雌激素受体β1在白介素6介导肺鳞癌免疫反应中的作用及机制研究

Lung cancer immunotherapy contained huge potential clinical benefit is key issues and challenges. Numerous studies have confirmed that interleukin-6 (IL-6), one of the most important immune mediators of lung cancer, shows a significant correlation with Estrogen (Estrogen, E2, the main function of the estrogen receptor subtypes in lung cancer is ERβ, and the key isoform is ERβ1). So it is possible to increase lung cancer immune effect IL-6-mediated by E2 intervention, while the underlying mechanisms are poorly understood. ERβ1 is highly correlated with IL-6 in lung squamous cell immunity, but how to participate in IL-6-mediated immune responses in lung cancer has not been reported. On the basis of pre-experiment and the long-period study on the mechanism of which ERβ1 and IL-6, we propose several hypotheses: ERβ1 is highly correlated with IL-6 in lung squamous cell carcinoma (LSCC), and E2 may promote the secretion of IL-6 in patients with LSCC through ERβ1, thus contributing to the progress of LSCC. Firstly, the project will expand the samples of LSCC to test the expression of ERβ1 and IL-6. Furthmore, we will establish a stable high or low expression of ERβ1 in LSCC cell in vitro experiments to detect proliferation, apoptosis, migration and other cell effects. At the same time, we will also detect IL-6 promoter activity and the expression of key signaling pathway molecule, to explore the different roles of ERβ1 and IL-6 in LSCC, and to explore the effect of ERβ1 in to explore IL-6-mediated immune responses. Finally, we will establish stable mouse model of LSCC which steadily expresses ERβ1 (high or low) by plasmid, viral infections, and subcutaneous xenografts, to further verify the above conclusions. This project contributes to clarify the role and mechanism of ERβ1 in IL-6-mediated immune responses of LSCC, and to find more precise targets for LSCC immunotherapy.

肺癌免疫治疗潜在的巨大临床收益是目前国内外研究的重点和难点，大量研究表明雌激素（E2）与肺癌最重要的免疫介质之一白细胞介素6（IL-6）具有显著的相关性，但E2主要功能受体亚型ERβ1参与IL-6介导肺癌免疫反应的作用及机制尚无报道。我们在前期研究ERβ1与IL-6基础上，结合最新预实验结果提出假说：肺鳞癌中ERβ1与IL-6表达高度相关，E2可能通过ERβ1促进肺鳞癌患者IL-6的分泌，进而促进肺鳞癌进展。本项目扩大样本检测肺鳞癌组织中ERβ1与IL-6的表达；建立稳定高或低表达ERβ1肺鳞癌细胞株，检测细胞效应和IL-6启动子活性及关键分子表达，重点探讨ERβ1在IL-6介导肺鳞癌免疫反应中的作用；通过质粒构建、慢病毒感染和皮下移植瘤建立稳定高或低表达ERβ1小鼠肺鳞癌模型验证上述结论。本项目阐明ERβ1在IL-6介导肺鳞癌免疫反应中的作用及机制，为肺鳞癌的免疫治疗寻找更为精确的靶点。

大量研究表明，雌激素受体（Estrogen receptor，ER）和白介素6受体（Interleukin 6 receptor，IL-6R）两条信号通路在包括肺癌在内的众多肿瘤中存在相互作用，但ERβ亚型与IL-6R在肺癌进展中的相互作用仍无报道。包括我们在内的前期研究发现，ERβ1/2/5在肺癌中均存在表达，且ERβ5表现出的生物学功能不同于其它亚型。同时，作为IL-6R的主要信号转导分子糖蛋白130(Glycoprotein 130,GP130)在非小细胞肺癌(NSCLC，Non-small cell lung cancer)的进展中也有重要作用。因此，本文着重探讨ERβ1/2/5与IL-6R在NSCLC进展中有无相互作用及其可能机制。首先，我们通过组织芯片-免疫组化分析ERβ1/2/5、IL-6和GP130在NSCLC患者和肺良性病变(BPL, benign pulmonary lesions)患者中的表达。我们发现与BPL患者和肺鳞癌患者相比，肺腺癌患者中ERβ5、IL-6和GP130的表达明显增多（P<0.001），且ERβ5的过表达与NSCLC患者的TNM分期有关，GP130的过表达在腺癌患者中较多见，且ERβ5和GP130共同过表达患者的5年生存率较单一表达患者显著降低（P=0.0315）。在体外实验中，我们发现将ERβ5和IL-6同时作用于A549细胞，能够显著增加细胞的增殖和侵袭能力（P<0.01）。而在转染ERβ5的基础上加入Niclosamide（NIC，STAT3抑制剂）处理A549细胞，细胞凋亡明显增多(P<0.05)，细胞周期在G1期明显阻滞（P<0.05）。另外，ERβ5和IL-6同时作用于A549细胞，其关键信号分子ERβ5、GP130、p-AKT和p-44/42 MAPK的表达也明显升高(P<0.05)。这些结果表明ERβ5与GP130可协同促进NSCLC进展，并能联合作为患者预后不良的独立因素。另外，这些结果也为ERβ5和GP130在肺腺癌的联合靶向治疗提供了一定理论依据。