MS-275 sensitizes osteosarcoma cells to Fas ligand-induced cell death by increasing the localization of Fas in membrane lipid rafts

Fas expression is inversely correlated with the metastatic potential of osteosarcoma (OS) cells to the lungs. Fas⁺ cells are rapidly eliminated when they enter the lungs via their interaction with constitutive Fas ligand (FasL) on the lung epithelium, whereas Fas OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Upregulation of Fas expression in established OS lung metastases results in tumor regression. Here, we demonstrate that treatment of Fas OS cells with the histone deacetylase inhibitor MS-275 results in the upregulation of Fas mRNA and sensitizes these cells to FasL-induced apoptosis. However, flow cytometry analysis revealed that Fas cell surface protein expression was not significantly increased. Rather, we observed increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent-insoluble lipid raft fractions and colocalization with GM1' lipid rafts. We had previously shown that MS-275 treatment inhibited expression of the anti-apoptotic cellular FLICE-inhibitory protein (c-FLIP). Here, we demonstrated that transfection of cells with short hairpin RNA to c-FLIP also resulted in the localization of Fas to lipid rafts. Overall, our studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlates with the c-FLIP-dependent distribution of Fas to lipid rafts.