FSTL1诱导结直肠癌相关成纤维细胞活化的分子机制研究

Cancer-associated fibroblasts are important mesenchymal cells in tumor microenvironment, but the process and mechanism of their activation and the effect on the tumor progression have not been fully clarified. In the previous study, the applicant found that the colorectal cancer cells over expressed and secreted follistatin like 1 (FSTL1). Our preliminary experiment showed that the distribution of CAFs in colorectal cancer tissues was consistent with the expression level of FSTL1. Additionally, recombinant human cytokine FSTL1 had obvious chemotactic effect on primary normal fibroblasts, and induce them to activate into CAFs. Therefore, we propose the hypothesis that FSTL1 secreted by colorectal cancer cells can induce activation of NFs into CAFs, which further promotes cancer progression. In this study, we will explore the downstream signal pathway and key nodes of FSTL1 regulation of CAFs activation via Western blot, immunofluorescence, immunoprecipitation, GST pull-down, High-throughput sequencing, and then verified through in vivo and in vitro experiments. Our study aims to elucidate the mechanism of colorectal cancer cells to activate CAFs by secreting FSTL1 to promote cancer progression, and to provide a reliable theoretical basis for the activation of CAFs as a therapeutic target for colorectal cancer.

癌相关成纤维细胞(CAFs)是肿瘤微环境中重要的间质细胞，其被活化的过程、具体机制及对肿瘤进展的影响尚未完全明确。申请人在前期研究中发现：结直肠癌肿瘤细胞高表达并分泌卵泡抑素样蛋白1（FSTL1）促进肿瘤侵袭转移，且预实验提示微环境中CAFs分布与FSTL1表达具有一致性，rhFSTL1可趋化NFs并诱导其活化为CAFs。因此我们提出结直肠癌细胞分泌的FSTL1能够诱导NFs活化为CAFs，活化的CAFs进一步促进肿瘤进展的研究假说。本课题拟利用Western blot、免疫荧光、免疫共沉淀、质谱分析及GST pull-down、高通量测序与染色质免疫共沉淀等技术阐明FSTL1诱导NFs活化为CAFs的下游信号通路及关键节点；结合体内、体外实验验证结直肠癌细胞通过分泌FSTL1诱导NFs活化为CAFs进而促进肿瘤进展，为以CAFs活化过程为结直肠癌治疗靶点提供理论依据。

近年来结直肠癌发病率呈逐年上升趋势。研究表明，肿瘤的进展不仅是肿瘤细胞基因突变或过度生长的独立事件，而且是肿瘤细胞与肿瘤微环境之间相互协同作用的结果。癌相关成纤维细胞是肿瘤微环境中重要的间质细胞，其被活化的过程、具体机制及对肿瘤进展的影响尚未完全明确。本项目通过免疫组织化学方法检测结直肠癌患者癌组织和配对的癌旁正常组织中FSTL1 表达和 CAFs 的浸润分布情况，结合公共数据库与患者临床病例资料分析，发现结直肠癌患者肿瘤组织中FSTL1高表达与CAFs在肿瘤微环境中的浸润程度正相关，并与患者肿瘤浸润深度及不良预后相关。通过体外细胞生物学实验发现FSTL1诱导CCD-18Co活化为 CAFs后增殖能力无明显变化，但细胞迁移能力增强。Western blot、RNAseq、免疫共沉淀、免疫荧光共定位等实验结果发现，FSTL1可通过与其受体TLR4相互作用，诱导CCD-18Co活化为 CAFs，活化后TGFβ-Smad2/3信号通路被激活，并使下游的促纤维化基因COL1A1、YKL40、α-SMA表达上调，同时ZFP36出现下调，使得MMP2的mRNA的稳定性增加，其蛋白水平上调；细胞外基质稳态被破坏，促进肿瘤细胞侵袭转移。通过CCK8、划痕实验、Transwell 迁移、Transwell 侵袭实验发现，FSTL1诱导活化的 CCD-18Co能够使肿瘤细胞的迁移及侵袭能力均明显增强，进一步促进结直肠癌进展。我们的研究结果明确了结直肠癌微环境中，肿瘤细胞分泌FSTL1与NFs上的受体TLR4结合后激活TGFβ-Smad2/3信号通路，诱导其活化为CAFs，并下调ZFP36蛋白水平，促进肿瘤细胞侵袭转移。这一分子机制可为以 CAFs活化过程为靶向策略的结直肠癌肿瘤治疗提供新思路。