EB病毒潜伏膜蛋白LMP1诱导鼻咽癌去分化的表观遗传学机制及应用

Undifferentiation is a prominent clinical feature of nasopharyngeal carcinoma (NPC) in China. The metastasis to cervical lymph nodes represents a frequent initial manifestation of undifferentiated NPC. Epstein-Barr Virus latent membrane protein 1 (LMP1) is the key viral oncoprotein to induce undifferentiated phenotype of NPC. But little is known about the molecular mechanisms underlying the transforming process. Epigenetic reprogramming induced by LMP1 is a critical control point for NPC transformation. Here we aim to determine the molecular mechanisms governing epigenetic reprogramming of NPC induced by LMP1. We have established CNE1-L and HNE2-L cell lines which stably expressed LMP1 based on high-differentiated NPC cell line CNE1 and medial-differentiated NPC cell line HNE2. LMP1 significantly facilitated the ability of proliferation and migration, induced dedifferentiation, and upregulated epigenetic factors DNMT1 and EZH2 of these cells. We further determined the genome-scale DNA methylation status of these cells through Reduced Representation Bisulfite Sequencing (RRBS) base on Next Generation Sequencing (NGS). Thousands of genes were hypermethylated or hypomethylated. The Gene Ontology (GO) Enrichment Analysis indicated that these genes are mainly involved in development, cell differentiation, cell morphogenesis and hormone stimulus. The KEGG Pathway Analysis indicated that Focal adhesion pathway, Wnt signaling pathway, Hedgehog signaling pathway and Notch signaling pathway are activated. We will further investigate the LMP1-associated dedifferentiated functional genes and signaling pathways,and determine the epigenetic mechanisms. These results will provide us the theoretical evidence and potential therapeutic targets for differentiation therapy of nasopharyngeal carcinoma.

未分化是我国鼻咽癌在临床上最显著的特征。EB病毒潜伏膜蛋白1（LMP1）是诱导鼻咽癌去分化的关键蛋白。表观遗传调控在LMP1诱导鼻咽癌去分化过程中的分子机制尚不清楚。申请人在肿瘤基因调控方面已具备坚实的基础（J Biol Chem,2012）。本课题组目前已建立LMP1诱导鼻咽癌去分化的细胞模型。发现LMP1上调了DNMT1和EZH2。通过全基因组简化亚硫酸氢盐甲基化测序（RBSS）发现LMP1诱导了鼻咽癌基因组大范围的异常甲基化。GO富集分析显示这些基因主要与发育、细胞分化、细胞形态和荷尔蒙刺激有关；KEGG信号通路分析首次发现Hedgehog/ Wnt/ Focal adhesion/ Notch等重要通路被甲基化修饰所激活。本项目将会继续深入研究这几条通路被异常甲基化激活的机制及其在调控鼻咽癌去分化方面的功能。本项目的研究结果将为鼻咽癌诱导分化治疗提供理论依据及潜在干预靶点。

诱导分化治疗在急性早幼粒细胞白血病中早已被证实是一种行之有效的肿瘤治疗方案，但是在实体肿瘤中尚未取得重要进展。未分化是我国鼻咽癌在临床上的一个最显著的特征，华南地区97%的鼻咽癌属于未分化的鼻咽癌。未分化的鼻咽癌容易发生颈部淋巴结转移。鼻咽癌的这一未分化的重要特征提示诱导分化治疗可能是治疗鼻咽癌的一个重要策略。阐明鼻咽癌未分化特征获得的分子机制是一个亟待解决的科学问题，对发现鼻咽癌诱导分化治疗靶点至关重要。.本课题从鼻咽癌去分化特征入手，建立鼻咽癌去分化研究模型，着眼于表观遗传相关信号通路的调控机理，借助高通量测序平台及生物信息分析手段，重点研究EB病毒潜伏膜蛋白LMP1调控鼻咽癌去分化的作用及分子机制，明确LMP1 能够诱导鼻咽癌去分化促进鼻咽癌细胞生长、转移、侵袭以及裸鼠成瘤；发现ATOH8是LMP1下游的关键靶蛋白；证实LMP1通过改变ATOH8启动子上H3K4me3和H3K27me3甲基化表观遗传修饰抑制ATOH8的表达，诱导鼻咽癌去分化；发现在去分化鼻咽癌中表观遗传调控因子组蛋白去甲基化酶KDM5A和组蛋白甲基转移酶EZH2在差异表达基因的启动子中显著富集。研究结果提示表观遗传网络的调控是鼻咽癌去分化的关键机制和干预靶点，为鼻咽癌的分化诱导治疗提供理论基础和指导方向。.本项目按照计划书规定任务积极开展研究工作，已经按时完成了计划书中的研究内容并达到预期目标。课题执行期间在IF>5的国际杂志上共发表SCI论文4篇；申请专利1项。