Regulation of invadopodia formation in breast cancer cells

肿瘤细胞转移是乳腺癌患者死亡的主要原因。癌细胞通过形成富含肌动蛋白的丝状突起侵入周围组织和血管，这种突起被称为浸润状突起，可降解细胞外基质（ECM）。乳腺癌研究的一个主要挑战是阐明侵过足形成的机制。皮质蛋白是一种肌动蛋白结合蛋白，可促进乳腺癌细胞的浸润形成和转移。接触蛋白的这些特性因其酪氨酸磷酸化而增强。我们已经证明，粘附在ECM上可以激活整合素粘附受体，从而刺激精氨酸酪氨酸激酶。反过来，精氨酸磷酸化接触蛋白，促进动态侵殖样细胞边缘突起。整合素1、3和精氨酸在高侵袭性乳腺癌细胞中均上调，抑制这些细胞中的精氨酸或接触功能会损害其侵袭性。我们的研究将阐明整合素-精氨酸接触轴控制侵入性足形成和功能的机制。我们的第一个目的是测试整合素1和整合素3是否通过Arg和接触发出信号，调节侵过泡形成、ECM侵袭性和肿瘤转移。我们将确定ECM粘附是否会刺激具有不同程度侵袭性的乳腺癌细胞的接触磷酸化，并测试整合素¿1或¿3或Arg的敲低是否会损害这一过程。我们将使用组织培养试验来确定是否敲低/敲除整合素¿1或¿3，Arg或接触破坏。整合素1、3和精氨酸在高侵袭性乳腺癌细胞中均上调，抑制这些细胞中的精氨酸或接触功能会损害其侵袭性。我们的研究将阐明整合素-精氨酸接触轴控制侵入性足形成和功能的机制。我们的第一个目的是测试整合素1和整合素3是否通过Arg和接触发出信号，调节侵过泡形成、ECM侵袭性和肿瘤转移。我们将确定ECM的粘附是否会刺激称为侵过足的接触性突起，从而降解细胞外基质（ECM）。乳腺癌研究的一个主要挑战是阐明侵过膜形成的机制。皮质蛋白是一种肌动蛋白结合蛋白，可促进乳腺癌细胞的浸润形成和转移。接触蛋白的这些特性因其酪氨酸磷酸化而增强。成纤维细胞与ECM的粘附激活整合素粘附受体，从而刺激精氨酸酪氨酸激酶。反过来，精氨酸磷酸化接触蛋白，促进动态侵殖样细胞边缘突起。有趣的是，整合素1和3、精氨酸和皮质蛋白在高侵袭性乳腺癌细胞中都是上调的，抑制精氨酸或皮质蛋白的功能会损害它们的侵袭性。利用我们的专业知识和我们的合作者，我们处于一个独特的位置来描述整合素-精氨酸接触蛋白信号轴在确定乳腺癌侵袭性和转移通过侵过性形成的相关性。我们有三个具体目标：目标2。了解Arg如何与接触相互作用以促进肌动蛋白聚合。皮质蛋白刺激肌动蛋白相关蛋白2/3 （Arp2/3）复合体促进肌动蛋白丝成核的能力，并稳定由此产生的分支的f -肌动蛋白网络。然而，目前尚不清楚这些接触的活动是如何被调节以促进侵入性胚形成的。我们的初步研究提供了证据，表明精氨酸结合和磷酸化可调节联系蛋白促进f -肌动蛋白突出的能力。我们将通过以下途径确定Arg调控联系的生化机制：A.绘制Arg和联系的相互作用域。精氨酸通过至少两种不同的蛋白质与接触物结合：蛋白质相互作用。我们将绘制Arg和接触中的哪些表面介导这些相互作用，并测试这些表面的破坏如何影响Arg：接触相互作用。B.确定精氨酸-接触蛋白相互作用在f -肌动蛋白成核中的意义。我们将测试Arg结合和磷酸化是否会影响其通过Arp2/3复合物刺激肌动蛋白丝成核和促进肌动蛋白分支稳定性的能力。目标3。了解接触磷酸化如何调节invadopodium的形成/功能。在对人类蛋白质组中几乎所有（115个中的100个）Src同源2 （SH2）结构域的大规模筛选中，我们发现了一个小子集与选择性磷酸化连接蛋白（cortactin-P）结合。我们假设这些蛋白与接触蛋白- p协同作用，促进f -肌动蛋白核心的组装和稳定性。我们将通过以下方法确定这些蛋白如何调节内联蛋白促进侵入样体形成/功能的能力：A.测试侵入样体形成中磷酸化位点的要求。精氨酸磷酸化三个酪氨酸残基（Y421, Y466, Y482）。我们将使用Y421、Y466和Y482接触点突变体来确定侵入adopodium形成所需的必要磷酸化位点。B.测定接触蛋白- p相互作用蛋白的亲和力和特异性。我们将使用同一组突变体确定接触蛋白中磷酸酪氨酸残基的单个SH2结构域的特异性。C.接触蛋白- p结合蛋白在侵殖虫形成中的作用。我们将使用活细胞成像和rnai介导的蛋白敲低来检查接触蛋白- p结合蛋白是否定位于入侵足并调节其形成。

Tumor cell metastasis is the leading cause of mortality in breast cancer patients. Cancer cells invade surrounding tissue and blood vessels by forming filamentous- (F-) actin-rich protrusions called invadopodia that degrade the extracellular matrix (ECM). A major challenge in breast cancer research is the elucidation of the mechanisms that underlie invadopodia formation. Cortactin, an actin-binding protein, nucleates invadopodia formation and promotes breast cancer cell metastasis. These properties of cortactin are potentiated by its tyrosine phosphorylation. We have shown that adhesion to ECM activates integrin adhesion receptors to stimulate the Arg tyrosine kinase. In turn, Arg phosphorylates cortactin to promote dynamic invadopodia-like cell edge protrusions. Integrins ¿1 and ¿3, and Arg are all upregulated in highly invasive breast cancer cells, and inhibiting Arg or cortactin function in these cells compromises their invasiveness. Our proposal will elucidate the mechanisms by which the integrin-Arg-cortactin axis controls invadopodia formation and function. Our first aim is to test whether integrins ¿1 and ¿3 signal through Arg and cortactin to regulate invadopodia formation, ECM invasiveness, and tumor metastasis. We will determine whether ECM adhesion stimulates cortactin phosphorylation in breast cancer cells with varying degrees of invasiveness and test if knockdown of integrins ¿1 or ¿3, or Arg compromise this process. We will use tissue culture assays to determine whether knockdown/knockout of integrins ¿1 or ¿3, Arg, or cortactin disrusions. Integrins ¿1 and ¿3, and Arg are all upregulated in highly invasive breast cancer cells, and inhibiting Arg or cortactin function in these cells compromises their invasiveness. Our proposal will elucidate the mechanisms by which the integrin-Arg-cortactin axis controls invadopodia formation and function. Our first aim is to test whether integrins ¿1 and ¿3 signal through Arg and cortactin to regulate invadopodia formation, ECM invasiveness, and tumor metastasis. We will determine whether ECM adhesion stimulates cortactrich protrusions called invadopodia that degrade the extracellular matrix (ECM). A major challenge in breast cancer research has been to elucidate the mechanisms that underlie invadopodium formation. Cortactin, an actin-binding protein, nucleates invadopodia formation and promotes breast cancer cell metastasis. These properties of cortactin are potentiated by its tyrosine phosphorylation. Adhesion of fibroblasts to ECM activates integrin adhesion receptors to stimulate the Arg tyrosine kinase. In turn, Arg phosphorylates cortactin to promote dynamic invadopodia-like cell edge protrusions. Interestingly, integrins ¿1 and ¿3, Arg, and cortactin are all upregulated in highly invasive breast cancer cells, and inhibiting Arg or cortactin function compromises their invasiveness. Utilizing our expertise and that of our collaborators, we are in a unique position to delineate the relevance of the integrin-Arg-cortactin signaling axis in determining breast cancer invasiveness and metastasis via invadopodia formation. We have three specific aims: Aim 2. To understand how Arg interacts with cortactin to promote actin polymerization in invadopodia. Cortactin stimulates the ability of the Actin-related protein 2/3 (Arp2/3) complex to promote actin filament nucleation and also stabilizes the resulting branched F-actin networks. However, it remains unclear how these activities of cortactin are regulated to promote invadopodium formation. Our preliminary studies provide evidence that Arg binding to and phosphorylation of cortactin regulates cortactin's ability to promote F-actin protrusions. We will determine the biochemical mechanisms by which Arg regulates cortactin by: A. Mapping the interaction domains in Arg and cortactin. Arg binds to cortactin via at least two distinct sets of protein:protein interactions. We will map which surfaces in Arg and cortactin mediate these interactions and test how disruption of these surfaces affects Arg:cortactin interactions. B. Determining the significance of Arg-cortactin interactions in F-actin nucleation. We will test whether Arg binding to and phosphorylation of cortactin affect its ability to stimulate actin filament nucleation by the Arp2/3 complex and promote actin branch stability. Aim 3. To understand how cortactin phosphorylation regulates invadopodium formation/function. In a large-scale screen of nearly all (100 out of 115) Src homology 2 (SH2) domains in the human proteome, we have identified a small subset that bind selectively phospho-cortactin (cortactin-P). We hypothesize that these proteins synergize with cortactin-P to promote assembly and stability of the invadopodium F-actin core. We will determine how these proteins regulate cortactin's ability to promote invadopodium formation/function by: A. Testing requirement of phosphorylation sites in invadopodium formation. Arg phosphorylates cortactin on three tyrosine residues (Y421, Y466, Y482). We will use Y421, Y466, and Y482 cortactin point mutants to identify essential phosphorylation sites required for invadopodium formation. B. Measuring affinity and specificity of cortactin-P interacting proteins. We will determine the specificity of individual SH2 domains for the phosphotyrosine residues in cortactin using the same set of mutants. C. Characterizing roles for cortactin-P-binding proteins in invadopodium formation. We will use live cell imaging and RNAi-mediated protein knockdown to examine whether the cortactin-P-binding proteins localize to invadopodia and regulate their formation.