Focal Adhesion Kinase (FAK) is a 125 kDa tyrosine kinase that is localized to contact points between cells and their extracellular matrix. We have demonstrated that FAK overexpression and upregulation occurred in early stages of tumorigenesis. We have also shown that FAK overexpression suppresses apoptosis, thus providing a survival signal to human cancer cells. Furthermore, our data has recently shown that the amino-terminus of FAK can induce apoptosis in breast cancer cells and can bind to a death domain containing serine-threonine kinase, Receptor Interacting Protein, (RIP). In addition, we have identified peptides that bind to the carboxy-terminus of FAK and cause apoptosis. 1 of these peptides contained a sequence homologous to the vascular endothelial growth receptor 3 (VEGFR-3) protein that we have shown to bind FAK. This proposal focuses on the biology of FAK in the development of cancer and on the biological mechanism by which attenuation of amino-terminal and carboxy-terminal FAK signaling causes tumor cell apoptosis. The first specific aim is to define the critical components of the amino terminus of FAK (FAKNT) that are responsible for its pro-apoptotic properties. We will define the portion(s) of FAK-NT that induce loss of adhesion in breast cancer cells and bind to RIP. Next, we will perform functional analyses of different peptides that bind the carboxy-terminus of FAK (FAK-CD), inhibit FAK function, and induce apoptosis in breast cancer cells. We will define their biological effects in different tumor cell lines to determine if they can induce apoptosis, perform initial binding studies using NMR spectroscopy, and perform site directed mutagenesis to determine which amino acids are critical for interaction with FAK-CD. Finally, we will define the physical interaction between FAK and VEGFR-3, determine the mechanism of survival signaling mediated by this interaction, and examine the relationship between levels of expression of FAK and VEGFR-3 in translational studies of primary human breast cancers. By defining the mechanism of interaction between FAK and its critical binding partners, we will identify novel sites for molecular targeting to induce apoptosis in human breast cancer cells.
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