Ezrin is a critical regulator of plasma membrane-cytoskeleton interaction as well as an important component of signal transduction pathways. These functions may suggest a role for ezrin in cancer progression and metastasis. We have previously demonstrated that ezrin is necessary for metastasis in murine models of osteosarcoma and rhabdomyosarcoma and that high expression of ezrin was associated with poor outcome in patients with either osteosarcoma or rhabdomyosarcoma. It is generally accepted that the activation of ezrin results from phosphorylation of a critical c-terminal threonine (T567) residue that results in the release of intramolecular and/or intermolecular head-to-tail associations of ezrin. The “open” active conformation of ezrin allows it link the plasma membrane with the actin-cytoskeleton. Previous studies have reported at least three kinases (PKCα, PKCθ and Rho-kinase) to be involved in this threonine phosphorylation in vitro or in vivo. The mechanisms associated with ezrin regulation and activation in cancer are not understood. To determine if ezrin activation was associated with these kinases in cancer, we evaluated ezrin T567 phosphorylation in the murine K7M2 osteosarcoma, RH30 rhabdomyosarcoma and B16 melanoma cells. K7M2 cells were exposed to PKC inhibitors (BIM, Ro31-8220, Go6976) and a Rho-kinase inhibitor (Y27632) at multiple time course and dose combinations. The phosphorylation of ezrin T567 was dependent on PKC but not Rho-kinase. Similar results were seen with all cell lines studied. The expression of PKC isoforms was then characterized by Western analysis in these cancer cells. There was no detectable PKCθ, but high expression of PKCα and γ in all cells. To begin to investigate the role of PKC on ezrin-dependent metastasis, we examined cell migration using the in vitro wound closure assay in K7M2 cells. The PKC inhibitor Go6976 resulted a reduction in cell motility in the wound assay, supporting the role of classical PKC pathway on cell motility. Similarly, K7M2 cells transfected with antisense ezrin, resulting in marked suppression of ezrin, had delayed wound closure (motility) compared to wild-type K7M2 cells and transfections control. The investigation of the direct association of PKC, ezrin T567 phosphorylation, cell migration and tumor metastasis is underway.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]