ILT4在非小细胞肺癌细胞中表达的作用及其上调VEGF-C表达的分子机制

Immunoglobulin-like transcript 4(ILT4) play an important role in the immune tolerance maintenance for pregnancy, inflammation and transplantation rejection. One recent study indicates an unexpected functional significance of ILT4 in support of leukemia development. However, ILT4 expression and its roles on non-myeloid tumor cells have not been reported. Our previous studies found that ILT4 was expressed on non-small cell lung cancer (NSCLC) cells. More importantly, its expression on the cancer cells was positively correlated with VEGF-C expression, micro-vessel and lymphatic vessel density, lymph node metastasis and poor prognosis. And the expression level of ILT4 in metastatic lymph nodes was much higher than in the primary stoves. Highly expressed ILT4 on NSCLC cancer cells could enhance the cell proliferation, invasion and migration and up-regulate the expression of angiogenesis and lymphangiogenesis factors. In addition, ILT4 could up-regulate vascular endothelial growth factor (VEGF)-C expression by activating extracellular regulated protein kinase (ERK) signaling pathway. We hypothesize that ILT4 could promote NSCLC invasion and metastasis through various links. In this study, we will use stable infection technique to obtain ILT4-overexpressed A549 and ILT4-knockdowned A549, to explore the role of ILT4 in promoting cancer cell proliferation, adhesion, migration and invasion, and to define the molecular mechanisms that ILT4 up-regulates VEGF-C expression. We will collect clinical data and establish the xenografts SCID mice model with ILT4-overexpressed and ILT4-knockdowned A549 cells, to observe the relationship between ILT4 expression and tumor invasion and metastasis, Lymphatic vessel and micro-vessel density. This study will define the role of ILT4 in NSCLC invasion and metastasis, angiogenesis and lymphangiogenesis, and clarify the molecular mechanisms that ILT4 up-regulates VEGF-C expression on cancer cells.

免疫球蛋白转录子(ILT)4在维持母胎和移植免疫耐受方面发挥重要作用。最近研究发现它在支持白血病细胞生长中亦起重要作用，而在非髓系肿瘤细胞中的表达及作用未见报道。我们前期研究发现ILT4在非小细胞肺癌（NSCLC）细胞中表达；且与VEGF-C表达呈正相关；ILT4阳性表达者的微淋巴管与微血管密度增高、淋巴结转移率增加、生存期缩短；ILT4高表达可增强肿瘤细胞增殖和迁移力、并可能通过ERK信号通路上调VEGF-C表达。故推测ILT4可通过多个环节促NSCLC侵袭转移。拟建立稳定高/低表达ILT4的NSCLC细胞株A549，明确ILT4促细胞增殖、粘附、侵袭迁移及上调VEGF-C表达的机制；拟建立稳定高/低表达ILT4的SCID小鼠NSCLC皮下移植瘤和淋巴道转移模型，并收集临床资料，观察ILT4与VEGF-C表达、侵袭转移、微淋巴管与微血管密度关系，明确ILT4在NSCLC侵袭转移中的作用。

免疫球蛋白样转录子（ILT）4属于免疫抑制性受体，在维持母胎和移植免疫耐受方面发挥重要作用。既往研究发现ILT4在血液系统肿瘤细胞中有异常高表达，2012年Nature研究报道ITT4在白血病细胞生长中发挥重要作用，但其在非髓系肿瘤细胞中的表达及作用未见报道。本课题组前期研究表明ILT4在非小细胞肺癌（NSCLC）细胞中高表达且与预后不良相关；与VEGFC表达呈正相关；ILT4阳性表达者的微淋巴管与微血管密度增高、淋巴结转移率增加、生存期缩短；ILT4 高表达可增强肿瘤细胞增殖和迁移力。本项目在前期研究的基础上，从体外细胞实验到动物体内实验，利用细胞生物学、分子生物学、基因组学和动物模型等手段和方法，从多基因、多角度研究了ILT4对肿瘤细胞的恶性生物学特性及肿瘤微环境的的影响。结果示ILT4通过ERK信号通路上调VEGF-C的表达等促进肿瘤细胞增殖、侵袭迁移等多种恶性生物学行为。此外，在NSCLC细胞中ILT4可通过激活PI3K/AKT/mTOR信号通路上调B7-H3的表达，并抑制肿瘤免疫细胞TILs的数量。而上述作用也可能基于ILT4与其配体（HLA-G及Angptl2）的相互调节作用的自分泌现象，共同促进肿瘤进展。ILT4配体Angptl2可诱导肿瘤相关巨噬细胞M2型分化，进一步参与NSCLC进展。本研究从理论机制上揭示了ILT4在NSCLC 疾病进展中的重要作用，动物体内实验证实了抑制NSCLC细胞内ILT4表达后肿瘤的生长和转移受到显著抑制，提示ILT4可以成为 NSCLC 潜在的治疗靶点，对于认识ILT4在恶性实体肿瘤进展中发挥的作用提供了重要依据，并为进一步探讨ILT4对肿瘤微环境中免疫细胞的作用提供了前期线索。