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。侵入性癌细胞使用称为Invadopodia的富含F-肌动蛋白的突起来降解迁移的细胞外基质（ECM）屏障。我们已经表明，整联蛋白介导的粘附刺激ARG非受体酪氨酸激酶与非癌细胞中F-肌动蛋白介导的细胞边缘缘的新生位点的肌动蛋白聚合调节剂Cortactin，N-WASP和VAV2相互作用。这些蛋白质中的每一个都将其定位于Invadopodia，这是侵袭功能所必需的。实际上，我们发现ARG介导的皮质磷酸化触发触发肌动蛋白在人类乳腺癌细胞Invadopodia中的聚合，从而导致其稳定并获得基质降解活性。我们将阐明整联蛋白：ARG：Cortactin：N-WASP：VAV2轴控制乳腺癌入侵和转移的侵袭性功能的机制，以及这些调节剂之间关键相互作用的抑制剂作为药物开发的铅化合物。 我们的第一个目的是了解ARG如何在Invadopodia中进行局部和监管。 ARG使用不同的结构域直接与F-肌动蛋白，微管和整合素bind。我们的初步工作强烈地表明，这些相互作用调节了该关键调节剂在Invadopodia中的定位和激活。我们将使用ARG和整联蛋白的RNAi敲低，用相互作用的ARG和整联蛋白突变体营救，并使用定量的免疫荧光和基质降解测定法来确定哪些相互作用将ARG ARG ARG在侵入性的人类乳腺癌细胞中介导了Invadopodia内部和活性。 我们的第二个目的是确定这些相互作用抑制剂的Invadodia功能和屏幕最关键的相互作用。除了结合皮质素外，ARG还使用一个不同的结构域来结合和激活N-WASP。 ARG介导的皮质蛋白磷酸化还促进了其与肌动蛋白聚合调节剂VAV2的结合。我们假设ARG会在Invadopodia内辅助Cortactin，N-WASP和VAV2的激活和组装，以触发ARP2/3复合介导的肌动蛋白聚合。我们将使用类似于AIM 1的敲低/互补方法来确定哪些相互作用对于侵袭性功能至关重要。我们还将执行高吞吐量的小分子筛选，以识别破坏这些蛋白质之间关键相互作用的化合物，并测试其阻断乳腺癌细胞侵入性的能力。 我们的第三个目的是测试关键的侵袭肌动蛋白调节剂的破坏如何影响乳腺癌的侵袭和转移。 Invadodia介导了基质屏障在体外的渗透，但是尚未严格测试它们是否以及如何介导体内乳腺癌的侵入性。我们将使用SCID小鼠异种移植物和MMTV-生态瘤中间T乳腺癌模型来确定这些侵袭性调节剂的破坏如何影响乳腺癌细胞的侵袭和体内转移。