Regulation of invadopodia formation in breast cancer cells

乳腺癌细胞侵袭伪足形成的调节

基本信息

批准号：
7847676
负责人：
Anthony J Koleske
金额：
$ 30.91万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-21 至 2011-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7847676
关键词：
Actin-Binding Protein Actins Adhesions Affect Affinity Binding Binding Proteins Biochemical Biochemical Pathway Biological Assay Blood Circulation Blood Vessel Tissue Breast Cancer Cell Cancer Patient Cells Complex Extracellular Matrix F-Actin Fibroblasts Human Individual Integrins Invaded Knock-out Knockout Mice Life Maps Measures Mediating Microfilaments Neoplasm Metastasis Organ Pathway interactions Phosphorylation Phosphorylation Site Phosphotransferases Phosphotyrosine Positioning Attribute Process Property Protein Tyrosine Kinase Proteins Proteome RNA Interference Regulation Research Role Set protein Signal Transduction Specificity Surface Testing Tissues Transgenic Organisms Tumor Cell Invasion Tumor Escape Tyrosine Tyrosine Phosphorylation Xenograft procedure actin-related protein 3 adhesion receptor anticancer research cancer cell cancer invasiveness cellular imaging human EMS1 protein malignant breast neoplasm mortality mouse model mutant polymerization protein protein interaction receptor tissue culture tumor

项目摘要

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 and , 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 disrupts invadopodium formation and how this affects overall breast cancer invasiveness and metastasis. Our second aim is to elucidate the role of tyrosine phosphorylation of cortactin in mediating invadopod formation. Integrin-mediated adhesion stimulates Arg-dependent cortactin phosphorylation on three tyrosine residues (Y421, Y466, Y482). Tyrosine phosphorylated cortactin (cortactin-P) has increased ability to promote invadopodium formation and cancer metastasis. We identified several proteins that bind selectively and with high affinity to cortactin-P, but not to nonphosphorylated cortactin. We will further characterize the specificity and affinity of cortactin-P for these proteins in vitro and in cells. We will use 4D live cell imaging and RNAi-mediated knockdown of the cortactin-P-interacting proteins to test their roles in invadopodia formation.

肿瘤细胞转移是乳腺癌患者死亡率的主要原因。癌细胞通过形成称为Invadopodia的丝状蛋白质来侵入周围的组织和血管，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了Invadodia形成的机制。 Cortactin是一种肌动蛋白结合蛋白，核Invadopodia形成并促进乳腺癌细胞转移。皮质素的这些特性是通过其酪氨酸磷酸化的。我们已经表明，对ECM的粘合剂激活整联蛋白粘附受体以刺激ARG酪氨酸激酶。反过来，ARG磷酸化皮尔塔克蛋白以促进动态侵袭性细胞边缘蛋白。整联蛋白和和ARG在高度浸润性的乳腺癌细胞中都进行了更新，并且在这些细胞中抑制ARG或皮质素的功能会损害其侵入性。我们的建议将阐明整联蛋白 - 阿尔格 - 软骨轴控制Invadopodia的形成和功能的机制。我们的第一个目的是测试整联蛋白1和3信号是否通过ARG和Cortactin来调节Invadodia形成，ECM侵入性和肿瘤转移。我们将确定ECM是否粘附在不同程度的侵入性和测试整联蛋白1或3或ARG损害此过程的情况下刺激乳腺癌细胞中皮尔塔克蛋白磷酸化。我们将使用组织培养测定法来确定整联蛋白1或3，ARG或Cortactin的敲低/敲除是否会破坏Invadopodium的形成，以及这如何影响整体乳腺癌的侵入性和转移。我们的第二个目的是阐明皮质素酪氨酸磷酸化在介导不体动物形成中的作用。整联蛋白介导的依从性刺激了三种酪氨酸残留物（Y421，Y466，Y482）上的ARG依赖性皮质磷酸化。酪氨酸磷酸化的皮质素（Cortactin-P）具有促进侵袭性形成和癌症转移的能力。我们确定了几种有选择性结合并与Cortactin-P高亲和力结合的蛋白质，但不能与非磷酸化的cortactin结合。我们将进一步表征Cortactin-P在体外和细胞中对这些蛋白质的特异性和亲和力。我们将使用4D活细胞成像和RNAi介导的Cortactin-P-P相互作用蛋白的敲低来测试其在Invadopodia形成中的作用。