新型G蛋白耦联雌激素受体（GPER）上调雌激素受体β亚型1（ERβ1）促进肺腺癌进展及机制研究

Extensive studies elucidates that estrogen receptor β (ERβ) is the main receptor activated by estrogen in lung adenocarcinoma progression. Significant inhibitory effects were found in researches by blocking ERβ in lung cancer. However, limited effects were observed in clinical trials. G protein coupled receptors (GPER) was found to be another signaling promoting lung adenocarcinoma in our recent study. Restricted efficacy of clinical trials could be attributed to trans-activation of GPER by ERβ inhibitors. Especially we found GPER may play a potential regulatory role in ERβ-mediated lung adenocarcinoma development. Therefore the effects of anti-estrogen treatment in lung adenocarcinoma may be improved by co-targeting both GPER and ERβ..　　According to previous studies and our results, we suppose that GPER may up-regulate ERβ1, the only one functionally completed subtype of ERβ compared with others, in lung adenocarcinoma. This mechanism may include GPER binding to promoter of ERβ1directly or activating ERK1/2 and PI3K/Akt and et al pathways and downstream transcription factors indirectly. Base on this hypothesis, co-inhibition of both receptors will exhibit better efficacy than blocking ERβ1 alone..　　In this project, firstly 200 lung adenocarcinoma tissues will be selected from Biological Sample Library of Thoracic Surgery of Tongji Hospital. GPER and ERβ1 expression and their correlation will be analysed by IHC assay. Secondly, the ERβ1 transcription upregulation by GPER and related mechanism, including binding to promoter directly and activating other pathways and downstream transcription factors indirectly, will be detected by real-time PCR, western blotting, dual-luciferase arrays, ChIP on chips and protein-protein interaction microarrays in cell lines interfered by plasmid vector and shRNA, expressing GPER and ERβ1 differently. Furthermore, the influence to cancer development of ERβ1 up-regulating by GPER will be detected in GPER, ERβ1 or both knockout lung adenocarcinoma mouse model. In addition, effects of co-inhibition by both GPER and ERβ1 will be compared with single blocking ERβ1 in vitro and in vivo..　　Our project will provide evidence that GPER and ERβ1 signaling are not simply independent pathways, GPER may up-regulate ERβ1 via binding to promoter directly or activating other pathways and downstream transcription factors indirectly. For this reason, the combined use of ERβ1 and GPER inhibitors may exhibit more effective inhibition than treatment with a single ERβ1 inhibitor. Our project heralds new opportunities for mechanism discovery estrogen-induced lung cancer. Furthermore, GPER can be considered a potential new endocrine target for improving anti-estrogen-based therapies for lung adenocarcinoma.

研究证实雌激素受体β（ERβ）是雌激素促进肺腺癌进展主要功能受体，但抑制ERβ治疗肺癌临床试验疗效有限。新型G蛋白耦联雌激素受体（GPER）通路是我们最近发现的另一雌激素促进肺腺癌新途径，抑制ERβ效果有限可能与反向激活GPER有关。更重要的是GPER除独立作用外可能还对ERβ具有调控作用，双靶点联合抑制可能是抗雌激素治疗肺腺癌的新方向。我们结合前期研究和预实验提出：肺腺癌中GPER可能直接结合启动子或间接激活其他转录因子上调ERβ唯一全长功能亚型ERβ1，联合抑制可能优于ERβ1单独抑制。本项目检测肺腺癌组织中GPER和ERβ1表达相关性，建立GPER和ERβ1高、低表达肺腺癌细胞系和GPER、ERβ1基因敲除鼠原位肺腺癌模型，检测并探讨GPER对 ERβ1上调和机制、二者联合抑制是否优于ERβ1单独抑制。本项目为阐明雌激素促进肺腺癌进展机制、解决ERβ抑制临床试验困境提出新方向。

项目背景：大量研究证实雌激素受体β（ERβ）是雌激素促进肺腺癌进展的主要功能受体，但以抑制ERβ为基础，抗雌激素治疗肺腺癌的临床试验效果有限，其原因和机制尚不清楚。GPER途径是我们发现的ERβ之外另一条雌激素促进NSCLC的新途径，GPER和ERβ除了独立作用外可能还存在某种未知的相关性。抑制ERβ治疗肺腺癌临床试验效果有限可能与GPER的激活有关，但关于GPER上调ERβ表达的机制及联合抗GPER和ERβ抑制肺腺癌进展方面，尚无相关研究。.研究内容：依据前期研究发现GPER可上调ERβ表达的现象，我们通过组织标本、体外实验、体内实验三个角度验证GPER上调ERβ表达的机制及联合抗GPER和ERβ抑制肺癌增殖、转移的效果。.重要结果：.1、首先通过对组织标本的收集和利用IHC方法检测标本，结果显示：肺腺癌中GPER过表达，主要分布在胞质和胞膜中，胞核中低或无表达。ERβ在肺腺癌组织中过表达，主要表达在胞浆和胞核中均有表达。其中cGPER与ERβ的表达水平呈现正相关，但nGPER与ERβ无表达相关性。.2、在体外实验中，我们通过RNA-seq、CHIP、双荧光素梅报告基因实验证实GPER可激活ERK/NF-kb途径上调ERβ的表达。.3、建立高、低表达GPER肺腺癌细胞，检测增殖、迁移、侵袭等效应。GPER过表达后，细胞增殖、迁移、侵袭能力增强，GPER敲降后，细胞增殖、迁移、侵袭能力减弱。相较于单独敲降GPER/ERβ，联合敲降GPER和ERβ在抑制肿瘤细胞增殖、迁移、侵袭方面效果更优。.4、通过慢病毒感染建立GPER单独敲降、ERβ单独敲降、联合敲降GPER和ERβ的细胞系，利用肺腺癌小鼠皮下瘤模型进一步验证上述结论。.科学意义：本项目明确了GPER通过ERK/NF-Κb途径上调ERβ表达，深入阐述对雌激素促进肺腺癌发展的机制，进一步揭示雌激素促进肺腺癌进展背后复杂的通路网络和关键信号分子。联合抗GPER和ERβ 的“多靶点治疗”可能是抗雌激素抑制肺腺癌发展的新方向。