Niclosamide suppresses cell migration and invasion in enzalutamide resistant prostate cancer cells via Stat3-AR axis inhibition

Abstract

Purpose: It is known that over expression of IL6 in prostate cancer cells confer enzalutamide resistance and that this may occur through constitutive Stat3 activation. Additionally, recent pre-clinical studies suggested enzalutamide might have the potential adverse effect of inducing metastasis of prostate cancer cells via Stat3 activation. This study is aimed to target Stat3 activation and improve enzalutamide therapy.

Experimental design: Sensitivity of prostate cancer cells to enzalutamide was tested using cell growth assays and clonogenic assays. Wound healing and invasion assays were performed to determine cell migration and invasion in vitro. Quantitative reverse transcription-PCR, ELISA and Western blotting were performed to detect expression levels of PSA, c-Myc, survivin, Stat3, and AR. ChIP assay was performed to examine recruitment of AR to the PSA promoter.

Results: In the present study, we found niclosamide, a previously identified novel inhibitor of androgen receptor variant (AR-V7), inhibited Stat3 phosphorylation, and expression of downstream target genes. Niclosamide synergistically reversed enzalutamide resistance in prostate cancer cells and combination treatment of niclosamide with enzalutamide significantly induced cell apoptosis and inhibited cell growth, colony formation, cell migration and invasion. Knock down of Stat3 abrogated enzalutamide resistance resulting in reduced recruitment of AR to the PSA promoter in prostate cancer cells expressing IL6. Moreover, niclosamide reversed enzalutamide resistance by down-regulating Stat3 target gene expression Stat3and abrogating recruitment of AR to PSA promoter resulting in PSA inhibition.

Conclusions: This study demonstrated the IL6-Stat3-AR axis in prostate cancer is one of the crucial mechanisms of enzalutamide resistance. Niclosamide has the potential to target the IL6-Stat3-AR pathway to overcome enzalutamide resistance and inhibit migration and invasion in advanced prostate cancer.

Keywords: Stat3; enzalutamide; interleukin-6; niclosamide; prostate cancer.

Figure 1. Niclosamide inhibited Stat3 activation in prostate cancer cells

A) LNCaP, C4-2B or DU145 cells were treated with DMSO, 0.5 μM or 1 μM niclosamide overnight, cells were then stimulated with 10 ng/mL of IL6 for 30 minutes. Whole cell lysates were prepared and subjected to Western blot analysis using antibodies as indicated. B) Niclosamide inhibited endogenous Stat3 activation. LNCaP-s17 and LNCaP-Stat3C cells were treated with DMSO, 0.5 μM, or 1 μM niclosamide overnight, Whole cell lysates were prepared and subjected to Western blot analysis using antibodies as indicated. C) Niclosamide inhibited Stat3 activity in LNCaP cells. LNCaP cells were transient transfected with 0.5 μg of pLucTKS3 reporter plasmid containing specific responsive elements for Stat3 or the control plasmid with or without 10 ng/mL IL6 for 24 hours. Cells were then treated with 0.5 μM or 1 μM niclosamide overnight and Luciferase activity was measured. D-F) DU145, LNCaP-s17 or LNCaP-Stat3C cells were transiently transfected with 0.5 μg of pLucTKS3 reporter plasmid containing specific responsive elements for Stat3 or the control plasmid for 24 hours, cells were then treated with 0.5 μM or 1 μM niclosamide overnight and Luciferase activity was measured. Results are presented as means ± SD of 3 experiments performed in duplicate. * P<0.05 Nic: Niclosamide.

Figure 2. Niclosamide inhibited cell migration, invasion and colony formation of prostate cancer cells

A) DU145, LNCaP-S17 or LNCaP-Stat3C cells were treated with 0.1μM, 0.25 μM or 0.5 μM niclosamide in media containing FBS after wound were introduced, photographs of the cells were taken and wound closure was quantified at the time indicated. B) Representative photographs of invasive LNCaP-Stat3C and DU145 cells. The cells were allowed to migrate through matrigel coated membranes with 8 mm pores for 48 hours in the presence of 0.1μM, 0.25 μM or 0.5 μM niclosamide. Invasive cells were counted under microscope (right panel). C) Colony formation was examined by clonogenic assay. LNCaP-s17 and LNCaP-Stat3C cells were treated with 0.25 μM or 0.5 μM niclosamide and equal amounts of cells were plated in 10cm dishes. The colonies were stained with 0.5% crystal violet after 3 weeks, and colony number was counted. Results are presented as means ± SD of 3 experiments performed in triplicate. * P < 0.05

Figure 3. Niclosamide synergistically reversed enzalutamide resistance in prostate cancer cells

A) Representative pictures of cell morphology changes in LNCaP-s17 cells after treatment with different regimens as indicated. LNCaP-s17 cells were treated with 0.25 μM or 0.5 μM niclosamide with or without 20 μM enzalutamide in media containing FBS. Cell numbers were counted after 48 h (B) and apoptosis was analyzed by Cell death ELISA (C). Results are presented as means ± SD of 3 experiments performed in duplicate. D) LNCaP-s17 cells were treated with 0.25 μM niclosamide with or without 20 μM enzalutamide in media containing FBS and cell numbers were counted after 2, 4 and 7 days. Results are presented as means ± SD of 3 experiments performed in duplicate. E) Colony formation was examined by clonogenic assay. LNCaP-s17 cells were treated with 0.25 μM niclosamide with or without 20 μM enzalutamide and equal amount cells were plated in 10cm dishes. The colonies were stained with 0.5% crystal violet after 3 weeks and colonies numbers were counted. F) LNCaP-Stat3C cells were treated with 0.25 μM niclosamide with or without 20 μM enzalutamide and equal amount cells were plated in 10cm dishes. The colonies were stained with 0.5% crystal violet after 3 weeks and colony number was counted. Results are presented as means ± SD of 3 experiments performed in duplicate.* P< 0.05 Enza: Enzalutamide, Nic: Niclosamide.

Figure 4. Co-treatment with enzalutamide and niclosamide inhibit prostate cancer cell migration and invasion

A) LNCaP-s17 and LNCaP-Stat3C cells were treated with 0.25 μM niclosamide with or without 20 μM enzalutamide in media containing FBS. After wounds were introduced, photographs of the cells were taken and wound closure was quantified at the time indicated. B) Representative photographs of invasive LNCaP-Stat3C cells. The cells were allowed to migrate through matrigel coated membranes with 8 μm pores for 48 hours in the presence of 0.25 μM niclosamide with or without 20 μM enzalutamide. Invasive LNCaP-Stat3C cells were counted under microscope (right panel). Results are presented as means ± SD of 3 experiments performed in triplicate. * P< 0.05

Figure 5. Stat3 activation induced enzalutamide resistance linked to the recruitment of AR to the PSA promoter

A) LNCaP, C4-2B, LNCaP-s17, LNCaP-Stat3C or PZ-HPV7 cell protein was harvested and immunoblotted with the antibody indicated. B) LNCaP-s17 cells were transfected with siRNA specific to Stat3 or control siRNA and whole cell lysate was extracted and immunoblotted with antibody indicated. Whole cell lysate from LNCaP-neo and LNCaPStat3C cells was extracted and immunoblotted with the antibody indicated. C) LNCaP-s17 cells were transfected with siRNA specific to Stat3 or control siRNA and were treated with or without 20 μM enzalutamide for 2 days and whole cell protein was extracted and immunoblotted with the antibody indicated. D) Recruitment of AR to AREs in the PSA promoter was analyzed by ChIP assay, LNCaP-s17 cells was transfected with siRNA specific to Stat3 or control siRNA and were treated with or without 20 μM enzalutamide overnight. Recruitment of AR to AREs in the PSA promoter was analyzed by ChIP assay (left) and the supernatant was collected for PSA ELISA analysis (right). * P< 0.05 Enza: Enzalutamide.

Figure 6. Co-treatment with enzalutamide and niclosamide down regulate Stat3 target gene expression and inhibit AR transcriptional activity

A) LNCaP-s17 and LNCaP-Stat3C cells were treated with 0.25 μM niclosamide with or without 20 μM enzalutamide in media containing FBS for 48 hours. Whole cell protein was extracted and immunoblotted with the antibody indicated. B) LNCaP-s17 cells were treated with 0.25 μM niclosamide with or without 20 μM enzalutamide overnight. Recruitment of AR to AREs in the PSA promoter was analyzed by ChIP assay. C) The supernatant was collected for PSA ELISA analysis. Results are presented as means ± SD of 3 experiments performed in triplicate. D) Proposed pathway of enzalutamide resistance in constitutively active Stat3 prostate cancer cells. * P< 0.05