR DNA-binding and transcriptional activity.

Figure 5

Bortezomib enhances CpdA ligand properties as a selective modulator of GR. (A) Semiquantitative RT-PCR analysis of GR-target gene expression in LNCaP-GR treated as in Figure 4A (24 h). (B.1) Analysis of GR promoter occupancy for SGK1, BIP/HSPA5 and CHOP /GADD153 in LNCaP-GR treated as in Figure 4A (16 h). (B.2) Positive control (Sup. 3): GAPDH promoter occupancy by Pol II; PCR products of ChIP-ed GAPDH promoter fragments and whole-cell Pol II analyzed by western blot. (B.3) Input: whole-cell GR analyzed by western blotting and PCR products of SGK1 and CHOP promoters before ChIP (see also Sup. 3). As a negative control, DNA ChIP-ed with non-specific rabbit IgG was used as described in Materials and Methods.

Figure 6

Bortezomib enhances CpdA ability to induce GR transrepression. (A) Evaluation of NFκB (A.1) and AP-1 (A.2) activity by Luciferase assay in LNCaP-GR cells. Cells were transfected with 3 × κB-Luciferase and AP-1-Luciferase reporters and pre-treated with 0.01% DMSO (control), 5 × 10−7 CpdA and 10−8 BZ for 16 h and, where it is indicated, induced with 1 µg/mL IL-1 for 12 h. *p < 0.05 statistically significant difference compared with control; #p < 0.05 statistically significant difference compared with CpdA alone. (B) Comparison of CpdA, BZ and BZ + CpdA effects on NFκB and AP-1 in LNCaP-V and LNCaP-GR cells induced with IL-1 and transfected as in Figure 6A. *p < 0.05 statistically significant difference compared with LNCaP-V; Note: (1) Bortezomib enhances CpdA ability to inhibit activity of NFκB and AP-1; (2) effect of CpdA, BZ and CpdA+BZ on NFκB and AP-1 is GR-dependent and more pronounced in LNCaP-GR cells.

Figure 7

Treatment with Bortezomib and CpdA cooperatively induces ERS through upregulation of CHOP /GADD153. (A and B) Western blot analysis of ERS regulators and SGK1/2 (anti-SGK1 Abs recognized both SGK1 and SGK2) in nuclear and cytoplasmic fractions from LNCaP-GR (A and B.1) and LNCaP-V (B.2) cells and whole-cell lysate from PC3 (B.3) and LNCaP-GR cells (B.4) treated as in Figure 4. (A) for 24 h. (C.1) Immunostaining of GR and CHOP /GADD153 in LNCaP-GR and LNCaP-GR-CHOP cells treated with 10−9 M BZ and 5 × 10−7 M CpdA for 48 h. Effect of CHOP expression on cell proliferation (C.2) and apoptosis (C.3). CHOP expression and PARP cleavage evaluated by western blot in in LNCaP-GR-shNS, LNCaPGR-CHOP and LNCaP-GR-shCHOP treated with 5 × 10−7 M CpdA, 10−7 M FA and 10−9 M BZ for 72 h (C.2) and 48 h (C.3). *p < 0.05 statistically significant difference compared with control; #p < 0.05 statistically significant difference compared with LNCaP-GR-shNS cells. Note: (1) in contrast to FA+BZ, CpdA+BZ upregulated BIP/HSPA5, CHOP /GADD153 and ATF2 but did not induce SGK1/2; (2) CHOP /GADD153 is important for CpdA+BZ anti-PC effect.