巨噬细胞中STK4等抑癌基因调控炎症紊乱和肝细胞癌变的机理研究

The observations on patients and animals demonstrate that uncontrolled chronic inflammation disorder will increase the rate of tumor occurrence. Hepatocellular carcinoma (HCC) is the well-documented human chronic inflammatory disorder-induced cancer, which is often associated with pathogen infection. The development of pathogen invasion induced chronic hepatitis into hepatic sclerosis and subsequent HCC is considered as one typical example of chronic inflammation induced carcinoma in human beings. Although multiple tumor suppressor genes were defined to inhibit HCC in a cell-intrinsic manner, it is largely unknown whether these tumor suppressors play dual roles by either targeting immune cells to inhibit pro-carcinogenic inflammation or blocking inflammation-driven tumorigenicity at the early stage of HCC. We screened a group of key tumor suppressor genes in macrophages previously. Similar to the expression profiles as p53 and LKB1 that were recently discovered to suppress inflammation, the levels of STK4, RapL, Rap1, Trim24 and RB were reduced in TLR3/4 agonist or bacterial treated macrophages. Although STK4, RapL and Rap1 could form a signaling complex to regulate immune cell adhesion, it is not clear whether they could inhibit inflammation and the development of inflammation-driven HCC. Furthermore, we set up a mouse HCC model by using DEN plus CCl4. The dual roles of STK4, RapL, and Rap1 in pro-carcinogenic inflammation and inflammation-driven tumorigenicity will be investigated with STK4 or RapL deficient mice and the samples from HCC patients. We will further investigate by which manner that STK4, RapL and Rap1 cooperate with different TLRs pathways to suppress inflammation-driven HCC. This project will uncover the cross-talk between tumor associated macrophages and hepatocytes in tumor microenvironment and deepen our understanding of modulation of inflammation into carcinogenesis in HCC development, which will help us to identify new diagnostic targets for inflammation-driven HCC at the early stage.

大量临床数据和动物实验证实非可控性、持续性的炎症反应会诱导肿瘤发生。慢性肝炎发展为肝细胞癌 (HCC) 被认为是慢性炎症紊乱诱发癌症的典型例子。此外，抑癌基因表达降低或功能缺失也是诱发癌症的重要因素。已知部分抑癌基因可被肝癌细胞和巨噬细胞（MФ）共表达，但不清楚它们是否同时调控肝炎和HCC发生。我们筛选了在这两种细胞中共表达的一组抑癌基因，并建立了DEN和CCl4诱发肝癌小鼠模型。发现STK4表达在肝癌小鼠的MФ和HCC病人单核细胞中都下调。本项目拟利用基因敲除小鼠结合HCC临床病理样本分析，研究STK4等基因通过调控MФ中TLR受体介导的信号通路而抑制肝炎和HCC的新功能。这将有助于揭示HCC肿瘤微环境中MФ与肿瘤细胞之间相互作用机制，加深对肝炎-肝癌演变过程的理解。由于MФ具有迁移能力，通过检测癌旁组织或淋巴、血液循环中MФ相关的抑癌分子的异常表达，将可能为筛查HCC提供新诊断标志。

在影响肝细胞癌（HCC）肿瘤微环境的各种生理因素中，天然免疫细胞尤其是肿瘤相关的巨噬细胞（TAM）对于肿瘤微环境的形成和肿瘤发生具有关键作用。临床数据显示许多肝癌的癌组织中存在经典肿瘤抑制基因突变或表达降低的现象，这些基因在HCC肿瘤微环境中的作用有待揭示。.我们收集并分析了上百例处于恶性期的肝癌病人的临床样本，包括肝癌组织、癌旁组织、外周血等。此外，我们利用致癌剂DEN、CCl4结合LPS或E.coli建立了肝炎-肝癌转变的小鼠模型，模拟慢性肝损伤转化成肝癌的病理进程。我们也比较了野生型和靶基因敲除小鼠在肝炎、肝硬化、肝癌阶段的炎症反应能力与肝组织病理改变及肝功能变化的相关性。通过高通量基因表达谱芯片分析，我们发现丝氨酸和苏氨酸激酶STK4的表达在癌组织和病人外周血中显著下调。我们还发现STK4通过结合和磷酸化IRAK1，促进IRAK1降解，从而抑制NF-kB的活化及下调促炎因子IL-6、IL-1和肿瘤坏死因子α（TNFα）的产生。而且，我们还发现肝癌病人的肿瘤组织出现大量的TAM浸润，并且肿瘤浸润巨噬细胞或外周血中的巨噬细胞中低水平的STK4与高水平的IRAK1的表达亦呈现负相关；与血清IL-6低水平的肝癌病人相比，仅血清IL-6高水平的病人的巨噬细胞特异性的下调STK4的表达。在慢性炎症相关肝癌的小鼠模型中，在巨噬细胞特异性敲除STK4的小鼠显著促进炎症因子的表达，加重肝功能损伤，进而在晚期引起小鼠肝纤维化，促进肝肿瘤形成。更重要的发现是利用IRAK1抑制剂处理的小鼠，明显降低由于STK4缺失导致的慢性炎症和肝癌的发生发展。此工作指出将STK4-IRAK1作为阻断炎性肝癌发生的新靶点，并首次提出通过监测外周血巨噬细胞内STK4水平，辅助早期诊断炎性肝癌的新策略，相关成果被发表在国际著名临床医学杂志J Clin Invest（2015;125(11):4239-54）上。