B7-H3通过与ENO1相互作用促进肺癌细胞恶性增殖并介导免疫逃逸机制研究

Immune escape is the key factor of tumorigenesis and progression; however, the mechanism of immune escape is so intricate that it has not been completely clarified. Recent studies show that either tumor’s aberrant metabolism or immunosuppression caused by B7 family members could result in tumor immune escape, while the correlation and causal relationship of these two mechanisms have never been reported. So Pull-down and LC-MSMS are applied for our preliminary investigation, the results of which confirmed the presence of the interaction between B7-H3 and ENO1 in lung cancer cell lines. Accordingly, we will (1)use RT-PCR, Western Blot assay, etc to analyze the correlation between B7-H3 and ENO1 in clinical lung cancer tissues; (2)use FRET，etc to investigate the accurate cellular localization and protein chemical modification of the interaction;(3)use cell biological methods and animal experiments to analyze the alteration of tumor’s metabolism and immunosuppression caused by different expression of B7-H3 and ENO1, and we hope that our research will illuminate that the interaction between B7-H3 and ENO1 bridges tumor’s aberrant metabolism and immunosuppression, and further reveal the crucial role of the interaction between these two proteins on progression and immune escape of lung cancer, eventually lay a solid foundation concerning experiments for exploring a new target of immune-based antitumor therapies.

免疫逃逸是肿瘤发生、发展的关键因素，机理复杂且未完全阐明。研究显示，肿瘤的异常代谢和B7分子免疫抑制作用均是造成肿瘤免疫逃逸的重要原因。但二者在肿瘤免疫逃逸网络中确切作用机制、是否存在关联及其相互作用的因果关系未见报道。课题组联合蛋白质Pull-down技术与质谱鉴定技术，在肺癌细胞中发现B7-H3与烯醇酶1 (enolase1,ENO1)存在直接作用关系。为此，本课题拟应用⑴RT-PCR及Western Blot等方法分析B7-H3与ENO1在肺癌标本中表达相关性；⑵荧光共振能量转移等技术研究其相互作用的亚细胞水平定位、蛋白化学修饰；⑶细胞学及动物实验分析B7-H3与ENO1在差异表达情况下引起肿瘤代谢及免疫抑制效应的改变，阐明两者的相互作用在免疫逃逸机制中所起的桥梁作用，明确B7-H3与ENO1的相互作用影响肿瘤代谢及其与免疫逃逸发生的关联机制，为开发肿瘤免疫治疗新靶点奠定基础。

本课题组前期应用肺癌细胞系进行蛋白质Pull-down技术与质谱鉴定（LC-MSMS）技术，经筛选并进行体内、外实验后发现B7-H3与ENO1存在直接相互作用关系。为此，本项目将对B7-H3与ENO1相互作用机制做进一步深入研究，并探讨二者的相互作用是否影响肿瘤代谢及生物学特性，继而改变机体对肿瘤细胞的免疫效应，最终导致肿瘤细胞免疫逃逸。本研究着重探讨：（1）B7-H3与ENO1相互作用对肿瘤细胞生物学特性及肿瘤代谢的影响；（2）B7-H3与ENO1相互作用对下游信号通路的具体调控机制。我们的研究发现：（1）①过表达B7H3、ENO1可促进肺癌细胞增殖、迁移能力，但对细胞凋亡及周期无显著影响；干涉B7H3、ENO1可抑制肺癌细胞增殖、迁移及凋亡，但对周期无影响。②肺癌细胞中B7-H3与ENO1直接相互作用，B7-H3通过影响ENO1活性，增强肿瘤的Warburg效应，促进肿瘤进展。③抑制ENO1活性可以减弱B7-H3对肺癌细胞增殖和迁移能力的促进作用。（2）① 过表达B7-H3上调了PI3K-p85α，Akt，mTOR的磷酸化水平，沉默B7-H3/ENO1下调p-PI3K-p85α，p-Akt，p-mTOR的蛋白表达；然而，对Akt 和 mTOR总蛋白表达没有影响。② 应用ENO1抑制剂处理B7H3过表达细胞后上述信号改变逆转。③ 靶向沉默B7H3或ENO1可抑制巨噬细胞M0向M2型分化。本研究分析体内B7-H3与ENO1的相互作用对于肿瘤生物学特性、代谢途径及肿瘤免疫的影响，阐明B7-H3与ENO1的相互作用影响肿瘤代谢作用的调控机制，为开发肿瘤免疫治疗新靶点奠定实验基础。