The long-term goal of our research is to establish a model of the invasion of the normal brain by malignant astrocytoma tumors that permits the development of specific and effective therapies. The goal of this proposal is to test the hypothesis that focal adhesion kinase (p125FAK) and the associated adaptor protein p130 CAS mediate malignant astrocytoma cell migration/invasion of normal brain. We have established that integrins alphavbeta3 and alphavbeta5 mark the malignant astrocyte phenotype and mediate attachment and migration of these cells toward relevant ligands, such as vitronectin, vitronectin attachment of malignant astrocytoma cells result in activation of p125FAK, a cytoplasmic tyrosine kinase that is a key attachment of malignant astrocytoma cells result in activation of p125FAK, a cytoplasmic tyrosine kinase that is a key component of integrin-associated signalling cascades in other cell types; and over-expression of p125FAK in malignant astrocytoma cells results in increased vitronectin-directed migration. We propose to 1) determine the role of p125FAK in the migration and invasion of malignant astrocytoma cells in vitro, 2) determine the role of p130CAS in the migration and invasion of malignant astrocytoma cells in vitro, and 3) determine the biologic role of p125FAK and p130 CAS in the intracerebral acid mouse model of malignant astrocytoma cells by injecting and propagating malignant astrocytoma cells by injecting and propagating malignant astrocytoma cells over- expressing wild type and mutant constructs of p125FAK and p130CAS. The expression and level of phosphorylation of endogenous p125FAK and p130CAS in U-251 MG and U-87 MG human malignant astrocytoma cells, and rat C6 glioma cells, as well as normal neonatal rat astrocytes will be determined, followed by determination of the binding of p125FAK to Src, p130 CAS and Grb2, and the binding of p130CAS to Crk, Nck, Src and p125 FAK. Wild-type and mutant constructs of p125 FAK and p130 CAS will be over-expressed in these neoplastic and normal astrocytes followed by evaluation of their adhesive, migratory, invasive and proliferative properties in vitro, and their proliferative and invasive properties in vivo. The expression and level of phosphorylation of exogenous (transfected) p125 FAK and p130CAS, as well as the binding of p125FAK to p130CAS, Src, and Grb2, and the binding of p130 CAS to Src, Nck and p125FAK will be determined in these cells. Completion of this work will establish a model of p125 FAK and p130CAS signaling after ligation of integrins on astrocytoma cells, which will lead to the development of therapy directed toward specific steps in the invasion process.
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