TGF-β调控S100A8促进结直肠癌侵袭转移的机制研究

Metastasis is the main cause of death in patients with colorectal cancer, and tumor microenvironment and epithelial mesenchymal transition play important roles in metastasis. TGF-β, enriched in tumor microenvironment, can pormote EMT. By microdissection and Affeymetrix U133 Plus 2.0 array, we found the expression of S100A8 in stroma of tumor invasive front was differ from that of tumor center. Predicted by the bioinformatics software, there maybe existed binding sites of USF2 in the promoter regions of S100A8. In previous study, our group found that TGF-β could upregulate the expression of USF2 and S100A8, and USF2 and S100A8 could induce invasion and migration in colorectal cancer, then downregulating USF2 or S100A8 could rescue the role of TGF-β in invasion and migration. Furthermore, overexpression of USF2 could active the expression of S100A8. In this study, we will clarify whether TGF-β could initiate EMT through upregulation the expression of USF2 and S100A8, and whether USF2 regulated the transcription of S100A8 by the binding to promotor regions of S100A8, and how did S100A8 regulate the program of EMT. The experimental methods include in vitro experiments, in vivo animal experiments, and detection in tissue samples. It will provide a theory basis on the relation between tumor inflammatory microenvironment and metastasis.

转移是危及肿瘤患者生命的主要原因，肿瘤微环境和上皮间质转化（EMT）是影响转移的重要环节。TGF-β是肿瘤微环境中重要的调节分子。S100A8是我们利用显微切割技术筛选出的在肿瘤前缘与肿瘤主体间质中存在差异表达的基因，经软件预测在S100A8启动子区域存在USF2的结合位点。我们前期实验结果表明，TGF-β可以上调USF2及S100A8的表达，USF2和S100A8可以促进结直肠癌细胞侵袭与迁移，干扰S100A8或者USF2可以抑制TGF-β引起的侵袭与迁移，过表达USF2上调S100A8表达。因此，本项目拟以结直肠癌为研究对象，通过体外细胞系实验、体内动物实验及人体组织样本检测，阐释TGF-β是否可以通过上调细胞内USF2及S100A8的表达来促进EMT，USF2是否直接结合在S100A8启动子区调控其表达，S100A8又是如何调控EMT的。从而为阐明肿瘤微环境与转移的关系提供新的角度。

上皮间质转化（epithelial-mesenchymal transition,EMT）是肿瘤细胞侵袭与转移的重要机制之一，TGF-β是EMT的重要调控因子，可以促进EMT发生，但其调控机制还有很多不明之处。. 分析TCGA结直肠癌转录组数据库（COAD与READ），发现TGF-β升高时S100A8也表达高。免疫组化检测407例结直肠癌组织样本中S100A8的表达，发现肿瘤细胞中S100A8表达阳性时患者预后较差、更易发生淋巴结转移和远处转移、TNM分期较高。小鼠尾静脉注射肺转移模型也表明S100A8可以促进肿瘤细胞转移。细胞学实验证明S100A8可以通过EMT促进肿瘤细胞迁移、侵袭。预测分析发现在S100A8启动子区存在转录因子上游刺激因子2（upstream stimulatory factor 2,USF2）的结合位点。荧光素酶报告基因实验与染色质免疫共沉淀实验证实USF2可结合在S100A8的启动子区、转录性上调S100A8表达。USF2可促进EMT，且USF2胞浆阳性时患者预后较差。. 课题组之前还发现在肿瘤间质中S100A8+细胞数量越丰富患者预后越好，与肿瘤细胞内的S100A8蛋白相反。我们用人重组S100A8蛋白刺激结直肠癌细胞，发现外源性S100A8负反馈性抑制胞内USF2/S100A8通路并抑制EMT。. 为了明确USF2/S100A8通路在TGF-β诱导的EMT中的作用，用TGF-β处理结直肠癌细胞，发生EMT的同时S100A8表达上调、USF2入核增加；如敲降USF2，则TGF-β上述作用被抑制。. 总之，TGF-β可通过激活结直肠癌细胞中USF2/S100A8信号通路促进EMT和转移，而胞外的S100A8可抑制胞内USF2/S100A8，USF2是S100A8在胞内和胞外作用相反的重要节点。我们的研究指出了对蛋白功能的研究需要结合定位，为阐明肿瘤微环境对肿瘤进展的影响提供了新角度；揭示了USF2/S100A8在结直肠癌转移中的重要作用，为结直肠癌的临床治疗提供了新依据、新靶点。