RESUMEN
Receptor tyrosine kinases (RTKs) are cell surface transmembrane proteins responsible for intracellular signal transduction. They are expressed in several cell types and, after activation by growth factor binding, trigger a series of intracellular pathways, leading to a wide variety of cell responses (e.g., differentiation, proliferation, migration and invasion, angiogenesis, survival). Over-expression and/or structural alteration of RTKs family members are often associated to human cancers and tumor cells are known to use RTK transduction pathways to achieve tumor growth, angiogenesis and metastasis. Therefore, RTKs represent pivotal target in approaches of cancer therapy. A number of small molecules acting as RTK inhibitors have been synthesized by pharmaceutical companies and are under clinical trials, are being analyzed in animal models or have been successfully marketed. Ligand-dependent downregulation of RTKs is a critical step for modulating their activity and the adaptor Cbl has been indicated as the key protein involved in negative regulation of RTKs, such as EGF and HGF receptors. These data suggest novel potential pharmacological targets for the treatment of human malignancies associated to oncogenic activation of RTKs.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Activación Enzimática/efectos de los fármacos , Proteínas de Fusión bcr-abl , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Humanos , Neoplasias/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Transducción de Señal , Factor de Células Madre/antagonistas & inhibidoresRESUMEN
1. Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive. 2. We show here that human recombinant MSP (hrMSP) evokes a dose-dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte-derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N-formylmethionyl-leucyl-phenylalanine and similar to phorbol myristate acetate-evoked one. 3. To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c-Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3-kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP-evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3-kinase. We also show that MAP kinase and p38 signalling pathways are involved. 4. These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages.
Asunto(s)
Proteínas de Caenorhabditis elegans , Sustancias de Crecimiento/farmacología , Factor de Crecimiento de Hepatocito , Macrófagos/efectos de los fármacos , Proteínas Proto-Oncogénicas , Superóxidos/metabolismo , Adulto , Androstadienos/farmacología , Animales , Proteínas Portadoras , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Insectos , Macrófagos/metabolismo , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteína Quinasa C/metabolismo , Piridinas/farmacología , Receptores de Droga/metabolismo , Estallido Respiratorio , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , WortmaninaRESUMEN
Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Mesotelioma/virología , Proteínas Proto-Oncogénicas c-met/metabolismo , Virus 40 de los Simios/fisiología , Replicación Viral , Animales , Antígenos Virales de Tumores/genética , Comunicación Autocrina , Células COS , Ciclo Celular , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Perros , Activación Enzimática , Epitelio/metabolismo , Epitelio/virología , Expresión Génica , Humanos , Modelos Biológicos , Fase S , Virus 40 de los Simios/metabolismoRESUMEN
Oncogenic activation of the Ron tyrosine kinase (Macrophage Stimulating Protein receptor) relies on substitutions of two highly conserved residues in the catalytic domain (D1232V and M1254T), which result in ligand-independent activation of the receptor, in vivo tumorigenesis and metastasis. We show here that the Y/F conversion of the Y1317 residue in the kinase domain impairs tumorigenic and metastatic properties of Ron activated by the MEN2B-like mutation (RonM1254T), but not by other two oncogenic substitutions. Furthermore, RonM1254T lacking the multifunctional docking site retains transforming and metastatic activity. These data reveal that the transforming activity of RonM1254T mutant is dependent on Y1317 phosphorylation, suggesting a shift in intramolecular substrate specificity. Consistently, a shift of RonM1254T kinase substrate specificity was observed by in vitro peptide phosphorylation assays and in vivo receptor auto-phosphorylation. The Y1317 phosphorylation elicits by itself activation of PI-3K/Akt and MAPK signalling pathways. Our data indicate that the accomplishment of the full oncogenic phenotype of RonM1254T requires the phosphorylation both of the canonical C-terminal docking site and of the unique Y1317 residue in the tyrosine kinase domain.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Superficie Celular/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasia Endocrina Múltiple Tipo 2b/genética , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusión , Transducción de Señal , Especificidad por SustratoRESUMEN
Ron (the receptor for Macrophage Stimulating Protein) has never been implicated before in human malignancies or in cell transformation. In this report we show that Ron can acquire oncogenic potential by means of two amino acid substitutions-D1232V and M1254T-affecting highly conserved residues in the tyrosine kinase domain. The same mutations in Kit and Ret have been found associated with two human malignancies, mastocytosis and Multiple Endocrine Neoplasia type 2B (MEN2B), respectively. Both mutations caused Ron-mediated transformation of 3T3 fibroblasts and tumour formation in nude mice. Moreover, cells transformed by the oncogenic Ron mutants displayed high metastatic potential. The Ron mutant receptors were constitutively active and the catalytic efficiency of the mutated kinase was higher than that of wild-type Ron. Oncogenic Ron mutants enhanced activation of the Ras/MAPK cascade with respect to wild type Ron, without affecting the JNK/SAPK pathway. Expression of Ron mutants in 3T3 fibroblasts led to different patterns of tyrosine-phos-phorylated proteins. These data show that point mutations altering catalytic properties and possibly substrate specificity of the Ron kinase may force cells toward tumorigenesis and metastasis.