食管癌相关成纤维细胞通过 CNTN-1/游离泛素调节食管癌放射敏感性的机制研究

Interplay between esophageal cancer cells and their microenvironment has shown great influence on tumor growth, invasion and radiosensitivity. Documents have shown that esophageal tumor-associated fibroblasts (TAF) is a major regulator of the microenvironment of esophageal cancer, however, the effect of TAF on esophageal cancer radiosensitivity remains unknown. In our preliminary study, we isolated TAF from esophaeal cancer tissues and found that neural cell adhesion molecule Contactin 1 (CNTN-1) was highly expressed in esophageal cancer-derived TAF. Overexpression of CNTN-1 promoted the secretion of free ubiquitin and increased the radioresistance of co-cultured esophageal cancer cells. However, the specific function and the mechanism of CNTN-1 in esophageal cancer radiosensitivity remains unclear. This project is proposed based on previous work using a variety of molecular biological approaches to illustrate the mechanism of CNTN-1 in stimulating the secretion of the free ubiquitin from esophageal cancer-derived TAF. We will also investigate the mechanism of free ubiquitin in modulating radioresistance through CXCR4 and downstream DNA damage repair pathways in esophageal cancer cells. The project aims to further illustrate the role of CNTN-1 in esophageal cancer microenvironment and reveal the mechanism that CNTN-1 regulates esophageal cancer radioresistance by activating the free ubiquitin secretion from TAF. This project is likely to illustrate the novel mechanism of CNTN-1 in modulating the radiosensitivity of cancer microenvironment and provide new therapeutic targets and strategies for esophageal cancer radiosensitization.

食管癌细胞与肿瘤间质微环境的相互作用对肿瘤细胞生长、侵袭以及放射敏感性等有重要影响。文献表明食管癌间质中存在的食管癌相关成纤维细胞（TAF）是食管癌微环境的主要调节细胞，但食管癌TAF如何影响微环境中食管癌细胞的放射敏感性尚不清楚。本项目组前期分离了食管癌TAF，发现神经细胞粘附分子1 (CNTN-1)在食管癌TAF中高表达，能促进TAF向微环境中分泌游离泛素，调节共培养的食管癌细胞放射敏感性，但具体机制不详。基于文献报道及前期工作基础，本课题拟在细胞水平及动物模型上运用多种分子生物学技术揭示CNTN-1通过调控转录因子C/EBP促进食管癌TAF向微环境中分泌游离泛素，激活邻近食管癌细胞膜上CXCR4受体通路后启动DNA损伤修复，从而调节食管癌放射敏感性的机制。本项目有望揭示CNTN-1调节食管癌微环境影响肿瘤放射敏感性的新机制，并为食管癌的放射增敏提供新的靶点和策略。

食管癌是全球死亡率最高的恶性肿瘤之一，食管癌细胞的放射抗拒性是影响食管癌放疗疗效的关键因素。前期研究发现，游离泛素能增加食管癌细胞的辐射抗拒性，但具体机制尚不明确。课题组研究发现，泛素（UB）在食管鳞癌（ESCC）组织中过表达，血清UB表达水平与淋巴结转移、TNM分期相关。同时，血清UB、SCC、CEA联合检测ESCC诊断敏感性较各个指标单独检测明显升高。采用MTT、克隆形成实验及Transwell实验发现下调UB表达水平减弱ESCC细胞侵袭能力。此外，采用蛋白质组学分析在UB沉默组和对照组，ESCC细胞中蛋白表达差异。差异表达的蛋白包括ferritin light chain， ferritin heavy chain，CRABP2，和CDT1等。另一方面，我们采用ELISA法检测X射线照射后，观察不同时间点小鼠、恒河猴、肿瘤病人血清中free UB的表达变化。采用MTT法、克隆形成实验发现，游离泛素可以减少肠道细胞的死亡，促进其增殖和克隆形成能力。CNTN-1在ESCC中表达明显高于正常食管组织，表达水平与TNM分期和淋巴结转移、淋巴管浸润相关。CNTN-1与VEGF-C、HIF-1α的表达呈正相关。最后，我们分离食管成纤维细胞（NF）和食管癌相关成纤维细胞（TAF）。通过二维电泳-质谱研究发现，CNTN-1表达在TAF中增高3.12倍。通过siRNA下调食管癌TAF中CNTN-1后可增加TE-1细胞放射敏感性。采用RT-PCR和Western blot及ELISA法发现，UBC的激活可影响TAF分泌游离泛素水平。上调CNTN-1表达可促进TAF中游离泛素的分泌。采用MTT法，结果显示TAF中CNTN-1低表达可抑制与之共培养的TE-1细胞增殖。游离泛素的加入能显著提高TE-1细胞的放射抗拒性。采用免疫荧光检测H2AX的foci，结果表明，游离泛素可增强受照射的食管癌细胞的DNA损伤修复能力。游离泛素能激活CXCR4下游的Akt和Erk1/2通路。. 总之，本课题研究了CNTN-1通过调节TAF游离泛素分泌影响食管癌放射敏感性的机制，为食管癌放射增敏提供新的靶点。