抑癌基因EYA2及其突变体在肝癌发生中的作用和分子机制

Inactivation of tumor suppressor genes is one of the important mechanisms of tumorigenesis. Eyes absent (EYA) is a transcription activation factor with tyrosine phosphatase activity as well. In the process of the development, EYA2 plays roles in DNA damage, apoptosis and so on. Previously we found a novel mutation (A510E) of EYA2 using whole exome sequencing of HCC. Transcriptional and translational analysis showed that EYA2 is down-regulated in human HCC tissues compared with adjacent non-tumor HCC tissues. Combining with functional and animal experiments, we found EYA2 negatively regulates the malignant phenotype of HCC, suggesting that EYA2 serves as a tumor suppressor. However, the mutant EYA2 (A510E) loses the tumor suppressor function. Meanwhile, the protein of mutant EYA2 (A510E) is detected degradation. Based on these research findings, we will investigate the molecular mechanisms of tumor suppressor gene EYA2 and its mutant in the hepatic carcinogenesis, and the gene expression regulation system using hepatocyte-specific EYA2-knockout mice, systems medical biology and molecular cell biology analysis strategies. We try to explain the down-regulation mechanisms (DNA-methylation modification and protein degradation) of EYA2 and its mutant in HCC progress. Our study will clarify a new tumor suppression gene, riching the gene mutation genetic and epigenetic mechanisms in HCC occurrence, which may provide new target and novel ideas for targeted therapy of liver cancer.

抑癌基因失活是肿瘤发生的重要机制之一。EYA（eyes absent）是一个转录共激活因子，同时具有酪氨酸磷酸酶活性，在机体发育过程中参与DNA损伤修复、细胞凋亡等。我们前期通过HBV相关肝癌样本外显子组测序分析，发现了EYA2的一个新的突变位点A510E；通过转录和蛋白水平检测证实，EYA2在人肝癌组织较癌旁组织呈低表达；功能研究和动物试验表明，EYA2负性调节肝癌恶性表型，即具有抑癌功能；而其突变体A510E的抑癌功能丢失，且突变体蛋白发生降解。本课题拟在此基础上，运用肝细胞特异性敲除EYA2基因小鼠模型、系统医学生物学分析和分子细胞生物学等手段，深入研究EYA2作为新的抑癌基因、负性调控肝癌发生的作用和分子机制，阐明EYA2及其突变体在肝癌中下调表达的甲基化修饰和蛋白降解机制。本课题的实施将丰富肝癌形成过程中的基因突变遗传学机制和表观遗传学机制，为肝癌靶向治疗提供新靶标、新思路。

在肝细胞癌（HCC）进展过程中，机体细胞中的多基因体细胞突变或表观遗传学修饰改变的共同累积是诱发肿瘤的重要分子驱动，同时也是肝癌异质性差异的主要原因，其关键在于抑癌基因的突变失活或其启动子区的高甲基化修饰，以及原癌基因突变后的过度激活或启动子区的低甲基化修饰。我们通过HCC的全外显子组测序数据，筛选出肝癌新颖的体细胞突变EYA2(A510E)。位点功能性预测发现，A510E突变在多物种中高度保守，且具有致病性。通过转录和蛋白水平检测证实，与癌旁组织相比，EYA2在人肝癌组织呈低表达。临床资料相关性分析发现，EYA2表达与肝癌临床进展呈负相关性，并可作为肝癌潜在的一个独立预后标志物。功能实验发现，EYA2能在抑制肝癌细胞的侵袭、迁移、增殖和克隆形成能力和体内裸鼠皮下成瘤能力。我们构建了人肝癌裸鼠原位移植模型，通过尾静脉注射2×107TU的EYA2慢病毒，发现其对肝原位肿瘤有良好的抑制作用。甲基化质谱发现，EYA2第一个内含子区的高甲基化抑制了EYA2基因的表达。EYA2(A510E)突变激活未折叠蛋白反应，从而依赖于溶酶体降解途径或非泛素化的蛋白酶体降解途径而被降解。机制上，EYA2能结合DACH1，通过上调SOCS3基因的转录，抑制JAK/STAT信号通路，从而抑制肝癌的进展。.结论：我们鉴定并验证了EYA2在HCC中是一个抑癌基因，为肝癌的发病机制提供了新的见解。