抗LMP1抗体抑制鼻咽癌肿瘤干细胞的作用及机制研究

Advanced-stage NPC might have chemotherapy resistance and radio-resistance. The prognosis remains poor in a significant number of NPC patients with relapse or metastatic diseases. Most cancers are believed to be initiated from and maintained by a small population of cancer stem cells (CSC) that are responsible for tumor relapse and chemotherapeutic resistance. In addition, CSC undergoing metastasis often expresses epithelial-to-mesenchymal transition (EMT). The interaction between stemness characteristics and EMT expression has shown that EMT acquires CSC properties, increases cancer cell tumorigenicity and shows a crucial link to metastasis. EBV latent membrane protein 1 (latent membrane protein 1, LMP1) is one of the most important oncogene protein, which is associated with human malignancies, especially nasopharyngeal carcinoma (NPC), promotes tumor cell invasion and metastasis. Meanwhile, LMP1 induce the cell proliferation associated factor, raise the anti-apoptosis gene, and up-regulate tumor invasion and metastasis related gene expression. Our previous studies have shown that the human anti-LMP1 antibody significantly inhibited proliferation of LMP1 + NPC cells (HNE2/LMP1) and nasopharyngeal carcinoma xenograft tumor growth in Balb / c mice. The antigen LMP1 is an ideal target for the treatment of nasopharyngeal carcinoma. On the basis of the preliminary studies, in this study, we enriched CSC by the sphere formation, and provided an efficient model for further experiments. Using this method, we examined the effects of anti LMP1 Fab antibody (htesFab) regulating the nasopharyngeal carcinoma (NPC) CSC and attempted to elucidate the possible mechanisms. The study will elucidate the molecular mechanism of Fab targeted inhibit LMP1 biological activity.

晚期的鼻咽癌患者存在较强的化疗耐药性及放疗抵抗，肿瘤干细胞自我更新、致瘤和上皮基质转化特性决定了恶性肿瘤无限制的生长、复发和转移的表型特征。较好的诠释了恶性肿瘤对治疗抵抗、治疗后复发和远处转移的生物学特性。因此，肿瘤干细胞可能成为鼻咽癌靶向治疗的新突破口。LMP1能调控细胞增殖相关因子，抗细胞调亡基因，及肿瘤浸润转移相关基因的表达，从而导致鼻咽癌细胞增殖活跃。我们前期研究表明，人源抗LMP1抗体封闭LMP1胞内效应区，明显抑制鼻咽癌细胞的增殖生长。抗原LMP1可能是抑制鼻咽癌肿瘤干细胞的理想标靶。在前期研究的基础上，本研究拟利用在前期抗LMP1的Fab片段的基础上，进行抗体的亲和力成熟，并构建Fab抗体的真核表达系统，分别在细胞和个体水平上，分析该抗体对鼻咽癌干细胞及其移植瘤的生长及转移的抑制作用，探讨抗体抑制鼻咽癌干细胞细胞生长的分子机制。

1.项目背景.鼻咽癌是我国南方和东南亚地区常见的恶性肿瘤之一,复发率高。虽然放化疗联合治疗已取得一定进展，但鼻咽癌的5年生存率不理想，复发率高达82%。这些患者缺乏有效的治疗方法，且预后不佳，越来越多的证据表明肿瘤干细胞在鼻咽癌的发生、维持、再生和复发中起重要作用。LMP1是唯一参与鼻咽癌细胞分化、转化和恶性肿瘤调控的潜伏蛋白。LMP1和抗LMP1-Fab抗体在鼻咽癌发病机制中的致癌信号转导特性和生物学功能尚未完全阐明。.2.主要研究内容.本研究观察抗LMP1抗体抑制鼻咽癌肿瘤干细胞的体内外的作用，初步探讨其中的信号转导的分子机制研究。.3.重要结果及关键数据.抗 LMP1全人源基因工程抗体真核表达系统的构建及抗体的大量表达纯化。首次证实鼻咽癌细胞系HNE2细胞球具有较强的类癌干细胞特性，可作为鼻咽癌干细胞的良好模型。通过微球形成、CCK-8、集落形成、Transwell及划痕实验试验证实LMP1在维持鼻咽癌的肿瘤干性中起重要作用。此外，通过CCK-8、TUNEL、qRT-PCR和Western blot观察到LMP1-Fab抗体调节PI3K/AKT信号通路抑制鼻咽癌干细胞的增殖和诱导细胞凋亡。在体内实验中LMP1-Fab抗体通过调节PI3K途径抑制鼻咽癌裸鼠移植瘤的生长。通过Transwell及划痕实验证实LMP1-Fab抗体抑制鼻咽癌细胞在LMP1+HNE2细胞中的迁移、侵袭和上皮间质转变。此外，LMP1-Fab抗体通过Western blot和qRT-PCR法增加E-cadherin上皮标志物的表达，降低N-cadherin、Vimentin和EMT相关转录因子Slug在LMP1阳性HNE2肿瘤干细胞中的表达。同时，通过体内成像观察到LMP1-Fab抗体通过调节wnt/β-catenin途径抑制裸鼠肺转移和肝转移。.4.科学意义.LMP1维持鼻咽癌干细胞特性方面的作用。LMP1-Fab抗体在鼻咽癌特别是复发性鼻咽癌的治疗中提供新思路。