Regulation of Invadopodia Formation in Breast Cancer Cells

乳腺癌细胞侵袭伪足形成的调控

基本信息

批准号：
8969667
负责人：
Anthony J Koleske
金额：
$ 31.05万
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 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8969667
关键词：
Actins Adhesions Affect Attenuated Binding Binding Proteins Biochemical Pathway Biological Assay Biological Models Blood Circulation Breast Cancer Cell Breast Cancer Model Breast cancer metastasis C-terminal Cells Chemicals Complex Cytoplasmic Tail Data Disseminated Malignant Neoplasm EMS1 gene Extracellular Matrix F-Actin Fibroblasts GTP Phosphohydrolase Activators Genetic Models Human Imatinib Immunofluorescence Immunologic In Vitro Integrins Invaded Lead Maps Measures Mediating Microtubules Mouse Mammary Tumor Virus Mus Neoplasm Metastasis Organ Penetration Phosphorylation Phosphotransferases Polyomavirus Positioning Attribute Process Protein Tyrosine Kinase Proteins RNA Interference Regulation STI571 Site Testing Tissues Tumor Escape Viral Tumor Antigens Work Xenograft procedure base cancer cell cancer invasiveness drug development high throughput screening in vitro activity in vivo inhibitor/antagonist kinase inhibitor knock-down malignant breast neoplasm matrigel metastatic process migration mortality mutant novel strategies polymerization rho GTP-Binding Proteins screening small molecule tumor progression

项目摘要

DESCRIPTION (provided by applicant): 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 ¿1-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 ¿1: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 ¿1. 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 ¿1, rescue with interaction-defective Arg and integrin ¿1 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.

描述（由申请人提供）：转移并发症是乳腺癌死亡的主要原因。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因子 cortactin、N-WASp 和 Vav2 相互作用，这些蛋白位于非癌细胞中 F-肌动蛋白介导的细胞边缘突出的新生位点。事实上，我们发现精氨酸介导的皮质蛋白磷酸化会触发肌动蛋白聚合。在人乳腺癌细胞侵袭伪足中，导致其稳定并获得基质降解活性，我们将阐明整合素的机制。 1:Arg:cortactin:N-WASp:Vav2 轴控制乳腺癌侵袭和转移过程中的侵袭足功能，并筛选这些调节剂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 如何定位和调节。在侵袭伪足内，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 ¿ 1. 我们的初步工作强烈表明，这些相互作用调节了入侵伪足中这一关键调节因子的定位和激活。我们将使用 RNAi 敲低 Arg 和整合素 ¿ 1，用相互作用缺陷的Arg和整合素拯救¿ 1 突变体，并使用免疫荧光和基质降解测定来确定这些相互作用中的哪一种相互作用介导侵袭性人乳腺癌细胞中侵袭伪足内的精氨酸定位和定量活性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侵袭伪足的抑制剂。这些相互作用除了结合 cortactin 之外，还使用独特的结构域来结合和激活 N-WASp 介导的 cortactin 磷酸化也促进其与 Vav2 的结合。我们勇敢地认为 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。确定哪些相互作用对侵袭足功能最关键，我们还将进行高通量小分子筛选，以确定破坏这些蛋白质之间关键相互作用的化合物，并测试它们阻断乳腺癌细胞的能力。我们的第三个目标是测试关键侵袭足肌动蛋白调节因子的破坏如何影响体外乳腺癌侵袭和转移，但它们是否以及如何介导体内乳腺癌侵袭性尚未得到严格测试。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。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education.

Abl-1 激酶对于 NK 细胞抑制信号传导是可有可无的，并且不参与小鼠 NK 细胞的培养。

DOI：
发表时间：
2017-09
期刊：
影响因子：
3.7
作者：
Ganesan, S;Luu, T T;Chambers, B J;Meinke, S;Brodin, P;Vivier, E;Wetzel, D M;Koleske, A J;Kadri, N;Höglund, P
通讯作者：
Höglund, P

The repeat region of cortactin is intrinsically disordered in solution.

Cortactin 的重复区域在溶液中本质上是无序的。

DOI：
发表时间：
2017-12-01
期刊：
影响因子：
4.6
作者：
Li, Xiaofeng;Tao, Yeqing;Murphy, James W;Scherer, Alexander N;Lam, TuKiet T;Marshall, Alan G;Koleske, Anthony J;Boggon, Titus J
通讯作者：
Boggon, Titus J

The Src kinases Hck, Fgr and Lyn activate Arg to facilitate IgG-mediated phagocytosis and Leishmania infection.

Src 激酶 Hck、Fgr 和 Lyn 激活 Arg 以促进 IgG 介导的吞噬作用和利什曼原虫感染。