Molecular Mechanisms of Invadopodia Formation and Function

描述（由申请人提供）：肿瘤转移是癌症死亡的主要原因。转移性癌细胞利用侵过性丝状膜突起降解并侵入周围细胞外基质（ECM）。然而，入侵虫介导的入侵仍未得到充分研究，对入侵虫形成和功能的分子机制知之甚少。我的长期目标是阐明入侵介导的入侵机制。这个申请有两个目的：(1)为我从博士后过渡到癌症生物学/转移领域独立成功的科学家提供必要的培训，(2)确定浸润性诱导肿瘤细胞粘附于ECM的细胞机制，这是我追求研究目标的下一个合乎逻辑的步骤。该建议的中心假设是入侵癌细胞的独特细胞粘附（我们称之为入侵前粘附）的信号事件启动了侵过体组装。这一假设源于强有力的初步数据，并基于我在实验室开发的独特的活细胞模型系统而制定，该模型系统用于同时可视化和分析ECM降解过程中侵殖细胞的形成和功能

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.