Reprogramming of intracellular metabolism is common in liver cancer cells. Understanding the mechanisms of cell metabolic reprogramming may present a new basis for liver cancer treatment. In our previous study, we reported that a novel oncogene eukaryotic translation initiation factor 5A2 (EIF5A2) promotes tumorigenesis under hypoxic condition. Here, we aim to investigate the role of EIF5A2 in cell metabolic reprogramming during hepatocellular carcinoma (HCC) development. In this study, we reported that the messenger RNA (mRNA) level of EIF5A2 was upregulated in 59 of 105 (56.2%) HCC clinical samples (P = 0.015), and EIF5A2 overexpression was significantly associated with shorter survival time of patients with HCC (P = 0.021). Ectopic expression of EIF5A2 in HCC cell lines significantly promoted cell growth and accelerated glucose utilization and lipogenesis rates. The high rates of glucose uptake and lactate secretion conferred by EIF5A2 revealed an abnormal activity of aerobic glycolysis in HCC cells. Several key enzymes involved in glycolysis including glucose transporter type 1 and 2, hexokinase 2, phosphofructokinase liver type, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase M2 isoform, phosphoglycerate mutase 1 and lactate dehydrogenase A were upregulated by overexpression of EIF5A2. Moreover, EIF5A2 showed positive correlations with FASN and ACSS2, two key enzymes involved in the fatty acid de novo biosynthetic pathway, at both protein and mRNA levels in HCC. These results indicated that EIF5A2 may regulate fatty acid de novo biosynthesis by increasing the uptake of acetate. In conclusion, our findings demonstrate that EIF5A2 has a critical role in HCC cell metabolic reprogramming and may serve as a prominent novel therapeutic target for liver cancer treatment.

Cancer was hypothesized to be driven by cancer stem cells (CSCs), but the metabolic determinants of CSC-like phenotype still remain elusive. Here, we present that hexosamine biosynthetic pathway (HBP) at least in part rescues cancer cell fate with inactivation of glycolysis. Firstly, metabolomic analysis profiled cellular metabolome in CSCs of hepatocellular carcinoma using CD133 cell-surface marker. The metabolic signatures of CD133-positive subpopulation compared to CD133-negative cells highlighted HBP as one of the distinct metabolic pathways, prompting us to uncover the role of HBP in maintenance of CSC-like phenotype. To address this, CSC-like phenotypes and cell survival were investigated in cancer cells under low glucose conditions. As a result, HBP inhibitor azaserine reduced CD133-positive subpopulation and CD133 expression under high glucose condition. Furthermore, treatment of N-Acetylglucosamine in part restores CD133-positive subpopulation when either 2.5 mM glucose in culture media or glycolytic inhibitor 2-deoxy-D-glucose in HCC cell lines was applied, enhancing CD133 expression as well as promoting cancer cell survival. Together, HBP might be a key metabolic determinant in the functions of hepatic CSC marker CD133.

Background and objectives: Hepatocellular carcinoma (HCC) is the primary liver malignancy with an extremely low survival rate. HCC progression is frequently associated with accelerated glucose consumption that confers growth advantage to tumor cell through two essential metabolic pathways - glycolysis and the pentose phosphate pathway (PPP). Glycolysis utilizes glucose for ATP production, while the PPP converts glycolytic intermediates to generate NADPH, the cellular antioxidant, and ribose-5-phophate, the nucleotide precursor. Although glycolysis has been extensively studied in HCC, how PPP supports HCC growth remains largely unknown. Our study aims at delineating the clinical significance, regulation and functions of PPP in HCC development and explore the therapeutic potential of PPP inhibitors for HCC therapy. Methods: The expression and abundance of the the PPP genes were examined in 16 pairs of human HCC tumors and adjacent non-tumors by transcriptome sequencing. Level of reactive oxygen species (ROS) and glucose uptake were measured by CM-H2DCFDA and 2-NBDG stainings, respectively. Metabolomics study was performed with CE-TOF-MS analysis. Metabolic flux analysis was conducted using UPLC-MS/MS. Results: Transcriptome sequencing data showed that enzymes in the PPP were frequently upregulated in HCC. Transketolase (TKT), the gene encodes the reversible enzyme connecting PPP and glycolysis, is the most abundant and most overexpressed PPP gene in HCC. Overexpression of TKT was confirmed in an expanded sample cohort at the mRNA and protein level. Meanwhile, TKT overexpression was significantly correlated with venous invasion, microsatellite formation, tumor size and absence of tumor encapsulation. Notably, TKT expression was controlled by NRF2/KEAP1/BACH1 pathway, which is a major transcription regulator for redox homeostasis. CHIP assay revealed that NRF2 and BACH1 competitively bound to the same antioxidant responsive elements (ARE) in TKT. Functionally, knockdown of TKT in HCC cells retarded cell growth, attenuated glucose uptake and NADPH production, increased intracellular ROS, and induced oxidative stress-associated cell cycle delay. In line with these findings, knockdown of TKT greatly suppressed tumor growth in vivo. Metabolomics and metabolic flux analysis revealed that loss of TKT disrupted the PPP and subsequently reduced NADPH production. Intriguingly, genetic knockdown and pharmacological inhibition of TKT enhanced the efficacy of Sorafenib, the only FDA-approved drug for HCC treatment, both in vitro and in vivo. Conclusion: Our study suggested the clinical significance of TKT in HCC and illustrated the anti-oxidative role of TKT in HCC progression. We also proposed that disrupting the metabolic machinery by TKT inhibition might be a novel therapeutic strategy for HCC treatment. Citation Format: Ming Jing Xu, Kit Ho Lai, Shu Hai Lin, Pui Wah Tse, David Kung Chun Chiu, Hui Yu Koh, Cheuk Ting Law, Chun Ming Wong, Zong Wei Cai, Carmen Chak Lui Wong, Irene Oi Lin Ng. Targeting pentose phosphate pathway (PPP) represents a novel therapeutic strategy for hepatocellular carcinoma (HCC) treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1058.