Ezrin Inhibition Up-regulates Stress Response Gene Expression

Haydar Çelik,Gülay Bulut,Jenny Han,Garrett T Graham,Tsion Z Minas,Erin J Conn,Sung-Hyeok Hong,Gary T Pauly,Mutlu Hayran,Xin Li,Metin Özdemirli,Ayşe Ayhan,Michelle A Rudek,Jeffrey A Toretsky,Aykut Üren

doi:10.1074/jbc.m116.718189

Abstract

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes.

Highlights

**Department of Biostatistics, Bioinformatics, and Biomathematics, Georgetown University, Washington, D
Ezrin has been validated as a key determinant in the development of metastatic osteosarcoma, which supports the development of small molecule ezrin inhibitors [12, 19]
We demonstrated that NSC305787 treatment but not NSC668394 inhibited the lung metastasis in Osx-Creϩp53fl/flpRBfl/fl transgenic mice

Summary

Experimental Procedures

Cell Lines and Culturing—Human MG63.3 osteosarcoma, mouse K7M2 osteosarcoma, and canine MCKOS, SKKOS, and CSKOS osteosarcoma cells were maintained in DMEM supplemented with 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C. The human MG63.3 and mouse K7M2 cell lines were kindly provided from Dr C. K7M2 cells express higher levels of ezrin protein, which leads to a greater potential to metastasize to the lungs than K12 cells [25]. Total RNA from osteosarcoma cells and mouse peripheral blood mononuclear cells (PBMCs) were extracted using the RNeasy Mini Kit Total RNA was reverse transcribed using a transcriptor first-strand cDNA synthesis kit (Roche) according to the manufacturer’s protocol. Immunoblotting—To verify ezrin knockdown, MG63.3 human osteosarcoma cells were lysed on ice for 30 min in phospholysis buffer (50 mmol/liter HEPES, pH 7.9, 100 mmol/liter NaCl, 4.0 mmol/liter sodium pyrophosphate, 10 mmol/liter EDTA, 10 mmol/liter sodium fluoride, and 1% Triton X-100) containing 2.0 mmol/liter sodium vanadate, 1.0 mmol/liter PMSF, 4.0 ␮g/ml aprotinin, 4.0 ␮g/ml leupeptin, and 1.0 ␮g/ml calyculin A.

Gene symbol

Reverse primer

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Results

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Discussion