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. 2021 Sep;40(35):5379-5392.
doi: 10.1038/s41388-021-01914-2. Epub 2021 Jul 16.

ARVib suppresses growth of advanced prostate cancer via inhibition of androgen receptor signaling

Chengfei Liu  1   2 , Cameron M Armstrong  1 , Shu Ning  1 , Joy C Yang  1 , Wei Lou  1 , Alan P Lombard  1 , Jinge Zhao  1 , Chun-Yi Wu  2   3 , Aiming Yu  2   3 , Christopher P Evans  1   2 , Clifford G Tepper  2   3 , Pui-Kai Li  4 , Allen C Gao  5   6   7
Affiliations

ARVib suppresses growth of advanced prostate cancer via inhibition of androgen receptor signaling

Chengfei Liu et al. Oncogene. 2021 Sep.

Abstract

Targeting androgen signaling with the second-generation anti-androgen drugs, such as enzalutamide (Enza), abiraterone (Abi), apalutamide (Apal), and darolutamide (Daro), is the mainstay for the treatment of castration-resistant prostate cancer (CRPC). While these treatments are effective initially, resistance occurs frequently. Continued expression of androgen receptor (AR) and its variants such as AR-V7 despite AR-targeted therapy contributes to treatment resistance and cancer progression in advanced CRPC patients. This highlights the need for new strategies blocking continued AR signaling. Here, we identify a novel AR/AR-V7 degrader (ARVib) and found that ARVib effectively degrades AR/AR-V7 protein and attenuates AR/AR-V7 downstream target gene expression in prostate cancer cells. Mechanistically, ARVib degrades AR/AR-V7 protein through the ubiquitin-proteasome pathway mediated by HSP70/STUB1 machinery modulation. ARVib suppresses HSP70 expression and promotes STUB1 nuclear translocation, where STUB1 binds to AR/AR-V7 and promotes its ubiquitination and degradation. ARVib significantly inhibits resistant prostate tumor growth and improves enzalutamide treatment in vitro and in vivo. These data suggest that ARVib has potential for development as an AR/AR-V7 degrader to treat resistant CRPC.

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Conflict of interest statement

PKL and ACG are co-inventors of a patent application of the small molecule inhibitors of androgen receptor variants (ARVib). All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Synthesis of niclosamide analogs and identification of potent inhibitors of AR/AR variants.
A The chemical structure of niclosamide and newly synthesized ARVibs. The red box indicates the structures of #7 and #31, the green box indicates chemical structures of #1 and #34. B CWR22Rv1 cells were treated with 1 µM ARVib for 16 h and whole-cell lysates were collected and subjected to western blot. C C4-2B MDVR and CWR22Rv1 cells were treated with different doses of ARVibs for 16 h and whole-cell lysates were collected and subjected to western blot. D CWR22Rv1 cells were treated with enzalutamide with or without ARVibs (#1, #34, #7, and #31), total cell numbers were determined at 0, 3, 5 days. E The CDI of enzalutamide or abiraterone with ARVibs in CWR22Rv1 and C4-2B MDVR cells were calculated. *p < 0.05. Results are the mean of three independent experiments (±S.D.). AR-FL: full-length AR, AR-Vs AR-Variants, Enza enzalutamide, Abi abiraterone, Nic niclosamide, CDI: coefficient of drug interaction.
Fig. 2
Fig. 2. ARVib disrupts the AR/AR-V7 gene expression program in enzalutamide-resistant prostate cancer cells.
A Venn diagram of RNA-seq results depicting the overlap of differentially expressed genes (DEGs) in C4-2B MDVR cells treated with 1.5 µM ARVib-7 (vs. DMSO) or 1.5 µM ARVib-31 (vs. DMSO) for 24 h. B Hierarchical clustering and heatmap visualization of the differentially expressed genes (DEGs) in ARVib-treated C4-2B MDVR cells with fold change (FC) > 1.2, as compared to vehicle (DMSO). The genes are displayed in rows and the normalized expression counts per sample are displayed in columns. Red and blue coloring indicates upregulated and downregulated relative expression levels, respectively. Middle and right, AR-FL and AR-V7 activity-signature genes that were altered in expression are displayed [5]. C GSEA of C4-2B MDVR cells treated with ARVibs (relative to DMSO control) demonstrates enrichment for suppressed expression of genes comprising PID-AR pathway (left) and the AR-V7 (right) gene signatures. The latter signature was defined by genes that are preferentially upregulated by AR-V7 [7]. D qRT-PCR analysis of the indicated genes in C4-2B MDVR cells treated with DMSO or 1 µM ARVibs for 48 h. E C4-2B MDVR cells were treated with DMSO or 1 µM ARVibs in FBS conditions for 48 h, PSA level was determined by PSA ELISA. F C4-2B cells were transiently transfected with pcDNA with or without 1 nM DHT, AR-V1, AR-V3, AR-V7, AR-V9, and AR-V12 with PSA E/P-luciferase plasmids in CS-FBS conditions, and then treated with DMSO, 5 µM abiraterone, 20 µM enzalutamide, 20 µM apalutamide, or 1 µM ARVibs for 16 h. PSA luciferase activity was examined. G 293 cells were transiently transfected with vector, AR-V7, mutant AR plus PSA promoter luciferase plasmids in FBS condition, and then treated with anti-androgens or ARVibs for 16 h. PSA luciferase activity was determined. *p < 0.05. Results are the mean of three independent experiments (±S.D.). Apal apalutamide, Bica bicalutamide.
Fig. 3
Fig. 3. ARVib inhibits AR/AR-V7 protein expression via HSP70/STUB1-mediated ubiquitin-proteasome system regulation.
A C4-2B MDVR cells were treated with 50 µg/mL cycloheximide with or without ARVibs, after 0, 2, 4, and 8 h, whole-cell lysis was collected and subjected to western blot, half-life of AR-V7 was calculated. B C4-2B MDVR cells were treated with 1 μM Nic, ARVib-7 or ARVib-31 for 16 h with 5 μM MG132 for 6 h, total cell lysates were immunoblotted with anti-AR-V7 and AR antibodies. C C4-2B MDVR cells were treated with ARVibs, whole-cell lysis was immunoprecipitated with AR antibody and blotted with ubiquitin and AR antibodies. D C4-2B MDVR cells were treated with different doses of Nic, ARVib-7, or ARVib-31 for 24 h, whole-cell lysates were collected and subjected to western blot. E GSEA demonstrates strong enrichment of the HSP70 inhibition signature in resistant cells treated with ARVibs. The signature was defined by genes with significant expression changes by HSP70 inhibition in prostate cancer cells [27]. F 293 cells were co-transfected with AR-V7, HSP70, and Flag-STUB1 for 3 days, and then treated with 1 μM Nic, ARVib-7, or ARVib-31 for 24 h, AR-V7 and STUB1 were visualized by dual immunofluorescence staining. White arrows indicate the typical staining of cells in each group. Nuclei were stained by DAPI. Scale bar 20 µm. G AR-V7, HSP70, and Flag-STUB1 were overexpressed in 293 cells, which were then treated with DMSO, Nic, ARVib-7, ARVib-31, or VER for 16 h. Total cell lysates were immunoprecipitated with anti-AR-V7 antibody and immunoblotted with anti-HA-Ub, Flag-STUB1, and AR-V7 antibodies. H CWR22Rv1 cells were treated with DMSO, 0.5 or 1.0 μM ARVib-7 for 16 h and 5 μM MG132 for additional 6 h, cytosolic and nuclear proteins were extracted and subjected to western blot. I CWR22Rv1 cells were treated with DMSO, 0.5 or 1.0 μM ARVib-7 for 16 h and 5 μM MG132 for additional 6 h, cytosolic and nuclear protein were extracted and immunoprecipitated with AR-V7 antibody and blotted with anti-Ub and AR-V7 antibodies. J CWR22Rv1 cells were transiently transfected with STUB1 siRNA for 3 days, and then treated with different doses of ARVib-7 for 16 h. Whole-cell lysates were collected and subjected to western blot. K CWR22Rv1 cells were transiently transfected with STUB1 siRNA for 3 days and then treated with different doses of ARVib-7 for 3 days, total cell numbers were determined. *p < 0.05. Results are the mean of three independent experiments (±S.D.).
Fig. 4
Fig. 4. ARVib suppresses resistant prostate cancer cell growth and induces apoptosis.
A, B CWR22Rv1 and C4-2B MDVR cells were cultured in FBS conditions and treated with DMSO, 5 µM abiraterone, 20 µM enzalutamide, 20 µM apalutamide or different doses (0.25, 0.5, 1.0 µM) of ARVibs for 3 days. Total cell numbers were determined. C CWR22Rv1 and C4-2B MDVR cells were cultured in FBS conditions and treated with DMSO, 5 µM abiraterone, 20 µM enzalutamide, 20 µM apalutamide, or 0.5 µM ARVibs. Total cell numbers were determined at 0, 3, and 5 days. D CWR22Rv1 and C4-2B MDVR cells were treated with DMSO and different doses (0.5 and 1.0 µM) of ARVibs. The colony numbers were determined. E GSEA of the HALLMARK_apoptosis pathway in C4-2B MDVR cells treated with ARVibs, as compared to DMSO. F CWR22Rv1 and C4-2B MDVR cells were treated with DMSO and different doses (0.5 and 1.0 µM) of ARVibs. Cell death rate was determined by cell death ELISA kit. *p < 0.05. Results are the mean of three independent experiments (±S.D.).
Fig. 5
Fig. 5. ARVib has improved bioavailability and suppresses advanced prostate tumor growth.
A 200 mg/Kg niclosamide, ARVib-7 or ARVib-31 were orally administrated to SD rats and rat plasma levels of each drug was determined at different time point. B Mice bearing CWR22Rv1 xenografts were treated with vehicle control, niclosamide (25 mg/Kg i.p.), ARVib-7 (25 mg/Kg i.p) for 3 weeks (n = 6). Tumor volumes were measured twice weekly. Tumors were photographed and weighed. Scale bar 1 cm. Data represent means ± S.D. from six mice per group. C Mice bearing LuCaP 35CR xenografts were treated with vehicle control, niclosamide (150 mg per Kg p.o.), ARVib-7 (150 mg per Kg p.o.) for 4 weeks (n = 7). Tumor volumes were measured twice weekly. Scale bar 1 cm. Data represent means ± S.D. from seven mice per group. D Tumors were weighed. E Kaplan–Meier curves of niclosamide and ARVib-7 treatment in LuCaP 35CR tumors. F PSA expression in mice serum was examined in different treatment groups. G Body weight was determined. H IHC staining of Ki67 and AR-V7 in each group was performed and quantified. H&E staining of kidney and liver from each group was performed. *p < 0.05.
Fig. 6
Fig. 6. ARVib improves enzalutamide treatment in vitro and in vivo.
A, B CWR22Rv1 cells were treated with DMSO, 20 µM enzalutamide, 0.5 µM ARVibs or their combination. Cell growth was determined at 3 days and colony formation ability was examined by clonogenic assay. C Mice bearing VCaP xenografts were castrated and the relapsed tumors were treated with vehicle control, enzalutamide (25 mg per Kg p.o), ARVib-7 (75 mg per Kg p.o.) or their combination for 24 days (n = 8). Tumor volumes were measured twice weekly. D Tumors were photographed. Scale bar 1 cm. Data represent means ± S.D. from eight mice per group. E Tumors weight. F PSA expression in mice serum was examined. G Kaplan–Meier curves showing survival benefits of ARVib-7 single treatment, ARVib-7 and enzalutamide combination treatment in relapsed VCaP tumors. H Body weight was determined. I IHC staining of Ki67, AR-V7, and HSP70 in each group was performed and quantified. *p < 0.05.
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References

    1. Fizazi K, Shore N, Tammela TL, Ulys A, Vjaters E, Polyakov S, et al. Darolutamide in nonmetastatic, castration-resistant prostate cancer. N. Engl J Med. 2019;380:1235–46. doi: 10.1056/NEJMoa1815671. - DOI - PubMed
    1. Smith MR, Saad F, Chowdhury S, Oudard S, Hadaschik BA, Graff JN, et al. Apalutamide treatment and metastasis-free survival in prostate cancer. N Engl J Med. 2018;378:1408–18. doi: 10.1056/NEJMoa1715546. - DOI - PubMed
    1. de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, et al. Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011;364:1995–2005. doi: 10.1056/NEJMoa1014618. - DOI - PMC - PubMed
    1. Scher HI, Fizazi K, Saad F, Taplin M-E, Sternberg CN, Miller K, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012;367:1187–97. doi: 10.1056/NEJMoa1207506. - DOI - PubMed
    1. Guo Z, Yang X, Sun F, Jiang R, Linn DE, Chen H, et al. A novel androgen receptor splice variant is up-regulated during prostate cancer progression and promotes androgen depletion-resistant growth. Cancer Res. 2009;69:2305–13. doi: 10.1158/0008-5472.CAN-08-3795. - DOI - PMC - PubMed

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