The Combination of Sorafenib and Everolimus abrogates mTORC-1 and mTORC2 up-regulation in preclinical models of Osteosarcoma.

PURPOSE: The multikinase inhibitor sorafenib displays antitumor activity in preclinical models of osteosarcoma. However, in sorafenib-treated metastatic-relapsed osteosarcoma patients, disease stabilization and tumor shrinkage were short-lived and drug resistance occurred. We explored the sorafenib treatment escape mechanisms to overcome their drawbacks. Experimental design: Immunoprecipitation, Western blotting, and immunohistochemistry were employed to analyze the mammalian target of rapamycin (mTOR) pathway (mTORC1 and mTORC2). Cell viability, colony growth, and cell migration were evaluated in different osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2OS, SJSA-1, SAOS-2) after scalar dose treatment with sorafenib (10-0.625 muM), rapamycin-analog everolimus (100-6.25 nM), and combinations of the two. Cell cycle, reactive oxygen species (ROS) production, and apoptosis were assessed by flow cytometry. NOD/SCID mice injected with MNNG-HOS cells were utilized to determine anti-tumor and anti-metastatic effects. Angiogenesis and vascularization were evaluated in vitro by exploiting endothelial branching morphogenesis assays and in vivo in xenografted mice and chorioallantoic membranes. RESULTS: After sorafenib treatment, mTORC1 signaling was reduced (downstream target P-S6) while mTORC2 was increased (phospho-mTOR Ser2481) in MNNG-HOS xenografts compared to vehicle-treated mice. Combining sorafenib with everolimus resulted in complete abrogation of both mTORC1 (through ROS-mediated AMPK activation) and mTORC2 (through complex disassembly). The sorafenib/everolimus combination yielded: 1) enhanced anti-proliferative and pro-apoptotic effects, 2) impaired tumor growth, 3) potentiated anti-angiogenesis, and 4) reduced migratory and metastatic potential. CONCLUSION: mTORC2 activation is an escape mechanism from sorafenib treatment. When sorafenib is combined with everolimus, its anti-tumor activity is increased by complete inhibition of the mTOR pathway in the preclinical setting.