A novel androgen receptor antagonist JJ-450 inhibits enzalutamide-resistant mutant ARF876L nuclear import and function

Background: Castration-resistant prostate Cancer can develop resistance to enzalutamide because of Androgen Receptor (AR) point mutations, AR overexpression, constitutively active AR splice variants, and/or elevated intratumoral androgen synthesis. The point mutation AR^F876L was reported to be stimulated, instead of inhibited, by enzalutamide, thus contributing to enzalutamide resistance. We have recently developed JJ-450 as a novel AR antagonist with the potential to treat enzalutamide-resistant castration-resistant prostate Cancer (CRPC).

Methods: We employed several assays to determine the impact of JJ-450 and enzalutamide on prostate Cancer cell lines expressing green fluorescent protein (GFP)-AR^F876L . These assays include a prostate-specific antigen enhancer/promoter-based luciferase assay to determine AR transcriptional activity, a quantitative real-time polymerase chain reaction assay, and Western blot analysis to detect expression of AR-target genes at the messenger RNA and protein level, fluorescence microscopy to show AR subcellular localization, and a 5-bromo-2'-deoxyuridine assay to measure prostate Cancer cell proliferation.

Results: As expected, enzalutamide inhibited wild-type (WT) AR but not AR^F876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and AR^F876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-AR^F876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-AR^F876L . To further evaluate JJ-450 inhibition of AR^F876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-AR^F876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-AR^WT , but not in the C4-2-GFP-AR^F876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-AR^WT , but not of the C4-2-GFP-AR^F876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines.

Conclusion: JJ-450 inhibits enzalutamide-resistant AR^F876L mutant nuclear translocation and function. Our findings suggest that JJ-450 and its analogs should be further developed to provide a potential new approach for the treatment of enzalutamide-resistant CRPC.

C4-2; CRPC; JJ-450; LNCaP; PC3; prostate cancer.