Glucocorticoid receptor antagonism reverts docetaxel resistance in human prostate cancer

Jan Kroon, Martin Puhr, Jeroen T Buijs, Geertje van der Horst, Daniëlle M Hemmer, Koen A Marijt, Ming S Hwang, Motasim Masood, Stefan Grimm, Gert Storm, Josbert M Metselaar, Onno C Meijer, Zoran Culig, Gabri van der Pluijm, Jan Kroon, Martin Puhr, Jeroen T Buijs, Geertje van der Horst, Daniëlle M Hemmer, Koen A Marijt, Ming S Hwang, Motasim Masood, Stefan Grimm, Gert Storm, Josbert M Metselaar, Onno C Meijer, Zoran Culig, Gabri van der Pluijm

Abstract

Resistance to docetaxel is a major clinical problem in advanced prostate cancer (PCa). Although glucocorticoids (GCs) are frequently used in combination with docetaxel, it is unclear to what extent GCs and their receptor, the glucocorticoid receptor (GR), contribute to the chemotherapy resistance. In this study, we aim to elucidate the role of the GR in docetaxel-resistant PCa in order to improve the current PCa therapies. GR expression was analyzed in a tissue microarray of primary PCa specimens from chemonaive and docetaxel-treated patients, and in cultured PCa cell lines with an acquired docetaxel resistance (PC3-DR, DU145-DR, and 22Rv1-DR). We found a robust overexpression of the GR in primary PCa from docetaxel-treated patients and enhanced GR levels in cultured docetaxel-resistant human PCa cells, indicating a key role of the GR in docetaxel resistance. The capability of the GR antagonists (RU-486 and cyproterone acetate) to revert docetaxel resistance was investigated and revealed significant resensitization of docetaxel-resistant PCa cells for docetaxel treatment in a dose- and time-dependent manner, in which a complete restoration of docetaxel sensitivity was achieved in both androgen receptor (AR)-negative and AR-positive cell lines. Mechanistically, we demonstrated down-regulation of Bcl-xL and Bcl-2 upon GR antagonism, thereby defining potential treatment targets. In conclusion, we describe the involvement of the GR in the acquisition of docetaxel resistance in human PCa. Therapeutic targeting of the GR effectively resensitizes docetaxel-resistant PCa cells. These findings warrant further investigation of the clinical utility of the GR antagonists in the management of patients with advanced and docetaxel-resistant PCa.

Keywords: docetaxel; glucocorticoid receptor; prostate cancer; therapy resistance.

© 2016 The authors.

Figures

Figure 1
Figure 1
Glucocorticoid receptor (GR) expression is enhanced in docetaxel-treated patients and docetaxel-resistant cell lines and is functionally involved in chemotherapy resistance. (A) Tissue microarray (TMA) analysis revealed the GR overexpression in prostate cancer (PCa) patient tissues treated with neoadjuvant docetaxel compared with chemonaive patients. **P<0.01. (B) Protein expression analysis revealed that GR is overexpressed in docetaxel-resistant cell lines PC3-DR, DU145-DR, and 22Rv1-DR compared with their parental counterparts. *P<0.05 vs parental and **P<0.01 vs parental. (C) Transient CRISPR/CAS9-directed deletion of the GR-1 results in reduced GR expression. **P<0.01 vs PC3-DR. (D) Enhanced docetaxel sensitivity in PC3-DR GR CRISPR cells. *P<0.05 vs PC3-DR.
Figure 2
Figure 2
Dexamethasone (DEX)-induced transcriptional activity of the GR-target genes GILZ and FKBP5 is inhibited by RU-486 and CPA. Expression of the GR-target genes GILZ and FKBP5 in PC3-(DR) and DU145-(DR) cells upon treatment with DEX and/or RU-486 or CPA. *P<0.05 vs vehicle; **P<0.01 vs vehicle; ***P<0.001 vs vehicle; $P<0.05 vs 1.0 μM DEX; $$P<0.01 vs 1.0 μM DEX; and $$$P<0.01 vs 1.0 μM DEX.
Figure 3
Figure 3
Therapeutic targeting of the glucocorticoid receptor (GR) resensitizes docetaxel-resistant cell lines to docetaxel. (A) GR antagonism with RU-486 or CPA resensitizes PC3-DR cells for docetaxel treatment after 72 h of treatment. *P<0.05 vs vehicle; **P<0.01 vs vehicle; and ***P<0.001 vs vehicle. (B) Dose-dependent (72 h) and (C) time-dependent (30 nM) antitumor effect of docetaxel upon simultaneous treatment with 3 μM RU-486/10 μM CPA and docetaxel in PC3-(DR) and DU145-(DR) cells. $P<0.05 vs parental; $$P<0.01 vs parental; $$$P<0.001 vs parental; **P<0.01 vs docetaxel-resistant line; and ***P<0.001 vs docetaxel-resistant line. (D) GR antagonism with RU-486 or CPA resensitizes AR-positive cell line 22Rv1-DR for docetaxel treatment after 72 h of treatment. $P<0.05 vs parental; $$P<0.01 vs parental; $$$P<0.001 vs parental; *P<0.05 vs docetaxel-resistant line; **P<0.01 vs docetaxel-resistant line; and ***P<0.001 vs docetaxel-resistant line. (E) Time-dependent (30 nM) antitumor effect of docetaxel upon simultaneous treatment with 3 μM RU-486 or 10 μM CPA. *P<0.05 vs vehicle; **P<0.01 vs vehicle; and ***P<0.001 vs vehicle. A full colour version of this figure is available at http://dx.doi.org/10.1530/ERC-15-0343.
Figure 4
Figure 4
Glucocorticoid receptor (GR) antagonism downregulates the expression of antiapoptotic Bcl-2 and Bcl-xL proteins. (A) Docetaxel-resistant cells undergo apoptosis upon treatment with RU-486 (3 μM) and docetaxel (30 nM). ***P<0.001 vs vehicle. (B) Bcl-2 and Bcl-xL are upregulated in docetaxel-resistant cell lines. Treatment with RU-486 (3 μM) reverses the elevated expression of Bcl-2 and Bcl-xL in docetaxel-resistant prostate cancer cells. (C) Co-incubation with an antagonist for Bcl-2 and Bcl-xL, ABT-263 (9 μM), sensitizes PC3-DR and DU145-DR cells to docetaxel treatment. *P<0.05; **P<0.01; ***P<0.001; $P<0.05 vs vehicle; $$P<0.01 vs vehicle; and $$$P<0.001 vs vehicle.
Figure 5
Figure 5
RU-486 and CPA outperform verapamil at dosages with similar P-glycoprotein (P-gp) inhibitory action. (A) P-gp mRNA expression and (B) basal activity in chemo-resistant and sensitive prostate cancer cells. *P<0.05; **P<0.01; and ***P<0.001. (C) Head-to-head comparison of the GR antagonists and verapamil on P-gp activity. *P<0.05 vs vehicle; **P<0.01 vs vehicle; and ***P<0.001 vs vehicle. (D) Head-to-head comparison of the GR antagonists and verapamil on cell viability. *P<0.05 vs vehicle; **P<0.01 vs vehicle; ***P<0.001 vs vehicle; and $$$P<0.001 vs verapamil. A full colour version of this figure is available at http://dx.doi.org/10.1530/ERC-15-0343.

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