Purpose: The multikinase inhibitor sorafenib displays antitumor activity in preclinical models of osteosarcoma. However, in sorafenib-treated patients with metastatic-relapsed osteosarcoma, 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 used to analyze the mTOR pathway [mTOR complex 1 (mTORC1) and mTOR complex 2 (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, and SAOS-2) after scalar dose treatment with sorafenib (10-0.625 mu mol/L), rapamycin-analog everolimus (100-6.25 nmol/L), and combinations of the two. Cell cycle, reactive oxygen species (ROS) production, and apoptosis were assessed by flow cytometry. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice injected with MNNG-HOS cells were used to determine antitumor and antimetastatic 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), whereas mTORC2 was increased (phospho-mTOR Ser2481) in MNNG-HOS xenografts compared with vehicle-treated mice. Combining sorafenib with everolimus resulted in complete abrogation of both mTORC1 [through ROS-mediated AMP-activated kinase (AMPK) activation] and mTORC2 (through complex disassembly). The sorafenib/everolimus combination yielded: (i) enhanced antiproliferative and proapoptotic effects, (ii) impaired tumor growth, (iii) potentiated antiangiogenesis, and (iv) reduced migratory and metastatic potential.Conclusion: mTORC2 activation is an escape mechanism from sorafenib treatment. When sorafenib is combined with everolimus, its antitumor activity is increased by complete inhibition of the mTOR pathway in the preclinical setting. Clin Cancer Res; 19(8); 2117-31. (C) 2013 AACR.

The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models

Pignochino, Ymera;Bruno, Stefania;Sangiolo, Dario;Torchiaro, Erica;D'Ambrosio, Lorenzo;Fagioli, Franca;Aglietta, Massimo;
2013

Abstract

Purpose: The multikinase inhibitor sorafenib displays antitumor activity in preclinical models of osteosarcoma. However, in sorafenib-treated patients with metastatic-relapsed osteosarcoma, 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 used to analyze the mTOR pathway [mTOR complex 1 (mTORC1) and mTOR complex 2 (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, and SAOS-2) after scalar dose treatment with sorafenib (10-0.625 mu mol/L), rapamycin-analog everolimus (100-6.25 nmol/L), and combinations of the two. Cell cycle, reactive oxygen species (ROS) production, and apoptosis were assessed by flow cytometry. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice injected with MNNG-HOS cells were used to determine antitumor and antimetastatic 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), whereas mTORC2 was increased (phospho-mTOR Ser2481) in MNNG-HOS xenografts compared with vehicle-treated mice. Combining sorafenib with everolimus resulted in complete abrogation of both mTORC1 [through ROS-mediated AMP-activated kinase (AMPK) activation] and mTORC2 (through complex disassembly). The sorafenib/everolimus combination yielded: (i) enhanced antiproliferative and proapoptotic effects, (ii) impaired tumor growth, (iii) potentiated antiangiogenesis, and (iv) reduced migratory and metastatic potential.Conclusion: mTORC2 activation is an escape mechanism from sorafenib treatment. When sorafenib is combined with everolimus, its antitumor activity is increased by complete inhibition of the mTOR pathway in the preclinical setting. Clin Cancer Res; 19(8); 2117-31. (C) 2013 AACR.
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AMP-Activated Protein Kinases; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Cell Cycle; Cell Survival; Chick Embryo; Everolimus; Female; Flow Cytometry; Humans; Immunohistochemistry; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Inbred NOD; Mice, SCID; Multiprotein Complexes; Neovascularization, Pathologic; Niacinamide; Osteosarcoma; Phenylurea Compounds; RNA Interference; Reactive Oxygen Species; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases; Up-Regulation; Xenograft Model Antitumor Assays
Pignochino, Ymera; Dell'Aglio, Carmine; Basiricò, Marco; Capozzi, Federica; Soster, Marco; Marchiò, Serena; Bruno, Stefania; Gammaitoni, Loretta; Sangiolo, Dario; Torchiaro, Erica; D'Ambrosio, Lorenzo; Fagioli, Franca; Ferrari, Stefano; Alberghini, Marco; Picci, Piero; Aglietta, Massimo; Grignani, Giovanni
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/1869289
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