N-乙酰基半乳糖胺转移酶7介导黏蛋白5B的O-GalNAc糖基化通过激活肿瘤相关巨噬细胞参与肾癌进展的机制研究

The glycosylation of proteins plays important role in their correct packaging, transportation, stability and protein-protein recognition. Altered glycosylation of transmembrane or secreted proteins restrict proteins’ normal functions and thus affect cell-to-cell adhesion as well as interactive signaling. Hence the aberrant glycoprotein secreted by tumor cell, which have been observed in most human malignancies, can modulate the functional phenotype of immune cells that extensively reside in tumor microenvironment. These immune cells “functional phenotype switch” promote tumor progression and resistance to certain chemotherapy as a case of immune surveillance dysfunction and immune escape. Our previous works have proved that the N-acetygalactosaminyl transferase family（GALNT）, which contains key enzymes catalyzing O-glycan initiation, has a key role in tumor progression and drug resistance through different signaling pathways. In this research, we found that increased GALNT7 expression levels in surgically resected clear-cell renal cell carcinoma (ccRCC) tumor tissues are higher than paired peritumor tissues, the intratumoral GALNT7 expressions correlated significantly with clinical TNM stages, post-sunitinib treatment survivals, local macrophages infiltrations and their tumor-associated molecular phenotypes. Furthermore, the secretions of high-GALNT7 tumor cells reduce the exocytosis of chymase from mast cells in tumor stroma, while chymase have been found to crop and degrade several cytokines/chemokines, which may lead to the TAMs protumoral phenotypes switch that we observed above. Based on these preliminary data, our current project proposal is designed to further elucidate the regulatory effect and functional significance of GALNT7-mediated O-glycosylation in ccRCC tumor progressions, especially its regulatory effect on TAMs phenotype activations through special glycoproteins and cytokines, mainly focus on the molecular mechanism and functional significance of the glycosylation-immune crosstalk through O-glycoprotein such as mucins and tumor-promoting cytokines such as IL4/CCL2. The intrinsic model of molecular regulation pathway established by this research may help to develop O-glycan-based biomarker to improve preclinical diagnose sensitivity, postsurgical sunitinib administration decision or survival prediction, and pave the way to the clinical investigation and drug discovery based on GALNT7-mediated immune changes, especially the immune cell associated targeted therapy in current clinical managements of high-risk ccRCC patients.

蛋白质糖基化修饰改变是肿瘤的重要特征之一，其与肿瘤的发生发展互为因果。项目申请人前期研究发现肿瘤细胞蛋白质糖基化修饰改变和肿瘤浸润免疫细胞参与肿瘤发生发展和治疗抵抗过程。本项目申请前期发现肾癌患者肿瘤组织中N-乙酰基半乳糖胺转移酶GALNT7高表达并与肿瘤进展、舒尼替尼治疗生存获益及瘤内巨噬细胞浸润程度和功能表型密切相关，黏蛋白mucin5B可能介导肾癌细胞GALNT7表达调控巨噬细胞促癌功能表型转化。本项目申请拟在此基础上阐述肾癌细胞GALNT7表达介导黏蛋白mucin5B的O-GalNAc糖基化修饰参与肾癌细胞与肿瘤相关巨噬细胞相互作用的分子机制及其在肾癌发生发展和治疗抵抗中的功能意义，建立肾癌发生发展和治疗抵抗的黏蛋白O-GalNAc糖基化修饰-免疫细胞功能表型和相关细胞因子分泌调控模型，为开发针对相应异常免疫细胞功能表型及其分泌细胞因子的肾癌患者分子靶向和免疫治疗方案奠定理论基础。

在本项目资助下，项目申请人围绕肿瘤免疫调节与免疫耐受方向，深入研究肿瘤细胞促癌信号活化促进肿瘤免疫逃逸形成的细胞和分子机制，并取得一系列研究成果，以通讯作者在European Urology、Gut、Nature Communications、Annals of Oncology、Clinical Cancer Research、Cancer Research、Journal for Immunotherapy of Cancer、European Journal of Cancer、International Journal of Cancer、Annals of Surgery等肿瘤学领域国际权威期刊发表SCI论文13篇，其中ESI高被引论文2篇，影响因子10以上论文11篇。研究发现，N-乙酰基半乳糖胺转移酶（polypeptide N-acetylgalactosaminyltrans ferases，GALNTs）在肾透明细胞癌的发生和发展中发挥了关键作用。其中，GALNT7异常高表达的肾癌患者常伴有BAP1的突变。GALNT7的高表达通过促进CD8+ T细胞向耗竭型CXCL13+CD8+T细胞分化，同时削弱DCs细胞的抗原提呈功能，导致患者的肿瘤进展和不良的术后总体生存。此外，GALNT7还通过催化其底物黏蛋白mucin5B的O-GalNAc糖基化修饰，诱导肿瘤相关巨噬细胞向促癌功能表型转变，介导肾癌患者对舒尼替尼的治疗抵抗。本项目阐明了CD8+T、肿瘤相关巨噬细胞等肿瘤浸润免疫细胞功能表型变化在肿瘤细胞促癌信号诱导免疫逃逸形成过程中的关键作用及其细胞分子机制，这些发现对于认识肿瘤免疫逃逸形成机制具有重要意义，同时也为肿瘤免疫治疗提供了新的靶点。