Background and Objective: Excessive accumulation of oxidative stress/reactive oxygen species (ROS) can be harmful to cancer cells. Hypoxia or O2 deprivation, which is commonly found in hepatocellular carcinoma (HCC), is a crucial factor that contributes to elevated ROS level in HCC cells as hypoxia causes inefficient transfer of electrons in the mitochondria. To survive, HCC cells need to devise strategies to counteract and balance hypoxia-induced oxidative stress. While it is known that hypoxia inducible factors (HIFs) are essential to metabolic reprogramming in HCC cells under hypoxia, there are significant gaps in knowledge about underlying mechanisms and transcriptional targets of HIFs.

Experimental Procedures: Gene profiling of HCC cell lines (exposed to 20% and 1% O2) was analyzed by transcriptome sequencing to identify novel candidate responsible for counteracting hypoxia-induced oxidative stress. ShRNA-mediated gene silencing and gene activation by CRISPR-dCas9 system were used to modify transcriptional expression of HEY1 for different functional assays. Transmission electron microscopy was used to visualize the mitochondrial structure. Orthotopic and subcutaneous HCC implantation models were used to evaluate the role of HEY1 in HCC progression. Transcriptome sequencing and ChIP assay were performed to identify novel transcriptional targets of HEY1.

Results: We showed that transcriptional repressor HEY1 was induced under hypoxia and directly regulated by HIF-1α. Overexpression of HEY1 was associated with poor overall survival in HCC patients. Importantly, we identified PINK1 as a novel repression target of HEY1. PINK1 is known to protect cells against mitochondrial dysfunction. We demonstrated that HEY1 actively repressed PINK1 and downregulation of PINK1 led to loss of mitochondrial mass and impaired mitochondrial cristae formation, subsequently decreasing intracellular ROS level. Downregulation of PINK also associated with poor overall survival and decrease-free in HCC patients. Genetic ablation of HEY1 in HCC cells profoundly reduced tumor growth and lung metastasis while genetic ablation of PINK1 in HCC cells reversely promoted HCC growth. Strikingly, HEY1 and PINK1 expressions reversely correlated in human HCC tissues.

Conclusion: This study unprecedentedly identifies an upstream regulatory mechanism of PINK1, which controls the oxidative stress in HCC cells. It also reveals a novel molecular mechanism by which ablation of HEY1 leads to elevation of oxidative stress, making HCC cells more vulnerable. Targeting HEY1 represents an attractive therapeutic approach against HCC.

Citation Format: David Kung-Chun Chiu, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Dicky Cheuk-Ting Law, Vincent Wai-Hin Yuen, Larry Lai Wei, Hui-Yu Koh, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. HEY1 counteracts hypoxia-induced oxidative stress via transcriptionally repressing PINK1 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2415.