Metastatic cancers, once established, are the primary cause of mortality associated with cancer. Previously, we used a genomic approach to identify metastasis-associated genes in cancer. From this genomic data, we selected ezrin for further study based on its role in physically and functionally connecting the actin cytoskeleton to the cell membrane. In a mouse model of osteosarcoma, a highly metastatic pediatric cancer, we found ezrin to be necessary for metastasis. By imaging metastatic cells in the lungs of mice, we showed that ezrin expression provided an early survival advantage for cancer cells that reached the lung. AKT and MAPK phosphorylation and activity were reduced when ezrin protein was suppressed. Ezrin-mediated early metastatic survival was partially dependent on activation of MAPK, but not AKT. To define the relevance of ezrin in the biology of metastasis, beyond the founding mouse model, we examined ezrin expression in dogs that naturally developed osteosarcoma. High ezrin expression in dog tumors was associated with early development of metastases. Consistent with this data, we found a significant association between high ezrin expression and poor outcome in pediatric osteosarcoma patients.

Rapamycin and several analogs, such as CCI-779 and RAD001, are currently undergoing clinical evaluation as anticancer agents. In this study, we show that inhibition of mammalian target of rapamycin (mTOR) signaling by rapamycin leads to an increase of Akt phosphorylation in Rh30 and RD human rhabdomyosarcoma cell lines and xenografts, and insulin-like growth factor (IGF)-II-treated C2C12 mouse myoblasts and IGF-IIoverexpressing Chinese hamster ovary cells. RNA interference-mediated knockdown of S6K1 also results in an increase of Akt phosphorylation. These data suggest that mTOR/S6K1 inhibition either by rapamycin or small interfering RNA (siRNA) triggers a negative feedback loop, resulting in the activation of Akt signaling. We next sought to investigate the mechanism of this negative feedback regulation from mTOR to Akt. Suppression of insulin receptor substrate (IRS)-1 and tuberous sclerosis complex-1 by siRNAs failed to abrogate rapamycininduced upregulation of Akt phosphorylation in both Rh30 and RD cells. However, pretreatment with h7C10 antibody directed against insulin-like growth factor-1 receptor (IGF-1R) led to a blockade of rapamycin-induced Akt activation. Combined mTOR and IGF-1R inhibition with rapamycin and h7C10 antibody, respectively, resulted in additive inhibition of cell growth and survival. These data suggest that rapamycin mediates Akt activation through an IGF-1R-dependent mechanism. Thus, combining an mTOR inhibitor and an IGF-1R antibody/inhibitor may be an appropriate strategy to enhance mTOR-targeted anticancer therapy.