RESUMEN
UNLABELLED: Activation of v-akt murine thymoma viral oncogene homolog (AKT) and Ras pathways is often implicated in carcinogenesis. However, the oncogenic cooperation between these two cascades in relationship to hepatocellular carcinoma (HCC) development remains undetermined. To investigate this issue, we generated a mouse model characterized by combined overexpression of activated forms of AKT and neuroblastoma Ras viral oncogene homolog (N-Ras) protooncogenes in the liver by way of hydrodynamic gene transfer. The molecular mechanisms underlying crosstalk between AKT and N-Ras were assessed in the mouse model and further evaluated in human and murine HCC cell lines. We found that coexpression of AKT and N-Ras resulted in a dramatic acceleration of liver tumor development when compared with mice overexpressing AKT alone, whereas N-Ras alone did not lead to tumor formation. At the cellular level, concomitant up-regulation of AKT and N-Ras resulted in increased proliferation and microvascularization when compared with AKT-injected mice. Mechanistic studies suggested that accelerated hepatocarcinogenesis driven by AKT and N-Ras resulted from a strong activation of mammalian target of rapamycin complex 1 (mTORC1). Furthermore, elevated expression of FOXM1/SKP2 and c-Myc also contributed to rapid tumor growth in AKT/Ras mice, yet by way of mTORC1-independent mechanisms. The biological effects of coactivation of AKT and N-Ras were then recapitulated in vitro using HCC cell lines, which supports the functional significance of mTORC1, FOXM1/SKP2, and c-Myc signaling cascades in mediating AKT and N-Ras-induced liver tumor development. CONCLUSION: Our data demonstrate the in vivo crosstalk between the AKT and Ras pathways in promoting liver tumor development, and the pivotal role of mTORC1-dependent and independent pathways in mediating AKT and Ras induced hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/fisiopatología , Factores de Transcripción Forkhead/fisiología , Neoplasias Hepáticas/fisiopatología , Proteína Oncogénica v-akt/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Proteína Forkhead Box M1 , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Técnicas In Vitro , Hígado/efectos de los fármacos , Hígado/patología , Hígado/fisiopatología , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos , Neovascularización Patológica/fisiopatología , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
We have previously shown that metoprolol can inhibit carnitine palmitoyltransferase-1 catalytic activity and decrease its malonyl CoA sensitivity within 30 min, suggesting the importance of a covalent modification. The aim of this study was to characterize the effects of PTMs on CPT-1 in the heart. Mitochondria were isolated from the hearts of male Wistar rats and incubated with kinases of interest (protein kinase A, CAMK-II, p38 MAPK, Akt) or with peroxynitrite and sodium nitroprusside. PKA decreased CPT-1 malonyl CoA sensitivity, associated with phosphorylation of CPT-1A, whereas CAMK-II increased malonyl CoA sensitivity by phosphorylating CPT-1B. p38 bound to CPT-1B and stimulated CPT-1 activity. The association of CPT-1 with these kinases and their scaffolding proteins was confirmed in co-localization studies. Peroxynitrite and sodium nitroprusside reversibly stimulated CPT-1 activity, and the change in CPT-1B activity was most consistently associated with glutathiolation of CPT-1B. These studies have identified a new regulatory system of kinases, scaffolding proteins and thiol redox chemistry which can control cardiac CPT-1 in vitro.