激活NOD1/2信号通路肿瘤外泌体的主要成分及其介导肿瘤转移的生物学机制

The metastasis is a leading cause of cancer patients. NOD1 and NOD2 (NOD1/2) are critical for immune homeostasis and tissue repairing. Abnormal NOD1/2 signals are involved in multiple inflammatory diseases including cancer. However, the endogenous ligands of NOD1/2 activation remain unknown. Our results have indicated that exosomes secreted by both Lewis lung cancer cell (LLC) and breast cancer cell MCF7 are able to activate NOD1/2 signals; antagonists of NOD1 are capable of blocking LLC-Exo- and MCF7-exo- resulted NOD1 activation. Proteinase treatment of MCF7-exosomes fully blocked, and lipase treatment partially reduced the NOD1 activation, but neither DNase treatment nor RNase treatment did. These results strongly implied that the lipoproteins or lipopeptides in MCF-exo are the endogenous ligand(s) of NOD1/2. This project aims to further precisely determine and confirm the lipoproteins or lipopeptides by using quantitative LS-MS technology, and to investigate the molecular mechanisms with which the endogenous NOD1/2 ligands activate thier signals, promote the development of preMN and subsequently cause tumor metastasis. Therefore, this project will provide theoretic supports for deep understanding NOD1/2 signaling pathways and for development of antitumor drugs targeting NOD1/2.

肿瘤转移是癌症患者死亡的最主要原因。NOD1/2在平衡炎症反应和组织修复过程中起关键作用，其信号通路异常与包括癌症在内的多种炎性疾病密切相关。但是肿瘤发生和转移过程中活化NOD1/2活化的内源性配基仍不清楚。我们的实验结果表明，Lewis肺癌细胞LLC和乳腺癌细胞MCF7产生的外泌体（LLC-Exo和MCF7-Exo）均可活化NOD1/2信号；NOD1/2特异性拮抗剂可降低其活性。重要的是，蛋白酶或酯酶可完全或部分抑制外泌体所致NOD1活化，酯酶可显著降低其活化能力，提示NOD1/2很可能是由肿瘤细胞外泌体中的脂肽或脂蛋白激活。本课题拟在此基础上，利用定量质谱技术分析和鉴定NOD1/2的内源性配基，探讨其激活NOD1/2、诱导转移前微环境形成并最终导致肿瘤转移的分子和细胞生物学机制，为深入了解NOD1/2信号通路和开发以NOD1/2为靶标的抗肿瘤药物提供理论基础。

本项目研究发现，结直肠癌细胞来源的EVs（CRC-EVs）比正常结肠细胞来源的EVs携带更高含量的小G蛋白42（CDC42）。CRC-EVs进入巨噬细胞后，CDC42会以GTP-CDC42活化形式介导核苷酸结合寡聚化结构域蛋白1（NOD1）信号通路的激活，诱导细胞分泌炎症因子和趋化因子，促进结直肠癌发生转移。进一步研究发现结直肠癌肝转移（CRC-LM）患者血浆EVs中的较高含量CDC42也参与了免疫细胞中NOD1信号通路的激活。研究进一步发现NOD1在结直肠癌原发灶和肝转移灶肿瘤组织中均表达增高，且肝转移患者的原发灶肿瘤组织中NOD1表达量显著高于非转移患者肿瘤组织；肿瘤组织中NOD1的高表达与临床预后差具有相关性。我们推测肿瘤组织中NOD1表达量越高越有利于结直肠癌发生肝转移，导致患者预后差。本项目研究结果揭示了CRC-EVs携带高含量的CDC42蛋白，被巨噬细胞摄取后，介导NOD1信号通路激活，诱导炎症相关因子产生，形成有利于结直肠癌转移的炎症微环境，促进肿瘤转移，为临床上治疗和诊断CRC-LM提供理论基础。