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Published on: November 15, 2013
A novel androgen receptor antagonist JJ-450 inhibits enzalutamide-resistant mutant ARF876L nuclear import and
Zeyu Wu1,2, Ke Wang2, Zhenyu Yang1,2
1Department of Urology, Xiangya School of Medicine, Xiangya School of Medicine, Central South University, Changsha, China.
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 ARF876L 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)-ARF876L . 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 ARF876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and ARF876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-ARF876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-ARF876L . To further evaluate JJ-450 inhibition of ARF876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-ARF876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-ARWT , but not in the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-ARWT , but not of the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines.
Conclusion:
JJ-450 inhibits enzalutamide-resistant ARF876L 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.
Insights
A new drug, JJ-450, effectively inhibits the F876L mutation in androgen receptors (AR) that causes resistance to enzalutamide in prostate cancer. This finding suggests JJ-450 as a promising treatment for enzalutamide-resistant castration-resistant prostate cancer (CRPC).
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Castration-resistant prostate cancer (CRPC) can develop resistance to enzalutamide through androgen receptor (AR) mutations, overexpression, splice variants, or increased androgen synthesis.
- The AR F876L mutation confers enzalutamide resistance by being stimulated rather than inhibited by the drug.
- JJ-450 is a novel AR antagonist developed to address enzalutamide-resistant CRPC.
Purpose of the Study:
- To evaluate the efficacy of JJ-450 against enzalutamide-resistant prostate cancer cells harboring the AR F876L mutation.
- To compare the activity of JJ-450 and enzalutamide on AR transcriptional activity, nuclear localization, gene expression, and cell proliferation.
Main Methods:
- Prostate cancer cell lines expressing wild-type (WT) AR or AR F876L were used.
- Assays included luciferase reporter assays for AR transcriptional activity, quantitative real-time PCR and Western blot for AR-target gene expression, fluorescence microscopy for AR subcellular localization, and BrdU assays for cell proliferation.
Main Results:
- Enzalutamide inhibited WT-AR but not AR F876L activity, whereas JJ-450 inhibited both WT-AR and AR F876L.
- JJ-450, unlike enzalutamide, inhibited nuclear translocation and AR-target gene expression in cells with the AR F876L mutation.
- JJ-450 effectively inhibited proliferation of both WT-AR and AR F876L expressing prostate cancer cells, while enzalutamide only inhibited WT-AR cells.
Conclusions:
- JJ-450 demonstrates potent inhibition of enzalutamide-resistant AR F876L mutant nuclear translocation and function.
- JJ-450 and its analogs represent a potential new therapeutic strategy for treating enzalutamide-resistant CRPC.
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