Supplement to Regulation of invadopodia formation in breast cancer cells

乳腺癌细胞侵袭伪足形成调节的补充

• 批准号: 8652002
• 负责人: Anthony J Koleske
• 金额: $ 2.3万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-21 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8652002
• 关键词: Actins; Adhesions; Affect; Binding; Biochemical Pathway; Biological Assay; Blood Circulation; Breast Cancer Cell; Breast Cancer Model; Cells; Complex; Disseminated Malignant Neoplasm; Extracellular Matrix; F-Actin; Immunofluorescence Immunologic; In Vitro; Integrins; Lead; Malignant Neoplasms; Mediating; Microtubules; Mouse Mammary Tumor Virus; Mus; Neoplasm Metastasis; Organ; Penetration; Phosphorylation; Polyomavirus; Protein Tyrosine Kinase; Proteins; RNA Interference; Regulation; Site; Testing; Tissues; Tumor Escape; Work; Xenograft procedure; cancer cell; cancer invasiveness; drug development; human EMS1 protein; in vivo; inhibitor/antagonist; malignant breast neoplasm; migration; mortality; mutant; polymerization; small molecule

ABSTRACT Complications from metastasis are the leading cause of mortality from breast cancer. Invasive cancer cells use F-actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) barriers to migration. We have shown that integrin -mediated adhesion stimulates the Arg nonreceptor tyrosine kinase to interact with the actin polymerization regulators cortactin, N-WASp, and Vav2 at nascent sites of F-actin-mediated cell edge protrusion in non-cancerous cells. Each of these proteins localizes to invadopodia and is required for invadopodial function. Indeed, we find that Arg-mediated cortactin phosphorylation triggers actin polymerization within human breast cancer cell invadopodia, leading to their stabilization and acquisition of matrix-degrading activity. We will elucidate the mechanisms by which the integrin :Arg:cortactin:N-WASp:Vav2 axis controls invadopodia function during breast cancer invasion and metastasis and screen for inhibitors of key interactions between these regulators as lead compounds for drug development. Our first aim is to understand how Arg is localized and regulated within invadopodia. Arg uses distinct domains to bind directly to F-actin, microtubules, and integrin . Our preliminary work strongly suggests that these interactions regulate localization and activation of this key regulator at invadopodia. We will use RNAi knockdown of Arg and integrin , rescue with interaction-defective Arg and integrin  mutants, and use quantitative immunofluorescence and matrix degradation assays to determine which of these interactions mediates Arg localization to and activity within invadopodia in invasive human breast cancer cells. Our second aim is to identify the interactions most critical for invadopodia function and screen for inhibitors of these interactions. In addition to binding cortactin, Arg uses a distinct domain to bind and activate N-WASp. Arg-mediated cortactin phosphorylation also promotes its binding to Vav2, a regulator of actin polymerization. We hypothesize that Arg coordinates the activation and assembly of cortactin, N-WASp, and Vav2 within invadopodia to trigger Arp2/3 complex-mediated actin polymerization. We will use a knockdown/complementation approach similar to Aim 1 to identify which interactions are most critical for invadopodial function. We will also perform high throughput small molecule screens to identify compounds that disrupt key interactions between these proteins and test their ability to block breast cancer cell invasiveness. Our third aim is to test how disruption of key invadopodial actin regulators affect breast cancer invasion and metastasis. Invadopodia mediate penetration of matrix barriers in vitro, but whether and how they mediate breast cancer invasiveness in vivo has not been rigorously tested. We will use scid mouse xenograft and MMTV-polyoma middle T breast cancer models to determine how disrupting these invadopodial regulators affects breast cancer cell invasion and metastasis in vivo.

抽象的 转移并发症是乳腺癌死亡的主要原因。侵袭性癌细胞利用富含 F-肌动蛋白的突起（称为侵袭伪足）来降解细胞外基质 (ECM) 的迁移屏障。我们已经证明，整合素 1 介导的粘附刺激 Arg 非受体酪氨酸激酶与肌动蛋白聚合调节剂 cortactin、N-WASp 和 Vav2 在非癌细胞中 F-肌动蛋白介导的细胞边缘突出的新生位点相互作用。这些蛋白质中的每一种都定位于侵袭足，并且是侵袭足功能所必需的。事实上，我们发现精氨酸介导的皮质蛋白磷酸化会触发人乳腺癌细胞侵袭伪足内的肌动蛋白聚合，从而导致其稳定并获得基质降解活性。我们将阐明整合素 1:Arg:cortactin:N-WASp:Vav2 轴在乳腺癌侵袭和转移过程中控制侵袭伪足功能的机制，并筛选这些调节因子之间关键相互作用的抑制剂，作为药物开发的先导化合物。 我们的首要目标是了解 Arg 如何在侵袭伪足中定位和调节。 Arg 使用不同的结构域直接与 F-肌动蛋白、微管和整合素  结合。我们的初步工作强烈表明这些相互作用调节了入侵伪足的这一关键调节因子的定位和激活。我们将使用 RNAi 敲低精氨酸和整合素 ，用相互作用缺陷型精氨酸和整合素  突变体进行拯救，并使用定量免疫荧光和基质降解测定来确定这些相互作用中哪些相互作用介导了侵袭性侵袭伪足中精氨酸的定位和活性。人类乳腺癌细胞。 我们的第二个目标是确定对侵袭伪足功能最关键的相互作用并筛选这些相互作用的抑制剂。除了结合 cortactin 之外，Arg 还使用独特的结构域来结合和激活 N-WASp。 Arg 介导的 cortactin 磷酸化也促进其与肌动蛋白聚合调节因子 Vav2 的结合。我们假设 Arg 协调侵袭伪足内皮质蛋白、N-WASp 和 Vav2 的激活和组装，以触发 Arp2/3 复合物介导的肌动蛋白聚合。我们将使用类似于目标 1 的敲低/互补方法来确定哪些相互作用对于侵袭足功能最关键。我们还将进行高通量小分子筛选，以识别破坏这些蛋白质之间关键相互作用的化合物，并测试它们阻止乳腺癌细胞侵袭的能力。 我们的第三个目标是测试关键的侵袭足肌动蛋白调节因子的破坏如何影响乳腺癌的侵袭和转移。侵袭伪足在体外介导基质屏障的穿透，但它们是否以及如何在体内介导乳腺癌侵袭性尚未经过严格测试。我们将使用 scid 小鼠异种移植物和 MMTV-多瘤中 T 乳腺癌模型来确定破坏这些侵袭足调节因子如何影响体内乳腺癌细胞的侵袭和转移。