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丝状(F-)肌动蛋白的突起来侵袭周围的组织和血管，这种突起被称为内陷，它会降解细胞外基质(ECM)。乳腺癌研究中的一个主要挑战是阐明内陷形成的机制。Cortactin是一种肌动蛋白结合蛋白，它能形成内陷，促进乳腺癌细胞的转移。皮质蛋白的这些特性被它的酪氨酸磷酸化所增强。我们已经证明，与ECM的黏附激活整合素黏附受体，从而刺激Arg酪氨酸激酶。反过来，Arg磷酸化Cortactin以促进动态的内陷足样细胞边缘突起。在高侵袭性的乳腺癌细胞中，整合素、和Arg都表达上调，抑制这些细胞中的Arg或Cortactin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-皮质蛋白轴控制内翻足形成和功能的机制。 我们的第一个目标是测试整合素1和3是否通过精氨酸和Cortactin信号调节内翻足的形成、细胞外基质侵袭和肿瘤转移。我们将确定细胞外基质黏附是否刺激具有不同侵袭力的乳腺癌细胞中皮质肌动蛋白的磷酸化，并测试整合素1或3或精氨酸的敲除是否影响这一过程。我们将使用组织培养分析来确定整合素1或3、精氨酸或皮质素的敲除/敲除是否扰乱了不定形的形成，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皮质肌动蛋白酪氨酸磷酸化在调节英索多德形成中的作用。整合素介导的黏附刺激三个酪氨酸残基(Y421，Y466，Y482)上依赖Arg的皮质动蛋白磷酸化。酪氨酸磷酸化皮质蛋白(Cortactin-P)具有促进内陷形成和肿瘤转移的能力。我们鉴定了几种与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内进一步鉴定Cortactin-P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相互作用蛋白的敲除来测试它们在内陷足形成中的作用。

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.