Cell interactions with extracellular matrix (ECM) regulate cell proliferation, migration, and differentiation. A hallmark of cancer cells is loss of productive cell-ECM interactions. We have recently shown that human HT1080 fibrosarcoma cells show a Ras-related suppression of integrin activity that prevents assembly of fibronectin into fibrillar matrix. Matrix formation can be stimulated by treatment with dexamethasone or by directly activating integrins with exogenous agents. Inhibition of signaling from Ras to ERK MAP kinases also induces fibronectin fibril formation indicating that constitutively active N-ras in HT1080 cells down-regulates integrin function. As dexamethasone action is dependent on gene expression, a cDNA microarray screen to identify responsive genes is being developed. Our primary goal is to identify components of integrin regulatory pathways that modulate cell adhesion in human cancers; a second goal is to learn if these newly identified components have a role in human cancer.
Aims : (1) cDNA microarrays will be used to monitor the mRNA expression of about 40,000 transcripts during integrin-mediated matrix assembly. mRNAs from HT1080 cells treated or not with dexamethasone or other agents that stimulate matrix assembly will be used to generate targets. A set of genes that respond in common to these treatments will be extracted by cluster analysis and related techniques. (2) By monitoring gene expression across 40,000 transcripts and three different tumor cell lines that produce fibronectin matrix in response in dexamethasone or other treatments, we will identify consistent expression patterns related to matrix assembly. The associated transcripts will be identified and incorporated in a focused """"""""integrin-ECM pathway microarray."""""""" (3) A subset of genes will be identified by detailed screening with the integrin-ECM array of diverse tumor cell lines across different treatments, and different time periods. This subset will be prioritized for further analyses by identifying genes downstream of Ras or genes that encode proteins associated with integrin signaling or cytoskeletal connections. Northern analysis, immunoblotting, and other functional tests will be performed to elucidate specific roles in integrin and ECM regulation. (4) To determine the relationship between changes in integrin regulation, gene expression, and tumor progression, we will perform in situ hybridization and immunohistochemistry on a wide range of human tumor samples. This combined approach will help us to determine the molecular mechanisms underlying down-regulation of cell- ECM interactions in tumors.
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