N-乙酰基半乳糖胺转移酶5通过调控Mucin4的O-糖基化及HER2信号参与胃癌发生发展的分子机制和临床意义研究

Aberrant O-linked glycosylation status catalyzed by polypeptide N-acetygalactosaminyl transferases has been reported to be associated with the development of many malignant tumors. Our previous study has identified that the polypeptide N-acetygalactosaminyl transferase 5 (GALNT5) was reduced in gastric cancer tissues compared with non-tumor mucosa and correlated with deteriorated tumor cell differentiation, aggravated tumor invasion, lymph node metastasis ,advanced TNM stage and reduced overall survival. However, the precise mechanisms underlying the role of GALNT5 in pathogenesis of gastric cancer remain unknown. The ErbB family is composed by ErbB1/EGF receptor (EGFR), ErbB2/HER2/Neu, ErbB3 and ErbB4 which are closely related to tumor differentiation, proliferation, survival, migration and anti-apoptosis etc. Following ligand binding and receptor activation, these receptors are endocytosed and transported to lysomes where the receptor is degraded. The ability of receptors retention on cell membrane and formation of homo-dimers or hetero-dimers are key determinants of signal activity. HER2 mainly acts as coreceptor with other receptors for lacking specific ligand, and HER3 also need heterdimered with other receptor to transmit signaling for owning a kinase dead c-tetmainl. MUC4 is a membrane binding mucin modified by GalNAc-Ts. Muc4 can interact with HER2 via EGF domain in the extracellular section. GALNT5 play important role in modifying the O-glycosylation of Muc4 to affect their conformation and reducing constitutive endocytosis. GALNT5 mediated Muc4 O-glycosylation blocked the formation of HER2 homodimers and/or HER2-HER3 heterodimers by competitive interation with HER2. Reduction of GALNT5 expression facilitated the interaction of HER2 and other receptors, enhancing downstream pathway activation, including ras/raf/MEK/Erk and PI3K/AKT signaling. This project proposal for grant application is designed to elucidate the regulatory effect and functional role of the mechanism of glycosylation of MUC4 post-translation and its function on HER2 signal transduction based on human gastric cancer tissues and cells . From the point of view in GALNT5 prompting cell differentiation and cancer glycobiology, our present research project will focus on the molecular mechanism and functional significance for O-glycosylation-mediated alteration of membrane HER2 heter/homo dimer formation by abnormally expressed GALNT5 and will establish a model of molecular regulation and therapeutic intervention of HER2 receptor O-glycosylation change on gastric cancer cells membrane, which paves the way to clinical investigation and drug discovery based on molecular therapy targeting aberrant O-glycosylation and HER2 signal activation in gastric cancer patients，and constructs a GALNT5 based molecular prognosis model.

糖基转移酶异常介导的蛋白质糖链结构变化与恶性肿瘤发生发展互为因果。项目申请人前期研究发现，作为介导Mucin样O-糖基化修饰的N-乙酰基半乳糖胺转移酶家族的重要成员，N-乙酰基半乳糖胺转移酶5（GALNT5）在胃癌中的低表达与肿瘤进展相关，并可作为独立预后因子增强TNM分期预测患者术后生存的准确性（Brit J Cancer，2014），然而其发挥肿瘤抑制作用的分子机制及功能意义尚不清楚。胃粘膜高表达的黏蛋白Mucin4可通过类表皮生长因子域与表皮生长因子受体2(HER2)结合抑制其二聚化及信号转导，GALNT5可能在调控Mucin4的O-糖链合成及功能发挥中起重要作用。本申请项目拟在前期预实验基础上，研究GALNT5调控Mucin4的O-糖链合成进而影响HER2信号的分子机制及功能意义，建立其参与胃癌发生发展的分子调控和干预治疗模型，为胃癌术后生存评估分子分型和靶向治疗药物开发奠定基础。

在本项目资助下，项目负责人围绕胃癌分子分型与靶向治疗研究方向、在肿瘤微环境研究领域展开研究工作并取得一系列研究成果，以通讯作者在Gastroenterology、Gut、Cancer Res、Ann Surg、Br J Surg、Gastric Cancer 等肿瘤学及外科学国际权威期刊上发表18-篇SCI论文。我们发现GALNT5的低表达可以通过活化 EGFR/PAK1信号抑制胃癌细胞肿瘤抗原的表达，促进免疫逃逸，抑制抗肿瘤免疫反应。研究发现胃癌组织中GALNT5 低表达的患者，其肿瘤组织中发挥抗肿瘤效应的肿瘤浸润中性粒细胞(Ann Surg. 2018)及肿瘤浸润肥大细胞(Br J Surg. 2017)数量显著减少，而发挥免疫逃逸作用的M2型肿瘤相关巨噬细胞(Gastric Cancer. 2015)浸润显著增多，该研究首次确认GALNT5可作为干预胃癌进展的潜在治疗靶点。我们还发现作为一种特定亚型的肿瘤相关巨噬细胞，表达CXCL8的肿瘤相关巨噬细胞一方面可以通过激活RACK1介导的miRNA-302c表观抑制，促进胃癌细胞侵袭转移等恶性生物学行为(Cancer Research. 2015)，同时可以诱导肿瘤相关巨噬细胞表达PD-L1促进免疫逃逸，抑制抗肿瘤免疫反应(Gut. 2019，accepted)，该研究首次揭示表达CXCL8的肿瘤相关巨噬细胞在在胃癌发生发展过程中的肿瘤促进作用及其作为潜在的预后因子及治疗靶点的临床应用价值；我们还发现CLEC2可抑制AKT及糖原合酶激酶-3β的激活，从而抑制胃癌细胞的侵袭及上皮间质转化(Gastroenterology. 2016)，该研究首次阐述胃癌细胞中 CLEC2/AKT/Snail信号参与肿瘤细胞恶性生物学行为发展的分子机制，为胃癌患者个体化治疗提供了新的潜在靶点。