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的主要原因，癌细胞通过形成富含丝状 (F-) 肌动蛋白的突起（称为侵入伪足）来侵入周围组织和血管，从而降解细胞外基质 (ECM)，这是乳腺癌面临的主要挑战。癌症研究旨在阐明侵袭伪足形成的机制，Cortactin 是一种肌动蛋白结合蛋白，使侵袭伪足形成成核并促进乳腺癌细胞。 Cortactin 的这些特性通过其酪氨酸磷酸化而增强。和  和 Arg 在高度调节中均上调侵袭性乳腺癌细胞，抑制这些细胞中的精氨酸或皮质蛋白功能会损害其侵袭性。我们的建议将阐明整合素-精氨酸-皮质蛋白轴控制侵袭性伪足形成和功能的机制。我们的首要目标是测试整合素 1 和 3 是否通过 Arg 和 cortactin 发出信号来调节侵袭伪足的形成、ECM 侵袭性和肿瘤转移。我们将确定 ECM 粘附是否会刺激不同程度侵袭性的乳腺癌细胞中的 cortactin 磷酸化。如果整合素 1 或 3 或 Arg 的敲低破坏了这一过程，我们将使用组织培养。确定整合素 1 或 3、Arg 或 cortactin 的敲低/敲除是否会破坏侵袭伪足的形成以及这如何影响整体乳腺癌的侵袭和转移。我们的第二个目标是阐明 cortactin 酪氨酸磷酸化在介导入侵足形成中的作用，整合素介导的粘附刺激 Arg 依赖性 cortactin 在三个酪氨酸残基（Y421、Y466、Y482）上的磷酸化增加。促进侵袭伪足形成和癌症转移的能力。鉴定了几种与 cortactin-P 选择性结合且具有高亲和力的蛋白质，但不与非磷酸化的 cortactin 结合。我们将在体外和细胞中进一步表征 cortactin-P 对这些蛋白质的特异性和亲和力。 RNAi 介导的 cortactin-P 相互作用蛋白敲低，以测试它们在侵袭伪足形成中的作用。