Role of PLCgamma and NFAT Signaling Pathway in Carcinoma

本研究的目的是了解癌细胞中磷脂酶CGamma(PLCGamma)和NFAT信号通路的激活，以及它如何参与整合素依赖的侵袭。PLCGamma是脂质信号通路中的一个关键酶，负责细胞内第二信使IP3、钙离子和DAG的产生。PLCGamma参与了许多需要肌动蛋白细胞骨架重塑的生理反应，如趋化作用。在免疫细胞中，PLCGamma对于转录因子NFAT(激活的T细胞核因子)的激活是必不可少的，但关于PLCGamma/NFAT在肿瘤中的表达和功能作用尚不清楚。整合素α6β4在癌细胞中的表达导致癌细胞侵袭和迁移速度的增加。该提案中提出的初步研究表明，PLCGamma活性对于介导不同癌细胞系的侵袭是必要的，也是充分的。此外，我们发现NFAT在这些细胞中既有表达又有转录活性，并且它在这些细胞的侵袭过程中起着至关重要的作用。据我们所知，这是第一次证明这种转录因子在癌症中的功能作用。在该提案中，实验旨在专门测试α6beta4整合素对PLCGamma的激活，以及磷脂酶、SH2、SH3和PH结构域在介导NFAT的入侵、迁移和激活中的相对贡献。关于NFAT在上皮细胞或癌细胞中的调控机制尚不清楚，因此，我们将评估PLCGamma、钙钙调神经磷酸酶和GSK-3调节NFAT活性和侵袭的机制，以及这如何与α6β4整合素的激活有关。还将评估PLCGamma和NFAT在导致板层形成和迁移的细胞骨架重塑中的作用。最后，还将研究新的NFAT-5转录因子的调节机制，因为初步研究表明，它也是介导侵袭性表型的关键。这些研究的结果将提供关于以前未知的信号通路的重要新信息，该信号通路由整合素激活，并导致依赖于PLCGamma和NFAT的肿瘤侵袭。我们预计，生物化学和分子遗传学方法的结合将有助于更好地了解控制癌症进展的机制，因此，基于这些发现，未来有可能开发新的癌症治疗干预措施。

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.