The role of actin polymerization in tumor metastasis

描述：(申请人提供)本申请的长期目标是 了解肿瘤转移的分子机制。以前的工作有 证实了染色体11q13的扩增，这种现象经常发生 在乳腺癌、头颈部鳞癌和膀胱癌方面，结果 在蛋白质的重要底物Cortactin或EMS-1的过度表达中 可能与肌动蛋白细丝结合的酪氨酸激酶Src。病人 伴有皮质蛋白基因扩增的患者预后往往较差，且增高 复发的可能性。然而，皮质肌动蛋白的作用机制 对肿瘤的进展仍不清楚。有越来越多的证据表明 皮质酮参与细胞骨架变化的调节 与细胞运动和细胞形状变化有关。我们最近的研究进一步 证明了Cortactin在肌动蛋白聚合中起着重要作用 与Arp2/3复合体相互作用，Arp2/3复合体是启动肌动蛋白的关键蛋白质机制 在细胞内聚合。此外，Cortactin还调节细胞的活性 Arp2/3复合体，肌动蛋白成核和肌动蛋白分支的两个重要步骤 细胞前沿结构的形成。我们还发现， 野生型Cortactin基因的过表达可增强体外培养细胞的运动能力 促进体内肿瘤转移，而Cortactin过表达 酪氨酸磷酸化或Arp2/3结合突变均可损害细胞 迁移和骨转移。基于这些观察，我们假设 Arp2/3复合体和Cortactin介导的肌动蛋白聚合 在肿瘤转移中起重要作用。为了检验这一假设，我们建议 描绘Arp2/3复合体和Cortactin之间的详细相互作用，探索 Src、PIP2等细胞对Cortactin/Arp2/3复合体的调节 因素，并检验是否抑制肌动蛋白的假说 通过破坏这些相互作用进行聚合将能够有效地 损害体内的转移。因此，该应用程序的具体目标是 包括：(1)皮质肌动蛋白激活血管紧张素转运蛋白的机制的表征 Arp2/3复合体对肌动蛋白成核和分支的活性。我们会 描述Cortactin和Cortactin之间相互作用的结构基础 Arp2/3复合体，研究皮质肌动蛋白增强肌动蛋白的机制 成核，促进和稳定肌动蛋白分支。(2)研究 Cortactin/Arp2/3介导的肌动蛋白核化和分支的调控。我们会 评估Src和PIP2对肌动蛋白成核和分支的影响 由Cortactin/Arp2/3复合体在体外介导，并寻找其他细胞 Src、PIP2、Cdc42和Rac可能通过其调节细胞外信号调节因子(S) Cortactin/Arp2/3复合体。(3)突变体的效应分析 Arp2/3和Cortactin对肿瘤转移的影响。我们将介绍使用 来源于Arp2/3和皮质素的逆转录病毒功能肽可以 干扰或增强肌动蛋白聚合为MDA-MB-231肿瘤细胞。接下来，我们 将评估这些细胞的运动性和转移潜力 体外和体内。

DESCRIPTION: (provided by applicant) The long-term goal of this application is to understand the molecular mechanism ot tumor metastasis. Prior works have established that amplification of the chromosome 11q13, which occurs frequently in breast cancer, head and neck squamous carcinomas and bladder cancer, results in overexpression of cortactin or EMS 1, a prominent substrate of protein tyrosine kinase Src with potential to associate with actin filaments. Patients with gene amplification of cortactin tend to have poor prognosis and increased possibility of relapse. However, the mechanism by which cortactin contributes to tumor progression is still unknown. There has been accumulated evidence that cortactin is implicated in the modulation of cell cytoskeletal changes associated with cell motility and cell shape changes. Our recent study further demonstrated that cortactin plays an important role in actin polymerization via interaction with Arp2/3 complex, a key protein machinery to initiate actin polymerization within cells. Furthermore, cortactin modulates the activity of Arp2/3 complex for actin nucleation and actin branching, two important steps in the formation of cell leading edge structures. We also found that overexpression of wild-type cortactin can enhance cell motility in vitro and facilitate tumor metastasis in vivo, whereas overexpression of cortactin mutants either in tyrosine phosphorylation or Arp2/3 binding can impair cell migration and bone metastasis. Based on these observations, we hypothesize that actin polymerization mediated by Arp2/3 complex and cortactin plays an important role in tumor metastasis. To test this hypothesis, we propose to delineate the detailed interactions among Arp2/3 complex and cortactin, explore the regulation of cortactin/Arp2/3 complex by Src, PIP2 and other cellular factors, and to test the hypothesis whether or not inhibition of actin polymerization by disruption of these interactions would be effectively able to compromise metastasis in vivo. Thus, the specific aims for this application include: (1) Characterization of the mechanism by which cortactin activates the activity of Arp2/3 complex for actin nucleation and branching. We will characterize the structural basis for the interactions between cortactin and Arp2/3 complex, examine the mechanism by which cortactin enhances actin nucleation and promotes and stabilizes actin branching. (2) Study of the regulation of cortactin/Arp2/3-mediated actin nucleation and branching. We will assess the effect of Src and PIP2 on the actin nucleation and branching mediated by cortactin/Arp2/3 complex in vitro, and search for other cellular factor(s) through which Src, PIP2, Cdc42 and Rac may regulate the function of cortactin/Arp2/3 complex. (3) Analysis of the effects of the mutants derived from Arp2/3 and cortactin on tumor metastasis. We will introduce using retrovirus functional peptides derived from Arp2/3 and cortactin that can disrupt or enhance actin polymerization into MDA-MB-23 1 tumor cells. Next, we will evaluate the motility and metastatic potentials of these cells both in vitro and in vivo.