FMNL2通过促进EGFL6旁分泌调控结直肠癌血管生成和转移的分子机制

Our previous study showed that prometastatic factor FMNL2 regulated tumor angiogenesis, but the mechanism remains unclear. EGFL6 was screened out as an FMNL2 interacting protein using yeast two-hybrid technology. FMNL2 promoted the exocytosis and combination of EGFL6 with endothelial cell membrane receptor CKAP4 by coimmunoprecipitation and mass spectrometry assays. Based on literature review and preliminary experiments, we propose that FMNL2 inhibits degradation and promotes extracellular secretion of EGFL6, which combines with CKAP4 and promotes angiogenesis and metastasis of CRC by activating ERK1/2 signal pathway. To validate this hypothesis, in vitro and in vivo experiments were performed in this study: (1) to confirm the regulation relationship between FMNL2 and EGFL6; (2) to explore the role of EGFL6 in angiogenesis and related molecular signal pathway; and (3) to confirm the mechanism of FMNL2 in promoting angiogenesis and metastasis of CRC by regulating EGFL6. Our study aims to illustrate the mechanism of FMNL2 in promoting angiogenesis and metastasis of CRC, and to provide a novel target and strategy for prognosis evaluation and treatment of CRC.

前期工作中我们发现转移促进因子FMNL2可以调控肿瘤血管生成，但其机制尚不清楚。利用酵母双杂交筛选出其相互作用蛋白EGFL6，发现FMNL2可促进EGFL6的胞外分泌，利用免疫共沉淀及质谱鉴定初步筛查出EGFL6可能与内皮细胞膜受体CKAP4结合，结合文献我们提出：结直肠癌细胞高表达的FMNL2通过与EGFL6结合，抑制其降解，并促其分泌至胞外，随后与内皮细胞膜受体CKAP4结合，活化ERK1/2信号通路，从而促进血管生成和转移。为证实这一假设，本研究拟从细胞水平和动物水平，（1）明确FMNL2与EGFL6的调控关系；（2）探讨EGFL6在结直肠癌血管生成中的作用及其相关分子信号通路；（3）阐明FMNL2通过调控EGFL6促进结直肠癌血管形成和转移的机制。以深入揭示FMNL2参与肿瘤血管微环境形成进而诱导结直肠癌转移的重要作用，为结直肠癌的预后评估及治疗提供新的靶点和策略。

前期工作中我们发现转移促进因子FMNL2可以调控肿瘤血管生成，但其机制尚不清楚。利用酵母双杂交筛选出其相互作用蛋白EGFL6，利用免疫荧光、Western blot、CO-IP和GS-Pull Down实验证实FMNL2与EGFL6存在直接相互作用且具有正向调控关系；通过透射电镜和Western blot实验发现结直肠癌细胞以旁分泌途径分泌囊泡，囊泡中含有EGFL6；利用Transwell、小管形成和鸡胚尿囊膜实验证实FMNL2通过EGFL6促进血管形成。利用裸鼠盲肠原位移植实验证实FMNL2通过EGFL6促进结直肠癌的肝转移且血管生成；通过Western blot发现，FMNL2/EGFL6通过激活ERK1/2通路促进血管生成。综上所述，结直肠癌细胞高表达的FMNL2通过与EGFL6结合，抑制其降解，并促其分泌至胞外，活化ERK1/2信号通路，从而促进血管生成和转移。以深入揭示FMNL2参与肿瘤血管微环境形成进而诱导结直肠癌转移的重要作用，为结直肠癌的预后评估及治疗提供新的靶点和策略。