Metastatic spread of cancer cells to other tissues and organs is the primary cause of mortality from breast cancer. Metastatic cancer cells invade surrounding tissues and blood vessels by forming F-actin rich protrusions called invadopodia that degrade the extracellular matrix and enable invasion through it. A major challenge in breast cancer research is to elucidate the mechanisms that underlie invadopodia formation and function. The non-receptor tyrosine kinase Pyk2 is highly expressed in invasive cancers, but the signaling mechanism through which Pyk2 regulates invasion and metastasis are largely unknown. This proposal aims to understand whether and how signaling via Pyk2 regulates invadopodia formation and function in metastatic breast cancer cells. Our first goal in the proposed research would be to determine whether and how the integrin-Pyk2-Src axis drives invadopodial protrusion and invasiveness in breast cancer cells. Following elucidation of the role of Pyk2 in invadopodia, we will compare the similar or differential roles of Pyk2 vs. FAK in Src localization and in invadopodia regulation. Using a high throughput proteomic approach utilizing purified proteins, we will then explore additional substrates and interactors of Pyk2 in invadopodia, in order to elucidate new signaling pathways in which the kinase is involved. Following the experiments described above, we will use mice models combined with cutting-edge single cell resolution intravital imaging of mammary tumors in mice, in order to verify the effect of Pyk2 on invadopodia regulation and cancer metastasis in vivo. The proposed research aims to elucidate the molecular, cellular, and whole organism mechanisms that contribute to the tissue invasiveness and metastatic potential of breast cancer cells. We hope to translate the findings of this research into novel strategies for predicting the invasive potential of breast cancer carcinoma, blocking breast cancer metastasis, and reducing mortality.