The objective of this proposal is to understand at the molecular level the activation of the phospholipase type C-gamma (PLC-gamma) signaling pathway by integrins, and how this relates to carcinoma invasion. PLC-gamma is a key enzyme in lipid signaling pathways, and is responsible for the generation of two important intracellular second messengers, Ins-1,4,5-P3 and diacylglycerol. PLC-gamma is implicated in a multitude of cellular processes and deregulation of this pathway is often associated with cell transformation. PLC-gamma has also been implicated in many physiological responses which require remodeling of the actin cytoskeleton, such as chemotaxis and formation of focal adhesions. Preliminary studies presented in this proposal show that PLC-gamma is tyrosine phosphorylated following ligation of the alpha6beta4, but not alpha6beta1 integrin. Similarly, I show that a PLC-gamma antagonist is a potent inhibitor of alpha6beta4-initiated carcinoma invasion. These studies suggest that the PLC-gamma pathway is required for mediating the invasive phenotype of carcinoma cells, specifically through the alpha6beta4 integrin. In the proposal, experiments are designed to demonstrate that PLC-gamma subtypes 1 and 2 are tyrosine phosphorylated following alpha6beta4 engagement. Similarly, activation of PLC-gamma in MDA-MEB-435 mock and beta4-transfected cells will be determined by monitoring the release of Ins-1,4,5-P3 and DAG in vivo. This will provide evidence of integrin specificity in the activation PLC-gamma. The mechanism of activation of PLC-gamma will also be explored, to determine if there is a physical association with the beta4 subunit, and whether PI 3-K is required for activation of PLC-gamma. Experiments are also proposed to directly asses the role of PLC-gamma in alpha6beta4-mediated carcinoma invasion in both MDA-MB-435 breast cancer cells as well as clone A colon cancer cells. A combination of chemical inhibitors, dominant negative and constitutively active PLC-gamma mutants will be used in in vivo studies to determine the importance of this pathway in invasion. Finally, the role of PLC-gamma in mediating cell spreading, lamellapodia formation and association with focal adhesion complexes will be investigated. The results of these studies will provide a framework with which to asses the relative importance of these signaling pathways in mediating invasion downstream of alpha6beta4. This biochemical and molecular genetic approach is likely to provide many clues as to how the alpha6beta4 integrin is a potent mediator of carcinoma invasion. A better understanding of PLC-gamma signaling in these processes has implications for the understanding of cell function and may provide a novel therapeutic approach for the treatment of metastatic carcinoma.
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