Recurrence and metastasis of HCC are still primary causes of patients' death and these factors are closely related to the proliferation and invasion capacities of tumor cells. Recently, although a lot of progress has been made in the pathogenesis of HCC. Therefore, exploring new therapeutic targets for HCC is of great significance to improve postoperative survival rates and facilitate the survival prognosis of patients. As a key enzyme in the PPP process, the main role of G6PD is to provide sufficient reducing capacity to support cell growth and keep cells in redox homeostasis. Its product, NADPH, acts as a pro-oxidant, generates reactive oxygen species (ROS) and reactive nitrogen species (RNS), which ameliorate tumor cell proliferation and metastasis, cell cycle and apoptosis. Severe deficiency of G6PD can impair embryonic development and retard the organism's growth. Hence, altering the activity of G6PD is associated with pathophysiology such as autophagy, insulin resistance, infection, inflammation, diabetes, hypertension, etc. Besides, abnormal activation of G6PD can also lead to cell proliferation in many cancers(9). In the current study, G6PD activity is significantly increased in tumor tissues of gastric cancer, breast cancer, bladder cancer, cervical cancer, and colorectal cancer(10-14). This is consistent with the result that G6PD is highly expressed in pan-cancer in the TCGA data. Therefore, G6PD is also considered as a potentially effective target for tumor treatment(15-17).
As an independent predictor of prognosis for gastric cancer patients,G6PD expression was higher in gastric cancer tissues than in paired normal gastric mucosa groups, and high G6PD expression was closely associated with tumor size, depth of infiltration and tumor size, lymph node metastasis, distant metastasis, TNM stage and survival rate in previous studies(10). Zhang et al.(18)found that intracellular high expression of G6PD decreased matrix metalloproteinase expression through the G6PD /HIF-1α/ Notch1 axis and promoted the migration of tumor cells. Chen J et al.(19)showed that G6PD-based metabolic markers can be used as predictors of prostate cancer metastasis. G6PD was expressed at high levels in melanoma. In animal models, wild-type nude mice models had faster tumor growth, larger tumor size, and higher malignancy than G6PD-deficient nude mice(20). Hong et al. (21)found that high G6PD expression was significantly associated with poor prognosis in HCC, especially in HCC patients who are in the advanced stages of HCC treated with sorafenib after hepatocellular carcinoma surgery, high G6PD expression was significantly associated with worse PFS and OS. This may also have a link with PPP and tumor cell resistance. Yin X et al.(22) found that Inhibitor of Differentiation-1(ID-1) in HCC cells leads to reduce G6PD and NADPH activity and increase ROS production, and transfection of G6PD into ID-1 knockdown HCC cells reversed these changes and induced oxaliplatin resistance, and the above evidence also provides new ideas for the study of hepatocellular carcinoma in terms of resistance to sorafenib and oxaliplatin. In our research, G6PD expression is associated with histological grading, pathological stage, T-stage, vascular infiltration and AFP levels (P<0.05). HCC patients in the G6PD low expression group have more prolonged overall survival and better prognosis than the G6PD high expression group (P<0.05), and high G6PD expression is a potential biomarker for poor prognosis of HCC. In addition, we combined G6PD with age, AFP, and Child classification to construct columnar line graph prognostic plots to obtain a more accurate prognostic prediction model, and the C-index G6PD-related Cox model predicted OS of 0.686. The calibration plots show that the best agreement between the predictions of the column line graphs is associated with G6PD and the actual observations of the 1-year, 3-year and 5-year OS probabilities. Thus, our model can provide personalized scoring for HCC patients.
In this study, the PPI network was used to identify co-expressed proteins of G6PD, and we identified G6PD-related genes, including GAPDH, TPI1, PGLS, TP53, PGD, PKM, TALDO1, LDHB, and GPI. Among them, TP53 has become a key antitumor factor since its discovery, and TP53 can be activated under conditions of genotoxic stress, oncogene activation, ribosomal stress, hypoxic state, and abnormal energy metabolism(23). Furthermore, the MDM2-TP53 axis may be manifested in hepatocyte glycolipid metabolism by enhancing glycolipid catabolism, but can promote hepatocyte injury in the early and late stages of glycolipid metabolism disorders (24). Oxidative stress, steatosis and abnormal cell growth can be detected in hepatocytes with disorders of glucolipid metabolism, all of which may contribute to the development of HCC(24). GAPDH and LDHB are all glycolysis-related genes, and it has been shown that TFB2M activates aerobic glycolysis in hepatocellular carcinoma cells through NAD /SIRT3/HIF-1α Signaling pathway(25). Among them, TFB2M (mitochondrial transcription factor B2) is a core mitochondrial transcription factor, and its overexpression is significantly associated with the malignancy and prognosis of hepatocellular carcinoma(26). Enrichment analysis of differential genes in tumor tissues and normal tissues based on the DAVID database was performed, and the results of GO functional enrichment analysis showed that G6PD-related genes were enriched in the metabolism of pyridine compounds, metabolism of nicotinamide nucleotides, metabolism of pyridine nucleotides, carbohydrate-binding, monosaccharide binding, and NADP binding. Among them, nicotinamide ribonucleotide (NMN) is a precursor of coenzyme 1NAD+ (nicotinamide adenine dinucleotide), which is not only a coenzyme involved in intracellular redox reactions, but also involved as a substrate in regulating apoptosis, DNA repair, immune response, and many other physiological roles (27).Moreover, due to the high rate of tumor cell appreciation and DNA repair, the demand for NAD is increased, and some studies have shown an important role of nicotinamide phosphoribosyl transferase (NAMPT)-mediated NAD remediation pathway in the energy homeostasis of hepatocellular carcinoma cells and suggested that NAMPT inhibition is a potential therapeutic option for hepatocellular carcinoma (28). KEGG results suggest that G6PD-related genes are enriched in carbon metabolism, gluconeogenesis, and the pentose phosphate pathway (PPP), which is consistent with the function of G6PD. The pentose phosphate pathway (PPP) is the first step reaction of glycolysis, and its product NADPH has an important role in biosynthesis as well as in maintaining cellular redox homeostasis. ROS is a collective term for a variety of oxygen radicals in intracellular metabolic processes, which can promote normal cell proliferation when ROS levels are low, and trigger apoptosis when ROS levels are too high(29). The significance of PPP at this time lies largely in reducing excess ROS and maintains cellular energy metabolism in a redox homeostasis. In tumor cells, making PPP at high levels is able to resist ROS-induced apoptosis (30). In hepatocellular carcinoma cells, high expression of the G6PDH gene is closely related to the occurrence, development, metastasis and prognosis of hepatocellular carcinoma(31).
In addition, tumor cell metabolic reprogramming is involved in tumor immune regulation, and metabolic reprogramming plays a crucial role in antitumor immunotherapy. Tumor cells require large amounts of energy during proliferation, while body has to compete with tumor cells for these nutrients by tumor-infiltrating effector cells CD8+T lymphocytes (CD8TILs) in order to fight tumors. Studies have shown that sustained antitumor immunity can be triggered by upregulating glycolysis and oxidative phosphorylation in CD8TILs(32).
T cells also play an important role in the immune microenvironment against infections and tumors (33), Naïve T cells have low metabolic demand and rely mainly on oxidative phosphorylation for energy production, but with the onset of infections and tumors, Naïve T cells will be activated, and activated T cell energy metabolism will shift to aerobic glycolysis and increase oxidative phosphorylation, which is essential for effector T cell production and function(34, 35). Gu M et al.(36) found that the G6PD-NADPH redox system plays an important role in the stability and metabolism of hexokinase 2 (HK2) in activated T cells. However, there are few studies on the relationship between G6PD and immune cells in HCC. In the present study, the correlation between G6PD and immune cell infiltration was assessed using TIMER and ssGSEA. Our results showed that significant differences between G6PD and CD8+ T lymphocytes, CD4+ T lymphocytes, B cells, neutrophils, macrophages, dendritic cells, helper T cells, follicular helper T cells, Th1, Th2, Th17, regulatory T cells (Treg). This also provides new ideas for immunotherapy in HCC patients with elevated G6PD. However, there are some limitations in this study, such as the relatively small sample size in the TCGA database and the hypothesis of this study was not validated using animal models. Therefore, we will conduct further cellular assays to further prove in the following studies.