The combination of sorafenib and everolimus abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models

Ymera Pignochino, Carmine Dell'Aglio, Marco Basiricò, Federica Capozzi, Marco Soster, Serena Marchiò, Stefania Bruno, Loretta Gammaitoni, Dario Sangiolo, Erica Torchiaro, Lorenzo D'Ambrosio, Franca Fagioli, Stefano Ferrari, Marco Alberghini, Piero Picci, Massimo Aglietta, Giovanni Grignani

Research output: Contribution to journalArticle

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 mmol/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 vehicletreated 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.

Original languageEnglish
Pages (from-to)2117-2131
Number of pages15
JournalClinical Cancer Research
Volume19
Issue number8
DOIs
Publication statusPublished - Apr 15 2013

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Osteosarcoma
Up-Regulation
Methylnitronitrosoguanidine
Reactive Oxygen Species
Everolimus
sorafenib
S 6
Chorioallantoic Membrane
AMP-Activated Protein Kinases
SCID Mice
Sirolimus
Therapeutics
Growth
Morphogenesis
Immunoprecipitation
Drug Resistance
Heterografts
Cell Movement
Neoplasms
Cell Survival

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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The combination of sorafenib and everolimus abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models. / 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.

In: Clinical Cancer Research, Vol. 19, No. 8, 15.04.2013, p. 2117-2131.

Research output: Contribution to journalArticle

Pignochino, Y, Dell'Aglio, C, Basiricò, M, Capozzi, F, Soster, M, Marchiò, S, Bruno, S, Gammaitoni, L, Sangiolo, D, Torchiaro, E, D'Ambrosio, L, Fagioli, F, Ferrari, S, Alberghini, M, Picci, P, Aglietta, M & Grignani, G 2013, 'The combination of sorafenib and everolimus abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models', Clinical Cancer Research, vol. 19, no. 8, pp. 2117-2131. https://doi.org/10.1158/1078-0432.CCR-12-2293
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. / The combination of sorafenib and everolimus abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models. In: Clinical Cancer Research. 2013 ; Vol. 19, No. 8. pp. 2117-2131.
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T1 - The combination of sorafenib and everolimus abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models

AU - Pignochino, Ymera

AU - Dell'Aglio, Carmine

AU - Basiricò, Marco

AU - Capozzi, Federica

AU - Soster, Marco

AU - Marchiò, Serena

AU - Bruno, Stefania

AU - Gammaitoni, Loretta

AU - Sangiolo, Dario

AU - Torchiaro, Erica

AU - D'Ambrosio, Lorenzo

AU - Fagioli, Franca

AU - Ferrari, Stefano

AU - Alberghini, Marco

AU - Picci, Piero

AU - Aglietta, Massimo

AU - Grignani, Giovanni

PY - 2013/4/15

Y1 - 2013/4/15

N2 - 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 mmol/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 vehicletreated 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.

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