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Open Full Page Published OnlineFirst January 27, 2016; DOI: 10.1158/1078-0432.CCR-15-1987 Biology of Human Tumors Clinical Cancer Research NDUFA4L2 Fine-tunes Oxidative Stress in Hepatocellular Carcinoma Robin Kit-Ho Lai1, Iris Ming-Jing Xu1, David Kung-Chun Chiu1, Aki Pui-Wah Tse1, Larry Lai Wei1, Cheuk-Ting Law1, Derek Lee1, Chun-Ming Wong1,2, Maria Pik Wong1, Irene Oi-Lin Ng1,2, and Carmen Chak-Lui Wong1,2 Abstract Purpose: Hepatocellular carcinoma (HCC) lacks effective cura- Results: NDUFA4L2 was drastically overexpressed in human tive therapy. Hypoxia is commonly found in HCC. Hypoxia elicits HCC and induced by hypoxia. NDUFA4L2 overexpression was a series of protumorigenic responses through hypoxia-inducible closely associated with tumor microsatellite formation, absence factor-1 (HIF1). Better understanding of the metabolic adapta- of tumor encapsulation, and poor overall survival in HCC tions of HCC cells during hypoxia is essential to the design of new patients. We confirmed that NDUFA4L2 was HIF1-regulated in therapeutic regimen. HCC cells. Inactivation of HIF1/NDUFA4L2 increased mitochon- Experimental Design: Expressions of genes involved in the drial activity and oxygen consumption, resulting in ROS accu- electron transport chain (ETC) in HCC cell lines (20% and 1% O2) mulation and apoptosis. Knockdown of NDUFA4L2 markedly and human HCC samples were analyzed by transcriptome suppressed HCC growth and metastasis in vivo. HIF inhibitor, sequencing. Expression of NDUFA4L2, a less active subunit in digoxin, significantly suppressed growth of tumors that expressed complex I of the ETC, in 100 pairs of HCC and nontumorous liver high level of NDUFA4L2. tissues were analyzed by qRT-PCR. Student t test and Kaplan– Conclusions: Our study has provided the first clinical relevance Meier analyses were used for clinicopathologic correlation and of NDUFA4L2 in human cancer and suggested that HCC patients survival studies. Orthotopic HCC implantation model was used with NDUFA4L2 overexpression may be suitable candidates for to evaluate the efficiency of HIF inhibitor. HIF inhibitor treatment. Clin Cancer Res; 22(12); 3105–17. Ó2016 AACR. Introduction accompanied by the loss of normal metabolic functions in the liver and the acquisition of new metabolic functions which Hepatocellular carcinoma (HCC), a malignancy derived favor cancer growth. Exploration of the molecular contexts from hepatocytes, accounts for 90% of primary liver cancer. associated with these metabolic changes will help to identify It is the fifthmostcommoncancerandthethirdleadingcause novel targets for HCC treatment. of cancer deaths in the world (1). Majority of deaths in HCC are Hypoxia, or oxygen (O ) deprivation, is frequently found in attributed to its asymptomatic nature that delays diagnosis and 2 regions of HCC that are distant from functional vasculature. treatment. Most HCC patients are not suitable for the only Palliative therapies, such as transcatheter arterial (chemo) promising curative therapies, surgical resection, and liver trans- embolization (TAE/TACE) and hepatic artery ligation, that plantation. Sorafenib, an oral multikinase inhibitor and the involve restriction of blood supply to the tumors adversely only FDA-approved drug for advanced HCC patients, could induce hypoxia (4). To overcome the shortage of O , cells adapt modestly prolong the survival of patients for 3 months (2, 3). 2 to hypoxia through HIFs which are composed of the constitu- Liver is an organ responsible for many important metabolic tively expressed HIF1b subunit and the oxygen labile subunit functionsinthebody,suchastheCori-cycle,glycogenmetab- HIF1/2a (5). In the presence of O ,HIF1/2a is hydroxylated by olism, and blood glucose homeostasis. Hepatocarcinogenesis is 2 prolyl hydroxylases (6), facilitating binding of von Hippel– Lindau (VHL), which polyubiquitinates HIF1/2a for proteaso- mal degradation (7). In the absence of O2, stabilized HIF1/2a 1Department of Pathology, The University of Hong Kong, Hong Kong. dimerizes with HIF1b to initiate transcription of genes related 2State Key Laboratory for Liver Research, The University of Hong to hypoxia adaptive responses that advantage cancer develop- Kong, Hong Kong. ment (8). Although it is known that upregulation of HIF1 is Note: Supplementary data for this article are available at Clinical Cancer closely associated with poor clinical outcome in HCC patients Research Online (http://clincancerres.aacrjournals.org/). (4), the detailed molecular mechanisms by which HIF1 pro- Corresponding Authors: Carmen Chak-Lui Wong, The University of Hong Kong, motes HCC progression remain poorly understood. L-704 Laboratory Block, LKS Faculty of Medicine, 21 Sassoon Road, Pokfulam, HIF1a transcriptionally activates many metabolic genes, Hong Kong, Hong Kong. Phone: 852-39179014; Fax: 852-28195375l; E-mail: allowing the cells to adapt to hypoxia, by shunting the glucose [email protected]; Irene Oi-Lin Ng, [email protected] intermediates into glycolysis instead of the tricarboxylic acid doi: 10.1158/1078-0432.CCR-15-1987 (TCA) cycle (9). These metabolic genes include glucose trans- Ó2016 American Association for Cancer Research. porter (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase A www.aacrjournals.org 3105 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 27, 2016; DOI: 10.1158/1078-0432.CCR-15-1987 Lai et al. alpha subcomplex 4–like 2 (NDUFA4L2), to reduce complex I Translational Relevance activity and ROS production (23). Hepatocellular carcinoma (HCC) frequently experiences Currently, knowledge about NDUFA4L2, particularly on its hypoxia. Hypoxia results in an inefficient transfer of electrons roles in cancer development, is scarce. NDUFA4L2 was found to during oxidative phosphorylation leading to increased oxida- be one of the seven biomarkers that distinguish medullary tive stress. In this study, we demonstrated that a less active thyroid carcinoma from head and neck paraganglioma (PGL; complex I subunit in the electron transport chain, NDUFA4L2, ref. 24). Earlier study showed that NDUFA4L2 is overexpressed was significantly overexpressed in HCC and other human in PGL tumors that lack VHL (25). Nonetheless, its clinical cancer types. Overexpression of NDUFA4L2 was associated implications and in vivo roles in cancers, particularly in HCC, with aggressive clinical features in human HCC and shorter have never been thoroughly studied. This study uncovers the overall survival in HCC patients. A series of in vitro and in vivo clinical relevance and roles of NDUFA4L2 in REDOX homeo- assays converged to show that NDUFA4L2 reduced ROS- stasis in HCC. Furthermore, we have successfully demonstrated mediated apoptosis to confer HCC cells growth advantages. thattargetingHIF1/NDUFA4L2pathwaybyHIFinhibitor As NDUFA4L2 is a direct transcriptional target of HIF, we represents a novel therapeutic strategy for HCC. found that HIF inhibitor, digoxin, profoundly inhibited growth of tumors that expressed high level of NDUFA4L2 in orthotopic model. Our findings suggested that cancer patients Materials and Methods with NDUFA4L2 overexpression may be suitable candidates Patient samples for HIF inhibitor treatment. Human HCC and the corresponding paired nontumorous liver tissues were collected at Queen Mary Hospital, the University of Hong Kong during surgical resection. Human lung squamous cell carcinoma (SCC) and the corresponding paired nontumorous lung tissues samples were kindly provided by Dr. Maria P. Wong (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1). GLUT1 (the University of Hong Kong). Use of human samples was facilitates glucose uptake (10). HK2 catalyzes the phosphory- approved by the Institutional Review Board of the University of lation of glucose to glucose-6-phosphate, the first step of glycol- Hong Kong/Hospital Authority Hong Kong West Cluster. ysis (11, 12). LDHA converts pyruvate to lactate (13, 14). PDK1 inactivates pyruvate dehydrogenase to prevent pyruvate conver- Cell lines sion to acetyl CoA. These processes restrict the entrance of glucose Human HCC cell line, PLC/PRF/5, and human cervical cancer intermediates into the TCA cycle, thereby reducing the activity of cell line, HeLa, were obtained from the American Type Culture oxidative phosphorylation (OXPHOS) in the mitochondria (9). Collect (ATCC) and cultured according to ATCC recommenda- Interestingly, all these metabolic genes are involved in cancer tions. Metastatic human HCC cell line, MHCC97L, was a gift from progression. Dr. Z.Y. Tang (Fudan University of Shanghai). All the cell lines The OXPHOS system comprises four electron transport chain used were authenticated by the AuthentiFiler PCR Amplification (ETC) complexes, including complex I (NADH–ubiquinone oxi- Kit (Applied Biosystems) on September 1st, 2014. All cell lines doreductase), complex II (succinate:ubiquinone oxidoreductase), used in this article were thawed from the authenticated cell stock complex III (ubiquinol–cytochrome c reductase), and complex IV and used within four passages. (cytochrome c oxidase). Most ATP in the cells is produced when electrons transfer through these complexes to the ultimate elec- Clinicopathologic correlation and patients' survival analysis tron acceptor, O2. Complex I, a 1 MDa complex of 45 subunits, is the first step where OXPHOS takes place. Complex I catalyzes the The clinicopathologic features of HCC patients were ana- transfer of electrons from NADH to flavoprotein through eight lyzed by pathologist
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