RESUMO
Malignant gliomas are characterized by their ability to invade normal brain tissue. We have previously shown that the small GTPase Rac1 plays a role in both migration and invasion in gliomas. Here, we aim to identify Rac-activating guanine nucleotide exchange factors (GEFs) that mediate glioblastoma invasiveness. Using a brain tumor expression database, we identified three GEFs, Trio, Ect2, and Vav3, that are expressed at higher levels in glioblastoma versus low-grade glioma. The expression of these GEFs is also associated with poor patient survival. Quantitative real-time polymerase chain reaction and immunohistochemical analyses on an independent set of tumors confirmed that these GEFs are overexpressed in glioblastoma as compared with either nonneoplastic brain or low-grade gliomas. In addition, depletion of Trio, Ect2, and Vav3 by siRNA oligonucleotides suppresses glioblastoma cell migration and invasion. Depletion of either Ect2 or Trio also reduces the rate of cell proliferation. These results suggest that targeting GEFs may present novel strategies for anti-invasive therapy for malignant gliomas.
Assuntos
Glioblastoma , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Encéfalo/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica , Glioblastoma/patologia , Glioblastoma/fisiopatologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-vav/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The generation of contractile force mediated by actin-myosin interactions is essential for cell motility. Myosin activity is promoted by phosphorylation of myosin light chain (MLC). MLC phosphorylation in large part is controlled by kinases that are effectors of Rho family GTPases. Accordingly, in this study we examined the effects of ROCK and Rac1 inhibition on MLC phosphorylation in astrocytoma cells. We found that low concentrations of the ROCK inhibitor Y27632 increased the phosphorylation state of the Triton X-100 soluble fraction of MLC, whereas higher concentrations of Y27632 decreased soluble phospho-MLC. These effects of Y27632 were dependent on Rac1. The soluble form of phospho-MLC comprises about 10% of total phospho-MLC in control cells. Interestingly, ROCK inhibition led to a decrease in the phosphorylation state of total MLC, whereas Rac1 inhibition had little effect. Thus, the soluble form of MLC is differentially regulated by ROCK and Rac1 compared with MLC examined in a total cell extract. We also observed that astrocytoma migration is stimulated by low concentrations of the myosin II inhibitor blebbistatin. However, higher concentrations of blebbistatin inhibit migration leading us to believe that migration has a biphasic dependence on myosin II activity. Taken together, our data show that modulation of myosin II activity is important in determining optimal astrocytoma migration. In addition, these findings suggest that there are at least two populations of MLC that are differentially regulated.