Abstract A110: Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells

Androgen receptor (AR) over-expression, mutation and the emergence of AR splice variants (AR-Vs) promote disease-driving AR signaling in castration-resistant prostate cancer (CRPC). In addition to the sustained AR activation, apoptosis evasion adds extra hurdle for treating hormone refractory prostate cancer (PCa). To overcome these challenges, we firstly designed isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and then rationally combined ITC-ARi with glutathione (GSH) biosynthesis inhibitor buthionine sulphoximine (BSO) to efficiently downregulate AR/AR-V and induce ferroptosis in enzalutamide (Enz)-resistant PCa cells. Naturally occurring ITCs (e.g., sulforaphane and phenylethyl isothiocyanate) display pleotropic anti-PCa activities. A representative ITC-ARi 2-63 is designed by incorporating ITC into an AR ligand scaffold. The sulfhydryl reactivity of ITC is transiently masked as N-acetyl cysteine (NAC) conjugate that gradually releases parental free ITC in aqueous solution. 2-63 antagonizes AR transactivation, downregulates AR/AR-V7, and upregulates cellular stress markers Hsp70 and heme oxygenase-1 (HO-1), indicating the disruption of heat shock proteins (Hsps) and the activation of Nrf2 pathway. Because the anticancer effects of ITC/ITC-NAC conjugate are attenuated by GSH-participated conjugation or thiol exchange, we rationally combined 2-63 with BSO for enhanced anti-PCa activities. 2-63 and BSO synergistically reduce viability of multiple PCa cell lines (VCaP, C4-2 and 22Rv-1), and the noncancerous prostatic RWPE-1 cell is much less affected. Because 2-63 plus BSO does not upregulate typical apoptosis markers, we investigated alternative cell death mechanism in combinatorial treatment. Drug combination-caused cell viability loss is effectively rescued by iron chelator deferoxamine (DFO), antioxidants α-tocopherol (α-Toc) and ferrostatin-1 (Fer-1), or the inhibitor of HO-1 Zinc (II) protoporphyrin IX (ZnPP). Both Fer-1 and α-Toc are potent radical scavengers blocking lipid hydroperoxide generation, and ZnPP suppresses HO-1-catalyzed upregulation of intracellular Fe2+. The reverse of growth suppression by DFO, antioxidants and ZnPP supports the induction of ferroptosis, a regulated non-apoptotic cell death caused by reactive oxygen species (ROS)- and iron-dependent lipid peroxidation. 2-63 and BSO also more efficiently downregulate AR/AR-V than 2-63 alone in Enz-resistant PCa cells. Interestingly, ferroptosis-preventing DFO and Fer-1 do not rescue AR/AR-V7 and are unable to suppress the upregulation of Hsp70 and HO-1. Our results suggest that the synergism of 2-63 and BSO occurs through increasing drug accessibility to cellular targets, expanding availability of iron and potentially affecting glutathione peroxidase 4 (GPX4) activity, the most important cellular defense to eliminate lipid hydroperoxide. Combining ITC-ARi and GSH-depleting agents could be a new concept leading to effective CRPC treatment. Citation Format: Zhihui Qin, Liping Xu, Siyu Ou. Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A110. doi:10.1158/1535-7163.TARG-19-A110