Trop2激活β-catenin通过Wnt信号通路促进胃癌EMT和侵袭转移

The gastric cancer (GC) has the characteristics of high metastasis and high recurrence. Moreover, the targeted therapy for GC has not achieved obvious effect, it is urgent to find novel target for GC. Our previous study indicated that Trop2 was over-expressed in GC and associated with poor overall survival rates of GC patients, moreover, the relationship between the expression level of Trop2 and that of vimentin was positively correlated . We also found that Trop2 could induce proliferation, migration, invasion and EMT of GC in vitro. Bioinfomatic analysis and pull-down experiment indicated that Trop2 could bind with β-catenin and play a role in activating Wnt signaling pathway. Based on above results, we therefore hypothesize that Trop2 promotes epithelial–mesenchymal transition and metastasis of gastric cancer by activating Wnt /β-catenin signaling pathway. In this study, we would clarify the role of Trop2 in invasion and metastasis of GC both in vivo and in vitro. We plan to analysis the molecular mechanism of Trop2 to induce EMT and promote the invasion and metastasis of GC by activating Wnt/β-catenin signaling pathway. Up to now, there is no report about Trop2’s regulating Wnt/β-catenin signaling pathway of GC. Our study could provide Trop2 as a new potential biomarker for GC, bring a better understanding of the molecular mechanism of invasion and metastasis of GC, and may also provide the theoretical and experimental basis for precision treatment of GC.

由于胃癌的高转移、高复发性等特点，迫切需要寻找新的治疗靶点。前期研究发现Trop2在胃癌中高表达，与胃癌病人预后及vimentin的表达相关；细胞学实验发现Trop2能促进胃癌细胞的增殖、迁移、侵袭，并能诱导胃癌EMT现象；生物信息学分析和pull down实验提示Trop2和β-catenin存在物理结合，并可能激活Wnt信号通路。因此我们提出“Trop2激活β-catenin通过Wnt信号通路促进胃癌EMT和侵袭转移”这一科学假说。本项目拟结合体内外实验，证明Trop2在胃癌侵袭转移中的作用，应用Co-IP, CHIP等技术结合拯救实验，阐明Trop2激活β-catenin通过Wnt信号通路促进胃癌EMT和侵袭转移的分子机制。经文献检索，国内外尚未见相关报道，本研究有望明确Trop2作为胃癌新的治疗靶点，加深对胃癌侵袭转移分子机制的阐明，为胃癌的精准治疗提供理论基础和实验依据。

本研究阐明了Trop2结合β-catenin在肿瘤浸润，及促进胃癌上皮-间充质转化中的作用。应用多种实验方法分析Trop2在促进胃癌细胞EMT中的作用，通过体内和体外实验研究Trop2促进EMT的潜在分子机制；免疫组化法检测248例胃癌组织和86例相匹配的癌旁组织中Trop2表达与肿瘤转移的关系。结果发现，Trop2促进肿瘤细胞转移，诱导胃癌细胞转移EMT；Trop2可结合并激活β-catenin促进EMT。此外，Trop2增加β-catenin在细胞核中的积累，从而加速胃癌细胞的转移。抑制Trop2在胃癌细胞中的表达可阻止胃癌细胞在体内的迁移和侵袭。Trop2+/vimentin+在胃癌组织中表达高于癌旁组织中Ttro2+/vimentin+的表达，且与肿瘤细胞的分化、TNM分期和远处转移有关。Trop2/β-catenin可通过Wnt信号通路促进胃癌EMT和侵袭转移，检测Wnt下游靶基因TCF4/OELEF1/DKK1的表达情况；检测癌干调控基因Nanog/Lin28A/Sox2/Oct4的表达情况、MMP2/MMP9/VEGF、癌干细胞表面Marker CD44/CD54/CD133/EpCAM和SP细胞数量；进一步利用野生型MGC803，OE-TROP2稳转胃癌细胞株MGC803和MGC803EMT模型细胞株，进行ITRAQ实验，分离蛋白裂解为短肽进行质谱分析，将捕获的TROP2互作蛋白送蛋白质谱分析，共检测出28个和TROP2互相作用的蛋白，结果拓展了Trop2/β-catenin促进胃癌EMT和侵袭转移伴随其他信号通路，结合KEGG数据库对TROP2在胃癌中的功能进行基因富集分析(GSEA)，富集到TROP2 mRNA的差异表达与钙信号转导通路、MAPK信号通路、Wnt信号通路等呈正相关，与药物代谢及酶类呈负相关。利用STRING 数据库构建由TROP2预测互作蛋白组成的网络，结果显示，TROP2(TACSTD2)通过与转录因子ATF2结合，调控CTNNB1、HIF2A、VIM、PRKCA的转录，以上基因的转录产物与Wnt通路相关，进一步影响RUVBL2在核内的作用，调控DNA损伤修复应答。结果表明，在EMT和胃癌转移过程中，Trop2具有一个新的调控网络，Trop2可能是一个有用的诱导EMT和胃癌转移的标志物，我们的研究有助于更好地理解胃癌的发病机制。