Molecular Mechanisms of Invadopodia Formation and Function

描述(申请人提供)：肿瘤转移是癌症死亡的主要原因。转移性癌细胞通过细胞外基质(ECM)降解和侵袭。然而，介导性侵袭的研究仍然很少，对支配介导性介体形成和功能的分子机制也知之甚少。我的长期目标是阐明介导性侵袭的机制。这项申请具有双重目标：(1)提供我从博士后到癌症生物学/转移领域独立成功科学家所需的培训，以及(2)确定不定形诱导肿瘤细胞与细胞外基质黏附的细胞机制，这是我追求研究目标的下一步合乎逻辑的步骤。这一提议的中心假设是，内向组装是由侵袭性癌细胞的独特细胞粘连处的信号事件启动的，我们称之为内向前粘连。这一假说源于强大的初步数据，并基于我在实验室开发的独特的活细胞模型系统，用于同时可视化和分析内陷足的形成和在细胞外基质降解中的作用。 (Artym等人，癌症研究66：3034-43,2006)。利用三通道活细胞成像技术，我最近发现，首先，富含肌动蛋白和皮质蛋白的覆膜结构核心形成，然后，覆膜功能所需的金属蛋白酶MT1-MMP会在覆膜上积聚，从而触发细胞外基质的降解。我现在已经观察到在特定的粘连部位--侵袭前粘连--内翻足的启动和形成。 为了验证中心假说和实现这一应用的目标，提出了三个具体的目标：1)确定p1和(33)整合素调节不定足形成的机制；2)确定vinculin在不定足形成中的功能；3)在生理相关的3D体外系统中，确定ECM在不定足前粘连诱导不定足中的作用。将使用共聚焦和TIRF显微技术，并结合siRNA技术、蛋白质生物化学和电子显微镜。这项拟议的工作是创新的，因为它利用了我在实验室开发的实时活细胞模型，该模型允许在监测隐足动物组装和功能的同时表征蛋白质动力学。这项研究具有重要意义，因为它将扩大我们对恶性转化的认识，并为控制肿瘤细胞的侵袭和转移提供潜在的靶点。与公共卫生相关：了解内生生物的形成和功能的机制应该有助于深入了解肿瘤细胞生物学，从而为癌症转移提供潜在的治疗方法。

DESCRIPTION (provided by applicant): Tumor metastasis is the leading cause of cancer deaths. Metastatic cancer cells use invadopodia, specialized filament-like membrane protrusions, to degrade and invade through surrounding extracellular matrix (ECM). However, invadopodia-mediated invasion remains understudied, and little is known about the molecular mechanisms that govern invadopodia formation and function. My long-term goal is to elucidate mechanisms of invadopodia-mediated invasion. This application has the dual objective of: (1) providing training required for my transition from postdoctoral fellow to independent successful scientist in the field of cancer biology/metastasis, and (2) determining the cellular mechanisms of invadopodia induction upon tumor cell adhesion to ECM, which is the next logical step in pursuit of my research goal. The central hypothesis of this proposal is that invadopodia assembly is initiated by signaling events at unique cell adhesions of invading cancer cells that we call preinvadopodial adhesions. This hypothesis stems from strong preliminary data, and was formulated on the basis of the unique live-cell model system that I developed in our laboratory for simultaneous visualization and analysis of invadopodia formation and function in ECM degradation (Artym et al., Cancer Res. 66:3034-43, 2006). Using three-channel live-cell imaging, I recently discovered that first, structural cores of invadopodia rich in actin and cortactin are formed, and then, MT1-MMP, a metalloprotease required for invadopodia function, is accumulated at the invadopodia triggering ECM degradation. I have now observed invadopodia initiation and formation at specific adhesion sites, preinvadopodial adhesions. To test the central hypothesis and to accomplish the objective of this application, three specific aims are proposed: 1) Determine the mechanism of invadopodia regulation by p1 and (33 integrins; 2) Determine the function of vinculin in invadopodia formation; 3) Determine the role of ECM in regulation of invadopodia induction from preinvadopodial adhesions in physiologically relevant 3D in vitro system. Confocal and TIRF microscopy techniques in combination with siRNA technology, protein biochemistry, and electron microscopy will be used. The proposed work is innovative because it takes advantage of the real-time live-cell model developed by me in our laboratory that allows characterizing protein dynamics at invadopodia while simultaneously monitoring invadopodia assembly and function. The proposed research is significant because it will expand our knowledge of malignant transformation and provide potential targets for control of tumor cell invasion and metastasis. Relevance to Public Health: understanding the mechanisms governing the formation and function of invadopodia should provide insights into tumor cell biology that could lead to potential therapeutic approaches to cancer metastasis.