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。癌细胞通过形成称为侵袭伪足的富含丝状肌动蛋白的突起侵入周围组织和血管，所述突起降解细胞外基质（ECM）。乳腺癌研究的一个主要挑战是阐明侵袭伪足形成的机制。皮质激素是一种肌动蛋白结合蛋白，它能促进乳腺癌细胞侵袭伪足的形成并促进其转移。这些特性的coronin是加强其酪氨酸磷酸化。我们已经表明，粘附到ECM激活整合素粘附受体，刺激精氨酸酪氨酸激酶。反过来，精氨酸磷酸化coronin，以促进动态入侵样细胞边缘突起。整合素？1、？3和Arg在高度侵袭性的乳腺癌细胞中均上调，抑制这些细胞中的Arg或coronin功能会损害其侵袭性。我们的建议将阐明整合素-精氨酸-角蛋白轴控制侵袭伪足的形成和功能的机制。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cornein来调节侵袭伪足形成、ECM侵袭和肿瘤转移。我们将确定ECM粘附是否刺激具有不同程度侵袭性的乳腺癌细胞中的皮质蛋白磷酸化，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除/敲除整合素1或3、Arg或cornein。整合素？1、？3和Arg在高度侵袭性的乳腺癌细胞中均上调，抑制这些细胞中的Arg或coronin功能会损害其侵袭性。我们的建议将阐明整合素-精氨酸-角蛋白轴控制侵袭伪足的形成和功能的机制。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cornein来调节侵袭伪足形成、ECM侵袭和肿瘤转移。我们将确定ECM粘附是否刺激称为侵入伪足的皮质突起，其降解细胞外基质（ECM）。乳腺癌研究中的一个主要挑战是阐明invadopodium形成的机制。皮质激素是一种肌动蛋白结合蛋白，它能促进乳腺癌细胞侵袭伪足的形成并促进其转移。这些特性的coronin是加强其酪氨酸磷酸化。成纤维细胞与ECM的粘附激活整合素粘附受体以刺激Arg酪氨酸激酶。反过来，精氨酸磷酸化coronin，以促进动态入侵样细胞边缘突起。有趣的是，整合素1、？3、Arg和corneum在高度侵袭性乳腺癌细胞中均上调，抑制Arg或corneum功能会损害其侵袭性。利用我们的专业知识和我们的合作者，我们在一个独特的位置，以描绘的相关性，整合素-精氨酸-corneum信号轴在确定乳腺癌的侵袭性和转移通过侵袭伪足的形成。我们有三个具体目标：目标2。了解精氨酸如何与皮质素相互作用，促进侵袭伪足中肌动蛋白的聚合。Corpine刺激肌动蛋白相关蛋白2/3（Arp 2/3）复合物促进肌动蛋白丝成核的能力，并且还稳定所得分支的F-肌动蛋白网络。然而，目前还不清楚这些活动的corneum调节，以促进invadopodium形成。我们的初步研究提供的证据表明，精氨酸结合和磷酸化的coronin调节coronin的能力，促进F-肌动蛋白突起。我们将通过以下方式确定精氨酸调节皮质激素的生化机制：A.在Arg和coronin中绘制相互作用结构域。Arg通过至少两组不同的蛋白质：蛋白质相互作用与coronin结合。我们将映射哪些表面在精氨酸和coronin介导这些相互作用和测试如何破坏这些表面影响精氨酸：coronin的相互作用。B。确定F-肌动蛋白成核中Arg-角蛋白相互作用的重要性。我们将测试是否精氨酸结合和磷酸化的coronin影响其刺激肌动蛋白丝成核的Arp 2/3复合物和促进肌动蛋白分支稳定性的能力。目标3.了解coronin磷酸化如何调节invadopodium形成/功能。在几乎所有（100出115）Src同源性2（SH 2）结构域在人类蛋白质组的大规模筛选，我们已经确定了一个小的子集，选择性结合磷酸皮质素（皮质素-P）。我们推测，这些蛋白协同皮质素-P，以促进组装和稳定的侵袭足F-肌动蛋白核心。我们将通过以下方式确定这些蛋白质是如何调节皮质素促进侵袭足形成/功能的能力的：入侵伪足形成中磷酸化位点的检测要求。Arg磷酸化三个酪氨酸残基（Y 421，Y 466，Y 482）上的coronin。我们将使用Y 421，Y 466，和Y 482 corponin点突变体，以确定必要的磷酸化位点所需的invadopodium形成。B。测量皮质素-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单个SH 2结构域对coronin中磷酸酪氨酸残基的特异性。C.皮质素-P-结合蛋白在侵袭足形成中的特征作用。我们将使用活细胞成像和RNAi介导的蛋白质敲低来检查皮质素P结合蛋白是否定位于侵袭伪足并调节其形成。

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.