S100a8/S100a9调控Smad-Ids轴促进结直肠癌转移的机制研究

Our group previously discovered that colorectal tumor-associated macrophage (TAMs) and S100a8/S100a9 can promote colorectal cancer metastasis. And we found that the TAMs could express and secrete higher S100a8/S100a9 compared to the normal colorectal macrophage. Furthermore, our data from Affymetrix GeneChips revealed S100a8/S100a9 could activate the TGF-β/Smad signaling pathway and induce the expression of downstream genes of the signaling pathway - Ids genes. According to the previous reports, we knew that the upregulation of Ids was closely related to tumor invasion, metastasis and angiogenesis. Based on our previous data, we speculate that S100a8/S100a9 secreted by TAMs can promote colorectal cancer metastasis through Smad-Ids axis. Firstly, we will verify that TAMs can promote colorectal cancer metastasis and angiogenesis through its secretion of S100a8/S100a9 using the cell model and nude mouse orthotopic xenograft model. Then, migration assay, invasion assay, nude mouse orthotopic xenograft model and western blot will be used to examine the role of S100a8/S100a9, Ids and Smad signaling pathway in promoting colon cancer metastasis by reversing or not reversing the expression of Smad and Ids in S100a8/S100a9 over-expression and knockdown cell lines respectively. Immunoprecipitation and immunofluorescence will be employed to analyze the biochemical interaction between Smad1/5/8 and Ids. Finally, immunohistochemistry and statistics will be used to investigate the relationship between expression levels of F4/80,CD68,CD11b, S100a8/S100a9, Smad1 /5/8 and Ids in patient's colorectal cancer tissues and analyze the association between the expression levels of these molecular with the clinical stage, metastasis and survival rates of patients with colorectal cancer. This study will delineate the mechanism of S100a8/S100a9 secreted by tumor- associated macrophage in promotion of colorectal cancer metastasis through Smad-Ids axis and facilitate identification of novel potential therapeutic targets for colorectal cancer metastasis.

本课题组发现肠粘膜肿瘤相关巨噬细胞（TAMs）高表达并分泌S100a8/S100a9，S100a8/S100a9可能促进结直肠癌的转移。基因芯片初步提示在结直肠癌中TAMs分泌的S100a8/S100a9可能通过调控Smad-Ids轴发挥作用。本课题将利用细胞模型及裸鼠移植瘤活体成像技术证实TAMs通过S100a8/s100a9诱导结直肠癌的转移；利用逆转与非逆转Smad1/5/8及Ids的S100a8/S100a9过表达和沉默结肠癌细胞株，采用体内外转移实验及蛋白印迹等技术明确Smad及Ids是否为S100a8/S100a9促进转移的关键节点；采用IP技术探究Smad1/5/8与Ids的交互作用，并在结直肠癌组织中验证上述分子的表达及其与转移、生存率的关系，阐明结直肠癌中S100a8/S100a9-Smad-Ids轴促进转移和血管生成的机制，为结直肠癌转移诊治提供新靶点。

前期课题组发现肠粘膜肿瘤相关巨噬细胞（TAMs）高表达并分泌S100A8/S100A9可能促进结直肠癌的转移。本研究证实了S100A8/S100A9可通过调控Smad-Ids轴发挥促进结直肠炎-癌进展的作用，基本阐明了S100A8/S100A9影响结肠炎-癌发生发展的调控机制和作为潜在治疗靶点的应用前景。.课题组首先分析研究了S100A8和S100A9具有促进结肠癌细胞增殖的作用；发现Id3可促进结肠癌细胞增殖及细胞周期进展，阐明了S100A8可通过激活Akt1及Smad5通路活化Id3并下调p21的表达。采用裸鼠移植瘤模型验证过表达S100A8能够促进小鼠结肠癌细胞的转移，并证实anti-S100A9抗体干预可治疗小鼠并抑制肿瘤发展，揭示其潜在治疗效果。.进一步对S100A8和S100A9在人结直肠炎、腺瘤及腺癌组织中的表达情况以及结直肠癌患者血清S100A8和S100A9的表达及临床病理特征进行了分析。通过免疫组化技术明确了人结直肠癌组织中S100A8和S100A9的表达，发现S100A8/S100A9不仅在间质细胞中表达增高，其在肿瘤腺上皮细胞浆中亦高表达。同时利用ELISA法检测并分析了结直肠癌患者血清中S100A8和S100A9的表达水平，明确了其诊断结直肠癌的诊断效能及与临床病理特征之间的关系。.为了验证S100A8和S100A9在细胞内的作用机制，课题组构建了结肠癌细胞内S100A8和S100A9高表达模型并分析其诱导结肠癌细胞分泌炎症因子的功能，发现S100A8和S100A9可依赖炎症信号通路途径诱导CXCL5、GM-CSF、IL1a分泌，并初步阐明了细胞内S100A8和S100A9可通过CXCL5/CXCR2轴促进结肠癌细胞增殖侵袭的功能及机制。.最后课题组在建立S100A8和S100A9-HEK293过表达细胞株的基础上，通过亲和分离纯化结合质谱分析S100A8/S100A9相互作用蛋白，采用生物信息学分析方法发现S100A8和S100A9发生相互作用的蛋白主要参与糖代谢、细胞黏附、正向调控NF-κB转录因子活性、抗凋亡等多种生物学过程，并筛选验证了PKM2、14-3-3ε等蛋白是与S100A8和S100A9相互作用的蛋白。