Her2通过LKB1-AMPK-mTOR信号通路调控胃癌肿瘤干细胞自噬并影响其致瘤性及机制的研究

Establishing the relationships among autophagy-related genes, molecules and signal pathways in gastric cancer stem cells could contribute to understanding the role of autophagy in the tumorigenesis and development of gastric cancer, which needs culturing GCSC firstly and finding out the key links of regulation as the prerequisites. Our previous studies have identified gastric cancer stem cells successfully, and suggested that LKB1-AMPK-mTOR signal pathway mediated autophagy might be one of the possible reasons why Her2 had different effects on the tumorigenicity and invasion as well as self-renewal in gastric cancer stem cells. Based on these, whether Her2 regulates the autophagy of gastric cancer stem cells through LKB1-AMPK-mTOR signal pathway and subsequently influences its tumorigenicity? What is the molecular mechanism of Her2 in regulating LKB1-AMPK-mTOR signal pathway? need to be answered in this research. The research aims to investigate the role of Her2 in regulating autophagy of gastric cancer stem cell through LKB1-AMPK-mTOR signal pathway and subsequently influencing the tumorigenicity by RNA interference/overexpression, CRISPR/Cas9 gene knock out technic, transplanted tumor experiments and transgenic mice model etc, explore the molecular mechanism of Her2 in regulating LKB1 by CHIP, EMSA, luciferase assay and mass spectrometric analyses, and analyze the relationship between Her2-LKB1-AMPK-mTOR signal pathway and prognosis of gastric cancer patients by immunohistochemistry. This research might shed the lights on elucidating the potential mechanism how the autophagy is regulated in gastric cancer stem cell and subsequently influence the tumorigenicity.

在胃癌肿瘤干细胞(GCSC)中建立自噬相关基因、分子、信号通路间的联系有助于理清自噬对胃癌发生发展可能的作用，但须先培养出GCSC，并找到调控的关键环节。前期工作中，我们成功培养出了GCSC，并提示LKB1-AMPK-mTOR信号通路介导的自噬可能是Her2对GCSC侵袭更新与致瘤性作用不一致的原因之一。基于此，Her2是否通过上述通路调控GCSC的自噬，并最终影响其致瘤性？Her2调控该通路的分子机制为何？均有待回答。本项目拟通过RNA干扰/过表达、CRISPR/Cas9基因敲除、移植瘤实验、转基因小鼠模型等研究Her2通过LKB1-AMPK-mTOR通路在调控GCSC自噬并影响致瘤性方面所起作用。利用CHIP、EMSA、荧光素酶报告基因及质谱分析研究Her2调控LKB1的分子机理；利用免疫组化分析该通路与胃癌病人预后的关系。本项目或有助于揭示GCSC自噬调控及其影响致瘤性的可能机制。

本课题试图研究Her2是否通过LKB1-AMPK-mTOR信号通路调控胃癌肿瘤干细胞自噬，并影响其致瘤性及发挥作用机制。首先，课题组成功构建Her2过表达及干扰的胃癌干细胞进行CCK-8等实验发现Her2过表达时增殖、克隆形成和自我更新能力增强，而侵袭迁移能力减弱。其次利用Western Blot检测到Her2过表达的胃癌干细胞自噬相关、STING及SEC24C等蛋白表达降低。接下来，课题组利用定量PCR和Western Blot检测LKB1-AMPK-mTOR通路分子mRNA、蛋白表达水平变化，结果证明LKB1-AMPK-mTOR信号通路调控胃癌干细胞自噬相关性较低，用临床标本进一步验证时发现Her2、LKB1、mTOR在胃癌组织中低表达，AMPKa1在胃癌中高表达，但仅mTOR的表达差异具有统计学意义，且mTOR低表达时患者的预后较差。随后，课题组对Her2过表达及干扰胃癌干细胞进行全转录组测序寻找差异基因，利用RT-PCR和Western Blot在体外进行验证，证实CTSD基因表达差异较大；紧接着利用免疫组化在胃癌标本中进行验证。与此同时，课题组发现Pan-HER抑制剂PF299具有抑瘤作用，于是研究方向转为PF299对胃癌肿瘤干细胞生物学特性的影响及其机制。我们首先利用CCK-8法等检测到PF299处理后Her2表达降低，胃癌肿瘤干细胞的增殖、克隆形成、侵袭、自我更新能力等减弱，凋亡增多；并通过RT-PCR和免疫荧光检测移植瘤中Her2的mRNA及蛋白表达显著降低。对于相关机制的研究，首先利用Western Blot、免疫共沉淀检测不同浓度PF299处理时Her家族及下游相关信号通路蛋白、磷酸化水平的变化情况，发现PF299可能通过抑制异二聚体形成影响信号通路的传导。然后通过全转录组测序筛选出PF299处理前后差异表达基因，构建HBXIP过表达的胃癌肿瘤干细胞，检测到其增殖能力增强，细胞周期进程加快。通过RESCUE实验验证HBXIP介导的抗肿瘤作用后利用免疫共沉淀实验检测发现PF299抑制HBXIP-MYC异二聚体的形成，并通过RT-PCR发现MYC靶基因的转录降低。最后，免疫组化染色发现HBXIP和MYC表达具有正相关关系，且HBXIP、MYC在胃癌组织中表达高于正常组织，生存分析发现HBXIP、MYC高表达时患者的预后较差。