FMNL2/Shank2参与结直肠癌侵袭性伪足形成的分子机制

FMNL2 is a gene associated with metastasis of colorectal carcinoma,which is screened and verified in our former exprements. Our reliminary experiment showed that Shank2 was one of the interacting proteins of FMNL2. Shank2 was a binding protein of Cortactin, what's more Cortactin is one of the key proteins of invadopodia. Therefore, we propose a novel regulatory mechanism of FMNL2 for the invasion and metastasis of colorectal cancer, which is that the regulation of FMNL2 is dependent on the formation of invadopodia through shank2. To validate this mechanism, gene transfection, interference, immunofluorescence, gelatin zymography, live cell imaging, experiments in vitro and in vivo are performed:(1) To confirm that FMNL2 activate the cortactin via shank2 and the formation of invadopodia;(2) To detect the effects of EGF/cdc42 pathway on the formation of invadopodia induced by FMNL2;(3) To confirm that FMNL2 induces the invasion and metastasis of colorectal carcinoma by the formation of invadopodia and shank2. Our study aims to reveal a novel regulatory mechanism of FMNL2, and to provide the novel gene target for clinical diagnosis, treatment, and prognosis of colorectal carcinoma.

FMNL2 是一个我们前期筛选并验证的结直肠癌转移促进基因。预实验中，我们利用酵母双杂交筛选出其相互作用蛋白Shank2，由于Shank2是Cortactin结合蛋白，而Cortactin是侵袭性伪足的关键标志物，因此我们提出FMNL2依赖Shank2促进侵袭性伪足的形成，从而诱导结直肠癌的侵袭及转移的新假设。本项目拟验证这一机制，利用基因转染、干扰、免疫荧光、明胶酶谱、活细胞成像技术、体内外侵袭及转移实验等进行（1）确认FMNL2通过Shank2活化Cortactin促进侵袭性伪足的形成；（2）检测FMNL2介导的侵袭性伪足形成受EGF/cdc42信号通路的调控；（3）证实FMNL2依赖Shank2及侵袭性伪足诱导结直肠癌的侵袭及转移。旨在揭示一个新的FMNL2表达调控机制，为临床抗结直肠癌侵袭和转移提供新的靶点和治疗策略。

结直肠癌是常见的恶性肿瘤，已经成为我国三大癌症之一。三分之一的结直肠癌患者行手术和放化疗后发生肝、肺转移，危及生命。阐明结直肠癌转移的分子机制，控制转移是当前研究的重要方向。FMNL2 是一个我们前期筛选并验证的结直肠癌转移促进基因。我们利用酵母双杂交筛选出其相互作用蛋白Shank2，由于Shank2是Cortactin结合蛋白，而Cortactin是侵袭性伪足的关键标志物，因此我们提出FMNL2依赖Shank2促进侵袭性伪足的形成，从而诱导结直肠癌的侵袭及转移的新理论。本项目利用基因转染、干扰、免疫荧光、明胶酶谱、活细胞成像技术、体内外侵袭及转移实验等：1）确认FMNL2通过Shank2活化Cortactin促进侵袭性伪足的形成；2）检测FMNL2介导的侵袭性伪足形成受EGF/cdc42信号通路的调控；3）证实FMNL2依赖Shank2及侵袭性伪足诱导结直肠癌的侵袭及转移。本项目的主要研究结果显示：1）FMNL2通过支架蛋白SHANK2与cortactin作用，还直接与cortactin相互作用； FMNL2与SHANK2、cortactin三色共定位于胞膜和胞浆；FMNL2与SHANK2的表达存在显著正相关, FMNL2与cortactin的表达存在显著正相关；人结肠癌组织中癌中表达FMNL2、SHANK2和cortactin,三者的表达趋势较一致；2）FMNL2-CT 段与 SHANK2 直接结合，FMNL2-FH1 与 cortactin-SH3 直接结合；3）FMNL2促进人结直肠癌细胞侵袭性伪足的形成；4）SHANK2和cortactin是FMNL2诱导侵袭性伪足形成所必需的；5）hEGF/Cdc42信号通路在上游调控FMNL2诱导结直肠癌侵袭性伪足的形成；hEGF刺激后SiFMNL2/Cdc42Q61L细胞侵袭性伪足显著增多；细胞迁移率显著升高;给与AG1478抑制后FMNL2细胞侵袭性伪足显著减少；明胶降解率显著下降。本项目阐述了FMNL2与侵袭性伪足关键蛋白cortactin存在直接或间接作用，FMNL2通过SHANK2或cortactin促进结直肠癌细胞侵袭性伪足的形成，该作用受到上游hEGF/Cdc42信号通路的正向调节；为临床抗结直肠癌侵袭和转移提供新的靶点和治疗策略。

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