Mechanism of Tumor Cell Invasion

描述（由申请人提供）：Invadopodia直接细胞迁移到周围的细胞外匹配和组织中，这是通过它们从细胞向外延伸并粘附和降解基质的能力来完成的。研究人员提出了一种新的接触蛋白作用，即这种与肌动蛋白结合的c-Src底物参与了靶向囊泡的递送、对接和在侵过孔的回收，并且c-Src是形成含有接触蛋白的功能性侵过孔复合物所必需的。此外，我们还提出脾脏酪氨酸激酶Syk是一种肿瘤抑制因子，它通过对Src活性的影响来改变细胞运动和侵袭。Syk的缺失与Src和接触蛋白的表达和激活增加的相互作用可能通过共同影响主要的信号转导途径促进乳腺癌的进展。我们提出了四个具体目标。目的1，确定Src/ contact在靶膜进出侵足的转运过程中的要求。目的2，确定由Src/ contact调控的MT1-MMP进出外源性的转运。目的3，通过对Src的负调控来确定Syk对侵过体的调控。目的4：确定Src、皮质和Syk相互作用与体内肿瘤细胞侵袭的相关性，以及它们在治疗靶向、风险评估或诊断方面的潜力。膜和分子动力学的检测包括表达绿色或红色荧光嵌合体的活细胞的共聚焦成像。基质降解的位点将与侵入物相关分子共定位。免疫电镜将用于确定接触素相关囊泡和膜的超微结构。突变体、抑制剂或RNAi敲低将用于评估Src、接触和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/接触蛋白和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.