Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration-resistant prostate cancer

Abstract

Purpose: Enzalutamide, a second-generation antiandrogen, was recently approved for the treatment of castration-resistant prostate cancer (CRPC) in patients who no longer respond to docetaxel. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently. Androgen receptor (AR) splice variants such as AR-V7 have recently been shown to drive castration-resistant growth and resistance to enzalutamide. This study was designed to identify inhibitors of AR variants and test its ability to overcome resistance to enzalutamide.

Experimental design: The drug screening was conducted using luciferase activity assay to determine the activity of AR-V7 after treatment with the compounds in the Prestwick Chemical Library, which contains about 1,120 FDA-approved drugs. The effects of the identified inhibitors on AR-V7 activity and enzalutamide sensitivity were characterized in CRPC and enzalutamide-resistant prostate cancer cells in vitro and in vivo.

Results: Niclosamide, an FDA-approved antihelminthic drug, was identified as a potent AR-V7 inhibitor in prostate cancer cells. Niclosamide significantly downregulated AR-V7 protein expression by protein degradation through a proteasome-dependent pathway. Niclosamide also inhibited AR-V7 transcription activity and reduced the recruitment of AR-V7 to the PSA promoter. Niclosamide inhibited prostate cancer cell growth in vitro and tumor growth in vivo. Furthermore, the combination of niclosamide and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth, suggesting that niclosamide enhances enzalutamide therapy and overcomes enzalutamide resistance in CRPC cells.

Conclusions: Niclosamide was identified as a novel inhibitor of AR variants. Our findings offer preclinical validation of niclosamide as a promising inhibitor of AR variants to treat, either alone or in combination with current antiandrogen therapies, patients with advanced prostate cancer, especially those resistant to enzalutamide.

Figure 1

AR-V7 is constitutively active in prostate cancer cells. A. LNCaP, C4-2, CWR22Rv1 and VCaP cells were cultured in CS-FBS condition for 3 days. AR-V7 mRNA level were analyzed by qRT-PCR, whole cell protein was extracted and immunoblotted with indicated antibodies. Results are presented as means ± SD of 3 experiments performed in duplicate. B. PC-3 cells and LNCaP cells were cultured in CS-FBS condition and transiently transfected with WT-AR or AR-V7 plasmid for 3 days followed by treatment with 10nM DHT for another 24 hours and whole cell lysates were subjected to luciferase assay. C. C4-2 neo and C4-2 AR-V7 stable clone were cultured in CS-FBS condition, AR-V7 and PSA mRNA level was determined by qRT-PCR, the AR-V7 and PSA protein expression was detected by western blot (insert). D. CWR22Rv1 cells were transient transfected with control siRNA, AR exon7 siRNA or AR V7 siRNA in CS-FBS condition, cell numbers were counted on different days and the knock down efficiency was examined by western blot. Results are presented as means ± SD of 3 experiments performed in duplicate. * P<0.05

Figure 2

Niclosamide inhibited AR-V7 transcription activity. A. 293-AR-V7-PSA luciferase promoter stable clone was treated with 1.0 μM niclosamide or 20μM enzalutamide overnight in media containing 10% FBS or 10% CS-FBS and whole cell lysates were subjected to luciferase assay. B. LNCaP cells were co-transfected with PSA luciferase promoter and AR V7 in CS-FBS condition for 24 hours, followed by treatment with 1.0 μM niclosamide or 20μM enzalutamide overnight and whole cell lysates were subjected to luciferase assay. C. C4-2 neo and C4-2 AR-V7 cells were cultured in CS-FBS condition for 3 days, followed by treatment with 1.0 μM niclosamide or 20μM enzalutamide overnight and the supernatants were subjected to PSA ELISA.. D. C4-2 neo and C4-2 AR-V7 cells were cultured in CS-FBS condition for 3 days, whole cell lysis was subjected to ChIP assay (left). C4-2 AR-V7 cells were treated with 0.5 μM, 1.0 μM niclosamide or 20μM enzalutamide overnight and whole cell lysates were subjected to ChIP assay (right). Results are presented as means ± SD of 3 experiments performed in duplicate. * P<0.05. Enza: Enzalutamide, Nic: Niclosamide, RLU: relative luciferase unit.

Figure 3

Niclosamide inhibited AR-V7 protein expression through enhancing protein degradation. A. CWR22Rv1 cells were treated with 0 μM, 0.5 μM or 1.0 μM niclosamide in RPMI 1640 media containing 10% FBS overnight and the whole cell lysates were immunoblotted with the indicated antibodies (left). CWR22Rv1 cells were treated with 1.0 μM niclosamide in RPMI 1640 media containing 10% FBS, whole cell lysates were extracted at different time points and immunoblotted with the indicated antibodies (right). B. CWR22Rv1 cells were treated with 0 μM, 0.5 μM or 1.0 μM niclosamide in RPMI 1640 media containing 10% FBS overnight, total RNAs were extracted and AR or AR-V7 mRNA levels were analyzed by qRT-PCR. C. 50 μg/mL cycloheximide (CHX) was added with or without 2 μM Niclosamide (Nic) at time 0 hour. At specified time points, cells were harvested, and the levels of AR-V7 protein were measured by Western blot using antibodies specific against AR-V7. Plotted on semilog scale relative to respective time 0 AR-V7 value as 100%, dashed line indicates 50% half-life. D. Effect of MG132 on niclosamide–induced AR protein degradation. MG132 (5 μmol/L) was added to CWR22Rv1 cells together with cycloheximide (50 μg/mL) in the presence or absence of 2 μM niclosamide. The cell lysates were prepared at 8h. AR-V7 protein levels were determined by Western blot analysis using antibodies specifically against AR-V7 and tubulin as a control. Nic: Niclosamide, CHX: Cycloheximide.

Figure 4

Niclosamide inhibited prostate cancer cell growth and induced cell apoptosis. A. C4-2 neo, C4-2 AR-V7, CWR22Rv1 and PZ-HPV-7 cells were treated with 0.5 μM niclosamide in media containing FBS, cell numbers were counted and cell survival rate was calculated after 48 hours. B. C4-2 neo, C4-2 AR-V7, CWR22Rv1 and PZ-HPV7 cells were treated with 0.5 μM niclosamide in media containing FBS, and apoptosis was analyzed by Cell death ELISA after 48 hours. C. C4-2 neo, C4-2 AR-V7 or CWR22Rv1 cells were treated with 0, 0.5 μM or 1.0 μM niclosamide and clonogenic assays were performed. D. Colonies were counted and results are presented as means ± SD of 2 experiments performed in duplicate. Niclosamide inhibited colony formation in a dose dependent manner. * P<0.05

Figure 5

C4-2B cells chronically treated with enzalutamide express AR variants and are sensitive to Niclosamide. A. C4-2B parental or C4-2B MR cell was treated with 20μM enzalutamide in RPMI 1640 media containing 10% FBS and total cell numbers were counted at different time points as indicated. B. C4-2B parental cells and C4-2B MR cells were cultured in RPMI 1640 media containing 10% FBS for 3 days, total RNAs were extracted and AR-V1, AR-V7, AR1/2/2b, AR1/2/3/2b or AR full length mRNA levels were analyzed by qRT-PCR. AR-V7 protein level was examined by western blot (inside panel).C4-2B parental cells and C4-2B MR cells were cultured in media containing 10% CS-FBS for 3 days, the cells were harvested for preparation of cytosolic and nuclear fractions and analyzed by Western blotting using antibodies against AR-V7, AR, RNA polymerase II, or Tubulin (right panel). The expression of RNA polymerase II and tubulin were used as markers for the integrity of the nuclear and cytosolic fractions, respectively. C. C4-2B MR cells were cultured in media containing 10% FBS and treated with different concentrations of enzalutamide or niclosamide as indicated and total cell numbers were counted after 48 h. D. C4-2B MR cells were treated with DMSO, 10 μM or 20 μM enzalutamide, 0.5 μM or 1.0 μM niclosamide and clonogenic assays were performed. Colonies were counted and results are presented as means ± SD of 2 experiments performed in duplicate. * P<0.05. Enza: Enzalutamide, Nic: Niclosamide.

Figure 6

Niclosamide enhances enzalutamide effects both in vitro and in vivo. A. CWR22Rv1 cells or C4-2B MR cells were treated with 0.25 μM niclosamide with or without 20 μM enzalutamide in media containing FBS and cell numbers were counted after 3 and 5 days. Results are presented as means ± SD of 3 experiments performed in duplicate. B. CWR22Rv1 cells or C4-2B MR cells were treated with 0.25 μM niclosamide with or without 20 μM enzalutamide in media containing FBS and clonogenic assays were performed. Colonies numbers were counted and results are presented as means ± SD of 2 experiments performed in duplicate. C. Mice bearing CWR22Rv1 xenografts were treated with vehicle control, enzalutamide, niclosamide or their combination for 3 weeks, tumor volumes were measured twice every week and the tumors were collected and weighed. D. Ki67 was analyzed in tumor tissues by IHC staining and quantified as described in methods * P<0.05.