黏蛋白型O-聚糖异常表达和结直肠癌发病的关系研究

Colorectal cancer is a leading cause of cancer-related death around the world but there are no reliable treatments available. It is very hard to completely cure colorectal cancer especially for metastatic cancers. O-linked oligosaccharides (O-glycans) are the primary components of the intestinal mucus layer that overlies the gastrointestinal epithelium. Notably, altered intestinal O-glycan expression are seen in over 80% of colorectal cancers, however, whether or not this abnormal O-glycan expression contributes to the etiology of colorectal cancer is unknown. Our previous studies have created mice lacking intestinal O-glycans and we found that O-glycan-/- mice develop spontaneous colorectal adenocarcinoma. In this study, we intend to investigate the role of mucin-type O-glycans in the development and metastasis of colorectal cancer in O-glycan-/- mice. In the meanwhile, we aim to examine if mucin-type O-glycan-mediated O-glycosylation plays a contributory role in cancer cell apoptosis, proliferation, invasion by culturing LS174T cells harboring aberrant O-glycans. We also investigate the frequency of abnormal O-glycosylation in human primary and metastatic colorectal cancer, as well as the relationship between abnormal O-glycosylation and the clinical features. Once we get some significant results, we will specifically isolate Tn antigen positive tumor cells (which is a specific marker for abnormal mucin-type O-glycans) through laser capture microdissection (LCM) and FACS, respectively. Then, we are going to use exome capture sequencing, qRT-PCR, and analysis of the promoter methylation by MassARRAY to explore the underlying mechanisms related to abnormal expression of mucin-type O-glycans in these collected human tumor cells. Finally, we will use gene expression microarray to analyze the expression of the relevant genes and signaling pathways related to abnormal O-glycosylation. Our study will disclose some novel mechanisms regarding the role of O-glycosylation in the development and metastasis of colorectal cancer and provide some clues for their early diagnosis and targeted therapies in clinical settings.

结直肠癌是常见的恶性肿瘤，极易复发转移。黏蛋白型O-聚糖是肠道上皮细胞黏液层的主要成分，在结直肠癌的发病过程中常表达异常，但具体机制不明。本项目在前期工作基础上，（1）利用黏蛋白型O-聚糖缺陷的小鼠和细胞株（LS174T），以O-型糖基化反应为视角探索肿瘤的发生发展、转移及相关分子机制；（2）检测黏蛋白型O-聚糖在人结直肠癌原发灶和转移灶的异常表达及与临床转归的关系；（3）采用流式细胞分选和激光捕获显微切割（LCM）技术分离O-聚糖异常表达的人结直肠癌细胞，然后采用外显子捕捉测序、定量PCR和MassARRAY甲基化检测等技术探索O-聚糖异常表达的分子机制。我们还拟采用表达谱分析技术检测与O-聚糖异常表达相关的差异基因及分子事件，以阐明黏蛋白型O-聚糖生物学作用的分子机制及调控环节。本项目首次在糖生物学水平上探索结直肠癌发生及转移的机理，对于肿瘤早期诊断、病程监测、预后评估均有重要意义。

结直肠癌是临床常见的消化道恶性肿瘤，极易复发转移，但机制不明；蛋白糖基化修饰异常与肿瘤的发生和转移密切相关，其中O-型糖基化反应是蛋白糖基化修饰的主要方式，在细胞癌变过程中常发生异常，表现为肿瘤相关糖类抗原-Tn阳性表达；但目前尚不清楚黏蛋白型O-聚糖异常表达是细胞癌变中的一个病理生理现象抑或是促癌发生的一个重要因素。本项目在前期基础上，从临床结直肠癌病人、细胞和动物三个层面系统探讨黏蛋白型O-聚糖异常表达即Tn抗原阳性对结肠癌发生和转移的影响作用及相关机制。我们首先收集了数百例结肠癌病人的肿瘤组织，检测发现Tn抗原阳性比例高达85%以上，且与肿瘤转移和预后不良密切相关；进一步研究发现，Cosmc甲基化是导致糖基转移酶T-synthase活性异常和Tn抗原阳性的主要原因。与此同时，我们分别靶向敲除T-synthase和Cosmc制备了O-聚糖缺陷结肠癌细胞株，通过细胞实验和裸鼠成瘤实验，观察发现Tn抗原明显增强肿瘤细胞恶性程度，并增加了肿瘤细胞对TRAIL的耐药性，其机制与破坏下游糖蛋白DR4/5糖基化修饰有关，进而激活EMT信号通路。最为重要的是，封闭Tn抗原能明显抑制肿瘤细胞侵袭转移，提示O-聚糖可以作为肿瘤干预治疗的靶点。进一步研究发现，黏蛋白型O-聚糖可以影响内质网应激、凋亡、糖代谢重编程、免疫微环境等诸多分子事件或信号途径，以糖基化异常反应为切入点探讨结肠癌的发生和转移，可望在新的层面揭示肿瘤发生和干预的细致环节。本项目还采用蛋白组学、转录组和代谢组学技术筛查O-聚糖异常表达影响的关键分子或代谢物，有助于揭示糖基化异常反应的分子机制及调控环节，对肿瘤的早期诊断、病程监测和开发靶向干预策略等均有重要指导意义。