Ezrin Inhibition Up-Regulates Stress Response Gene Expression And Blocks Osteosarcoma Metastasis

Ezrin is a member of the ezrin, radixin, moesin (ERM) protein family of membrane-cytoskeleton linkers. Ezrin has been implicated in many essential cellular functions including cell adhesion, motility, maintenance and determination of cell shape, cell proliferation and apoptosis, regulation of ion channels, morphogenesis and signal transduction. Ezrin promotes invasive and migratory capabilities of cancer cells. A high level of ezrin expression is associated with poor clinical outcome and metastatic behavior of pediatric solid tumors including osteosarcoma and rhabdomyosarcoma as well as multiple other tumor types. Ezrin, therefore, could be a promising molecular target for the prevention and treatment of cancer metastasis. We previously discovered two small molecule inhibitors, NSC305787 and NSC668394, which bind directly to ezrin and inhibit its activity in mediating the invasive phenotype of osteosarcoma cells in multiple in vitro and in vivo assays. In this study, we expand on our previous findings by demonstrating that NSC305787-treatment but not NSC668394 significantly reduces pulmonary metastasis in a genetically engineered mouse model of osteosarcoma. We assessed the pharmacokinetics of compounds in mice and demonstrated that NSC305787 has a more favorable pharmacokinetic profile compared with NSC668394. In order to uncover ezrin-mediated biological pathways that can be used for a specific pharmacodynamic marker(s) of response to ezrin inhibition, we profiled global gene expression in osteosarcoma cells after treatment with inhibitors. We identified several commonly up-regulated genes with functional relevance to integrated stress response, implicating that a common underlying mechanism may be shared by these compounds. We further validated the microarray data through extensive testing using real-time qPCR and verified the specificity of the transcriptional response using another novel ezrin inhibitor MMV667492 that we have identified recently from the MMV400 “Malaria Box” library. The effect of ezrin inhibitors on the expression of stress genes was recapitulated by siRNA-mediated depletion of ezrin. The up-regulation of stress genes was much weaker in cells with reduced ezrin levels compared to wild-type cells, indicating the specificity of the compounds on ezrin-mediated cellular responses. Analysis of the expression of stress genes in white blood cells and skin of NSC305787-treated mice demonstrated up-regulation of the DDIT4/REDD1, suggesting that DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of response to ezrin inhibition. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive, may have important functions regulating gene expression and inhibition of ezrin activity by NSC305787 in osteosarcoma could be an attractive therapy to prevent clinically significant metastasis. Citation Format: Haydar Celik, Gulay Bulut, Jenny Han, Garrett T. Graham, Tsion Z. Minas, Erin J. Conn, Sung-Hyeok Hong, Gary T. Pauly, Mutlu Hayran, Xin Li, Metin Ozdemirli, Ayse Ayhan, Michelle A. Rudek, Jeffrey A. Toretsky, Aykut Uren. Ezrin inhibition up-regulates stress response gene expression and blocks osteosarcoma metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2446.