HBV增强Tim-3表达维持肝癌起始细胞干性促进肿瘤发生的机制研究

Tim-3, an immune checkpoint molecule, regulates the function of immune cells and participates in tumorigenesis, which makes it being an candidate target in tumor immunotherapy. We reported previously Tim-3 was involved in the pathogenesis of chronic hepatitis B and liver cancer by regulating the function of T cell and NK cell and the differentiation of macrophage. We also found that Tim-3 is highly expressed on malignant hepatocytes, which is positively correlated with HBsAg and pathologic grade and negatively correlated with patient survival. Tim-3 promotes the growth of liver cancer cells both in vitro and in vivo. Moreover, Tim-3+ hepatocytes have higher level of CD133 and stronger sphere formation ability. Thus, we hypothesized that HBV-enhanced Tim-3 maintains the stemness of tumor initiating cells and promotes the development of liver cancer. Here, we would utilize mutants analysis, mass spectrometry and functional blocking assay to study the mechanism of HBV enhancement on Tim-3 expression on hepatocytes, to demonstrate the function of Tim-3 in maintaining the stemness and promoting carcinogenesis, and to probe into the mechanisms by using in vitro and in vivo model and clinical HCC samples. This study would be helpful for clarifying the novel mechanisms for Tim-3 involvement in tumorigenesis and for the drug design and biological treatment based on Tim-3.

免疫检查点分子Tim-3调控多种免疫细胞功能，是肿瘤免疫治疗的重要候选分子。课题组已报道Tim-3通过调控CD8+T、NK细胞功能和巨噬细胞分化，参与慢乙肝和肝癌发生。前期研究还发现，Tim-3可高表达于肝癌细胞，与HBsAg、病理分级正相关，与生存期负相关，促进肝癌细胞体内外生长，且Tim-3+肝癌细胞表达更高CD133，具有更强的成球能力。据此，提出本课题假说：HBV上调肝细胞中Tim-3表达，维持肿瘤起始细胞干性特征，促进肝癌进程。本课题拟在前期工作基础上，利用突变体分析、质谱技术结合功能阻断实验，通过体内外实验和临床检测，进一步研究HBV上调肝细胞中Tim-3表达的机制，明确肝癌细胞Tim-3维持干性特征参与肿瘤发生的作用，探讨Tim-3维持肿瘤起始细胞干性特征的分子机制。课题的实施将阐释为Tim-3参与肿瘤发生的新机制，为以其为靶点的药物设计和生物学治疗提供更为确凿的实验依据。

恶性肿瘤发病率居高不下，严重危害健康，位居人类疾病死因第二位。肝细胞肝癌为第四位最常见恶性肿瘤，死亡率却位居第三位。近年来，新型免疫治疗策略在临床肿瘤实践，特别是在晚期肿瘤患者治疗中，取得了令人瞩目的突破性进展。T细胞免疫球蛋白及粘蛋白-3（T cell immunoglobulin and mucin domain-3, Tim-3）是Tim家族成员中一员，可表达于多种免疫细胞，并通过介导CD8+ T细胞和NK细胞功能耗竭、增强Treg细胞免疫抑制功能等多种方式，在慢性病毒性感染及肿瘤发生中发挥重要作用，是肿瘤免疫治疗的重要候选分子。课题组已报道Tim-3通过调控CD8+T、NK细胞功能和巨噬细胞分化，参与慢乙肝和肝癌发生。本课题首先利用免疫荧光染色分析了临床肝癌组织中Tim-3在实质细胞中的表达情况，结果发现与癌旁组织相比，肝癌实质细胞Tim-3表达显著升高；细胞系研究发现HBV及其编码蛋白、TGF-β单独或联合IL-6、IL-4可显著上调肝癌细胞中Tim-3表达；体内外实验进一步证实肝癌细胞内源性Tim-3表达可显著加速细胞周期进程、促进肿瘤细胞增殖，抑制细胞凋亡；机制研究发现，Tim-3表达通路激活肝癌细胞NF-κB通路、上调IL-6表达，进而促进肝癌细胞增殖；Tim-3中和抗体可通过阻断肝癌细胞内源性Tim-3通路进而显著抑制肿瘤生长；此外，肝癌细胞Tim-3表达可抑制T细胞增殖和功能，负向调节抗肿瘤免疫反应。同时，课题系统检测肝癌患者外周血和肿瘤组织中免疫细胞进一步明确了Tim-3分子在肝癌组织和患者外周血多种免疫细胞中高表达，各细胞亚群间Tim-3表达显著正相关，并与肿瘤进展、患者生存期密切相关；Tim-3中和抗体可显著促进CD4+T、 CD8+T、NK和NKT细胞中细胞因子的产生和杀伤活性，下调单核细胞中CD206表达，而CD4+T细胞在Tim-3介导的免疫调控中发挥关键性作用。上述研究成果为Tim-3参与肿瘤发生的新机制，为以其为靶点的药物设计和生物学治疗提供更为确凿的实验依据。