Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer
Shidong Lv, … , Wenhua Huang, Zhou Wang
Shidong Lv, … , Wenhua Huang, Zhou Wang
Published December 17, 2020
Citation Information: J Clin Invest. 2021;131(4):e141335. https://doi.org/10.1172/JCI141335.
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Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer

Abstract

Nuclear localization of the androgen receptor (AR) is necessary for its activation as a transcription factor. Defining the mechanisms regulating AR nuclear localization in androgen-sensitive cells and how these mechanisms are dysregulated in castration-resistant prostate cancer (CRPC) cells is fundamentally important and clinically relevant. According to the classical model of AR intracellular trafficking, androgens induce AR nuclear import and androgen withdrawal causes AR nuclear export. The present study has led to an updated model that AR could be imported in the absence of androgens, ubiquitinated, and degraded in the nucleus. Androgen withdrawal caused nuclear AR degradation, but not export. In comparison with their parental androgen-sensitive LNCaP prostate cancer cells, castration-resistant C4-2 cells exhibited reduced nuclear AR polyubiquitination and increased nuclear AR level. We previously identified 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) in a high-throughput screen for its inhibition of androgen-independent AR nuclear localization in CRPC cells. The current study shows that CPPI is a competitive AR antagonist capable of enhancing AR interaction with its E3 ligase MDM2 and degradation of AR in the nuclei of CRPC cells. Also, CPPI blocked androgen-independent AR nuclear import. Overall, these findings suggest the feasibility of targeting androgen-independent AR nuclear import and stabilization, two necessary steps leading to AR nuclear localization and activation in CRPC cells, with small molecule inhibitors.

Authors

Shidong Lv, Qiong Song, Guang Chen, Erdong Cheng, Wei Chen, Ryan Cole, Zeyu Wu, Laura E. Pascal, Ke Wang, Peter Wipf, Joel B. Nelson, Qiang Wei, Wenhua Huang, Zhou Wang

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Figure 5

CPPI enhanced nuclear AR polyubiquitination and degradation, increased AR association with MDM2 in the nucleus, and inhibited AR nuclear import.

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CPPI enhanced nuclear AR polyubiquitination and degradation, increased A...
(A) Quantification of tumor volume changes after CPPI treatment (50 mg/kg/d) in C4-2 xenograft tumors (n = 5). (B) Representative images of H&E, Ki-67 immunostaining, and AR immunofluorescent staining in C4-2 xenograft tumors treated with CPPI or vehicle (n = 3). (C) Patient-derived explants were treated with CPPI. Effects of CPPI on AR expression with representative sections are shown. (D) Representative fluorescent images of total (green signal) and ReAsH pulse-labeled (red signal) GFP-AR-4Cys in response to CPPI (30 μM) in LNCaP or C4-2 cells with or without MG132. (E) Western blot analysis of endogenous AR in nuclear and cytoplasmic extracts of LNCaP and C4-2 cells after CPPI treatment with or without MG132 as in (C). (F) Western blot analysis of ubiquitin and AR in C4-2 cells treated with or without CPPI for 24 hours in the presence of MG132. (G) IP samples shown in E were also analyzed by Western blot with additional indicated antibodies. (H) Western blot analysis of AR expression in MDM2 knockdown or control C4-2 cells after CPPI treatment. (I) Time course of GFP-AR localization in transfected COS-7 cells treated with or without CPPI in the presence of MG132 in CSS medium. Nuclear GFP-AR quantification data are shown at right (n = 6). (J) AR, AR S81, and PSA were detected by Western blot in LNCaP and C4-2 cells treated with CPPI. Quantitative data are presented as mean ± SD, and all data represent 1 of at least 2 independent experiments with consistent results. Unpaired t test (I) was used to determine statistical significance. **P < 0.01; ***P < 0.001. Original magnification, ×40.