Abstract 2914: Hexokinase 2-mediated metabolic reprogramming and apoptosis inhibition supports hepatocellular carcinoma radiation resistance
Authors: Fang Yuan , Zhan Yizhi , Li Yiyi , Wang Wei , Wu Dehua , Ding Yi 
Published: August 2019

Purpose: Hepatocellular carcinoma (HCC) cells are metabolically distinct from normal hepatocytes by selectively upregulating hexokinase 2 (HK2), a key enzyme that catalyzes the first committed step of aerobic glycolysis, which provides genetic proof of concept that HK2 can be inhibited to treat HCC without adverse physiological consequences. The present study was designed to investigate the role of HK2-mediated metabolic reprogramming and apoptosis inhibition in radiation resistance of HCC cells.

Experimental design: Acquired radiation-resistant cells were generated by sequential irradiation and recovery, and proteomics approach was used to illustrate the biological changes and molecular mechanisms involved. qRT-PCR was performed to evaluate the expression of glycolysis genes including HK2. TCGA and GEO analysis were used to investigate the prognostic value of HK2. Intrinsic radiation responsive and resistant HCC cells were also used to further identify the resistant role of HK2. Either lentivirus-mediated HK2 knockdown or pharmacological blockade by 3-bromopyruvate (BrPA) was used to inhibit HK2. Radiation resistance was evaluated by DNA damage response, in vitro cytotoxicity and colony formation assay. Apoptosis and ROS levels were measured with Annexin V and DC-FDA. Cytochrome c release and HK2 mitochondrial translocation were detected by immunofluorescence and western blots. 2-NBDG uptake, HK2 activity, lactate production, and 13C isotopomer tracing experiment were conducted to illustrate the glycolytic activities. HK2 translation efficiency was measured by polyribosome analysis. The upstream AKT-mTOR signaling were detected by western blots and rescued by pharmacological inhibitors. Xenografts were treated with radiation, BrPA, or a combination of radiation and BrPA.

Results: Aerobic glycolysis was significantly activated and was indispensable for cell survival in both acquired and intrinsic radiation-resistant HCC cells. Mechanically, both up-regulation of mRNA expression of hexokinase (HK2) after sequential irradiation and increased translation of HK2 mRNA after one-fractional irradiation were mediated by the activation of AKT-mTOR axis. In addition, high HK2 expression was significantly associated with poor overall survival of HCC patients. Inhibition of HK2 expression or its mitochondrial translocation re-sensitized HCC cells to radiation therapy by promoting mitochondrial cytochrome c release and inhibiting of glycolysis both in vitro and in vivo.

Conclusion: AKT-mTOR-HK2 axis plays crucial roles in both acquired and intrinsic radiation resistance of HCC cells by facilitating aerobic glycolysis and inhibiting cytochrome c release. Targeting HK2 could be an attractive and beneficial approach to improve the therapeutic effects of radiotherapy in HCC patients.

Citation Format: Yuan Fang, Yizhi Zhan, Yiyi Li, Wei Wang, Dehua Wu, Yi Ding. Hexokinase 2-mediated metabolic reprogramming and apoptosis inhibition supports hepatocellular carcinoma radiation resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2914.