Abstract
Purpose: Hepatocellular carcinoma (HCC) lacks effective curative therapy. Hypoxia is commonly found in HCC. Hypoxia elicits a series of protumorigenic responses through hypoxia-inducible factor-1 (HIF1). Better understanding of the metabolic adaptations of HCC cells during hypoxia is essential to the design of new therapeutic regimen.
Experimental Design: Expressions of genes involved in the electron transport chain (ETC) in HCC cell lines (20% and 1% O2) and human HCC samples were analyzed by transcriptome sequencing. Expression of NDUFA4L2, a less active subunit in complex I of the ETC, in 100 pairs of HCC and nontumorous liver tissues were analyzed by qRT-PCR. Student t test and Kaplan–Meier analyses were used for clinicopathologic correlation and survival studies. Orthotopic HCC implantation model was used to evaluate the efficiency of HIF inhibitor.
Results: NDUFA4L2 was drastically overexpressed in human HCC and induced by hypoxia. NDUFA4L2 overexpression was closely associated with tumor microsatellite formation, absence of tumor encapsulation, and poor overall survival in HCC patients. We confirmed that NDUFA4L2 was HIF1-regulated in HCC cells. Inactivation of HIF1/NDUFA4L2 increased mitochondrial activity and oxygen consumption, resulting in ROS accumulation and apoptosis. Knockdown of NDUFA4L2 markedly suppressed HCC growth and metastasis in vivo. HIF inhibitor, digoxin, significantly suppressed growth of tumors that expressed high level of NDUFA4L2.
Conclusions: Our study has provided the first clinical relevance of NDUFA4L2 in human cancer and suggested that HCC patients with NDUFA4L2 overexpression may be suitable candidates for HIF inhibitor treatment. Clin Cancer Res; 22(12); 3105–17. ©2016 AACR.
Hepatocellular carcinoma (HCC) frequently experiences hypoxia. Hypoxia results in an inefficient transfer of electrons during oxidative phosphorylation leading to increased oxidative stress. In this study, we demonstrated that a less active complex I subunit in the electron transport chain, NDUFA4L2, was significantly overexpressed in HCC and other human cancer types. Overexpression of NDUFA4L2 was associated with aggressive clinical features in human HCC and shorter overall survival in HCC patients. A series of in vitro and in vivo assays converged to show that NDUFA4L2 reduced ROS-mediated apoptosis to confer HCC cells growth advantages. As NDUFA4L2 is a direct transcriptional target of HIF, we 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 with NDUFA4L2 overexpression may be suitable candidates for HIF inhibitor treatment.
Introduction
Hepatocellular carcinoma (HCC), a malignancy derived from hepatocytes, accounts for 90% of primary liver cancer. It is the fifth most common cancer and the third leading cause of cancer deaths in the world (1). Majority of deaths in HCC are attributed to its asymptomatic nature that delays diagnosis and treatment. Most HCC patients are not suitable for the only promising curative therapies, surgical resection, and liver transplantation. Sorafenib, an oral multikinase inhibitor and the only FDA-approved drug for advanced HCC patients, could modestly prolong the survival of patients for 3 months (2, 3). Liver is an organ responsible for many important metabolic functions in the body, such as the Cori-cycle, glycogen metabolism, and blood glucose homeostasis. Hepatocarcinogenesis is accompanied by the loss of normal metabolic functions in the liver and the acquisition of new metabolic functions which favor cancer growth. Exploration of the molecular contexts associated with these metabolic changes will help to identify novel targets for HCC treatment.
Hypoxia, or oxygen (O2) deprivation, is frequently found in regions of HCC that are distant from functional vasculature. Palliative therapies, such as transcatheter arterial (chemo)embolization (TAE/TACE) and hepatic artery ligation, that involve restriction of blood supply to the tumors adversely induce hypoxia (4). To overcome the shortage of O2, cells adapt to hypoxia through HIFs which are composed of the constitutively expressed HIF1β subunit and the oxygen labile subunit HIF1/2α (5). In the presence of O2, HIF1/2α is hydroxylated by prolyl hydroxylases (6), facilitating binding of von Hippel–Lindau (VHL), which polyubiquitinates HIF1/2α for proteasomal degradation (7). In the absence of O2, stabilized HIF1/2α dimerizes with HIF1β to initiate transcription of genes related to hypoxia adaptive responses that advantage cancer development (8). Although it is known that upregulation of HIF1 is closely associated with poor clinical outcome in HCC patients (4), the detailed molecular mechanisms by which HIF1 promotes HCC progression remain poorly understood.
HIF1α transcriptionally activates many metabolic genes, allowing the cells to adapt to hypoxia, by shunting the glucose intermediates into glycolysis instead of the tricarboxylic acid (TCA) cycle (9). These metabolic genes include glucose transporter (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1). GLUT1 facilitates glucose uptake (10). HK2 catalyzes the phosphorylation of glucose to glucose-6-phosphate, the first step of glycolysis (11, 12). LDHA converts pyruvate to lactate (13, 14). PDK1 inactivates pyruvate dehydrogenase to prevent pyruvate conversion to acetyl CoA. These processes restrict the entrance of glucose intermediates into the TCA cycle, thereby reducing the activity of oxidative phosphorylation (OXPHOS) in the mitochondria (9). Interestingly, all these metabolic genes are involved in cancer progression.
The OXPHOS system comprises four electron transport chain (ETC) complexes, including complex I (NADH–ubiquinone oxidoreductase), complex II (succinate:ubiquinone oxidoreductase), complex III (ubiquinol–cytochrome c reductase), and complex IV (cytochrome c oxidase). Most ATP in the cells is produced when electrons transfer through these complexes to the ultimate electron acceptor, O2. Complex I, a 1 MDa complex of 45 subunits, is the first step where OXPHOS takes place. Complex I catalyzes the transfer of electrons from NADH to flavoprotein through eight iron–sulfur clusters, and finally to ubiquinone. Complex I is a major ROS-producing site. Low level of ROS has been shown to activate signaling pathways, such as MAPK-ERK, JNK, p38 MAPK, and FAK, to promote cancer cell survival and metastasis (15), whereas excessive ROS accumulation suppresses cancer cell growth through induction of G2–M cell-cycle arrest and apoptosis (16–18). Most of the chemotherapeutic and radiotherapeutic strategies against cancer cells are mediated through ROS induction (19, 20).
HIF1α regulates several ETC components to minimize ROS production and optimize mitochondrial respiration. HIF1α induces the switching of COX subunit 4 isoforms from COX4-1 to COX4-2 in complex IV to maximize the efficiency of OXPHOS under hypoxia (21). HIF1α induces expression of a miRNA, miR-210, which suppresses iron–sulfur cluster assembly proteins (ISCU1/2) in complexes I and III to reduce oxygen consumption, ROS production, and apoptosis during hypoxic stress (22). Mainly demonstrated in mouse embryonic fibroblasts, HIF1α induced ETC complex I subunit, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4–like 2 (NDUFA4L2), to reduce complex I activity and ROS production (23).
Currently, knowledge about NDUFA4L2, particularly on its roles in cancer development, is scarce. NDUFA4L2 was found to be one of the seven biomarkers that distinguish medullary thyroid carcinoma from head and neck paraganglioma (PGL; ref. 24). Earlier study showed that NDUFA4L2 is overexpressed in PGL tumors that lack VHL (25). Nonetheless, its clinical implications and in vivo roles in cancers, particularly in HCC, have never been thoroughly studied. This study uncovers the clinical relevance and roles of NDUFA4L2 in REDOX homeostasis in HCC. Furthermore, we have successfully demonstrated that targeting HIF1/NDUFA4L2 pathway by HIF inhibitor represents a novel therapeutic strategy for HCC.
Materials and Methods
Patient samples
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 (the University of Hong Kong). Use of human samples was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster.
Cell lines
Human HCC cell line, PLC/PRF/5, and human cervical cancer cell line, HeLa, were obtained from the American Type Culture Collect (ATCC) and cultured according to ATCC recommendations. Metastatic human HCC cell line, MHCC97L, was a gift from Dr. Z.Y. Tang (Fudan University of Shanghai). All the cell lines used were authenticated by the AuthentiFiler PCR Amplification Kit (Applied Biosystems) on September 1st, 2014. All cell lines used in this article were thawed from the authenticated cell stock and used within four passages.
Clinicopathologic correlation and patients' survival analysis
The clinicopathologic features of HCC patients were analyzed by pathologist as we previously described (26). The parameters included age, gender, tumor size, cellular differentiation by Edmondson grading, direct liver invasion, absence of tumor encapsulation, presence of tumor microsatellite formation, venous invasion, and background liver disease. Overall survival was calculated from the date of surgical resection to the date of death or last follow-up. The prognostic significance of NDUFA4L2 overexpression was determined by the Kaplan–Meier method followed by the log-rank test, as we previously described (27, 28). All statistical tests were performed by SPSS20.0. The demographic data of HCC patients were summarized in Supplementary Table S1.
Fluorescence imaging and flow cytometry analysis
Cells were grown on glass coverslips in 6-well culture plate and cultured in 20% and 1% O2 for 24 hours. Cells were stained with 2 μmol/L 5,5′,6,6′-tetrachloro-1,1′,3,3′ -tetraethyl-benzimidazolylcarbocyanine chloride (JC-1; Invitrogen). Images were captured and scanned under 20× magnification with LSM 510 Meta laser scanning microscope (Carl Zeiss) connected to Axiocam microscope camera (Carl Zeiss). For flow cytometry analysis, cells were stained with 2 μmol/L JC-1.
Oxygen consumption assay
Cells were grown on 96-well culture plate and cultured in 20% and 1% O2 for 24 hours. Cells were stained with oxygen-sensitive probe MitoXpress-Intra (NanO2) and washed according to the manufacturer's instruction (Luxcel Bioscience). Phosphorescence was measured by a multilabel reader Victor2 (Perkin-Elmer Life Sciences) with 340 nm excitation and 642 nm emission filters.
ROS measurement
Trypsinized cells were washed with PBS and stained with 2 μmol/L general ROS indicator chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Life Technologies) or 5 μmol/L mitochondrial ROS indicator MitoSOX (Molecular Probes) for 5 and 10 minutes, respectively, and analyzed by flow cytometer FACSCanto II Analyzer (BD Biosciences). For MitoSOX staining, cells were washed with staining buffer 3 times before flow cytometry analysis. Data from flow cytometry studies were analyzed by FlowJo software. For antioxidant treatment, 0.5, 2, or 5 mmol/L of N-acetyl-L-cysteine (NAC) or L-ascorbic acid (Sigma Aldrich) were incubated at 37°C for 24 hours prior to ROS measurement. For in vivo ROS measurement, tumors harvested from mice were dissociated with gentleMACS dissociator, and single-cell suspensions were stained with 2 μmol/L CM-H2DCFDA for flow cytometry analysis.
Apoptosis and cell proliferation assays
For apoptosis measurement, 2.5 × 105 cells were cultured in 20% and 1% O2 for 24 hours. Apoptosis was determined by flow cytometry after staining with propidium iodide (Calbiochem) and Annexin V (MBL International Corporation). For antioxidant treatment, 0.1 mmol/L glutathione ethyl ester (GSH-EE; Cayman Chemical) was added to MHCC97L-shL2 cells and cultured in 1% O2 for 40 hours. For cell proliferation measured by cell counting, 2 × 104 MHCC97L-NTC and -shL2 cells were grown on 12-well culture plate and cultured in 20% and 1% O2 for 4 days. Cells were trypsinized and counted with Z1 coulter particle counter (Beckman Coulter) every 24 hours. For cell proliferation measured by 5-bromo-2′-deoxyuridine (BrdUrd) assay, 2.5 × 104 MHCC97L-NTC and -shL2 cells were grown on 96-well culture plate and cultured in 20% and 1% O2 for 24 hours. BrdUrd labeling and colorimetric measurement were performed according to the manufacturer's instructions (Roche Life Science).
Animal studies
All animal studies were approved by the Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong, and followed under the Animals (Control of Experiments) Ordinance of Hong Kong. For orthotopic liver tumor injection in nude mice, 1 × 106 luciferase-labeled MHCC97L cells were injected into the left lobes of the livers of 6- to 8-week-old BALB/c nude mice. Six weeks after injection, mice were administered with 100 mg/kg D-luciferin by peritoneal injection 5 minutes before bioluminescent imaging (IVIS 100 Imaging System; Xenogen). Livers and lungs were harvested for ex-vivo imaging and histologic analysis. Livers were harvested for ex-vivo imaging and histologic analysis as described above. For subcutaneous tumor model, 1 × 106 MHCC97L cells were injected subcutaneously to the flanks of nude mice. Tumor volume was calculated with the formula: length (mm) × width (mm) × depth (mm) × 0.52 (mm3). For pharmacologic studies, drug administration began a week after orthotopic or subcutaneous injection. Mice were administered with 1.2 mg/kg/day digoxin or vehicles (saline) by intraperitoneal injection for 21 consecutive days.
Transcriptome sequencing
Transcriptome sequencing was performed in 16 pairs of HCC tissues and corresponding NT liver tissues or MHCC97L cells exposed to 20% and 1% O2 for 24 hours. TruSeq standard mRNA sample Prep kit (Illumina) was used for polyA + mRNA library preparation. Illumina HiSeq2000 was employed for 100 bp paired-end sequencing (Axeq Technologies), and data were analyzed by TopHat-Cufflinks pipeline (29). Values were indicated by FPKM (fragments per kilobase transcript sequence per million mapped reads). Pathway analysis was performed by the Database for Annotation, Visualization and Integrated Discovery (DAVID).
Statistical analysis
Statistical analysis was performed by two-tailed Student t test or Wilcoxon's signed rank test using GraphPad Prism 5.0 (GraphPad Software Inc.). All functional assays are representative of ≥3 independent experiments and expressed as mean ± SEM. P value less than 0.05 was considered to be statistically significant.
Quantitative real-time PCR, establishment of knockdown and knockout HCC cells, in vitro assays, and TCGA/Oncomine data
The following are described in Supplementary Materials and Methods: quantitative real-time PCR (qRT-PCR), chromatin immunoprecipitation (ChIP) assay, establishment of HIF or NDUFA4L2 knockdown and HIF knockout HCC cells, mitochondrial membrane potential, mitochondrial mass, migration assay, histology, and The Cancer Genome Atlas (TCGA)/Oncomine data.
Results
Mitochondrial NDUFA4L2 is frequently overexpressed in human HCC and other human solid cancers
To better understand the hypoxia-adaptation system in HCC, we performed transcriptome sequencing to compare the gene expression profiles of a HCC cell line, MHCC97L, that were exposed to normoxia (20% O2) and hypoxia (0.1% and 1% O2). Among the upregulated genes, mitochondrial NDUFA4L2 was one of the five most abundant genes in hypoxic conditions, indicating the biologic relevance of this gene in human HCC. Other well-characterized HIF target genes, including ANPTGL4 (30), PLOD2 (31), VEGF (32), CA9 (33), and P4HA2 (34), were on the top 20 of the list. Intriguingly, when we interrogated the expression of genes that are involved in the mitochondrial complex I, only the subunit NDUFA4L2 was distinctly and dramatically upregulated in hypoxia (both 0.1% and 1% O2), whereas all the other subunits remained unaffected or downregulated (Fig. 1A). We further examined the transcriptome sequencing data on 16 primary HCCs and their corresponding nontumorous (NT) livers and found that NDUFA4L2 was substantially overexpressed in HCC tissues (Fig. 1A). Moreover, overexpression of NDUFA4L2 mRNA was validated by qRT-PCR in a separate, larger cohort of 100 pairs of HCC samples (Fig. 1B). Furthermore, overexpression of NDUFA4L2 in HCC tumors was significantly associated with aggressive pathologic features, including presence of tumor microsatellite formation (Student t test; P = 0.002) and absence of tumor encapsulation (Student t test; P = 0.039; Fig. 1C and Supplementary Table S4). More importantly, overexpression of NDUFA4L2 tended to be associated with poorer overall survival of HCC patients (Student t test; P = 0.086; Fig. 1C). TCGA database, which includes transcriptome sequencing data of 49 pairs of HCC and NT liver tissues from an independent cohort, echoed with our in-house data (Fig. 1D). Intriguingly, when we studied the microarray data available in the Oncomine database, we noticed that NDUFA4L2 was also overexpressed in other human solid cancers, including renal cell carcinoma (RCC), lung SCC, as well as colorectal carcinoma (Supplementary Fig. S1). The overexpression of NDUFA4L2 mRNA in lung SCC was further confirmed in our 31 pairs of lung SCC tissues and their normal counterparts by qRT-PCR (Fig. 1E). Collectively, these results demonstrated the clinical relevance of NDUFA4L2 in human HCC and other human solid cancers.
NDUFA4L2 is overexpressed in human HCC and other human solid cancers. A, transcriptome sequencing data comparing FPKM values of genes encoding mitochondrial complex I subunits in MHCC97L cells cultured in (left) 0.1% O2 or (middle) 1% O2 relative to 20% O2 and (right) 16 pairs of human HCC and the corresponding NT tissues. B, mRNA expression levels of NDUFA4L2 normalized with hypoxanthine-guanine phosphoribosyltransferase in an expanded cohort of 100 cases of paired human HCC, and corresponding NT tissues were determined by qRT-PCR. Waterfall plot shows that NDUFA4L2 is overexpressed in 71% of human HCC samples by at least 2-fold. C, clinicopathologic analysis shows that NDUFA4L2 overexpression is closely associated with more aggressive features in HCC, including the presence of tumor microsatellite formation and the absence of tumor encapsulation. HCC patients with high NDUFA4L2 expression (≧median) tend to have lower 5-year overall survival rate (Student t test; P = 0.086). D, mRNA expression levels of NDUFA4L2 obtained from TCGA database of 49 paired human HCC and corresponding NT tissues (RSEM, RNA-Seq by Expectation Maximization). E, mRNA expression levels of NDUFA4L2 normalized with β-actin in 31 cases of paired human lung SCC, and corresponding NT tissues were determined by qRT-PCR. Waterfall plot shows that NDUFA4L2 is overexpressed in 65% of human lung SCC samples by at least 2-fold. (Wilcoxon's signed rank test; ***, P < 0.001 for B, D, and E. Student t test; *, P < 0.05; **, P < 0.01 for C).
NDUFA4L2 is overexpressed in human HCC and other human solid cancers. A, transcriptome sequencing data comparing FPKM values of genes encoding mitochondrial complex I subunits in MHCC97L cells cultured in (left) 0.1% O2 or (middle) 1% O2 relative to 20% O2 and (right) 16 pairs of human HCC and the corresponding NT tissues. B, mRNA expression levels of NDUFA4L2 normalized with hypoxanthine-guanine phosphoribosyltransferase in an expanded cohort of 100 cases of paired human HCC, and corresponding NT tissues were determined by qRT-PCR. Waterfall plot shows that NDUFA4L2 is overexpressed in 71% of human HCC samples by at least 2-fold. C, clinicopathologic analysis shows that NDUFA4L2 overexpression is closely associated with more aggressive features in HCC, including the presence of tumor microsatellite formation and the absence of tumor encapsulation. HCC patients with high NDUFA4L2 expression (≧median) tend to have lower 5-year overall survival rate (Student t test; P = 0.086). D, mRNA expression levels of NDUFA4L2 obtained from TCGA database of 49 paired human HCC and corresponding NT tissues (RSEM, RNA-Seq by Expectation Maximization). E, mRNA expression levels of NDUFA4L2 normalized with β-actin in 31 cases of paired human lung SCC, and corresponding NT tissues were determined by qRT-PCR. Waterfall plot shows that NDUFA4L2 is overexpressed in 65% of human lung SCC samples by at least 2-fold. (Wilcoxon's signed rank test; ***, P < 0.001 for B, D, and E. Student t test; *, P < 0.05; **, P < 0.01 for C).
HIF1α, but not HIF2α, positively regulates NDUFA4L2 in hypoxia
To investigate whether NDUFA4L2 expression was mediated by HIFs under hypoxia, we generated HCC cells, MHCC97L, that stably expressed shRNA against HIF1α (-shHIF1α) or HIF2α (-shHIF2α; Supplementary Fig. S2A). The hypoxia-induced NDUFA4L2 expression was markedly abolished when HIF1α, but not HIF2α, was knocked down (Fig. 2A). We further established HIF1α knockout cells by Transcription Activator-like Effector Nuclease approach and found that hypoxia-induced NDUFA4L2 expression was drastically abrogated (Fig. 2B and Supplementary Fig. S2B). Digoxin inhibited HIF1 protein synthesis and profoundly inhibited hypoxia-induced NDUFA4L2 expression in a dose-dependent manner (Fig. 2C). In addition, by in silico analysis, we identified two potential hypoxia response elements (HRE) containing the core HIF-binding sequence motif 5′-RCGTG-3′ at the promoter of NDUFA4L2 (Fig. 2D). ChIP assay in cell lines of different origins, MHCC97L and HeLa cells, indicated that DNA was enriched when HIF1α antibody was used, as compared with the IgG control (Fig. 2E and Supplementary Fig. S3). Intriguingly, we consistently detected a band beneath the expected protein size of NDUFA4L2. This protein was only expressed in normoxia but not in hypoxia (Figs. 2A, B, and C, and 3A and B). NDUFA4L2 shared 67% amino acid sequence with its paralogue NDUFA4, which is 6 amino acids shorter than NDUFA4L2. To confirm the identity of the unknown protein beneath NDUFA4L2, we knocked down NDUFA4 (-shA4-30 and -shA4-88) in MHCC97L cells and confirmed that the lower band was NDUFA4 (Supplementary Fig. S4A and S4B). These results demonstrated that NDUFA4L2 was preferentially expressed under hypoxia solely through HIF1α, whereas NDUFA4 protein was preferentially expressed under normoxia.
NDUFA4L2 is regulated by HIF1α but not HIF2α in HCC. A, mRNA and protein expression of NDUFA4L2 in MHCC97L-EV, -shHIF1α, and -shHIF2α cells cultured in 20% and 1% O2 for 24 and 48 hours, respectively. B, NDUFA4L2 protein expression in MHCC97L-WT (parental) and -HIF1α−/− (KO) cells cultured in 20% and 1% O2 for 48 hours. C, NDUFA4L2 mRNA and protein expression in MHCC97L cells treated with HIF inhibitor digoxin at the indicated doses. D, putative HREs are mapped in the promoter of NDUFA4L2. Core HIF binding sequences (5′-RCGTG-3′) are highlighted as bold with underlines, while ancillary sequence is highlighted as bold. E, ChIP assay was performed with IgG and HIF1α antibodies in MHCC97L cells cultured in 20% and 1% O2. qRT-PCR was performed after immunoprecipitation with anti-IgG or anti-HIF1α antibodies, represented as fold enrichment normalized to 20% O2 (results represent mean ± SEM. Student t test; N = 3 independent experiments: *, P < 0.05; **, P < 0.01; ***, P < 0.001 as indicated).
NDUFA4L2 is regulated by HIF1α but not HIF2α in HCC. A, mRNA and protein expression of NDUFA4L2 in MHCC97L-EV, -shHIF1α, and -shHIF2α cells cultured in 20% and 1% O2 for 24 and 48 hours, respectively. B, NDUFA4L2 protein expression in MHCC97L-WT (parental) and -HIF1α−/− (KO) cells cultured in 20% and 1% O2 for 48 hours. C, NDUFA4L2 mRNA and protein expression in MHCC97L cells treated with HIF inhibitor digoxin at the indicated doses. D, putative HREs are mapped in the promoter of NDUFA4L2. Core HIF binding sequences (5′-RCGTG-3′) are highlighted as bold with underlines, while ancillary sequence is highlighted as bold. E, ChIP assay was performed with IgG and HIF1α antibodies in MHCC97L cells cultured in 20% and 1% O2. qRT-PCR was performed after immunoprecipitation with anti-IgG or anti-HIF1α antibodies, represented as fold enrichment normalized to 20% O2 (results represent mean ± SEM. Student t test; N = 3 independent experiments: *, P < 0.05; **, P < 0.01; ***, P < 0.001 as indicated).
NDUFA4L2 reduces mitochondrial activity, oxygen consumption, and ROS production in hypoxia. A, mRNA and protein expression of NDUFA4L2 in MHCC97L-NTC and -shL2 cells exposed to 20% and 1% O2 for 24 and 48 hours, respectively (results represent mean ± SEM. Student t test; N = 3 independent experiments: ***, P < 0.001 as indicated). B, mRNA and protein expression of NDUFA4L2 in PLC-EV and -NDUFA4L2 cells cultured in 20% O2 for 24 and 48 hours, respectively (results represent mean ± SEM. Student t test; N = 3 independent experiments: ***, P < 0.001 as indicated). C, Left, representative fluorescent images of JC-1 stained MHCC97L-NTC and -shL2 cells that were exposed to 20% and 1% O2. Right, mitochondrial membrane potential was further determined with JC-1 staining by flow cytometry analysis (results represent mean ± SEM. Student t test; N = 5 independent experiments: ***, P < 0.001 as indicated). Scale, 50 μm. D, mitochondrial mass was measured with NAO staining by flow cytometry analysis (results represent mean ± SEM. Student t test; N = 5 independent experiments: **, P < 0.01 as indicated). E, oxygen consumption was measured with MitoXpress®-Intra (NanO2) in MHCC97L-NTC and -shL2 cells or MHCC97L-WT and -HIF1α−/− (KO) cells exposed to 20% and 1% O2 (results represent mean ± SEM. Student t test; N = 3 independent experiments: *, P < 0.05; **, P < 0.01 as indicated). F, ROS level was measured with CM-H2DCFDA or MitoSox staining by flow cytometry analysis in (i and ii) MHCC97L-NTC and -shL2 cells cultured in 20% and 1% O2, (iii) PLC-EV and -NDUFA4L2 cells cultured in 20% O2, and (iv) PLC-EV and -CA5 cells cultured in 20% O2 (results represent mean ± SEM. Student t test; N = 3 independent experiments: **, P < 0.01 as indicated). Cells for all metabolic assays were cultured for 24 hours. Values obtained in mitochondrial membrane potential, mass, and ROS were normalized to 20% O2 MHCC97L-NTC or PLC-EV.
NDUFA4L2 reduces mitochondrial activity, oxygen consumption, and ROS production in hypoxia. A, mRNA and protein expression of NDUFA4L2 in MHCC97L-NTC and -shL2 cells exposed to 20% and 1% O2 for 24 and 48 hours, respectively (results represent mean ± SEM. Student t test; N = 3 independent experiments: ***, P < 0.001 as indicated). B, mRNA and protein expression of NDUFA4L2 in PLC-EV and -NDUFA4L2 cells cultured in 20% O2 for 24 and 48 hours, respectively (results represent mean ± SEM. Student t test; N = 3 independent experiments: ***, P < 0.001 as indicated). C, Left, representative fluorescent images of JC-1 stained MHCC97L-NTC and -shL2 cells that were exposed to 20% and 1% O2. Right, mitochondrial membrane potential was further determined with JC-1 staining by flow cytometry analysis (results represent mean ± SEM. Student t test; N = 5 independent experiments: ***, P < 0.001 as indicated). Scale, 50 μm. D, mitochondrial mass was measured with NAO staining by flow cytometry analysis (results represent mean ± SEM. Student t test; N = 5 independent experiments: **, P < 0.01 as indicated). E, oxygen consumption was measured with MitoXpress®-Intra (NanO2) in MHCC97L-NTC and -shL2 cells or MHCC97L-WT and -HIF1α−/− (KO) cells exposed to 20% and 1% O2 (results represent mean ± SEM. Student t test; N = 3 independent experiments: *, P < 0.05; **, P < 0.01 as indicated). F, ROS level was measured with CM-H2DCFDA or MitoSox staining by flow cytometry analysis in (i and ii) MHCC97L-NTC and -shL2 cells cultured in 20% and 1% O2, (iii) PLC-EV and -NDUFA4L2 cells cultured in 20% O2, and (iv) PLC-EV and -CA5 cells cultured in 20% O2 (results represent mean ± SEM. Student t test; N = 3 independent experiments: **, P < 0.01 as indicated). Cells for all metabolic assays were cultured for 24 hours. Values obtained in mitochondrial membrane potential, mass, and ROS were normalized to 20% O2 MHCC97L-NTC or PLC-EV.
NDUFA4L2 reduces mitochondrial activity by decreasing oxygen consumption and prevents excessive ROS production under hypoxia
To investigate the functions of hypoxia-induced NDUFA4L2 in mitochondria in vitro, we generated NDUFA4L2 loss-of-function and gain-of-function HCC cell models. For NDUFA4L2 loss-of-function HCC cell model, shRNA against NDUFA4L2 (shL2) or nontarget control (NTC) was stably expressed in MHCC97L cells by lentiviral transfection approach (Fig. 3A). For NDUFA4L2 gain-of-function HCC cell model, NDUFA4L2 and empty vector (EV) were stably expressed in another HCC cell line, which expressed a lower level of NDUFA4L2, PLC/PRF/5 (PLC; Fig. 3B). As NDUFA4L2 is located in the first complex of the ETC, we asked if NDUFA4L2 would affect the mitochondrial activity by staining the cells with a probe, JC-1, which accumulates in active mitochondria to indicate mitochondrial potential. By fluorescence imaging, we showed that the mitochondrial membrane potential was decreased in hypoxia, but was elevated when NDUFA4L2 was knocked down (Fig. 3C). Flow cytometry analysis further confirmed the result (Fig. 3C). Similar observation was obtained with tetramethylrhodamine ethyl ester, another fluorescent probe that measures the mitochondrial membrane potential (Supplementary Fig. S5A). Consistently, mitochondrial mass was increased in NDUFA4L2 knockdown cells as indicated by the nonyl acridine orange (NAO) staining (Fig. 3D). Consistently, we found that oxygen consumption in the mitochondria was significantly elevated when NDUFA4L2 was knocked down under hypoxia (Fig. 3E). HIF1α knockout mirrored the effect of NDUFA4L2 knockdown in oxygen consumption (Fig. 3E), further supporting the regulatory role of HIF1α on NDUFA4L2. Hypoxia triggers an imbalanced electron flow through the ETC leading to generation of ROS in the mitochondria. Hence, we evaluated the intracellular ROS level by the general ROS indicator, chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), and mitochondrial ROS indicator, MitoSOX. ROS level was elevated when cells were exposed to hypoxia and was further induced when NDUFA4L2 was knocked down especially under hypoxic condition (Fig. 3F). Reversely, a significant reduction of ROS was found in NDUFA4L2-overexpressing HCC cells (Fig. 3F). Similar trend was observed in HCC cells that expressed the constitutively active form of HIF1α (CA5; Fig. 3F). To eliminate off-target effect, siRNAs targeting different sequences of NDUFA4L2 were transfected into MHCC97L cells (Supplementary Fig. S5B), and similar metabolic consequences were found as compared with stable knockdown of NDUFA4L2 (Supplementary Fig. S5C and S5D).
NDUFA4L2 maintains redox homeostasis and promotes cell proliferation and survival
Knockdown of NDUFA4L2 resulted in a dramatic increase of ROS, which we speculated would be harmful to HCC cells. To demonstrate the roles of ROS in HCC cells, we employed two antioxidants, NAC and L-ascorbic acid. ROS induced by NDUFA4L2 knockdown was rescued dose dependently by both antioxidants (Fig. 4A). To further demonstrate the regulation of HIF1α on NDUFA4L2 in ROS scavenging, we re-expressed NDUFA4L2 in HIF1α knockdown PLC cells. ROS was dramatically induced in HIF1α knockdown cells under hypoxia and was significantly rescued upon re-expression of NDUFA4L2 (Fig. 4B). Excessive accumulation of ROS can cause cell senescence and apoptosis, eventually leading to cell death (16–18). We therefore evaluated apoptosis by Annexin V–propidium iodide (PI) staining in MHCC97L-NTC and -shL2 cells. MHCC97L-NTC cells showed higher Annexin V and PI staining under hypoxic than normoxic condition, while knockdown of NDUFA4L2 further increased apoptosis (Fig. 4C), which could be rescued by extracellular GSH-EE, a modified and more permeable form of glutathione, an antioxidant that counteracts mitochondrial ROS (Supplementary Fig. S6A and S6B). To further confirm that increased apoptosis was caused by ROS, we exogenously treated the cells with H2O2, a common source of ROS (35). Apoptosis was elicited by H2O2 drastically in MHCC97L-NTC cells and was further elevated in -shL2 cells (Supplementary Fig. S6C). Consistently, both the cell counting and BrdUrd assays showed that cell proliferation was substantially reduced when NDUFA4L2 was knocked down especially under hypoxic condition (Fig. 4D and Supplementary Fig. S6D). More interestingly, cell proliferation was restored by NAC in the NDUFA4L2 knockdown HCC cells (Fig. 4E), suggesting that impairment of cell proliferation was associated with ROS. All these data converged to show that NDUFA4L2 was responsible to reduce ROS under hypoxia, thereby maintaining cell survival and promoting cell proliferation.
NDUFA4L2 reduces intracellular ROS to enhance HCC cell survival. A, CM-H2DCFDA staining showing the ROS levels of MHCC97L-NTC and -shL2 cells exposed to 1% O2 for 24 hours in the presence of indicated concentrations of NAC and ascorbic acid. Top, representative flow cytometry analysis showing the ROS levels in the indicated subclones. Bottom, histograms summarizing the ROS levels in the indicated subclones. The ROS level was relative to 1% O2 MHCC97L-NTC. B, CM-H2DCFDA staining showing the ROS levels of PLC-EV, -shHIF1α, and HIF1α knockdown with NDUFA4L2 overexpressing (-shHIF1α + NDUFA4L2) cells exposed to 1% O2 for 24 hours. The ROS level was relative to 20% O2 PLC-EV. Top, representative flow cytometry analysis showing the ROS levels in the indicated subclones. Bottom, histograms summarizing the ROS levels in the indicated subclones. C, Annexin V and PI staining showing the percentage of apoptotic cells in MHCC97L-NTC and -shL2 cells exposed to 20% and 1% O2 for 24 hours. D, cell proliferation of MHCC97L-NTC and -shL2 cells exposed to 20% and 1% O2 for 96 hours. E, BrdUrd assay of MHCC97L-NTC and -shL2 cells treated with indicated concentrations of NAC was exposed to 1% O2 for 24 hours (results represent mean ± SEM. Student t test; N = 3 independent experiments: *, P < 0.05; **, P < 0.01; ***, P < 0.001 as indicated).
NDUFA4L2 reduces intracellular ROS to enhance HCC cell survival. A, CM-H2DCFDA staining showing the ROS levels of MHCC97L-NTC and -shL2 cells exposed to 1% O2 for 24 hours in the presence of indicated concentrations of NAC and ascorbic acid. Top, representative flow cytometry analysis showing the ROS levels in the indicated subclones. Bottom, histograms summarizing the ROS levels in the indicated subclones. The ROS level was relative to 1% O2 MHCC97L-NTC. B, CM-H2DCFDA staining showing the ROS levels of PLC-EV, -shHIF1α, and HIF1α knockdown with NDUFA4L2 overexpressing (-shHIF1α + NDUFA4L2) cells exposed to 1% O2 for 24 hours. The ROS level was relative to 20% O2 PLC-EV. Top, representative flow cytometry analysis showing the ROS levels in the indicated subclones. Bottom, histograms summarizing the ROS levels in the indicated subclones. C, Annexin V and PI staining showing the percentage of apoptotic cells in MHCC97L-NTC and -shL2 cells exposed to 20% and 1% O2 for 24 hours. D, cell proliferation of MHCC97L-NTC and -shL2 cells exposed to 20% and 1% O2 for 96 hours. E, BrdUrd assay of MHCC97L-NTC and -shL2 cells treated with indicated concentrations of NAC was exposed to 1% O2 for 24 hours (results represent mean ± SEM. Student t test; N = 3 independent experiments: *, P < 0.05; **, P < 0.01; ***, P < 0.001 as indicated).
NDUFA4L2 promotes tumor growth, reduces oxidative stress, and enhances lung metastasis
To demonstrate the functions of NDUFA4L2 in tumor growth in vivo, we orthotopically injected the luciferase-labeled MHCC97L-NTC and -shL2 cells into nude mice. MHCC97L-NTC tumors grew substantially larger than the MHCC97L-shL2 tumors (Fig. 5A and 5B). To evaluate the proliferative output of MHCC97L-NTC and -shL2 cells in vivo, we stained the tumors with proliferative marker (Ki67). We found that knockdown of NDUFA4L2 markedly reduced Ki67 staining (Fig. 5C). We further dissociated the tumors and measured the ROS level of the tumors derived from MHCC97L-NTC and -shL2 cell lines. Consistent with our in vitro data, knockdown of NDUFA4L2 elevated ROS in vivo (Fig. 5D). Moreover, knockdown of NDUFA4L2 markedly repressed growth of metastatic lesions in the lungs of the tumor-bearing mice as indicated by Xenogen imaging (4 of 6 in MHCC97L-NTC and 1 of 6 in -shL2 group; Fig. 5E). We further found that knockdown of NDUFA4L2 significantly reduced cell-migratory ability of HCC cells in Transwell assay. Interestingly, the migratory ability of HCC cells was inversely correlated with the amount of intracellular ROS (Supplementary Fig. S7A and Fig. 3E), suggesting that ROS may also affect cell motility.
Knockdown of NDUFA4L2 suppresses tumor growth and induces oxidative stress in vivo. A, bioluminescence and (B) tumor volume of the orthotopic tumors derived from MHCC97L-NTC and -shL2 cells. Scale, 1 cm. C, Left, representative IHC pictures of Ki67 positivity in tumors. Cells were scored from 0 to 3 based on the staining intensity as represented by the arrows. Scale, 100 μm. Right, more than 1,000 cells were scored in each animal, and average percentage was calculated for each score. D, workflow of the measurement of intracellular ROS level in mouse model. Nude mice were orthotopically injected with MHCC97L-NTC and -shL2 cells. CM-H2DCFDA staining showing the ROS levels of the dissociated orthotopic tumors derived from MHCC97L-NTC and -shL2. E, bioluminescence of the lungs harvested from orthotopic tumor-bearing mice implanted with MHCC97L-NTC and -shL2 cells (Student t test; N = 6 per group: *, P < 0.05; **, P < 0.01).
Knockdown of NDUFA4L2 suppresses tumor growth and induces oxidative stress in vivo. A, bioluminescence and (B) tumor volume of the orthotopic tumors derived from MHCC97L-NTC and -shL2 cells. Scale, 1 cm. C, Left, representative IHC pictures of Ki67 positivity in tumors. Cells were scored from 0 to 3 based on the staining intensity as represented by the arrows. Scale, 100 μm. Right, more than 1,000 cells were scored in each animal, and average percentage was calculated for each score. D, workflow of the measurement of intracellular ROS level in mouse model. Nude mice were orthotopically injected with MHCC97L-NTC and -shL2 cells. CM-H2DCFDA staining showing the ROS levels of the dissociated orthotopic tumors derived from MHCC97L-NTC and -shL2. E, bioluminescence of the lungs harvested from orthotopic tumor-bearing mice implanted with MHCC97L-NTC and -shL2 cells (Student t test; N = 6 per group: *, P < 0.05; **, P < 0.01).
Pharmacologic inhibition of HIF suppresses progression of tumors that express high level of NDUFA4L2
So far, we found that hypoxia-induced NDUFA4L2 tightly regulated REDOX homeostasis and survival in HCC cells, and HIF1α is the central regulator of NDUFA4L2. We therefore reasoned that HIF inhibitor could repress the tumors that express high levels of NDUFA4L2 by damaging HCC cells through elevating ROS. We evaluated the ROS levels of MHCC97L-NTC and -shL2 cells treated with an HIF inhibitor, digoxin, under hypoxic condition. We observed that digoxin was more efficient in elevating the ROS level in MHCC97L-NTC cells than -shL2 cells (Fig. 6A). Next, we evaluated the effect of digoxin in vivo. We performed orthotopic implantation of the luciferase-labeled MHCC97L-NTC and -shL2 cells in nude mice. One week after implantation, mice were administered with 1.2 mg/kg/day digoxin or vehicle control (saline) through i.p. injection for 21 days. Digoxin markedly repressed growth of tumors with a more prominent effect on the MHCC97L-NTC group as compared with MHCC97L-shL2 group (Fig. 6B). Similarly, digoxin decreased the growth of subcutaneous tumors derived from MHCC97L-EV cells more significantly as compared with MHCC97L-shHIF1α cells (Supplementary Fig. S7B). Meanwhile, body weights of the animals were not affected by digoxin (Supplementary Fig. S7C). These results suggested that HIF inhibitor blocks HCC progression at least partially through HIF1α/NDUFA4L2.
HIF inhibitors suppress growth of tumors which express high level of NDUFA4L2. A, CM-H2DCFDA staining showing the ROS levels of MHCC97L-NTC and -shL2 cells exposed to 1% O2 for 24 hours in the presence of indicated concentrations of HIF inhibitor digoxin (results represent mean ± SEM. Student t test; N = 3 independent experiments: **, P < 0.01; ***, P < 0.001 as indicated). B, tumor volumes of orthotopic tumors derived from MHCC97L-NTC and -shL2 cells in mice that were administered with digoxin (1.2 mg/kg/day, i.p.) or vehicle (saline) for 21 consecutive days (Student t test; N = 6 per group: *, P < 0.05; ***, P < 0.001 as indicated). Scale, 1 cm. C, regulation and role of NDUFA4L2 in human HCC. i, hypoxia elicits an imbalanced electron flow through the ETC, leading to excessive ROS accumulation which is detrimental to HCC cells. ii, HIF1α overcomes by transcriptionally activating NDUFA4L2 in the mitochondrial complex I selectively to reduce the mitochondrial activity and the electron flux through ETC, thereby reducing ROS and ROS-induced apoptosis. This metabolic adaptation of oxidative stress mediated by HIF1α under hypoxia conferred survival advantage to the rapidly growing HCC cells which frequently experience hypoxia.
HIF inhibitors suppress growth of tumors which express high level of NDUFA4L2. A, CM-H2DCFDA staining showing the ROS levels of MHCC97L-NTC and -shL2 cells exposed to 1% O2 for 24 hours in the presence of indicated concentrations of HIF inhibitor digoxin (results represent mean ± SEM. Student t test; N = 3 independent experiments: **, P < 0.01; ***, P < 0.001 as indicated). B, tumor volumes of orthotopic tumors derived from MHCC97L-NTC and -shL2 cells in mice that were administered with digoxin (1.2 mg/kg/day, i.p.) or vehicle (saline) for 21 consecutive days (Student t test; N = 6 per group: *, P < 0.05; ***, P < 0.001 as indicated). Scale, 1 cm. C, regulation and role of NDUFA4L2 in human HCC. i, hypoxia elicits an imbalanced electron flow through the ETC, leading to excessive ROS accumulation which is detrimental to HCC cells. ii, HIF1α overcomes by transcriptionally activating NDUFA4L2 in the mitochondrial complex I selectively to reduce the mitochondrial activity and the electron flux through ETC, thereby reducing ROS and ROS-induced apoptosis. This metabolic adaptation of oxidative stress mediated by HIF1α under hypoxia conferred survival advantage to the rapidly growing HCC cells which frequently experience hypoxia.
Potential gene targets of NDUFA4L2
To explore novel functions of NDUFA4L2, we performed transcriptome sequencing to compare the global gene expression profiles of MHCC97L-NTC and MHCC97L-shL2 cells. The expressions of 629 genes were found to be decreased in MHCC97L-shL2 cells for more than 0.5-fold as compared with MHCC97L-NTC (Supplementary Table S5). The expressions of 683 genes were found to be increased for more than 2-fold in MHCC97L-shL2 cells relative to MHCC97L-NTC (Supplementary Table S6). Intriguingly, pathway analysis by DAVID suggested that NDUFA4L2 may regulate multiple biologic processes, including membrane potential and membrane depolarization (Supplementary Table S7). Of note, NRF2, a transcription factor that activates antioxidant genes, was found to be slightly induced (FPKM 29.246 in MHCC97L-NTC vs. FPKM 34.151 in -shL2), suggesting that a compensatory anti–oxidant-producing mechanism might be involved in response to the high ROS level upon NDUFA4L2 knockdown.
Discussion
ROS are fundamentally produced from O2 when it is consumed in different metabolic reactions. These reactions take place in the mitochondria at the ETC for ATP generation, endoplasmic reticulum during disulfide bond formation for protein folding, and peroxisomes during β-oxidation of fatty acids. Under hypoxia, these metabolic events cannot be carried out properly, leading to the generation of O2-containing free radicals. In the mitochondria that experiences shortage of the ultimate electron acceptor, O2, electrons cannot be efficiently pass through the ETC, leading to rapid accumulation of ROS in complexes I and III. Hypoxia is a common finding in HCC, and our study revealed the molecular mechanisms by which hypoxic HCC cells modulate ROS level in the mitochondria. Our study showed that HCC cells, by utilizing NDUFA4L2 in the complex I of the ETC, reduced the activity of the mitochondria to prevent ROS accumulation and apoptosis (Fig. 6C). Not only have we confirmed that this was dependent on HIF1α in HCC cells, more importantly, our study unprecedentedly revealed the clinical relevance of NDUFA4L2 in human HCC. Apart from HCC, we also showed that NDUFA4L2 was overexpressed in multiple solid cancers, suggesting that our findings do not limit to HCC but applies to other solid cancer models that frequently experience hypoxia. Notably, we and Tello and colleagues. consistently found that NDUFA4 and NDUFA4L2 have opposite expression patterns at normoxia and hypoxia in different cell models (23). Studies have documented that HIF1α could induce expression of siah E3 ubiquitin protein ligase 2 (SIAH2) and mitochondrial LON peptidase to degrade mitochondrial proteins such as α-ketoglutarate dehydrogenase and COX4-1 to reduce mitochondrial flux under hypoxia, respectively (21, 36). Whether NDUFA4 is degraded in a similar fashion remains an interesting topic to be addressed.
Prenatal lethality in NDUFA4L2 knockout mice has greatly limited the expansion of knowledge on the in vivo functions of this complex I subunit (23). Our study, using orthotopic liver cancer model, has provided the first in vivo evidence to confirm the protumorigenic functions of NDUFA4L2. Our study also demonstrated that digoxin profoundly blocked growth of orthotopic tumors that expressed high level of HIF1α/NDUFA4L2. Digoxin has been shown to inhibit HIF1α synthesis and is well-tolerated in patients (30). Digoxin has been used for the treatment of congestive heart failure and is currently under clinical trial for the treatment of breast cancer (ClinicalTrials.gov identifier: NCT01763931). Our study suggested that digoxin may be the most suitable candidate for patients whose primary HCCs express high levels of HIF1α or NDUFA4L2.
Worth mentioning, complex I inhibitors including metformin have promising antitumor effects in the clinical setting. Metformin has been the most frequently prescribed antidiabetic medication in the world. By reducing the ATP production in the ETC, metformin activates tumor suppressor 5′-AMP-activated protein kinase (AMPK; ref. 37). Reports have indicated that metformin reduced cancer incidence (38, 39). Metformin also reduced proliferative output of breast cancer cells in nondiabetic cancer patients (40). These interesting findings highlight an interesting dilemma—is energy or REDOX homeostasis more important for cancer survival? Accumulating studies suggested that ATP is not the limiting factor while excessive ROS is cell destructive. Unquestionably, cancer cells could not survive without energy; therefore, cancer cells need to acquire a tightly controlled homeostatic balance for the ETC, a place that generates both ATP and ROS, to sustain tumor growth. Drugs that could tip this balance merits exploration for their efficiency in cancer treatment.
REDOX homeostasis can be achieved by two mechanisms: (i) reduction of ROS production and (ii) elevation of antioxidant production. Our study showed that NDUFA4L2 directly decreased the generation of ROS in the mitochondria of HCC cells. Meanwhile, genes that are associated with the production and utilization of antioxidants, including NADPH, glutathione, and thioredoxin, have been implicated in HCC. Glucose 6 phosphate dehydrogenase, a critical enzyme in the pentose phosphate pathway generating NADPH, has been shown to be overexpressed in HCC in a PTEN-dependent manner (41). Overexpression of thioredoxin and thioredoxin-related REDOX molecules, glutaredoxin and peroxiredoxin, was documented in HCC and was associated with increased proliferative output, aggressive clinicopathologic parameters, presence of metastasis, and poor prognosis in HCC patients (42, 43). Elevation of cysteine/glutamate transporter, xCT, which is important for glutathione production, was found to be associated with poor clinical outcome in HCC, and disruption of xCT in HCC cells increased ROS-mediated autophagic cell death (44). A recent study has elegantly demonstrated that cotreatment of inhibitors blocking different pathways that synthesize antioxidants could synergistically prevent tumor growth (45). Along similar lines, our current study suggested that raising ROS level through HIF inhibitor, which suppressed HIF1α/NDUFA4L2 pathway, represents an attractive therapeutic strategy for HCC treatment. In the coming decade, combination therapies targeting multiple machineries that buffer ROS represent an exciting translation research direction.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: R.K.-H. Lai, I.O.-L. Ng, C.C.-L. Wong
Development of methodology: R.K.-H. Lai, C.C.-L. Wong
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.K.-H. Lai, I. M.-J. Xu, D.K.-C. Chiu, A.P.-W. Tse, M.P. Wong, C.C.-L. Wong
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.K.-H. Lai, D.K.-C. Chiu, A.P.-W. Tse, C.-T. Law, C.-M. Wong, C.C.-L. Wong
Writing, review, and/or revision of the manuscript: R.K.-H. Lai, D. Lee, M.P. Wong, I.O.-L. Ng, C.C.-L. Wong
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L.L. Wei, C.-T. Law, I.O.-L. Ng, C.C.-L. Wong
Study supervision: I.O.-L. Ng, C.C.-L. Wong
Acknowledgments
pQCXIN HIF1α (CA5) construct is a generous gift from Professor Gregg L. Semenza. The authors thank the Core Facility of LKS Faculty of Medicine for their technical support. They also thank Dr. Wilson Yick-Pang Ching and Dr. Yuan Zhou for the valuable advice and technical help.
Grant Support
This work was supported by the Small Project Funding of the University of Hong Kong 2012 and the Hong Kong Research Grants Council General Research Fund (HKU 781213M).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.