Mechanism of Tumor Cell Invasion

描述（由申请人提供）：侵袭伪足引导细胞迁移进入并穿过周围的细胞外基质和组织，这是通过它们从细胞向外延伸并粘附和降解基质的能力实现的。一个新的作用coronin的建议，在此肌动蛋白结合，c-Src基板参与有针对性的囊泡交付，对接和检索在invadopodia和c-Src是必需的功能invadopodial复合物含有coronin的形成。此外，有人提出，脾酪氨酸激酶，Syk，我们已经证明是一种肿瘤抑制剂，改变细胞的运动和入侵通过其影响Src的活性。Syk的缺失与Src和coronin的表达和激活增加的相互作用可能通过共同影响主要信号转导途径来促进乳腺癌的进展。我们提出了四个具体目标。目的1，确定Src/corneum在侵袭伪足靶向膜转运和从侵袭伪足靶向膜转运中的需求。目的2，确定Src/corpine调节的MT 1-MMP转运至侵袭伪足和从侵袭伪足转运的调节。目的3，确定Syk通过Src的负调节对侵袭性伪足的调节。目的4，确定Src、corneumn和Syk相互作用与体内肿瘤细胞侵袭的相关性，以及它们用于治疗靶向、风险评估或诊断的潜力。侵袭伪足的膜和分子动力学分析包括表达绿色或红色荧光嵌合体的活细胞的共聚焦成像。基质降解位点将与侵袭足相关分子共定位。免疫电子显微镜将用于确定皮质素相关囊泡和膜的超微结构在侵入伪足。将使用突变体、抑制剂或RNAi敲低来评估Src、corpine和Syk的分子需求。最后，原位杂交和免疫组织化学将用于确定人乳腺组织中Src、EMSI/coronin和Syk的水平。我们的长期目标是制定干预Src驱动转移的策略，并研究患者的风险评估策略。

DESCRIPTION (provided by applicant): Invadopodia direct cell migration into and through surrounding extraceilular matches and tissues and this is accomplished by their ability to extend outward from the cell and adhere to and degrade the matrix. A novel role for cortactin is proposed in which this actin-binding, c-Src substrate participates in targeted vesicle delivery, docking and retrieval at invadopodia and that c-Src is required for the formation of functional invadopodial complexes containing cortactin. Further, it is proposed that the spleen tyrosine kinase, Syk, that we have shown to be a tumor suppressor, alters cellular motility and invasion via its effect upon Src activity. The interaction of loss of Syk with increased expression and activation of Src and cortactin may promote breast cancer progression by collectively affecting major signal transduction pathways. We propose four specific aims. Aim1, Determine the requirement for Src/cortactin for targeted membrane transport to and from invadopodia. Aim2, Determine regulation of MT1-MMP transport to and from invadopodia regulated by Src/cortactin. Aim3, Determine regulation of invadopodia by Syk via negative regulation of Src. Aim4, Determine relevance of Src, cortactin, and Syk interaction for tumor cell invasion in vivo, and their potential for therapeutic targeting, risk assessment or diagnosis. Assays for membrane and molecular :lynamics at invadopodia include confocal imaging of live cells expressing green or red fluorescent chimeras. Sites of matrix degradation will be co-localized with invadopodia-associated molecules. Immuno-electron microscopy will be used to determine the ultrastructure of cortactin-associated vesicles and membranes at invadopodia. Mutants, inhibitors, or RNAi knockdown will be used to assess the molecular requirements for Src, cortactin and Syk. Finally, in situ hybridization and immunohistochemistry will be used to determine the levels of Src, EMSI/cortactin, and Syk in human breast tissues. Our long term goal is to develop strategies to interfere with Src-driven metastasis and to study risk assessment strategies for patients.