β1,6-N-乙酰氨基葡萄糖转移酶5糖基化修饰TGF-β受体参与调控胃癌细胞分化的分子机制及临床意义研究

The prognosis of gastric cancer correlated greatly with tumor differentiation.Our previously study showed β1,6-N-acetylglucosaminyltransferase V (MGAT5) has a negative correlation with gastric tumor cell differentiation using high-throughput tissue microarrays and find that high MGAT5 expression has better prognosis indicating MGAT5 was highly related to tumor cell differentiation and can be used as a prognostic marker. Aberrant MGAT5 expression in malignant tissues has been reported to be involved in the development of various cancers and their progression.MGAT5 can modifies the size and N-Glycans branching of glycoproteins in tumor microenvironment, especially the secreted proteins and membrane receptor proteins that have important functions in proliferation、differentiation、survive、anti-apoptosis etc. Transforming growth factor-β(TGFβ) signal plays a pivotal role in cell differentiation. The location of TGF-β receptor on cell membrane are key determinants of signal activity. Galectins are N-acetyllactosamine binding proteins that can bind the N-glycans of glycoprotein. Galectin 3 cross linked N-Glycans on transforming growth factor-β receptors at the cell surface and delayed their removal by constitutive endocytosis. As such, the lattice of interactions resulting from galectin/glycoprotein crosslinking limits receptor internalization and maintains downstream signaling sensitivity. Reduced galectin-3 binding to complex N-glycans on the TGF-β receptors in epithelial cells impaired the activation of TGF-β signal.MGAT5 may play a pivotal role in receptor N-Glycan modification. Low expression status of MGAT5 in gastric cancer cell may lead to the accelerated endocytosis of TGF-β receptor and weaken the ability of prompting cell differentiation. This project proposal for grant application is designed to elucidate the regulatory effect and functional role of the mechanism of glycosylation of TGF-β receptor post-translation and its function based on human gastric cancer tissues and cells . From the point of view in MGAT5 prompting cell differentiation and cancer glycobiology, our present research project will focus on the molecular mechanism and functional significance for N-glycosylation-mediated alteration of membrane TGF-β receptor protein endocytosis and stability conferred by abnormally expressed MGAT5 and will establish a model of molecular regulation and therapeutic intervention of TGF-β receptor N-glycosylation change on gastric cancer cells membrane, which paves the way to clinical investigation and drug discovery based on molecular therapy targeting aberrant N-glycosylation and TGF-β signal activation in gastric cancer patients，and constructs a MGAT5 based molecular prognosis model.

胃癌的预后与分化程度密切相关。我们前期研究发现N-糖基化修饰酶β 1,6-N-乙酰氨基葡萄糖转移酶V（MGAT5）在胃癌组织中的表达与分化呈负相关，高表达MGAT5患者预后好，提示其可能通过改变肿瘤组织中N-糖基化修饰状态调控分化进而影响预后。TGF-β信号通路在细胞分化过程中发挥重要作用，其受体的N-糖基化修饰可增加受体膜稳定性进而增强信号活化程度，MGAT5介导的TGF-β受体N-糖基化修饰可能在TGF-β信号通路的活化中起重要作用。本项目以胃癌组织和细胞及其异常表达的MGAT5为研究对象，从MGAT5调控细胞分化和肿瘤糖生物学的角度出发,研究MGAT5糖基化修饰TGF-β受体的分子机制及功能意义。构建MGAT5糖基化修饰TGF-β受体致其异常活化参与细胞分化与胃癌发生发展的分子调控和干预治疗模型，为开发针对N-糖基化修饰的分子靶向药物及建立MGAT5对胃癌预后的分子分型模式奠定基础。

本项目从肿瘤糖生物学及其可能介导的细胞因子、膜表面受体及肿瘤免疫微环境中的免疫细胞的角度出发。分别研究了C型凝集素样受体2（CLEC2）在胃癌中的表达及作用，介导肿瘤免疫细胞浸润的趋化因子SDF-1在胃癌组织中的表达，胃癌细胞表面可能受盐藻糖基化修饰的Notch1信号活化在肿瘤发生进展中的作用，以及可能受SDF-1趋化的肿瘤相关巨噬细胞浸润在胃癌进展中的作用及其预后意义。进一步探索了肿瘤浸润中性粒细胞在胃癌中的浸润情况，揭示了其可能参与调控胃癌化疗敏感性调控的可能调控作用。研究发现CLEC2表达主要通过抑制AKT活化促进肿瘤进展及转移。SDF-1可能通过癌旁组织的旁分泌作用于肿瘤组织中表面SDF-1受体的肿瘤细胞及相关间质细胞，促进肿瘤转移，参与调控疾病进展。进一步研究，我们发现，在肿瘤组织中含量最丰富的固有免疫细胞，肿瘤相关巨噬细胞，受趋化因子的调控，浸润到胃癌肿瘤组织中，并表现出不同的极化功能类型。其中M1型极化的肿瘤相关巨噬细胞具有促炎，抗肿瘤作用；而M2型极化的肿瘤相关巨噬细胞具有免疫抑制，促肿瘤作用。当肿瘤组织中M1型浸润为主时，患者具有较好的预后；而当M2型浸润为主时具有相对较差预后。作为调控肿瘤细胞干性表型的重要信号通路，Notch1信号的活化至关重要。在胃癌组织中，Notch1信号活化，即其下游ICN1的表达明显增高，与疾病进展及患者相对较差的预后相关。Notch1信号受体收盐藻糖基化修饰后可以增加在细胞膜表面的锚定，促进信号激活及下游信号通路的传导。具有Notch1信号活化的肿瘤细胞表现出更多的干细胞样表型，诱导肿瘤相关免疫细胞，尤其是M2型肿瘤相关巨噬细胞极化。针对Notch1信号的活化，采用阻断其下游γ-分泌酶抑制剂阻断下游ICN1的活化，可能会成为胃癌治疗新的靶点。