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Androgen receptor (AR) signalling drives neoplastic growth and therapy resistance in prostate cancer. Recent clinical data show that docetaxel combined with androgen deprivation therapy improves outcome in hormone-sensitive disease. We studied whether testosterone and AR signalling interferes with docetaxel treatment efficacy in castration-resistant prostate cancer (CRPC). We found that testosterone supplementation significantly impaired docetaxel tumour accumulation in a CRPC model, resulting in decreased tubulin stabilisation and antitumour activity. Furthermore, testosterone competed with docetaxel for uptake by the drug transporter OATP1B3. Irrespective of docetaxel-induced tubulin stabilisation, AR signalling by testosterone counteracted docetaxel efficacy. AR-pathway activation could also reverse long-term tumour regression by docetaxel treatment in vivo. These results indicate that to optimise docetaxel efficacy, androgen levels and AR signalling need to be suppressed. This study lends evidence for continued maximum suppression of AR signalling by combining targeted therapeutics with docetaxel in CRPC.

Introduction Castration resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, driven by adrenal precursors and potentially de novo steroid synthesis in other organ tissues including prostate. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1 and the AR has been demonstrated to prolong survival of CRPC patients. In this study, we created a co-culture model using human prostate and adrenal tumours to study abiraterone resistance. Materials and Methods Human androgen-dependent PC (VCaP) and CPRC clones were cultured with substrates for de novo androgen synthesis or with adrenal androgens, or cultured with human adrenal cells (H295R) and treated with either the CYP17A1 inhibitor abiraterone or the antiandrogen MDV3100. Male mice bearing VCaP tumours and human adrenal H295R xenografts were castrated and treated with placebo or abiraterone. Tumour response was assessed by tumour growth, PSA release, steroid quantitation by (LC/MS-MS), immunohistochemistry and mRNA expression analysis of steroidogenic enzymes and nuclear receptors. Results In vitro, physiological levels of adrenal androgen precursors DHEA and androstenedione induced cell growth in parental and CRPC VCaP sub clones, whereas precursor steroids pregnenolone and progesterone for de novo synthesis did not. In a co-culture model, abiraterone blocked H295R-induced growth of VCaP cells. Likewise, in vivo, H295R tumours stimulated castration-resistant VCaP growth. This stimulative effect was inhibited by abiraterone, reducing - but not fully blocking - growth and PSA production. In the absence of H295R tissue, VCaP xenografts grew slow but became castration resistant nonetheless. In contrast to the observed effects on VCaP growing in castrate animals bearing H295R tumours, abiraterone was unable to inhibit the slow VCaP growth and low PSA production in castrate mice without H295R xenografts. LC/MS-MS analysis of plasma and tumour tissue could not confirm increased de novo production of androgens. Castrate and abiraterone-resistant VCaP tumours were characterised by increased levels of AR, AR variants and glucocorticoid receptor (GR) expression, resulting in equal AR target gene expression levels. Conclusions Our data indicate that AR ligand dependent regrowth of CRPC is predominantly supported via adrenal steroid production. Abiraterone resistant disease of VCaP relies on AR overexpression, expression of ligand independent AR variants and GR signalling. Citation Format: Jan Matthijs Moll, Johannes Hofland, Wilma Teubel, Corrina M.A. de Ridder, Anne E. Taylor, Ralph Graeser, Wiebke Arlt, Guido W. Jenster, Wytske M. van Weerden. Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C97.

Uit klinisch en preklinisch onderzoek blijkt dat in castratieresistente prostaattumoren (CRPC) reactivatie van de androgeenreceptor (AR) plaatsvindt, ondanks serumtestosteronspiegels onder de castratiegrens.

De behandelmogelijkheden voor patienten met gemetastaseerd castratieresistent prostaatcarcinoom (mCRPC) zijn de laatste jaren sterk toegenomen door de introductie van nieuwe middelen als cabazitaxel, abiraterone en enzalutamide.

Castratieresistent prostaatcarcinoom (CRPC) kan worden behandeld met de hormoonsynthese (CYP17A1) remmer abiraterone, of het antiandrogeen enzalutamide.

Introduction: In the majority of castration resistant prostate cancers (CRPC), the AR is still active despite low serum testosterone levels. Among other mechanisms of AR-activation, intratumoral androgen synthesis, either via conversion of adrenal androgens or de novo synthesis from cholesterol, is a mechanism of castration resistance. In a previous study, we found that clinical CRPC samples only expressed markers for conversion of adrenal androgens and not for de novo synthesis. In this study, we compared growth stimulation of hormone-naïve prostate cancer (PC) and CRPC cell lines by adrenal androgens (conversion) or androgen precursors (de novo synthesis) and subsequently tested a novel co-culture model to mimic adrenal stimulation of (CR)PC. Materials and methods: VCaP and DuCaP CRPC lines were generated by long-term culturing in steroid stripped medium (DCC) with or without the anti-androgens bicalutamide or flutamide. To test cell growth stimulation, VCaP and DuCaP cells and their CRPC sublines were cultured in the presence of androgen precursors (pregnenolone and progesterone) or adrenal androgens (DHEA and androstenedione) at levels found in men. Cells were treated with either vehicle, the CYP17A1-inhibitor Abiraterone (0.1 μM) or the anti-androgen MDV3100 (1 μM). Cell proliferation was assessed by MTT-assay on day 9 with each experiment performed in triplicate. In the co-culture model, VCaP cells were cultured with human adrenal (H295R) cells in separate compartments between which only medium could diffuse freely, and treated with vehicle, Abiraterone (1 μM) or DHT (0.1 nM). Results: In VCaP and DuCaP and their CRPC derivatives, androgen precursors pregnenolone and progesterone did not induce additional cell growth compared to cell growth in DCC. In contrast, physiological levels of adrenal androgens stimulated cell growth, comparable to optimal stimulus by DHT. Adrenal androgen concentrations found in serum of H295R-bearing mice induced a similar growth-response. Treatment with Abiraterone could not block adrenal androgen induced growth, while MDV3100 blocked steroid-induced growth in all conditions. Co-culturing of VCaP cells with H295R cells induced optimal cell growth, which could be inhibited by treatment with Abiraterone. Conclusion: In VCaP and DuCaP cells and the CRPC sublines, the substrate for conversion of adrenal androgens induced growth, while precursors of de novo androgen synthesis did not. These data support our observations that growth of (CR)PC seems to rely more on conversion than on de novo androgen synthesis. Co-culturing PC cells with human adrenal cells using a 2-compartment system is a relevant model to test CRPC stimulation in vitro. We are currently generating an in vivo model by inoculating PC and H295R cells simultaneously in nude mice more accurately reflect human CRPC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C133. Citation Format: Jan Matthijs Moll, Wilma Teubel, Ian Hickson, Ralph Graeser, Guido Jenster, Wytske van Weerden. Adrenal stimulation of CRPC growth in an in vitro co-culture model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C133.