Mechanism of Tumor Cell Invasion

描述(申请人提供)：内翻足引导细胞向周围的细胞外基质和组织迁移，这是通过它们从细胞向外延伸并附着和降解基质的能力实现的。提出了Cortactin的一个新的作用，其中这种肌动蛋白结合的c-Src底物参与了不定足的靶向囊泡的递送、对接和取回，并且c-Src是形成含有Cortactin的功能性内向复合体所必需的。此外，我们已经证明了脾酪氨酸激酶Syk是一种肿瘤抑制因子，它通过影响Src的活性改变细胞的运动和侵袭。Syk的缺失与Src和Cortactin表达和激活的增加相互作用，可能通过共同影响主要信号转导通路而促进乳腺癌的进展。我们提出了四个具体目标。目的1、确定靶向膜转运和膜外转运对Src/Cortactin的需求。AIM2，确定由Src/Cortactin调控的MT1-MMP运入和出出侵袭足的调节。目的：确定Syk通过对Src的负性调控来调控不定足的发生。目的：确定Src、Cortactin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。膜和分子的分析：在inadodidia上，赖氨酸包括表达绿色或红色荧光嵌合体的活细胞的共聚焦成像。基质降解的部位将与内毒素相关分子共同定位。免疫电子显微镜将被用来确定皮质素相关囊泡和膜的超微结构。突变体、抑制剂或RNAi敲除将被用来评估对Src、Cortactin和Syk的分子需求。最后，将使用原位杂交和免疫组织化学方法来确定人类乳腺组织中Src、EMSI/Cortactin和Syk的水平。我们的长期目标是开发干预src驱动的转移的策略，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.