Glucocorticoid receptor activation inhibits chemotherapy-induced cell death in high-grade serous ovarian carcinoma

Abstract

Objectives: To test the hypothesis that glucocorticoid receptor (GR) activation increases resistance to chemotherapy in high-grade serous ovarian cancer (HGS-OvCa) and that treatment with a GR antagonist will improve sensitivity to chemotherapy.

Methods: GR expression was assessed in OvCa cell lines by qRT-PCR and Western blot analysis and in xenografts and primary human tumors using immunohistochemistry (IHC). We also examined the effect of GR activation versus inhibition on chemotherapy-induced cytotoxicity in OvCa cell lines and in a xenograft model.

Results: With the exception of IGROV-1 cells, all OvCa cell lines tested had detectable GR expression by Western blot and qRT-PCR analysis. Twenty-five out of the 27 human primary HGS-OvCas examined expressed GR by IHC. No cell line expressed detectable progesterone receptor (PR) or androgen receptor (AR) by Western blot analysis. In vitro assays showed that in GR-positive HeyA8 and SKOV3 cells, dexamethasone (100nM) treatment upregulated the pro-survival genes SGK1 and MKP1/DUSP1 and inhibited carboplatin/gemcitabine-induced cell death. Concurrent treatment with two GR antagonists, either mifepristone (100nM) or CORT125134 (100nM), partially reversed these effects. There was no anti-apoptotic effect of dexamethasone on chemotherapy-induced cell death in IGROV-1 cells, which did not have detectable GR protein. Mifepristone treatment alone was not cytotoxic in any cell line. HeyA8 OvCa xenograft studies demonstrated that adding mifepristone to carboplatin/gemcitabine increased tumor shrinkage by 48% compared to carboplatin/gemcitabine treatment alone (P=0.0004).

Conclusions: These results suggest that GR antagonism sensitizes GR+ OvCa to chemotherapy-induced cell death through inhibition of GR-mediated cell survival pathways.

Keywords: Chemotherapy; GR antagonist; Mifepristone; Ovarian cancer.

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Figure 1. GR expression by Western blot and qRT-PCR in ovarian cancer cell lines

A) GR protein expression in OvCa cell lines by Western blot (B) GR mRNA transcript levels in OvCa cell lines. Transcript levels were first normalized to Actin-B mRNA expression and are shown as a ratio compared to Monty-1 cell line normalized GR (NR3C1) transcript expression. MDA-MB-231 GR (NR3C1) mRNA expression was used as a control. The error bars represent ± standard error of the mean (SEM).

Figure 2. Evaluation of GR target genes SGK1 and MKP1 mRNA expression following dex, dex/mif, or dex/CORT125134 treatment

OvCa cell lines HeyA8 (A), Monty-1 (B), and SKOV3 (C) were treated for four hours with either vehicle (ethanol), dex (100 nM), dex (100 nM) with mif (100 nM) or dex with CORT125134 (100 nM). SGK1 and MKP1/DUSP1 mRNA expression was first normalized to Actin-B mRNA levels and is shown as a ratio relative to normalized transcript expression from vehicle-treated cells. * P

Figure 3. Continuous microscopic imaging analysis (Incucyte) of cell death following treatment with chemotherapy +/- mifepristone or +/- CORT125134

OvCa cell lines HeyA8 (A), SKOV3 (B), and IGROV-1 (C) were treated with dex (100nM) +/- mif (1 μM) or +/- CORT125134 (1 μM). This treatment was followed one hour later by gemcitabine (250 nM)/carboplatin (120 nM) treatment. Error bars represent ± SEM of triplicate wells.

Figure 4. Carboplatin/gemcitabine +/- mifepristone treatment of mice bearing HeyA8 tumor xenografts

Combined data from two independent in vivo experiments. Total tumor burden is indicated at days 38-39 after initial treatment, P=0.0004 between gem/carbo (0.36g, range 0.27g-0.58g) and mif/gem/carbo (0.17g, range 0.10g-0.26g) groups. Error bars represent ± SEM of tumor weights in grams, g.

Figure 5. Primary human HGS-OvCa GR expression by IHC