Ivermectin inhibits HSP27 and potentiates efficacy of oncogene targeting in tumor models
Lucia Nappi, Adeleke H. Aguda, Nader Al Nakouzi, Barbara Lelj-Garolla, Eliana Beraldi, Nada Lallous, Marisa Thi, Susan Moore, Ladan Fazli, Dulguun Battsogt, Sophie Stief, Fuqiang Ban, Nham T. Nguyen, Neetu Saxena, Evgenia Dueva, Fan Zhang, Takeshi Yamazaki, Amina Zoubeidi, Artem Cherkasov, Gary D. Brayer, Martin Gleave
Lucia Nappi, Adeleke H. Aguda, Nader Al Nakouzi, Barbara Lelj-Garolla, Eliana Beraldi, Nada Lallous, Marisa Thi, Susan Moore, Ladan Fazli, Dulguun Battsogt, Sophie Stief, Fuqiang Ban, Nham T. Nguyen, Neetu Saxena, Evgenia Dueva, Fan Zhang, Takeshi Yamazaki, Amina Zoubeidi, Artem Cherkasov, Gary D. Brayer, Martin Gleave
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Research Article Oncology

Ivermectin inhibits HSP27 and potentiates efficacy of oncogene targeting in tumor models

Abstract

HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti–androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.

Authors

Lucia Nappi, Adeleke H. Aguda, Nader Al Nakouzi, Barbara Lelj-Garolla, Eliana Beraldi, Nada Lallous, Marisa Thi, Susan Moore, Ladan Fazli, Dulguun Battsogt, Sophie Stief, Fuqiang Ban, Nham T. Nguyen, Neetu Saxena, Evgenia Dueva, Fan Zhang, Takeshi Yamazaki, Amina Zoubeidi, Artem Cherkasov, Gary D. Brayer, Martin Gleave

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Figure 3

IVM reduces EGFR phosphorylation levels via HSP27 regulation of SHPTP1.

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IVM reduces EGFR phosphorylation levels via HSP27 regulation of SHPTP1. ...
(A) Immunoblotting for p-EGFR and total and p-HSP27 after si-HSP27 (20 nM) or IVM (5 μM) in HCC-827 cells (left) or IVM (2.5 μM) before and after EGF stimulation (50 μg/mL) in SW48 cells (right). DMSO was used as control. (B) Top panels: Coimmunoprecipitation of HSP27 and EGFR with and without IVM (5 μM) for 24 hours in HCC-827 cells. IgGs were used as negative control. Bottom panels: Proximity ligation assay between HSP27 and EGFR in HCC-827 cells. Confocal microscopy was used to detect interaction (red dots). DNA was counterstained with DAPI (blue). Erlotinib was used as a negative control. Scale bars: 20 μm. (C) Left panel: Immunoblotting for SHPTP1 and p-HSP27 in HCC-827 cells as in A. Right panel: Immunoblotting for p-EGFR, SHPTP1, and p-HSP27 in wild-type and HSF1–/– MEF cells transfected with either empty (E) or HSP27 plasmid. (D) SHPTP1 phosphatase activity in HCC-827 cells after DMSO or IVM (5 μM). Data shown as mean ± SEM; ***P < 0.001 by unpaired 2-tailed Student’s t test with Welch’s correction; n = 3 independent experiments. (E) Cell survival of HCC-827 and SW48 cells measured by crystal violet assay after 4 days of treatment with DMSO, IVM, erlotinib, or cetuximab and their combination with IVM. Data shown as mean ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA with Bonferroni’s correction. (F) Immunoblotting for EGFR, p-EGFR, HSP27, p-HSP27, ERK, p-ERK, AKT, p-AKT, PARP, and cleaved PARP in HCC-827 cells treated as in E for 24 hours. (G) Tumor volume of HCC-827 xenografts treated with 15 mg/kg erlotinib, 10 mg/kg IVM, or a combination of both. Data shown as mean ± SEM; *P < 0.05, ****P < 0.0001 by ANOVA with Tukey’s correction; n = 8. (H) Immunohistochemical analysis of p-HSP27, p-EGFR, SHPTP1, and TUNEL staining of HCC-827 xenografts. Scale bars: 10 μm.