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学中的作用。PI 3-K-Akt信号传导轴已显示通过影响细胞存活和生长而对肿瘤进展至关重要。然而，关于Akt在控制细胞运动和侵入性迁移中的作用知之甚少。我们的研究表明，Akt亚型Akt 1和Akt 2对许多乳腺癌细胞系的运动性具有不同的影响，其中Akt 1作为侵入性迁移的抑制剂发挥作用，而Akt 2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，促进其降解和钝化转录活性。最后，我们还表明，转录因子FOXO 3a，也是Akt底物，增加运动性和Akt的磷酸化阻断这种表型。基于我们的研究，我们提出了Akt亚型对上皮癌细胞运动性有明显影响的假设。我们认为这是由Akt 1和Akt 2的细胞定位介导的，反过来这又决定了Akt 1介导的NFAT和FOXO 3a的调节和磷酸化，导致它们的核输出和降解。三个目标将检验这一模式： 在AIM 1中，我们将严格测试Akt 1，Akt 2和Akt 3在调节乳腺癌细胞运动性和肌动蛋白细胞骨架改变中的作用，使用功能丧失方法，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亚型的细胞定位的重要性，以及Akt磷酸酶PHLPP的贡献。 在AIM 2中，我们将确定Akt亚型控制NFAT转录活性，导致核输出的机制。我们还提出了实验，机械地评估Akt亚型如何介导泛素化和NFAT降解通过E3连接酶MDM 2，Skp 2和NEDD 4 -2。我们将评估ERK通路在Akt调节的侵袭性迁移中的作用。 在AIM 3中，我们将确定FOXO 3a，一种独特的Akt底物，是否促进上皮细胞获得运动表型，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断这种反应。 这些研究的结果将为Akt亚型通过两个关键效应物NFAT和FOXO 3a调节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为该领域提供一个范式转变，因为它们将表明通过Akt的信号传导对癌细胞的细胞运动性具有深远的影响。我们的研究结果也有可能为肿瘤进展的新型治疗干预措施的未来发展提供帮助。

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.