Selective Glucocorticoid Receptor Modulators (SGRMs) Delay Castrate-Resistant Prostate Cancer Growth

Abstract

Increased glucocorticoid receptor (GR) expression and activity following androgen blockade can contribute to castration-resistant prostate cancer (CRPC) progression. Therefore, we hypothesized that GR antagonism will have therapeutic benefit in CRPC. However, the FDA-approved nonselective, steroidal GR antagonist, mifepristone, lacks GR specificity, reducing its therapeutic potential. Here, we report that two novel nonsteroidal and highly selective GR modulators (SGRM), CORT118335 and CORT108297, have the ability to block GR activity in prostate cancer and slow CRPC progression. In contrast to mifepristone, these novel SGRMs did not affect androgen receptor (AR) signaling, but potently inhibited GR transcriptional activity. Importantly, SGRMs decreased GR-mediated tumor cell viability following AR blockade. In vivo, SGRMs significantly inhibited CRPC progression in high GR-expressing, but not in low GR-expressing xenograft models. Transcriptome analysis following AR blockade and GR activation revealed that these SGRMs block GR-mediated proliferative gene expression pathways. Furthermore, GR-regulated proliferation-associated genes AKAP12, FKBP5, SGK1, CEBPD, and ZBTB16 are inhibited by CORT108297 treatment in vivo Together, these data suggest that GR-selective nonsteroidal SGRMs potently inhibit GR activity and prostate cancer growth despite AR pathway inhibition, demonstrating the therapeutic potential of SGRMs in GR-expressing CRPC. Mol Cancer Ther; 16(8); 1680-92. ©2017 AACR.

Figure 1. Variable GR expression and transcriptional activity in PC cells is dependent on AR function

For A-C LAPC4 and CWR-22Rv1 cells were treated with vehicle (Veh), R1881 (R, 1nM) +/− dexamethasone (D, 100nM) for three days. A. Cell lysates were analyzed by Western blot for AR (full length (fl) and AR-V7 splice variant are indicated), GR, and β-actin. B. RNA was isolated and SGK1 and KLK3 mRNA levels were quantified by qRT-PCR. C. Supernatants were analyzed by ELISA to measure PSA (top panels) and cell lysates were analyzed by Western blot for SGK1, and β-actin (lower panels). D. Cells were treated as in A for 10 days and assessed for the total number of viable cells by trypan blue exclusion assay. Data is representative of 3 independent experiments. Error bars represent SEM. Statistical significance was determined by Student’s t-test, *p<0.05, **p<0.01.

Figure 2. Novel GR-selective modulators do not impact AR function

A. LAPC4 cells were treated with vehicle (Veh) or R1881 (R, 1nM) +/− enzalutamide (E, 10µM), mifepristone (Mif, 100nM), CORT108297 (297, 1µM), or CORT118335 (335, 1µM) for three days. RNA was isolated and SGK1 and KLK3 mRNA levels were quantified by qRT-PCR. B. Cells were treated as in A, and cell lysates were analyzed by Western blot for AR, SGK1, and β-actin (left panel) and supernatant was analyzed by ELISA to measure PSA (right panel). C. Cells were treated as in A for ten days and assessed for the total number of viable cells by trypan blue exclusion assay. Data are representative of 3 independent experiments. Error bars represent SEM. Statistical significance was determined by Student’s t-test, *p<0.05, **p<0.01.

Figure 3. SGRMs block GR activity subsequent to AR blockade with enzalutamide

For A-C LAPC4 and CWR-22Rv1 cells were treated with vehicle (Veh), or R1881 (R, 1nM) and enzalutamide (E, 10µM), +/− dexamethasone (D, 100nM), +/− CORT108297 (297, 1µM), or CORT118335 (335, 1µM) for three days. A. Cell lysates were utilized for Western blot analysis for AR (full length (fl) and AR-V7 splice variant are indicated), GR, SGK1, and β-actin. B. RNA was isolated and SGK1 and KLK3 mRNA levels were quantified by qRT-PCR. C. Supernatants were analyzed by ELISA to measure PSA. Data are representative of 3 independent experiments. Error bars represent SEM. Statistical significance was determined by Student’s t-test, *p<0.05, **p<0.01.

Figure 4. SGRMs inhibit PC growth following AR blockade

A. Cells were treated as in Figure 1 and 3 then assessed for the total number of viable cells by digital counting of nuclei every 4 hours over 7 or 10 days. Error bars represent SEM. Statistical significance was determined by one-way ANOVA, **p<0.01. B. Mice were castrated and implanted with testosterone pellets. One week later, mice were injected subcutaneously with LAPC4 or CWR-22Rv1 cells. Following the establishment of primary tumors, mice were castrated and following 7 or 14 days of recovery, and began treatments of vehicle (Veh), 20mg/kg CORT108297 (297), or CORT118335 (335). C. Kaplan-Meier survival curves are depicted showing time to CRPC development defined as doubling of tumor volume. *Denotes p<0.05 with Log-rank test. D. LAPC4 and CWR-22Rv1 tumors were dissected from mice prior to castration (UNTR), and at the endpoint (tumor doubling from size at castration) and formalin-fixed for immunohistochemistry. Serial sections of tumor tissue were labeled with antibodies for GR and AR. Representative images are shown.

Figure 5. SGRMs inhibit GR-activated PC cell proliferation pathways

A. LAPC4 and CWR-22Rv1 cells were treated with R1881 (R, 1nM) and enzalutamide (E, 10µM) for 3 days, then dexamethasone (D, 100nM) +/− CORT108297 (297, 1µM), or CORT118335 (335, 1µM) for 6 hours. RNA-seq data was analyzed by Ingenuity Pathway Analysis (IPA). Proliferation/growth and apoptosis pathways are shown. Pathways that are significantly altered (p<0.05) are shown as activated (red) or repressed (blue). Mice treated as in Fig. 4 were euthanized following 7 days of treatment (Veh n=6; 297 n=9) B or at tumor doubling C (Veh n=6; bottom quartile n=4; top quartile n=4), the RNA was isolated from dissected tumors and analyzed by qRT-PCR. Error bars represent SEM. Statistical significance was determined by Student’s t-test, *p<0.05, **p<0.01. D. Schematic showing SGRMs competitively binds to GR to block GR-mediated PC cell proliferation and PC progression subsequent to AR-blockade.