title: The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 μmol/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.
  
  
  CONCLUSIONS
  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.
authors: Sarcoma Group, Fondazione del Piemonte per l'Oncologia, Institute for Cancer Research and Treatment at Candiolo, Torino, Italy. ymera.pignochino@ircc.it
authors: Ymera
authors: Y
authors: Pignochino
authors: Carmine
authors: C
authors: Dell'Aglio
authors: Marco
authors: M
authors: Basiricò
authors: Federica
authors: F
authors: Capozzi
authors: Marco
authors: M
authors: Soster
authors: Serena
authors: S
authors: Marchiò
authors: Stefania
authors: S
authors: Bruno
authors: Loretta
authors: L
authors: Gammaitoni
authors: Dario
authors: D
authors: Sangiolo
authors: Erica
authors: E
authors: Torchiaro
authors: Lorenzo
authors: L
authors: D'Ambrosio
authors: Franca
authors: F
authors: Fagioli
authors: Stefano
authors: S
authors: Ferrari
authors: Marco
authors: M
authors: Alberghini
authors: Piero
authors: P
authors: Picci
authors: Massimo
authors: M
authors: Aglietta
authors: Giovanni
authors: G
authors: Grignani
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002642:Chick Embryo; D000068338:Everolimus; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D046912:Multiprotein Complexes / Q000378:metabolism*; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000517:prevention & control; D009536:Niacinamide / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D012516:Osteosarcoma / Q000098:blood supply / Q000188:drug therapy* / Q000473:pathology; D010671:Phenylurea Compounds / Q000008:administration & dosage / Q000494:pharmacology; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D020123:Sirolimus / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D000077157:Sorafenib; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects*; D023041:Xenograft Model Antitumor Assays
chemical_list: D046912:Multiprotein Complexes; D010671:Phenylurea Compounds; D017382:Reactive Oxygen Species; D009536:Niacinamide; D000068338:Everolimus; D000077157:Sorafenib; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D020123:Sirolimus