Transformation of epithelial cells is marked by a loss of the classic polarized epithelial phenotype and the acquisition of a migratory, invasive phenotype. Associated with this switch in cellular phenotype are changes in cellular interactions with the extracellular matrix. In particular, the alpha2beta1 integrin, a receptor for collagens and laminin, is an important regulator of breast cell phenotype. Members of the Ras family of small GTPases are also important regulators of cell phenotype and transformation. Activation of a specific family member, R-Ras, disrupts epithefial polarization of human breast cells, and instead promotes cell migration and invasion. Activated R-Ras specifically promotes breast cell adhesion, migration, and focal adhesion formation on collagen through the alpha5beta1 integrin, but not on fibronectin through the alpha2beta1 integrin. Activated R-Ras enhances the phosphorylation of FAK (focal adhesion kinase), p130Cas, and PI3-kinase downstream of the alpha2beta1 integrin, in a manner that differs from, but requires, the alpha2beta1 integrin. The long-term goal of this proposal is to understand signaling pathways related to R-Ras and integrins, and how they affect the choice of a cell to polarize or migrate. As our working hypothesis, we propose that R-Ras plays a normal physiologically relevant role in outside-in and inside-out signaling events related to the alpha2beta1 integrin in a controlled spatial and temporal manner, and that dysregulation of R-Ras disrupts epithelial polarization and alters cell migration. This hypothesis will be tested in the following Specific Aims: 1) Define R-Ras and alpha2beta1 integrin regulation of the epithelial vs. migratory phenotype of cells; 2) Determine the molecular basis for R-Ras participation in alpha2beta1 integrin signaling pathways; 3) Investigate molecular mechanisms by which R-Ras promotes tumor formation. This work should enhance our understanding of the molecular mechanisms regulating epithelial phenotype, migration, and tumorigenesis.
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