具核梭杆菌损伤肠屏障激活TLR4/NFκB通过表观遗传修饰致DNA错配修复缺陷促大肠癌发生发展的分子机制

Colorectal cancer is the third burden of morbidity among all malignant tumors worldwide. Although several mechanistic events have been identified that play key roles during colorectal carcinogenesis and development, only a few of these molecular targets are clinically-translatable. Identification of previously unrecognized mechanisms that drive cancer development would be of clinical relevance as these would allow development of robust biomarkers and effective drug targets that are urgently needed for the therapeutic management of CRC. However, the mechanisms of occurrence and development of CRC are far from being elucidated. Our previous study confirmed a strong induction of host cell Toll-like receptor (TLR)4 by lipopolysaccharide of Fusobacterium nucleatum (F.nucleatum), leading to activation of RAS/MAPK signaling through modulation of the miR21 target, RASA1 (Gastroenterology.2017;152(4):851-866.e24. IF = 18.392). Nevertheless, whether the presence of the pathogenic microorganism is a cause or a consequence of Tumor development is also largely unknown. Approximately 15-20% of CRCs develop because of defective function of the DNA mismatch repair (MMR) system. Deficient MMR (dMMR) was significantly associated with primary tumors of the proximal colon. CRC Patients with dMMR have reduced rates of tumor recurrence. Previous studies revealed that the proportion of F.nucleatum-high CRC gradually increases from rectum to cecum. Moreover, F.nucleatum-high CRC was found to be associated with dMMR positivity. We put forward the hypothesis that F.nucleatum induces the impairment of gut mucosal barrier and increases development of colorectal cancer by epigenetically stimulating DNA mismatch repair deficiency via activating TLRs/NFκB/miR21/MSH2 or TLRs/NFκB/DNMT1/MLH1-methylation, leading to activation of WNT and MAPK signaling pathway. Removal of F.nucleatum from gut mucosal may reduce its susceptibility to dMMR and improves patient outcomes. It is expected that this study may provide experimental evidences for the research on precise CRC typing and translational medicine based on gut microorganisms.

大肠癌发病率和死亡率居高不下，其发生发展机制尚未被完全阐明。申请人既往研究发现潜在致癌微生物具核梭杆菌感染肠上皮细胞可激活TLR4/NFκB及下游RAS/MAPK通路促大肠癌发生，然而，肠道微生态紊乱和遗传学改变二者在大肠癌进展中的逻辑关系仍不明朗。本项目基于“右半大肠癌DNA错配修复缺陷（dMMR）发生率更高且预后更差，而具核梭杆菌与dMMR正相关且在右半结肠中丰度更高”的现象，拟进一步探讨具核梭杆菌突破肠屏障激活TLR4/NFκB，通过表观遗传修饰（NFκB/miR21/MSH2或NFκB/DNMT1/MLH1）诱导dMMR，引起DNA损伤累积，形成促炎/促癌免疫微环境，激活WNT和MAPK级联信号，致患者辅助治疗失敏和较差预后，拮抗或清除具核梭杆菌可降低dMMR易感性，改善患者预后。本项目期望由此拓展防治大肠癌的新思路，为基于微生物的大肠癌精准分型和临床转化治疗提供实验依据。

本研究首先利用人体微生物组结合非靶向代谢组学的关联性挖掘了肠道疾病的特征，通过临床组织和粪便样本鉴别出健康人和年轻人大肠癌（CRC）患者的特征性肠道菌群以及相关代谢小分子，发现了粪便微生物和代谢分子可能成为大肠癌诊断的潜在无创靶点和不同解剖部位分型的标志物。其次，本研究显示具核梭杆菌（F.nucleatum）通过诱导肿瘤相关巨噬细胞（TAMs）的PD-L1表达促进CRC的肿瘤进展。临床样本分析发现肿瘤样本中F.nucleatum的DNA含量与肿瘤相关巨噬细胞呈正相关。动物实验和离体细胞实验发现F.nucleatum感染通过TLR4/MyD88/NF-κB途径引起TAMs中分泌IL-6，激活的IL-6/STAT3途径可增强TAM中PD-L1的表达。通过小鼠模型进一步发现IL-6抑制剂和抗PD-L1处理可导致F.nucleatum诱导的肿瘤形成的减弱。F.nucleatum感染状态可能可以改善微卫星稳定（pMMR）的CRC患者免疫治疗疗效。本研究建立起来的肠道微生态和代谢研究技术方法，将可能扩展到其他实体瘤研究领域。对于其他肿瘤的早诊早治方法研究有一定示范性作用。本研究成功建立了肠道微生态和代谢组学联合分析方法，并建立了肿瘤细胞、免疫细胞和肠道潜在致病菌F.nucleatum共培养细胞模型，以及F.nucleatum感染的肠癌模型，为相关功能学的研究以及药物筛选等提供了可视化细胞和动物模型。对于本研究发现的F.nucleatum影响pMMR患者免疫治疗疗效，可进一步设计针对该潜在致病菌的工程载体，探讨并验证其在大肠癌免疫治疗中的潜在价值，以期应用于临床转化。