The role of actin polymerization in tumor metastasis

描述：（由申请人提供）本申请的长期目标是 了解肿瘤转移的分子机制。以前的作品有 建立了染色体11 q13的扩增，这经常发生， 在乳腺癌、头颈部鳞状细胞癌和膀胱癌中， 在皮质素或EMS 1（蛋白质的主要底物）的过表达中 酪氨酸激酶Src具有与肌动蛋白丝结合的潜力。患者 coronin基因扩增者预后差， 复发的可能性。然而，coronin的作用机制 与肿瘤进展的关系尚不清楚。越来越多的证据表明 皮质激素参与细胞骨架变化的调节 与细胞运动性和细胞形状变化有关。我们最近的研究进一步 证明了coronin在肌动蛋白聚合中起着重要作用， 与Arp 2/3复合物相互作用，Arp 2/3复合物是启动肌动蛋白的关键蛋白质机制 细胞内的聚合。此外，皮质激素调节 Arp 2/3复合物对肌动蛋白成核和肌动蛋白分支的作用， 细胞前缘结构的形成。我们还发现 野生型coronin的过表达可以增强体外细胞运动性， 促进体内肿瘤转移，而过度表达corneum 酪氨酸磷酸化或Arp 2/3结合的突变可以损害细胞 迁移和骨转移。基于这些观察，我们假设， 由Arp 2/3复合物和coronin介导的肌动蛋白聚合作用， 在肿瘤转移中起重要作用。为了验证这一假设，我们建议 阐明Arp 2/3复合物与coronin的相互作用， Src、PIP 2等细胞因子对Corp 2/Arp 2/3复合物调控 因素，并测试假设是否抑制肌动蛋白 通过破坏这些相互作用的聚合将能够有效地 危及体内转移。因此，本申请的具体目的 包括：（1）corneum激活的机制的表征 Arp 2/3复合物对肌动蛋白成核和分支的活性。我们将 表征coronin和 Arp 2/3复合物，研究corneum增强肌动蛋白的机制， 成核并促进和稳定肌动蛋白分支。(2)研究 调节corp 1 n/Arp 2/3介导的肌动蛋白成核和分支。我们将 评估Src和PIP 2对肌动蛋白成核和分支的影响 在体外研究Corp 1 n/Arp 2/3复合物介导的细胞凋亡，并寻找其他细胞凋亡的分子机制。 Src、PIP 2、Cdc 42和Rac可通过其调节 Corp 2/Arp 2/3复合物。(3)突变体的效果分析 Arp 2/3和coronin对肿瘤转移的影响。我们将介绍使用 来源于Arp 2/3和corpine的逆转录病毒功能肽， 破坏或增强肌动蛋白聚合到MDA-MB-231肿瘤细胞中。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.