Invadopodia direct cell migration into and through surrounding extraceilular matches and tissues and this is accomplished by their ability to extend outward from the cell and adhere to and degrade the matrix. A novel role for cortactin is proposed in which this actin-binding, c-Src substrate participates in targeted vesicle delivery, docking and retrieval at invadopodia and that c-Src is required for the formation of functional invadopodial complexes containing cortactin. Further, it is proposed that the spleen tyrosine kinase, Syk, that we have shown to be a tumor suppressor, alters cellular motility and invasion via its effect upon Src activity. The interaction of loss of Syk with increased expression and activation of Src and cortactin may promote breast cancer progression by collectively affecting major signal transduction pathways. We propose four specific aims.
Aim1, Determine the requirement for Src/cortactin for targeted membrane transport to and from invadopodia.
Aim2, Determine regulation of MT1-MMP transport to and from invadopodia regulated by Src/cortactin.
Aim3, Determine regulation of invadopodia by Syk via negative regulation of Src.
Aim4, Determine relevance of Src, cortactin, and Syk interaction for tumor cell invasion in vivo, and their potential for therapeutic targeting, risk assessment or diagnosis. Assays for membrane and molecular :lynamics at invadopodia include confocal imaging of live cells expressing green or red fluorescent chimeras. Sites of matrix degradation will be co-localized with invadopodia-associated molecules. Immuno-electron microscopy will be used to determine the ultrastructure of cortactin-associated vesicles and membranes at invadopodia. Mutants, inhibitors, or RNAi knockdown will be used to assess the molecular requirements for Src, cortactin and Syk. Finally, in situ hybridization and immunohistochemistry will be used to determine the levels of Src, EMSI/cortactin, and Syk in human breast tissues. Our long term goal is to develop strategies to interfere with Src-driven metastasis and to study risk assessment strategies for patients.
