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及其下游靶标在癌症病因中的作用。PI3-K-Akt信号轴通过影响细胞的存活和生长而在肿瘤进展中起关键作用。然而，关于Akt在控制细胞运动和侵袭性迁移中的作用，人们知之甚少。我们的研究表明，Akt亚型Akt1和Akt2对许多乳腺癌细胞株的运动能力有明显的影响，Akt1作为侵袭性迁移的抑制因子，而Akt2可能作为增强剂发挥作用。我们进一步证明Akt可以磷酸化转录因子NFAT，促进其降解并钝化转录活性。最后，我们还证明了转录因子FOXO3a，也是Akt的底物，增加了运动性，而Akt的磷酸化抑制了这一表型。基于我们的研究，我们提出了Akt亚型对上皮性癌细胞的运动有明显影响的假说。我们认为这是通过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的调节和磷酸化，导致它们的核输出和降解。三个目标将测试这一模式： 在目标1中，我们将严格测试Akt1、Akt2和AKT3在调控乳腺癌细胞运动和肌动蛋白细胞骨架变化中的作用，使用功能丧失的方法，如siRNA，以及激活Akt等位基因的功能获得遗传方法。我们将研究Akt异构体的细胞定位的重要性，以及Akt磷酸酶PHLPP的作用。 在AIM 2中，我们将确定Akt亚型调控NFAT转录活性，导致核出口的机制。我们还建议进行实验，以机械地评估Akt亚型如何通过E3连接酶MDM2、Skp2和NEDD4-2介导NFAT的泛素化和降解。我们将评估ERK通路在Akt调控的侵袭性迁移中的作用。 在AIM 3中，我们将确定不同的Akt底物FOXO3a是否促进上皮细胞运动表型的获得，以及Akt介导的磷酸化、核输出、泛素化和蛋白酶体降解是否阻止这一反应。 这些研究结果将为Akt亚型通过NFAT和FOXO3a这两个关键效应因子调控癌细胞侵袭性迁移的机制提供重要的新见解。我们预计，这些研究的成功完成将带来该领域的范式转变，因为它们将表明，通过Akt的信号对癌细胞的细胞运动具有深远的影响。我们的研究结果也有可能为肿瘤进展的新的治疗干预措施的未来发展提供依据。

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.