The Histone Deacetylase Inhibitor, MS-275 (Entinostat), Downregulates c-FLIP, Sensitizes Osteosarcoma Cells to FasL, and Induces the Regression of Osteosarcoma Lung Metastases

Abstract

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas(+) cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas(-) OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas(-) metastatic OS cell lines with 2 µM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in the downregulation of the anti-apoptotic protein, c-FLIP (cellular FLICE-inhibitory protein), by inhibiting c-FLIP mRNA. Downregulation of c- FLIP correlated with caspase activation and apoptosis induction. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in tumor regression, increased apoptosis and a significant inhibition of c-FLIP expression in tumors. Histopathological examination of mice showed no evidence of significant toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a direct reduction in the expression of c-FLIP.