The objective of this proposal is to understand the activation of the phospholipase Cgamma (PLCgamma) and NFAT signaling pathway in carcinoma cells, and how it contributes to integrin-dependent invasion. PLCgamma is a key enzyme in lipid signaling pathways, and is responsible for the generation of the intracellular second messengers, IP3, Ca2+ and DAG. PLCgamma has been implicated in many physiological responses which require remodeling of the actin cytoskeleton, such as chemotaxis. In immune cells, PLCgamma is essential for the activation of the transcription factor NFAT (nuclear factor of activated T cells), but nothing is known about the expression and functional role of PLCgamma/NFAT in carcinoma. Expression of the alpha6beta4 integrin in carcinoma cells leads to an increase in their rate of invasion and migration. Preliminary studies presented in the proposal show that PLCgamma activity is both necessary and sufficient to mediate invasion in distinct carcinoma cell lines. Moreover, we show that NFAT is both expressed and transcriptionally active in these cells, and that it is critical for mediating invasion of these cells. This is the first demonstration, to our knowledge, of a functional role for this transcription factor in carcinoma. In the proposal, experiments are designed to specifically test the activation of PLCgamma by the alpha6beta4 integrin, and the relative contribution of the phospholipase, SH2, SH3 and PH domains in mediating invasion, migration, and activation of NFAT. Nothing is known about the regulation of NFAT in epithelial or carcinoma cells, and thus we will evaluate the mechanism by which PLCgamma, Ca2+ calcineurin and GSK-3 regulate NFAT activity and invasion and how this is linked to the activation of the alpha6beta4 integrin. The role of PLCgamma and NFAT in cytoskeletal remodeling leading to lamellae formation and migration will also be evaluated. Finally, the mechanism of regulation of the novel NFAT-5 transcription factor will also be investigated, as the preliminary studies suggest that it is also critical for mediating the invasive phenotype. The results of these studies will provide important new information about a previously uncharacterized signaling pathway which is activated by integrins, and which leads to carcinoma invasion dependent on PLCgamma and NFAT. We anticipate that the combination of biochemical and molecular genetic approaches will contribute to a greater understanding of the mechanisms which govern carcinoma progression, and thus there is the future potential for the development of novel therapeutic interventions for cancer based on these findings.
