Regulation of invadopodia formation in breast cancer cells

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Tumor cell metastasis is the leading cause of mortality in breast cancer patients. Cancer cells invade surrounding tissue and blood vessels by forming filamentous- (F-) actin-rich protrusions called invadopodia that degrade the extracellular matrix (ECM). A major challenge in breast cancer research is the elucidation of the mechanisms that underlie invadopodia formation. Cortactin, an actin-binding protein, nucleates invadopodia formation and promotes breast cancer cell metastasis. These properties of cortactin are potentiated by its tyrosine phosphorylation. We have shown that adhesion to ECM activates integrin adhesion receptors to stimulate the Arg tyrosine kinase. In turn, Arg phosphorylates cortactin to promote dynamic invadopodia-like cell edge protrusions. Integrins and , and Arg are all upregulated in highly invasive breast cancer cells, and inhibiting Arg or cortactin function in these cells compromises their invasiveness. Our proposal will elucidate the mechanisms by which the integrin-Arg-cortactin axis controls invadopodia formation and function. Our first aim is to test whether integrins 1 and 3 signal through Arg and cortactin to regulate invadopodia formation, ECM invasiveness, and tumor metastasis. We will determine whether ECM adhesion stimulates cortactin phosphorylation in breast cancer cells with varying degrees of invasiveness and test if knockdown of integrins 1 or 3, or Arg compromise this process. We will use tissue culture assays to determine whether knockdown/knockout of integrins 1 or 3, Arg, or cortactin disrupts invadopodium formation and how this affects overall breast cancer invasiveness and metastasis. Our second aim is to elucidate the role of tyrosine phosphorylation of cortactin in mediating invadopod formation. Integrin-mediated adhesion stimulates Arg-dependent cortactin phosphorylation on three tyrosine residues (Y421, Y466, Y482). Tyrosine phosphorylated cortactin (cortactin-P) has increased ability to promote invadopodium formation and cancer metastasis. We identified several proteins that bind selectively and with high affinity to cortactin-P, but not to nonphosphorylated cortactin. We will further characterize the specificity and affinity of cortactin-P for these proteins in vitro and in cells. We will use 4D live cell imaging and RNAi-mediated knockdown of the cortactin-P-interacting proteins to test their roles in invadopodia formation.