Kras突变介导IL-10糖基化修饰导致胰腺癌中Tregs富集的机制研究

Pancreatic cancer is one of the most lethal cancer with an extremely poor prognosis. There are two basic features: 1. Kras high-frequency mutation runs through the whole process of pancreatic cancer initiation and progression. 2. Rich in stroma and the local immune microenvironment has a significant impact on its biological behavior. Our previous studies found that (1) KrasG12D mutation correlates with high Treg infiltration in pancreatic cancer tissues; (2) KrasG12D mutation could regulate the expression of IL-10 and promote the conversion of CD4 + T cells into Tregs; (3) Protein glycosylation was more common in KrasG12D mutant pancreatic cancer, and there is a N-glycosylation modification site in the amino acid sequence of IL-10, furthermore, Mass Spectrometry identified IL-10 is modified by glycosylation. Therefore, we hypothesized that KrasG12D mutation promotes the stability of IL-10 by mediating its glycosylation, and activates downstream pathways, then enrichment of Tregs, lead to immunosuppressive tumor microenvironment. This project attempts to investigate the Cross-Talk between tumor cells and immune microenvironment, focusing on the potential mechanism of KrasG12D mutation regulate the IL-10 glycosylation. This work may explore the novel mechanism of KrasG12D mutation regulate tumor immune escape, and could provide a potential therapeutic strategy.

胰腺癌恶性程度高，预后极差。具有两大特征：1. Kras高频突变，贯穿胰腺癌发生发展过程；2. 富含间质，局部免疫微环境对其生物学行为影响显著。我们前期研究发现:（1）KrasG12D突变的胰腺癌组织中，免疫抑制细胞Tregs高比例浸润；（2）KrasG12D突变可以调控IL-10的表达，促进CD4+T细胞向Tregs转化；（3）KrasG12D突变的胰腺癌中蛋白糖基化现象较为普遍，IL-10的氨基酸序列中存在N-糖基化修饰位点，质谱鉴定证实IL-10受糖基化修饰。基于上述证据，我们推测KrasG12D突变可能通过介导IL-10的糖基化修饰，促进其稳定表达，进而活化下游通路，富集Tregs，导致免疫抑制肿瘤微环境。本课题拟研究Kras突变通过翻译后修饰、影响肿瘤细胞与免疫微环境之间的“Cross-Talk”，提出胰腺癌Kras突变与肿瘤免疫逃逸内在联系的具体机制，并探索可能的干预靶点。

胰腺癌恶性程度极高，预后差，对免疫治疗不获益。基因组不稳定性和抑制性肿瘤免疫微环境是导致胰腺癌进展迅速的重要因素，针对导致胰腺癌基因不稳定性的机制以及改善抑制性免疫微环境的方法进行深入研究意义重大。.本项目围绕胰腺癌的基因组不稳定性和抑制性肿瘤免疫微环境，通过基因突变鉴定、生信分析、体内外实验等方法，深度挖掘了导致胰腺癌的基因组不稳定性的分子机制，并通过遗传修饰定向诱导分化和嵌合抗原受体等技术探讨了改善抑制性肿瘤免疫微环境的方法。通过对胰腺癌患者外周血循环肿瘤DNA（ctDNA）中Kras的突变以及循环调节性T细胞（Tregs）的检测,明确了循环Tregs相关的Kras突变在晚期胰腺癌患者中的预后价值；通过对胰腺癌中相关突变特征的鉴定和分析，阐明了APOBEC3C异常表达在诱导胰腺导管腺癌特征性基因组不稳定性中的作用以及预后价值；通过体外定向诱导分化和嵌合抗原受体技术，成功弥补了Tfh细胞来源不足和浸润比例低的不足，显著改善了胰腺癌抑制性免疫微环境，并为提升传统CAR-T细胞治疗的疗效提出了可行性方案。