Recent evidence suggests that the use of potent androgen receptor (AR) antagonists have contributed to the increased prevalence of prostate cancer (PCa) tumors exhibiting therapy resistance. Androgen targeted therapies (ATT) inhibit the androgen/AR axis which PCa cells rely on for growth. ATT are initially effective however most men progress to metastatic castration resistant prostate cancer (mCRPC). Mechanisms of resistance include programs of epithelial plasticity (EP) such as epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD). EMT enables PCa cells to become more motile and invasive. PCa cells that undergo NEtD gain neuroendocrine (NE)-like traits. Patients with NE prostate cancer (NEPC) usually present with visceral metastasis, are refractory to ATT and have short response to chemotherapies. We aim to characterize models of therapy-induced NEtD (tNEPC) and to investigate the role of tumor plasticity in the progression to mCRPC.
To recapitulate ATT in vitro, androgen-dependent LNCaP cells were treated with either enzalutamide (Enz) (10μM) or shRNA targeting AR in culture medium containing fetal bovine serum (FBS) or charcoal stripped serum (CSS) with or without 10nM dihydrotestosterone (DHT). To model EMT, LNCaP cells were engineered to express doxycycline (Dox) inducible overexpression of EMT transcription factor Snail. Androgen deprivation in vitro induced expression of NE markers in LNCaP cells compared with FBS treated cells (mean fold increase ± SEM): neuron specific enolase (ENO2) (6.0 ± 2.5), neural cell adhesion molecule (NCAM) (10.4 ± 6.8) and midkine (MDK) (13.2 ± 2.4). Cells co-treated with Enz and DHT had increased expression of NE markers compared to DHT alone: ENO2 (2.7 ± 0.6), NCAM (2.9 ± 0.3) and MDK (2.3 ± 1.8). AR knockdown resulted in a potent induction of NE markers compared with control cells: ENO2 (8.3 ± 1.4), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). Dox-induced Snail enhanced androgen deprivation-mediated upregulation of NE markers compared with no-Dox cells: ENO2 (1.3 ± 0.1), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). To more accurately model a preclinical setting, LNCaP tumors were grown subcutaneously in severe combined immune deficiency mice. Once tumors grew to 200mm3, mice were castrated and upon tumor recurrence mice were treated with Enz (10mg/kg). At the ethical endpoint, expression of EMT and NE markers will be assessed.
Patients treated with ATTs show an increase in NE-like traits. Thus, there is a need to identify molecular mechanisms involved in this adaptive response. In vitro, we have modelled the acquisition of NE-like features in PCa cells using ATTs, EMT drivers and AR knockdown. Further investigation using these models may lead to the development of biomarkers and therapeutic targets for the clinical management of advanced PCa.
Citation Format: Ellca Ratther, Katrina G. Sweeney, Nataly Stylianou, Qiuhua Hu, Gregor Tevz, Colleen C. Nelson, Elizabeth D. Williams, Brett G. Hollier. Unravelling the role of androgen targeted therapies in epithelial plasticity during prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 818. doi:10.1158/1538-7445.AM2017-818