Regulation of Invadopodia Formation in Breast Cancer Cells

描述(申请人提供)：转移并发症是乳腺癌死亡的主要原因。侵袭性癌细胞使用富含F-肌动蛋白的突起，称为侵袭足，以降解细胞外基质(ECM)的迁移障碍。我们已经证明，整合素1介导的黏附刺激Arg非受体酪氨酸激酶与肌动蛋白聚合调节因子Cortactin、N-Wasp和Vav2在F-肌动蛋白介导的非癌细胞细胞边缘突起的新生部位相互作用。这些蛋白中的每一种都定位于不定足类，是不定足类功能所必需的。事实上，我们发现精氨酸介导的皮质肌动蛋白磷酸化触发了人乳腺癌细胞内的肌动蛋白聚合，导致它们稳定并获得基质降解活性。我们将阐明整合素？1：精氨酸：皮质酮：N-黄蜂：Vav2轴在乳腺癌侵袭和转移过程中控制失足功能的机制，并筛选这些调控因子之间关键相互作用的抑制剂作为药物开发的先导化合物。我们的第一个目标是了解Arg是如何在Inadoodia中被本地化和调控的。Arg使用不同的结构域直接与F-肌动蛋白、微管和整合素1结合。我们的初步工作强烈表明，这些相互作用调节着这个关键调控因子在内侧足底的定位和激活。我们将使用RNAi敲除Arg和整合素1，用相互作用缺陷的Arg和整合素1突变体拯救，并使用定量免疫荧光和基质降解分析来确定这些相互作用中的哪一种介导Arg在侵袭性人类乳腺癌细胞中的定位和活性。我们的第二个目标是确定对内侧足纲功能最关键的相互作用，并筛选这些相互作用的抑制物。除了结合Cortactin，Arg还使用一个不同的结构域来结合和激活N-WASP。精氨酸介导的皮质肌动蛋白磷酸化也促进其与肌动蛋白聚合调节因子Vav2的结合。我们假设，Arg协调Cortactin、N-Wasp和Vav2在inadodidia内的激活和组装，以触发Arp2/3复合体介导的肌动蛋白聚合。我们将使用类似于目标1的击倒/互补方法来确定哪些相互作用对内向心功能最关键。我们还将进行高通量小分子筛选，以确定破坏这些蛋白质之间关键相互作用的化合物，并测试它们阻止乳腺癌细胞侵袭的能力。我们的第三个目标是测试关键的肌动蛋白调节因子的干扰如何影响乳腺癌的侵袭和转移。在体外，内毒素介导基质屏障的穿透，但它们是否以及如何在体内介导乳腺癌的侵袭性尚未得到严格的测试。我们将使用SCID小鼠异种移植和MMTV-多瘤中T乳腺癌模型来确定干扰这些内源性调节因子如何影响乳腺癌细胞在体内的侵袭和转移。

DESCRIPTION (provided by applicant): Complications from metastasis are the leading cause of mortality from breast cancer. Invasive cancer cells use F-actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) barriers to migration. We have shown that integrin ¿1-mediated adhesion stimulates the Arg nonreceptor tyrosine kinase to interact with the actin polymerization regulators cortactin, N-WASp, and Vav2 at nascent sites of F-actin-mediated cell edge protrusion in non-cancerous cells. Each of these proteins localizes to invadopodia and is required for invadopodial function. Indeed, we find that Arg-mediated cortactin phosphorylation triggers actin polymerization within human breast cancer cell invadopodia, leading to their stabilization and acquisition of matrix-degrading activity. We will elucidate the mechanisms by which the integrin ¿1:Arg:cortactin:N-WASp:Vav2 axis controls invadopodia function during breast cancer invasion and metastasis and screen for inhibitors of key interactions between these regulators as lead compounds for drug development. Our first aim is to understand how Arg is localized and regulated within invadopodia. Arg uses distinct domains to bind directly to F-actin, microtubules, and integrin ¿1. Our preliminary work strongly suggests that these interactions regulate localization and activation of this key regulator at invadopodia. We will use RNAi knockdown of Arg and integrin ¿1, rescue with interaction-defective Arg and integrin ¿1 mutants, and use quantitative immunofluorescence and matrix degradation assays to determine which of these interactions mediates Arg localization to and activity within invadopodia in invasive human breast cancer cells. Our second aim is to identify the interactions most critical for invadopodia function and screen for inhibitors of these interactions. In addition to binding cortactin, Arg uses a distinct domain to bind and activate N-WASp. Arg-mediated cortactin phosphorylation also promotes its binding to Vav2, a regulator of actin polymerization. We hypothesize that Arg coordinates the activation and assembly of cortactin, N-WASp, and Vav2 within invadopodia to trigger Arp2/3 complex-mediated actin polymerization. We will use a knockdown/complementation approach similar to Aim 1 to identify which interactions are most critical for invadopodial function. We will also perform high throughput small molecule screens to identify compounds that disrupt key interactions between these proteins and test their ability to block breast cancer cell invasiveness. Our third aim is to test how disruption of key invadopodial actin regulators affect breast cancer invasion and metastasis. Invadopodia mediate penetration of matrix barriers in vitro, but whether and how they mediate breast cancer invasiveness in vivo has not been rigorously tested. We will use scid mouse xenograft and MMTV-polyoma middle T breast cancer models to determine how disrupting these invadopodial regulators affects breast cancer cell invasion and metastasis in vivo.