The transition from carcinoma in situ to invasive cancer is a defining step in metastatic progression. The overall hypothesis examined in this proposal is the D3 phosphoinositide metabolism is essential for carcinoma invasion. This hypothesis is based on the finding that the a6b4 integrin can promote carcinoma invasion by a mechanism that involves its ability to activate phosphoinositide 3-OH kinase (PI3K) and one of its downstream effectors, the small GTP-binding protein Rac. PI3K is a lipid kinase that phosphorylates the D3 position of inositol lipids to form the D3 phosphoinositides. A major function of the D3 phosphoinositides is to bind and recruit signaling molecules to the plasma membrane where they can interact with other regulatory and effector molecules to alter cellular functions. The involvement of a PI3K- dependent signaling pathway in invasion adds to previous data that have implicated PI3K in tumor promoting functions including transformation, cell survival, anchorage-independent growth, and motility. Taken together, these findings support a central role for PI3K and its lipid products in carcinoma progression and highlight the need to investigate in more detail how this pathway is regulated. The focus of this R21 proposal will be to elucidate the mechanisms by which the a6b4 integrin influences the production and metabolism of D3 phosphoinositide second messengers. The first specific aim will address how a6b4 activates PI3K to produce the D3 phosphoinositides by identifying sequence motifs in the b4 cytoplasmic domain that are essential for a6b4-dependent activation of PI3K. Biochemical and molecular approaches will be used to identify the sites that are involved in activating PI3K, as well as the signaling molecules that interact with them. The second specific aim will examine the involvement of a D3-dependent inositol polyphosphate 5'-phosphatase in a6b4-dependent signaling of invasion. The experiments outlined in this proposal will be performed in collaboration with Dr. Lewis Cantley, an expert on lipid kinases and D3 phosphoinositides. These studies will contribute to our understanding of the mechanism by which a6b4 activates signaling pathways to promote carcinoma invasion. The goal is to identify molecular targets for future metastasis related study.
