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。在高度侵袭性的乳腺癌细胞中，整合素β-内酰胺酶、β-内酰胺酶和Arg都上调，抑制这些细胞中的Arg或coronin功能会损害它们的侵袭性。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β 1和β 3是否通过Arg和corneum信号调节侵袭伪足形成，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coronin磷酸化，并测试是否敲低整合素β 1或β 3或Arg会损害这一过程。我们将使用组织培养试验来确定整合素β 1或β 3、Arg或cornea蛋白的敲除/敲除是否会破坏侵袭性伪足的形成，以及这如何影响整体乳腺癌的侵袭性和转移。 我们的第二个目的是阐明酪氨酸磷酸化的coronin介导的invadopod形成的作用。整合素介导的粘附刺激三个酪氨酸残基（Y 421、Y 466、Y 482）上的Arg依赖性corpine磷酸化。酪氨酸磷酸化的皮质素（cortactin-P）具有促进侵袭足形成和癌症转移的能力。我们确定了几种蛋白质，结合选择性和高亲和力的皮质素-P，但不非磷酸化cortocin。我们将进一步表征皮质素-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相互作用蛋白的敲低来测试它们在侵袭伪足形成中的作用。

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.