Tang C, Hou Y, Wang H, Wang K, Xiang H, Wan X, Xia Y, Li J, Wei W, Xu S, Lei Z, Pawlik T M, Wang H, Wu M, Shen F
Department of Hepatic Surgery, The Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Department of Hepatobiliary Surgery, The Daping Hospital, Third Military Medical University, Chongqing, China.
Oncogenesis. 2017 Jul 17;6(7):e362. doi: 10.1038/oncsis.2017.64.
The mechanism of aberrant mitochondrial genome and function in hepatocellular carcinoma (HCC) remains largely unknown. Our previous study demonstrated an increased expression of aspartate β-hydroxylase (ASPH) in HCC tissues, which was associated with tumor invasiveness and a worse prognosis. Currently, we unexpectedly observed the presence of ASPH in purified mitochondrial protein fraction. In addition, immunostaining of both exogenously and endogenously expressed ASPH showed a colocalization with mitochondrial biomarkers. This study aimed to investigate whether the mitochondrial ASPH is involved in mitochondrial malfunction in HCC. Our results showed that ASPH overexpression in HCC tissues was correlated with decreased copy numbers of displacement loop (D-loop) and NADH dehydrogenase subunit 1 (ND-1) and enhanced D-loop mutation, suggesting the disrupted mitochondrial DNA (mtDNA) stability. The reduced mtDNA copy numbers were associated with aggressive clinicopathological features of HCC. The loss of mtDNA integrity induced by enforced expression of ASPH was accompanied with mitochondrial dysfunction, which was characterized by the aberrant mitochondrial membrane potential, decreased ATP generation and enhanced reactive oxygen species. In contrast, knocking down ASPH by siRNA in HCC cell lines showed the opposite impact on mtDNA integrity and function. Mass spectrometry and co-immunoprecipitation further identified that ASPH interacted with histone H2A member X (H2AX). ASPH overexpression diminished the interaction between H2AX and mitochondrial transcription factor A (mtTFA), an important DNA-binding protein for mtDNA replication, which then reduced the binding of mtTFA to D-loop region. Collectively, our results demonstrate that ASPH overexpression disrupts the mtDNA integrity through H2AX-mtTFA signal, thereby affecting mitochondrial functions in HCC.
肝细胞癌（HCC）中线粒体基因组和功能异常的机制在很大程度上仍不清楚。我们之前的研究表明，天冬氨酸β-羟化酶（ASPH）在HCC组织中表达增加，这与肿瘤侵袭性和较差的预后相关。目前，我们意外地在纯化的线粒体蛋白组分中发现了ASPH的存在。此外，对外源和内源表达的ASPH进行免疫染色显示其与线粒体生物标志物共定位。本研究旨在探讨线粒体ASPH是否参与HCC中的线粒体功能障碍。我们的结果表明，HCC组织中ASPH的过表达与置换环（D-loop）和NADH脱氢酶亚基1（ND-1）的拷贝数减少以及D-loop突变增强相关，提示线粒体DNA（mtDNA）稳定性受到破坏。mtDNA拷贝数减少与HCC的侵袭性临床病理特征相关。ASPH强制表达诱导的mtDNA完整性丧失伴随着线粒体功能障碍，其特征为线粒体膜电位异常、ATP生成减少和活性氧增加。相反，在HCC细胞系中通过siRNA敲低ASPH对mtDNA完整性和功能产生相反的影响。质谱分析和免疫共沉淀进一步鉴定出ASPH与组蛋白H2A成员X（H2AX）相互作用。ASPH过表达减少了H2AX与线粒体转录因子A（mtTFA）之间的相互作用，mtTFA是mtDNA复制的重要DNA结合蛋白，进而减少了mtTFA与D-loop区域的结合。总体而言，我们的结果表明，ASPH过表达通过H2AX-mtTFA信号破坏mtDNA完整性，从而影响HCC中的线粒体功能。
