EBV-LMP1上调鼻咽癌Warburg效应在肿瘤相关免疫细胞分化与功能调节中的作用和机制

Our previous study identified that the latent membrane protein (LMP1) is an important oncogene and target antigen in Epstein-Barr virus associated cancers including nasopharyngeal carcinoma (NPC), which regulated the Warburg effect of tumor cells and the differentiation and function of cancer-associated immune cells. In addition, it is revealed that the Warburg effect of tumor cells is correlated with cancer immune tolerance, however, the association and its regulated mechanism between the expression of LMP1 and Warburg effect as well as cancer immune tolerance in NPC is still unclear. Based on the previous study, we proposed the hypothesis that the expression of LMP1 in NPC cells induced the imbalance of differentiation and function of cancer-associated immune cells, including MDSC, macrophage, Treg and CTL cells, through up-regulation of Warburg effect of tumor cells, which leading to the abnormal biological function of tumor cells including cytokine secretion. We determined the mechanisms of LMP1 promoting the aerobic glycolysis of NPC cells, and inducing the differentiation and functional impair of cancer-associated immune cell subsets by RNA expression microarray, overexpression or knock-down of target genes and cell coculture. We investigated the association between the LMP1, Warburg effect and tumor immune escape as well as their role in the disease progression of NPC by RNA interference, clinical relevance, and animal model. We aimed to find out that the molecular mechanism of the expression of EBV-LMP1 in NPC cells regulated the immune tolerance of NPC by regulating the reprogramming of glucose metabolism of tumor cells, and some target genes which maybe provide a potent value in immunotherapy of NPC.

我们前期研究发现鼻咽癌中EBV-LMP1参与了癌细胞Warburg效应及肿瘤相关免疫细胞分化及功能的调控；且肿瘤Warburg效应与免疫耐受的发生有密切关系，但鼻咽癌细胞表达LMP1与Warburg效应及肿瘤免疫耐受三者间的关联性及分子机制尚不清楚。我们提出鼻咽癌通过表达LMP1促进癌细胞发生Warburg效应，使癌细胞发生细胞因子分泌等生物学行为改变，诱导肿瘤微环境免疫细胞（MDSC，巨噬细胞，Treg，CTL等）的分化与功能失衡，导致肿瘤免疫逃逸的科学假设。我们首先通过细胞共培养、RNA表达谱芯片、靶基因过表达及敲低等方法分析LMP1表达对鼻咽癌细胞糖酵解及肿瘤相关免疫细胞分化与功能的作用和调控机制；其次通过靶基因干预和相关体内实验探讨癌细胞表达LMP1、糖代谢失调与免疫耐受在鼻咽癌临床进程和治疗中的作用，预期发现癌细胞表达LMP1调控糖代谢失调参与鼻咽癌免疫耐受的分子机制和干预靶点。

我们前期工作发现鼻咽癌肿瘤微环境存在免疫抑制，其中髓源性抑制细胞（Myeloid-derived suppressor cells，MDSC）的扩增是一个主要原因。本项目在前期工作的基础上，我们深入分析鼻咽癌细胞通过上调EBV抗原LMP1及相关分子如非编码RNA和免疫抑制分子Galectin-9等调控抑制性免疫细胞如MDSC在肿瘤微环境分化和富集的分子机制。首先，我们发现鼻咽癌微环境中，鼻咽癌细胞表达EBV抗原LMP1与MDSC数目呈正相关，进一步机制研究发现LMP1抗原表达可上调鼻咽癌细胞的有氧糖酵解的能力，进而激活NLRP3炎性小体，导致IL-1b，IL-6和GM-CSF分泌增加，诱导肿瘤相关的MDSC扩增，促进肿瘤微环境的免疫抑制。其次，我们发现鼻咽癌肿瘤微环境的缺氧，可诱导miR-24表达上调、并在鼻咽癌来源的exosome富集，miR-24及鼻咽癌exosome携带的Galectin-9蛋白都可抑制STING的表达水平，STING通路的失活可导致SOCS1诱导的STAT3磷酸化增强，诱导肿瘤微环境的IL-1b和IL-6分泌增多，最终导致细胞因子依赖的MDSC扩增，与鼻咽癌免疫逃逸相关。总的说来，本项目揭示了鼻咽癌微环境肿瘤细胞通过一些关键分子调控免疫细胞亚群分化，导致肿瘤微环境髓源性免疫抑制的分子机制，提出了包括LMP1、miR-24、肿瘤代谢相关分子和Galectin-9等一系列潜在的免疫治疗靶点。