Abstract 1188: HIF-1α/NDUFA4L2 promotes hepatocellular carcinoma progression through reducing oxidative stress
Authors: Lai Kit Ho , Xu Ming Jing , Tse Pui Wah , Chiu Kung Chun , Lai Wei , Wong Chun Ming , Wong Pik , Ng Oi Lin , Wong Chak Lui 
Published: August 2015

Background: Liver is well characterized as a major metabolic organ, the metabolic machineries driving liver cancer (hepatocellular carcinoma, HCC) progression remains poorly understood. Oxygen deprivation, hypoxia, is frequently found in regions of tumors with insufficient blood supply. Hypoxia stabilizes hypoxia-inducible factor-1α (HIF-1α) which transcriptionally activates genes that utilize glycolysis over oxidative phosphorylation for ATP production. This metabolic switch reduces mitochondrial reactive oxygen species (ROS) production that would otherwise occur due to electron imbalance through the electron transport chain (ETC) in hypoxic condition. In our transcriptome sequence analysis, among all the mitochondrial subunits in the complex I of the electron transport chain (ETC), only NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 gene (NDUFA4L2) was distinctly and significantly induced by hypoxia and over-expressed in human HCC. However, the clinical implications and functions of this gene in HCC progression remain unknown.

Methods: The mRNA expression of NDUFA4L2 in an expanded cohort of 100 HCC patients was evaluated by qRT-PCR. ChIP assay was performed to study the interaction of HIF-1α with NDUFA4L2. Stable knockdown by shRNA or knockout by TALEN of HIF-1α/NDUFA4L2 was established in HCC cells to explore the roles of NDUFA4L2 in HCC. ROS, mitochondrial membrane potential, and oxygen consumption rate were measured in the HIF-1α/NDUFA4L2 knockdown or knockout HCC cells. Orthotopic implantation model was employed to evaluate the in vivo effect of NDUFA4L2 and the efficiency of HIF inhibitors.

Results: NDUFA4L2 was significantly overexpressed in human HCC and was markedly induced by hypoxia. Overexpression of NDUFA4L2 in human HCC was significantly correlated with aggressive HCC clinicopathological parameters including absence of tumor encapsulation, formation of tumor microsatellite, and poorer overall survival. ChIP assay confirmed the binding of HIF-1α with hypoxia response elements (HREs) in NDUFA4L2 promoter and expression study showed that hypoxia-induced NDUFA4L2 was abolished in HIF-1αknockdown or knockout HCC cells. Genetic ablation of HIF-1α/NDUFA4L2 increased the mitochondrial activity that coupled with higher intracellular reactive oxygen species (ROS) and ROS-induced apoptosis. Knockdown of NDUFA4L2 markedly suppressed tumor growth in vitro and in vivo. HIF inhibitors, digoxin and Sorafenib, profoundly inhibited the growth of HCC that expressed high levels of NDUFA4L2.

Conclusion: Over-expression of NDUFA4L2 is associated with poor clinical outcome in HCC patients. Hypoxia-induced NDUFA4L2 reduced the mitochondrial ROS production and ROS-triggered apoptosis, thereby conferring HCC cells growth advantage in hypoxic environment. Targeting HIF-1α/NDUFA4L2 pathway by HIF inhibitors represent a new therapeutic strategy for HCC.

Citation Format: Kit Ho Lai, Ming Jing Xu, Pui Wah Tse, Kung Chun Chiu, Wei Lai, Chun Ming Wong, Pik Wong, Oi Lin Ng, Chak Lui Wong. HIF-1α/NDUFA4L2 promotes hepatocellular carcinoma progression through reducing oxidative stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1188. doi:10.1158/1538-7445.AM2015-1188