Role of PLCgamma and NFAT Signaling Pathway in Carcinoma

本提案的目的是了解癌细胞中磷脂酶Cgamma （PLCgamma）和NFAT信号通路的激活，以及它如何促进整合素依赖性侵袭。plcγ是脂质信号通路中的关键酶，负责细胞内第二信使IP3、Ca2+和DAG的产生。PLCgamma与许多需要重塑肌动蛋白细胞骨架的生理反应有关，例如趋化性。在免疫细胞中，PLCgamma是激活转录因子NFAT（活化T细胞的核因子）所必需的，但PLCgamma/NFAT在癌中的表达和功能作用尚不清楚。癌细胞中α 6beta4整合素的表达导致其侵袭和迁移速度的增加。提案中提出的初步研究表明，PLCgamma活性是介导不同癌细胞系侵袭的必要和充分条件。此外，我们发现NFAT在这些细胞中既表达又转录活跃，并且它对介导这些细胞的入侵至关重要。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α 6β 4整合素对PLCgamma的激活，以及磷脂酶、SH2、SH3和PH结构域在介导NFAT入侵、迁移和激活中的相对贡献。关于上皮细胞或癌细胞中NFAT的调节尚不清楚，因此我们将评估PLCgamma、Ca2+钙调神经磷酸酶和GSK-3调节NFAT活性和侵袭的机制，以及这与α 6β 4整合素的激活如何相关。plcγ和NFAT在细胞骨架重塑导致片层形成和迁移中的作用也将被评估。最后，我们还将研究新型NFAT-5转录因子的调控机制，因为初步研究表明它在介导侵袭表型中也很重要。这些研究结果将为整合素激活的一种以前未被表征的信号通路提供重要的新信息，该信号通路导致依赖于plcγ和NFAT的癌症侵袭。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理解控制癌症进展的机制，因此，基于这些发现，未来有可能开发出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.