ADAM-17/EGFR信号轴活化在次级胆汁酸诱导结肠上皮细胞凋亡抵抗中的作用

Nowadays the prevalence of colorectal cancers increases dramatically, and may closely correlate to diets high in fats, which result in elevated second bile acids, especially deoxycholic acid (DCA) in the intestine. Studies have found that long term exposure to DCA may result in apoptosis resistance of colonic epithelial cells and promote carcinogenesis; however, the mechanisms are not elucidated. Our previous study showed that DCA could increase the number and development of intestinal tumors in APCmin/+ mice. Moreover, DCA could transactivate epidermal growth factor receptor (EGFR),and result in apoptosis resistance, which required a disintegrin and metalloprotease-17 (ADAM-17) activity. It was reported that ADAM-17 can cleave some EGFR ligands and transactivate EGFR. Thus, we suppose that DCA can induce apoptosis resistance of colonic epithelial cells and promote carcinogenesis via ADAM-17/EGFR axis activation. Our proposal is firstly to evaluate the effects on the alteration of cell apoptosis and associated proteins after inhibiting EGFR activity using specific blockers and siRNA transfection assays, which will establish the role of EGFR transactivation in apoptosis resistance of colonic epithelial cells induced by DCA. The mechanisms that ADAM-17 activation by DCA may cleave EGFR ligands, and transactivate EGFR and its downstream signal pathways will be further investigated using ADAM-17-/- colonic epithelial cells transducted with wt ADAM-17 or vector only, and ADAM-17 deficient mice model. This project will elucidate the mechanisms how DCA induces apoptosis resistance and promotes carcinogenesis, and provide new potential targets for the treatment of bile acids-related colorectal cancers.

大肠癌发病率逐年增高，与高脂饮食关系密切，后者可引起肠腔内次级胆汁酸尤其脱氧胆酸（DCA）水平显著升高。持续暴露DCA可诱导结肠上皮细胞产生凋亡抵抗从而促进癌变，但机制不明。我们发现DCA能显著增加小鼠肠道肿瘤数量并诱发癌变，还可活化EGFR产生凋亡抵抗并依赖ADAM-17的活性。文献报道ADAM-17能切割EGFR的相应配体从而活化EGFR，故我们推断"DCA通过活化ADAM-17/EGFR信号轴进而诱导凋亡抵抗促进癌变"。本项目首先采用靶向阻断和siRNA转染等方法观察阻断EGFR后细胞凋亡及凋亡蛋白变化，以明确EGFR活化在DCA诱导凋亡抵抗中的作用；进而运用ADAM-17基因转导的ADAM-17-/-细胞及其基因缺陷鼠模型，探讨DCA活化ADAM-17切割EGFR配体激活EGFR及其下游通路的机制。本项目旨在探索DCA如何诱导凋亡抵抗促进癌变的机制，为其相关肠癌的防治提供新靶点。

近年来结直肠癌（Colorectal cancer, CRC）的发病率和死亡率在全球范围显著增加。除了遗传背景，约70%的散发性CRC与饮食因素有关。高脂饮食可引起肠腔内次级胆汁酸尤其脱氧胆酸（DCA）水平显著升高。持续暴露DCA可诱导结肠上皮细胞产生凋亡抵抗从而促进癌变，但机制不明。本项目从肠癌高发的重要环境因素之一次级胆汁酸（DCA）入手，着重探讨了去整合素-金属蛋白酶-17(A disintegrin and metalloprotease-17, ADAM-17)/表皮生长因子受体(Epidermal growth factor receptor，EGFR) 信号轴活化在DCA诱导结肠上皮细胞凋亡抵抗中起到的重要作用及其相关分子机制。我们的科学假说是：DCA通过活化ADAM-17/EGFR信号轴进而诱导凋亡抵抗促进癌变。主要的研究内容包括：采用靶向阻断和siRNA转染等方法观察阻断EGFR后细胞凋亡及凋亡蛋白的变化，明确了EGFR活化在DCA诱导凋亡抵抗中起到关键作用；进而运用ADAM-17基因转导的ADAM-17-/-细胞及其基因缺陷鼠模型，探讨了DCA活化ADAM-17切割EGFR配体激活EGFR及下游通路的分子机制。主要的研究结果包括：发现EGFR活化诱导肠上皮细胞凋亡抵抗可能是DCA促进肠道肿瘤发生的重要机制之一；DCA可刺激肠上皮细胞释放可溶性配体双调蛋白（AR）进而活化EGFR，并且这一过程依赖ADAM17；还发现肠粘膜屏障损伤在DCA促进肠道肿瘤细胞增殖、凋亡抵抗及癌变发生中发挥了重要作用。本课题主要特色与创新之处在于同时运用永生化结肠上皮细胞以及基因缺陷鼠为研究对象，围绕细胞凋亡抵抗的关键信号轴，开展次级胆汁酸促癌机制的探讨，较以往采用结肠癌细胞为研究对象的实验更具说服力，而且还为今后相关研究的开展提供了技术支持和实验模型参考。成果以论文的形式提交，已在SCI期刊发表论文8篇，1篇正在审稿，核心期刊4篇；培养了从事大肠肿瘤基础和临床研究的青年骨干2名，使4名博硕士研究生获得系统的科研技能训练。因此，通过本项目的实施，深入探究了DCA诱导肠上皮细胞产生凋亡抵抗效应的机制，进而破坏增殖/凋亡稳态最终促进肿瘤发生。这些新的认识对肠癌发病机制研究起到了推动作用，并为其临床防治新靶点的选择提供新思路，产生了一定潜在的社会效益。