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.
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