APOE2调控TAK1介导的IKK磷酸化在结直肠癌肝转移中的作用机制

The liver metastasis was associated with poor prognosis of colorectal cancer. Inflammatory tumor microenvironment and metastasis are closely related. In our preliminary work, apolipoprotein E (APOE) were found to inhibit the function of the colorectal cancer cells and lower APOE led to poorer outcome of the liver metastasis patients. The APOE subtype, APOE2, showed the most intense inhibition upon the colorectal cancer cells compared with other subtypes. The result of western-blot and GST-pulldown revealed that APOE2 directly combines with TAK1 kinase. APOE2 was found to suppress the phosphorylation of IKK through TAK1 and inhibit the tumor related inflammatory cytokine expression based on our pre-test results both in vivo and in vitro. Therefore, we assume that the phosphorylation of IKK through APOE2 regulating upon TAK1 undermines the expression of the inflammatory cytokine, thus suppresses the liver metastasis. To testify this assumption, the project plans to 1) investigating the correlation between APOE2/TAK1 and liver metastasis prognosis through clinical samples; 2) validating the regulation of APOE2 upon NF-КB pathway through in vitro and in vivo experiment; 3) investigating the binding sites between APOE2 and TAK1 via mutation and recovery experiments . The result of this study aims to clarify the mechanism of APOE2 regulating upon TAK1 and form a theoretical foundation for target treatment for CLM.

肝转移显著影响结直肠癌预后，而肿瘤炎症微环境与转移密切相关。申请人前期通过临床样本验证及体内外实验，发现载脂蛋白E——APOE与肝转移发生相关。进一步比较APOE不同亚型的功能，申请人首次提出APOE2具有最强的抑制转移作用。后续的Pulldown及co-IP实验表明APOE2能与NF-КB通路中的TAK1结合。最后申请人通过预实验，初步验证了APOE2能抑制TAK1下游的IKK磷酸化，并减少炎症因子表达。申请人据此推想APOE2通过调控TAK1介导的IKK磷酸化，减少肿瘤炎症因子产生，抑制结直肠癌肝转移。本项目拟通过临床样本验证，阐明APOE2、TAK1与肝转移预后的关系；通过细胞及动物实验，验证APOE2对NF-КB通路的调控作用；最后通过结合位点突变及回复实验，研究APOE2与TAK1的直接结合位点。阐明APOE2调控TAK1的作用机制，能为结直肠癌肝转移相关的靶向治疗奠定理论基础。

肝转移显著影响结直肠癌预后，而肿瘤炎症微环境与转移密切相关。申请人前期通过临床样本验证及体内外实验，发现载脂蛋白E——APOE与肝转移发生相关。进一步比较APOE不同亚型的功能，申请人首次提出APOE2具有最强的抑制转移作用，能抑制TAK1下游的IKK磷酸化，并减少炎症因子表达。本项目通过临床样本验证，阐明APOE2、TAK1与肝转移预后的关系，具体为：发生转移的结直肠癌，APOE2表达显著降低，下游IKK的磷酸化表达显著升高，同时低APOE2表达及IKK磷酸化水平升高与结直肠癌患者预后差显著相关。通过细胞及动物实验，本项目验证了APOE2通过NF-КB通路，与TAK1蛋白相结合，并能抑制下游IKK蛋白的磷酸化水平，同时减少炎性介质的表达，最终在体内环境减少NK细胞及巨噬细胞的富集。最后通过结合位点突变及回复实验，初步验证了APOE2通过与TAK1的2号亚基结合，发挥其调控作用。上述研究结果，能为结直肠癌肝转移相关的靶向治疗奠定理论基础。