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. 2016 May 31;7(22):32210-20.
doi: 10.18632/oncotarget.8493.

Niclosamide enhances abiraterone treatment via inhibition of androgen receptor variants in castration resistant prostate cancer

Chengfei Liu  1 , Cameron Armstrong  1 , Yezi Zhu  1   2 , Wei Lou  1 , Allen C Gao  1   2   3
Affiliations

Niclosamide enhances abiraterone treatment via inhibition of androgen receptor variants in castration resistant prostate cancer

Chengfei Liu et al. Oncotarget. .

Erratum in

Abstract

Considerable evidence from both clinical and experimental studies suggests that androgen receptor variants, particularly androgen receptor variant 7 (AR-V7), are critical in the induction of resistance to enzalutamide and abiraterone. In this study, we investigated the role of AR-V7 in the cross-resistance of enzalutamide and abiraterone and examined if inhibition of AR-V7 can improve abiraterone treatment response. We found that enzalutamide-resistant cells are cross-resistant to abiraterone, and that AR-V7 confers resistance to abiraterone. Knock down of AR-V7 by siRNA in abiraterone resistant CWR22Rv1 and C4-2B MDVR cells restored their sensitivity to abiraterone, indicating that AR-V7 is involved in abiraterone resistance. Abiraterone resistant prostate cancer cells generated by chronic treatment with abiraterone showed enhanced AR-V7 protein expression. Niclosamide, an FDA-approved antihelminthic drug that has been previously identified as a potent inhibitor of AR-V7, re-sensitizes resistant cells to abiraterone treatment in vitro and in vivo. In summary, this preclinical study suggests that overexpression of AR-V7 contributes to resistance to abiraterone, and supports the development of combination of abiraterone with niclosamide as a potential treatment for advanced castration resistant prostate cancer.

Keywords: abiraterone; androgen receptor variant; niclosamide; prostate cancer; resistance.

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Conflict of interest statement

Patent application: C. Liu, W. Lou, and A.C. Gao are co-inventors on a patent application covering the use of niclosamide.

Figures

Figure 1
Figure 1. Enzalutamide resistant prostate cancer cells are cross resistant to abiraerone
A. C4-2B parental and C4-2B MDVR cells were treated with different dose of abiraterone (5μM, 10 μM and 20 μM) or enzalutamide (10 μM and 20 μM) and total cell lysates were harvested and subjected to western blot. B. C4-2B parental and C4-2B MDVR cells were treated with different doses of abiraterone (5μM, 10 μM and 20 μM) or enzalutamide (10 μM and 20 μM) for 48 hours and total cell numbers were counted and cell survival rate was calculated. C. Colonogenic assay was performed. Pictures were taken under microscope (inside panel). D. The colonies were counted and results are presented as means ± SD of 2 experiments performed in duplicate. Results for other panels are presented as means ± SD of 3 experiments performed in duplicate. *P<0.05
Figure 2
Figure 2. AR-V7 confers abiraterone resistance in prostate cancer
A. CWR22Rv1, C4-2B and LNCaP cells were treated with different concentrations of abiraterone for 48 hours, total cell numbers were counted and cell survival rate was calculated. B. whole cell lysates from LNCaP, C4-2B and CWR22Rv1cells were extracted, AR-V7 and full length AR were examined by western blot. C–D. CWR22Rv1 or C4-2B MDVR cells were transiently transfected with AR-V7 siRNA and then treated with 10 μM abiraterone for 3 days, total cell numbers were counted and cell survival rate (%) was calculated and the AR-V7 knock down efficiency was examined by western blot. E. C4-2 neo or C4-2 AR-V7 cells were treated with different concentrations of abiraterone for 48 hours, total cell numbers were counted and cell survival rate (%) was calculated. F. C4-2 neo or C4-2 AR-V7 cells were treated with different concentration of abiraterone for 48 hours, whole cell lysates were subjected to western blot. Results are presented as means ± SD of 3 experiments performed in duplicate. *P<0.05 Abi: Abiraterone.
Figure 3
Figure 3. Niclosamide enhanced abiraterone treatment in vitro
A–B. CWR22Rv1 cells or C4-2B MDVR cells were treated with 0.5 μM niclosamide with or without 10 μM abiraterone in media containing FBS and cell numbers were counted after 48 hours. Results are presented as means ± SD of 3 experiments performed in duplicate. C. Clonogenic assays were performed. Colonies numbers were counted and results are presented as means ± SD of 2 experiments performed in duplicate. D. CWR22Rv1 and C4-2B MDVR cells were treated with DMSO, 10 μM abiraterone, 0.5 μM niclosamide or combination for 48 hours, total cell lysates were collected and subjected to western blot. *P<0.05 Abi: Abiraterone. Nic: Niclosamide.
Figure 4
Figure 4. C4-2B cells chronically treated with abiraterone acetate express AR-V7
A. C4-2B parental and C4-2B AbiR cells were treated with different concentrations abiraterone acetate for 3 days, total cell number was counted and cell survival rate was calculated. B. C4-2B parental, C4-2B AbiR, C4-2B MDVR and CWR22Rv1 cells were cultured in media containing FBS for 3 days, total cell lysates were collected and subjected to western blot. C. C4-2B were transiently transfected with AR-V7 siRNA and then treated with 5 μM abiraterone acetate for 3 days. Total cell numbers were counted and cell survival rate (%) was calculated. The AR-V7 knock down efficiency was examined by western blot. D. C4-2B AbiR cells were treated with 0.5 μM niclosamide with or without 5 μM abiraterone acetate in media containing FBS and cell numbers were counted after 48 hours. Results are presented as means ± SD of 3 experiments performed in duplicate. E. C4-2B AbiR cells were treated with 0.5 μM niclosamide with or without 5 μM abiraterone acetate in media containing FBS and clonogenic assays were performed, colony pictures were taken under microscope. F. C4-2B AbiR cells were treated with DMSO, 5 μM abiraterone acetate, 0.5 μM niclosamide or combination for 48 hours. Total cell lysates were collected and subjected to western blot. *P<0.05 Abi-acetate: Abiraterone acetate. Nic: Niclosamide.
Figure 5
Figure 5. Niclosamide enhanced abiraterone treatment in vivo
A. Mice bearing CWR22Rv1 xenografts were treated with vehicle control, abiraterone acetate (200mg/Kg orally), niclosamide (500 mg/Kg orally) or their combination for 3 weeks, tumor volumes were measured twice every week and the tumors were collected. B. Pictures of tumors from each group were taken after 3 weeks treatment. C–D. Each group tumor weight and body weight were measured and averaged. E. Ki67 was analyzed in tumor tissues by IHC staining and quantified as described in methods. *P<0.05 Abi-Acetate: Abiraterone Acetate.
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