Somatic mutations are involved in hepatocellular carcinoma (HCC) progression, but the genetic mechanism associated to hepatocarcinogenesis remains poorly understood. We report that Eyes absent homolog 2 (EYA2) suppresses the HCC progression, while EYA2(A510E) mutation identified by exome sequencing attenuates the tumor-inhibiting effect of EYA2. Whole-exome sequencing was performed on six pairs of human HCC primary tumors and matched adjacent tissues. Focusing on EYA2, expression level of EYA2 in human HCC samples was evaluated by quantitative real-time PCR, western blot and immunohistochemistry. Loss- and gain-of-function studies, hepatocyte-specific deletion of EYA2 (Eya2−/−) in mice and RNA sequencing analysis were used to explore the functional effect and mechanism of EYA2 on HCC cell growth and metastasis. EYA2 methylation status was evaluated using Sequenom MassARRAY and publicly available data analysis. A new somatic mutation p.Ala510Glu of EYA2 was identified in HCC tissues. The expression of EYA2 was down-regulated in HCC and associated with tumor size (P = 0.001), Barcelona Clinic Liver Cancer stage (P = 0.016) and tumor differentiation (P = 0.048). High level of EYA2 was correlated with a favorable prognosis in HCC patients (P = 0.003). Results from loss-of-function and gain-of-function experiments suggested that knockdown of EYA2 enhanced, while overexpression of EYA2 attenuated, the proliferation, clone formation, invasion, and migration of HCC cells in vitro. Delivery of EYA2 gene had a therapeutic effect on inhibition of orthotopic liver tumor in nude mice. However, EYA2(A510E) mutation led to protein degradation by unfolded protein response, thus weakening the inhibitory function of EYA2. Hepatocyte-specific deletion of EYA2 in mice dramatically promoted diethylnitrosamine-induced HCC development. EYA2 was also down-regulated in HCC by aberrant CpG methylation. Mechanically, EYA2 combined with DACH1 to transcriptionally regulate SOCS3 expression, thus suppressing the progression of HCC via SOCS3-mediated blockade of the JAK/STAT signaling pathway. In our study, we identified and validated EYA2 as a tumor suppressor gene in HCC, providing a new insight into HCC pathogenesis.

中文翻译：

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EYA2 通过 SOCS3 介导的 JAK/STAT 信号传导阻断来抑制肝细胞癌的进展

体细胞突变参与肝细胞癌（HCC）的进展，但与肝癌发生相关的遗传机制仍知之甚少。我们报告说，Eyes 缺失同源物 2 (EYA2) 抑制 HCC 进展，而外显子组测序鉴定的 EYA2(A510E) 突变减弱了 EYA2 的肿瘤抑制作用。对六对人类 HCC 原发肿瘤和匹配的邻近组织进行全外显子组测序。针对EYA2，通过实时定量PCR、蛋白质印迹和免疫组织化学评估了人HCC样本中EYA2的表达水平。采用功能丧失和获得研究、小鼠肝细胞特异性删除 EYA2 (Eya2−/−) 以及 RNA 测序分析来探讨 EYA2 对 HCC 细胞生长和转移的功能影响和机制。使用 Sequenom MassARRAY 和公开数据分析评估 EYA2 甲基化状态。在 HCC 组织中鉴定出 EYA2 的新体细胞突变 p.Ala510Glu。EYA2 的表达在 HCC 中下调，并与肿瘤大小 (P = 0.001)、巴塞罗那临床肝癌分期 (P = 0.016) 和肿瘤分化 (P = 0.048) 相关。高水平的 EYA2 与 HCC 患者的良好预后相关（P = 0.003）。功能丧失和功能获得实验的结果表明，EYA2 的敲低增强了体外 HCC 细胞的增殖、克隆形成、侵袭和迁移，而 EYA2 的过表达则减弱。EYA2基因的递送对裸鼠原位肝肿瘤的抑制具有治疗作用。然而，EYA2(A510E)突变导致未折叠蛋白反应导致蛋白质降解，从而削弱了EYA2的抑制功能。小鼠肝细胞特异性删除 EYA2 显着促进二乙基亚硝胺诱导的 HCC 发展。EYA2 在 HCC 中也因异常 CpG 甲基化而下调。从机制上讲，EYA2 与 DACH1 结合，转录调节 SOCS3 的表达，从而通过 SOCS3 介导的 JAK/STAT 信号通路阻断来抑制 HCC 的进展。在我们的研究中，我们鉴定并验证了 EYA2 作为 HCC 的抑癌基因，为 HCC 发病机制提供了新的见解。