The Akt and NFAT Pathway and the Regulaton of Cancer Cell Motility

描述（由申请人提供）：本提案的目的是确定蛋白激酶Akt及其下游靶点在癌症病因学中的作用。PI 3-K-Akt信号轴已被证明通过影响细胞存活和生长对肿瘤进展至关重要。然而，Akt在控制细胞运动和侵袭性迁移中的作用知之甚少。我们的研究表明，Akt亚型Akt1和Akt2对许多乳腺癌细胞系的移动性有不同的影响，其中Akt1作为侵袭性迁移的抑制剂，而Akt2可能作为增强剂。我们进一步发现Akt可以磷酸化转录因子NFAT，促进其降解并降低转录活性。最后，我们还发现转录因子FOXO3a（也是Akt的底物）可以增加运动性，Akt的磷酸化可以阻断这种表型。基于我们的研究，我们提出Akt亚型对上皮癌细胞的运动有不同影响的假设。我们认为这是由Akt1和Akt2的细胞定位介导的，而这反过来又决定了akt介导的NFAT和FOXO3a的调控和磷酸化，从而导致它们的核输出和降解。有三个目标将检验这一模型

DESCRIPTION (provided by applicant): The objective of this proposal is to define the role of the protein kinase Akt and its downstream targets in the etiology of cancer. The PI 3-K-Akt signaling axis has been shown to be critical for tumor progression by impacting cell survival and growth. Little is known, however, concerning the role of Akt in controlling cell motility and invasive migration. Our studies have shown that Akt isoforms Akt1 and Akt2 have distinct effects on the motility of a number of breast cancer cell lines, where Akt1 functions as an inhibitor of invasive migration, whereas Akt2 may function as an enhancer. We further show that Akt can phosphorylate the transcription factor NFAT, promote its degradation and blunt transcriptional activity. Finally, we also show that the transcription factor FOXO3a, also an Akt substrate, increases motility and that phosphorylation by Akt blocks this phenotype. Based on our studies, we propose the hypothesis that Akt isoforms have distinct effects on the motility of epithelial cancer cells. We propose that this is mediated by cellular localization of Akt1 and Akt2, and that in turn this determines the Akt-mediated regulation and phosphorylation of NFAT and FOXO3a, leading to their nuclear export and degradation. Three aims will test this model:- In AIM 1, we will rigorously test the contribution of Akt1, Akt2 and Akt3 in regulating breast cancer cell motility and alterations in the actin cytoskeleton, using both loss-of-function approaches, such as siRNA, as well as gain-of-function genetic approaches with activated Akt alleles. We will investigate the importance of cellular localization of Akt isoforms, and also the contribution of the Akt phosphatase PHLPP. In AIM 2, we will determine the mechanism by which Akt isoforms control NFAT transcriptional activity, leading to nuclear export. We also propose experiments to mechanistically evaluate how Akt isoforms mediate ubiquitination and degradation of NFAT through the E3 ligases MDM2, Skp2 and NEDD4-2. We will evaluate the contribution of the ERK pathway in Akt-regulated invasive migration. In AIM 3, we will determine if FOXO3a, a distinct Akt substrate, promotes the acquisition of a motile phenotype in epithelial cells, and whether Akt-mediated phosphorylation, nuclear export, ubiquitination and proteasomal degradation block this response. The results of these studies will provide important new insights into the mechanisms by which Akt isoforms modulate cancer cell invasive migration through two crucial effectors, NFAT and FOXO3a. We anticipate that the successful completion of these studies will provide a paradigm shift in the field because they will show that signaling through Akt has profound effects on cell motility of cancer cells. There is also the potential that the outcome of our studies will provide for the future development of novel therapeutic interventions for tumor progression.