CIP4靶向募集Cdc42参与侵袭性伪足形成促进结直肠癌侵袭转移的分子机制研究

CIP4 (Cdc42 - interacting protein 4) is a protein encoding by TRIP10 gene. We confirmed that CIP4 expression was upregulated in colorectal cancer (CRC) tissues and cells, and closely related to migrate and invasive ability of CRC cells. Studies found that CIP4 and Cdc42 were closely associated with tumor invadopodia formation, and interacted each other. Thus, we propose that CIP4 targeted to recruit Cdc42 involving in invadopodia formation promotes invasion and metastasis of CRC. In order to confirm our hypothesis, we evaluate the correlation between CIP4 , Cdc42 expression and CRC metastasis by IHC and tissue samples analysis. Vitro and vivo biological function assays will be used to clear the effect of CIP4 on biological functions of CRC cells related to metastasis. Deletion mutant construction and GST-pull down assay will be adapted to explore the interaction between CIP4 and active Cdc42. Eventually, Using GTP-bound GTPase pull-down and immunofluorescence to confirm that CIP4 targeted to recruit Cdc42 involving in invadopodia formation promotes invasion and metastasis of CRC.

CIP4（ Cdc42-interacting protein 4 ）是由TRIP10 基因编码的蛋白。我们前期研究初步发现结直肠癌组织和细胞中CIP4表达上调，与肿瘤细胞迁移侵袭能力密切相关。研究发现CIP4和Cdc42与肿瘤侵袭性伪足形成密切相关，且两者存在相互作用。因此，我们提出结直肠癌细胞通过CIP4将Cdc42靶向募集参与侵袭性伪足形成促进肿瘤侵袭转移的假说。本项目拟采用免疫组化和组织标本分析等方法评估CIP4和Cdc42表达与结直肠癌转移的相关性；从体内外水平明确CIP4对结直肠癌细胞转移生物学功能的影响；运用截短子载体构建和GST-pull down等技术验证CIP4与活化Cdc42相互作用关系；最后利用GTP-bound GTPase pull-down和免疫荧光等方法证实结直肠癌细胞通过CIP4将Cdc42靶向募集参与侵袭性伪足形成促进肿瘤侵袭转移的假说。

背景:CIP4 (Cdc42- interaction protein 4)是F-BAR家族中的一员，在细胞膜和肌动蛋白调控中发挥重要作用，已有报道与Cdc42相互作用，与肿瘤侵袭伪足的形成密切相关。但在结直肠癌(CRC)中，CIP4与Cdc42相互作用及其下游应答信号通路的具体机制尚不清楚，其对肿瘤浸润转移的影响值得探索。.方法:采用免疫组化和western blot检测CIP4和Cdc42的表达。进一步分析其与CRC临床病理特征的关系。创伤愈合实验、transwell迁移和侵袭实验检测了CIP4对体外细胞迁移和侵袭能力的影响，原位结直肠癌异种移植小鼠模型评价体内肿瘤转移。采用免疫荧光、扫描电镜(SEM)和基质降解试验评价侵袭伪足的形成和功能。通过共免疫沉淀(co-IP)和GST下拉试验证实了CIP4与Cdc42的相互作用。免疫荧光法观察CIP4、GTP-Cdc42和侵袭伪足的共定位情况。western blot和免疫荧光检测相关下游信号通路。.结果:CIP4在人结直肠癌组织中表达明显增高，与结直肠癌浸润深度、转移及患者生存率低相关。在培养的CRC细胞中，敲除CIP4可抑制体外细胞迁移和侵袭能力，抑制体内肿瘤转移，而过表达CIP4则通过促进细胞的侵袭伪足形成和基质降解能力而证实相反的情况。此外，我们发现GTP-Cdc42是CIP4的一个直接相互作用蛋白，上调CIP4可招募GTP-Cdc42于侵袭性伪足并参于伪足的形成。此外，在过表达CIP4的CRC细胞中发现了激活的NF-κB信号通路，促进了侵袭伪足的形成，而抑制CIP4或Cdc42均可抑制NF-κB通路，导致侵袭伪足数量减少。.结论:CIP4靶向招募GTP-Cdc42并与与之结合，并通过激活NF-κB信号通路促进侵袭伪足形成，从而促进结直肠癌的侵袭和转移。