Involvement of Akt/mTOR pathway in ezrin-mediated metastatic behavior in K7M2 osteosarcoma cells. Free

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We have previously demonstrated that ezrin expression is associated with spontaneous metastatic behavior in a murine model of osteosarcoma (K7M2). To investigate the mechanism of ezrin-related metastatic behavior, we studied the effects of ezrin expression on several signal transduction pathways. We linked ezrin-related metastatic behavior to activation of S6K1 and 4E-BP1two downstream targets of mTOR signaling. We found that antisense and siRNA-induced reduction of ezrin expression led to both decreased expression and decreased phosphorylation of S6K1 and 4E-BP1. The decreased expression of both S6K1 and 4E-BP1 were mediated via proteosomal degradation, since proteosomal inhibition by MG132 reversed the ezrin antisense-mediated decrease of S6K1 and 4E-BP1 protein. However, proteosomal block failed to affect ezrin antisense-mediated phosphorylation of S6K1 and 4E-BP1. Down-regulating ezrin expression also led to the inhibition of Akt phosphorylation and activity. Treatment of K7M2 cells with a PI3K inhibitor LY294002 completely inhibited Akt phosphorylation as well as S6K1 and 4E-BP1 phosphorylation. Furthermore, stable transfection of a dominant negative Akt (K179M mutant) into K7M2 cells led to both a reduction in Akt phosphorylation and also a reduction in S6K1 and 4E-BP1 phosphorylation, whereas transient transfection of activated Akt into ezrin-antisense transfected cells rescued the ezrin mediated inhibition of S6K1 and 4E-BP1 phosphorylation. Finally, treatment of K7M2 cells with rapamycin, an inhibitor of mTOR signalling, led to a significant decrease in cell migration and invasion in vitro and metastasis in vivo. These results indicate that ezrin-mediated metastatic behavior in K7M2 osteosarcoma cells may be linked to an Akt dependent mTOR/S6K1/4E-BP1 pathway.