GPER在三阴性乳腺癌细胞中的转录调节作用及其对侵袭转移行为的影响

Triple negative breast cancer (TNBC) presents potent malignancy and routinely can not accept endocrine and target therapy. It was revealed by our early NSFC-supported projects that the novel estrogen receptor, estrogen (G-protein coupled) recepor (GPER) was expressed and actively mediated estrogenic response in TNBC cell line. However, its pleiotropic effects triggered by transcriptional regulation need further study. Considering the metastatic nature of TNBC cells, it is supposed that GPER would mediate estrogenic regulation of gene transcription to activate a variety of signaling pathways, contributing to aggressive biological behaviours such as migration, invasion and metastasis in TNBC cells. Accordingly, blocking of GPER signaling has potential therapeutic belefits. In the present program, Gene interference would utilized to establish cell model in TNBC cells for further study. Microarray and bioinformatic analysis would be performed to identify target genes and related signal pathways of GPER. Invasion- and metastasis-related signaling would be chosen to validate its biological effects and mechanisms in TNBC cells by gene interfere, western blot et al and pharmacological study. Then, the potential therapeutic effects of GPER would be evaluated by its specific antagonist in vitro and in vivo. Accordingly, the biological significance of GPER in TNBC would be revealed and GPER targeted strategy probably applied as a novel endocrine therapy in TNBC.

三阴性乳腺癌（TNBC）恶性潜能高，临床尚无内分泌治疗和靶向治疗条件。前期NSFC项目发现新型雌激素受体GPER在TNBC组织及细胞中表达且具受体活性，可介导雌激素效应，但其转录调节所致多样化影响尚需深入研究。结合TNBC易转移之特性提出GPER在TNBC中介导雌激素的转录调节，通过多种机制影响肿瘤侵袭转移行为；抑制GPER激活具有治疗效果。项目拟在明确TNBC细胞中GPER具有受体活性的基础上通过基因干扰构建针对GPER研究的细胞模型，应用基因芯片技术及生物信息学分析明确TNBC细胞中GPER的靶基因及其介导的信号通路改变；选择相关信号通路，应用免疫印迹等技术结合药理学处理验证其在TNBC细胞侵袭转移多个步骤中的影响与机制；通过体内外研究明确抑制GPER激活对TNBC的治疗作用，揭示GPER对TNBC的生物学影响，探讨靶向TNBC中GPER的可能性，有望为TNBC开拓内分泌治疗的新领域。

三阴性乳腺癌（triple negative breast cancer，TNBC）侵袭性强，易远处转移，特别是致死性强的内脏转移，是乳腺癌中预后最差的一种亚型。由于TNBC在临床上尚无内分泌治疗和靶向治疗条件，寻找对其有治疗意义的分子靶标便成为该领域的研究焦点。本项目在前期工作明确相当部分TNBC组织及细胞系均表达G蛋白偶联雌激素受体（Estrogen （G-protein coupled）receptor，GPER）的基础上提出“GPER在TNBC中通过转录调节作用介导雌激素促转移效应”的科学假设。按项目计划，构建靶向干扰GPER基因的shRNA慢病毒，感染TNBC细胞（HCC1806、MDA-MB-468），通过筛选获得稳定干扰GPER蛋白的细胞系，成功构建针对GPER研究的TNBC细胞模型。应用雌二醇（estradiol，E2）、GPER特异性激动剂G-1、抑制剂G-36处理TNBC细胞（MDA-MB-468）后行mRNA及长链非编码RNA（lncRNA）芯片检测。mRNA芯片提示GPER在TNBC细胞中被激活后调节了CTGF、CRISP3等589个靶基因，首次证实GPER在TNBC细胞中有广泛的转录调节作用。同时lncRNA芯片结果提示lnc-OR2AJ1-1:1、lnc-NGEF-1:1等1512个lncRNA与GPER特异相关，首次揭示GPER在TNBC细胞系中介导的表观遗传学效应。用上述mRNA及相关lncRNA在DAVID网站进行生物信息学预测，发现Glutamatergic synapse、cAMP等38条信号通路因GPER激活而激活，涉及肿瘤转移过程中细胞迁移、焦点粘附等多方面功能。Transwell实验明确GPER在TNBC细胞中被激活可促进细胞迁移。初步证实GPER在TNBC细胞中可通过cAMP及VGLUT信号通路，促进细胞迁移，进而在TNBC转移过程中扮演一定角色。上述研究结果挑战了“TNBC为雌激素不敏感乳腺癌”的传统观念，不但明确GPER在TNBC细胞中介导雌激素转录调节效应及表观遗传效应，且由于GPER在TNBC细胞中激活可促进转移，靶向GPER的内分泌治疗可能在TNBC中具有治疗效应，其具体分子机制值得深入研究，其临床应用前景也值得期待。