This study found that EZH1 mRNA and protein were upregulated in HCC, and its overexpression was related to poor prognoses. This study is the first to comprehensively explore the clinicopathological significance and molecular basis of EZH1 in HCC using global multicenter RNA-seq datasets and microarray data. EZH1 is deemed to be relevant to the occurrence and development of tumors. Previous studies showed that in triple-negative breast cancer, the expression levels of EZH1 protein and mRNA show a down regulation (Peng et al. 2022), while EZH1 overexpression significantly eliminates the function of TRIM21 protein to induce apoptosis of cancer cells in gastric cancer (Ping et al. 2022). Therefore, the role of EZH1 in different tumors is complex. After the integration of multi-center microarray and sequencing data, we found that EZH1 mRNA showed a clear high expression in HCC tissues, suggesting that EZH1 was considerable for the occurrence and development of HCC. Differential gene expression assessment plays a fundamental role in exploring its effect in the development of tumors. Compared with other cancer single-gene detection, our exploration of EZH1 expression in HCC is a large sample using an advanced integration algorithm by calculating the SMD value and drawing a sROC curve, which has distinct advantages. In this study, 3926 HCC samples and 3428 normal liver tissue samples used to analyze EZH1 expression came from global public data, which was in line with the concept of evidence-based medicine. The results of the sROC curve, sensitivity, and specificity suggest the credibility of our work. In addition, IHC results also supported the high expression of EZH1 in HCC at the protein level. The K-M survival analysis implied that overexpression of EZH1 showed a lower survival rate of HCC patients compared to the control group.
This study further explored the transcriptional targets of EZH1 to clarify the mechanism of EZH1 in HCC. It is worth noting that a large number of transcriptional targets are enriched in the autophagy pathway. Studies have shown that metabolic changes are a sign that cancer cells are different from normal cells (Tan et al. 2021). We all know that two major cellular pathways are included in metabolic adaptation, one is the production of adenosine triphosphate from mitochondrial oxidative phosphorylation to glycolysis, and the other is the circulation of intracellular components through autophagy (Ferro et al. 2020). Autophagy is the process of degrading damaged organelles and misfolded proteins in cells, which is important for maintaining cell homeostasis. Autophagy may promote or inhibit tumor growth in different situations. For example, tumor-prone mouse models with RAS pathway activation show a lack of autophagy, which leads to pre-tumor lesions and increased tumor incidence, suggesting that cancer development may be prevented by autophagy, while tumor cells increase autophagy flux through a variety of mechanisms that contribute to their growth in pancreatic cancer (Miller et al. 2021). In HCC, the expression of ATG2A and ATG14 has been shown to drive hypoxia-induced autophagy and the malignant progression of HCC (Li et al. 2021). Therefore, we speculate that EZH1 may play a role in the development of HCC by regulating autophagy. In addition, recent studies have shown that autophagy is associated with drug resistance in anti-tumor therapy, such as in cervical cancer (Tao et al. 2023). And autophagy inhibitors were found to have a surprisingly effective therapeutic effect when combined with molecular targeted or chemotherapeutic drugs in HCC (Wu et al. 2021). However, there are still many difficulties in exploring the combination of autophagy regulation and existing HCC treatment regimens. The significant enrichment of EZH1 putative transcriptional targets in the autophagy-related pathway provides a new theoretical basis for the treatment of autophagy inhibition in HCC. In addition, EZH1 transcriptional targets have also been found to be enriched in multiple pathways related to another pathways of metabolic adaptation, in which tumor cells promote cell proliferation by increasing energy supply. For example, fat can induce glucose metabolism in untransformed hepatocytes to promote liver tumorigenesis. Mitochondrial dysfunction causes mitochondrial oxidative phosphorylation defects and reactive oxygen species production to promote HCC progression (Lee et al. 2021). As a homologue of EZH1, there are many overlaps in the functions of EZH1 and EZH2. It has been reported that the transformation of myeloproliferative tumors into leukemia can be mediated by the metabolism of branched-chain amino acids enhanced by EZH2 inactivation (Gu et al. 2019). EZH1 may be linked to metabolic recoding. However, although research on metabolism and cancer has been accumulated for decades, the progress of targeted metabolic pathways to achieve precise treatment of cancer is slow, and the clinical benefits of cancer patients are limited (Stine et al. 2022). More in-depth research needs to be carried out to develop effective drugs. The interaction between autophagy and energy metabolism was found to play a vital role in the tumorigenesis and development of HCC. For example, autophagy can promote metastasis and glycolysis by upregulating MCT1 expression and Wnt/β-catenin signaling pathway activation in HCC cells (Fan et al. 2018). Clinical advanced progression will occur in HCC because of the production of reactive oxygen species, which is mediated by oxidative phosphorylation and autophagy (Kudo et al. 2020). In this study, we found that EZH1 transcriptional targets were co-enriched in autophagy and energy metabolism-related pathways. We speculated that EZH1 may promote HCC progression by regulating autophagy/energy metabolism.
Verification of potential transcriptional targets is helpful in exploring the mechanism of EZH1. Further exploration of the molecular regulatory axis is crucial for elucidating the mechanism of tumorigenesis and development and developing therapeutic targets (Wu et al. 2023, Hao et al. 2023). Among the multiple transcriptional targets in the autophagy pathway, EZH1 was found to have obvious binding peaks at the transcription start sites of multiple genes. Interestingly, ATG7 appears in both autophagy and energy metabolism-related pathways. On one hand, ATG7 itself is an autophagy-related gene and has been shown to be essential for inducing autophagy. It has been reported that acute systemic loss of ATG7 in mice induces greater regression of KRAS-driven cancer, suggesting that host autophagy promotes tumor growth (Poillet-Perez et al. 2018). Studies have shown that loss of ATG7 function can mediate severe fatty liver by impairing autophagy metabolism, and cohort analysis shows that loss of ATG7 autophagy function increases the risk of cirrhosis and HCC (Baselli et al. 2022). This again verifies the dual nature of autophagy in tumorigenesis and development. On the other hand, ATG7 has been reported to be related to energy metabolism. Studies have shown that ATG7 can activate dendritic cells and accelerate glycolysis (Yu et al. 2022). Moreover, decreased expression of ATG7 has also been shown to reduce autophagy and fat production at the same time (Wang et al. 2020). ATG7 may mediate the occurrence and development of tumors by regulating autophagy and energy metabolism. This study demonstrated a positive correlation between EZH1 and ATG7, and both were significantly overexpressed. Patients with high expression showed worse survival rates in HCC. In summary, we speculate that EZH1 promotes HCC progression through the autophagy/energy metabolism pathway, and that the EZH1-ATG7 regulatory axis may play an important role in it.
Based on the fact that targeted inhibition of EZH1 treatment may bring clinical benefits to HCC patients, we further verified the role of EZH1 in HCC treatment through drug sensitivity analysis. The drug sensitivity data were derived from CellMiner™ database 2.4.2, and after data processing and analysis, high expression of EZH1 was positively linked to the drug resistance of various anti-tumor drugs, suggesting that the high expression of EZH1 in HCC will mediate the resistance of patients to various drugs or even no response. Most patients with HCC are clinically advanced at the time of initial diagnosis and are no longer suitable for surgical treatment (Xu et al. 2022). For advanced HCC or patients with poor liver function, surgical resection and other local treatments are not effective and are no longer suitable for use. In this case, systemic drugs are a more effective choice (Yang et al. 2022). For example, a variety of targeted drugs, such as tyrosine kinase inhibitors, have been developed to continuously improve overall survival (Llovet et al. 2022b). Immune-checkpoint inhibitors have completely changed the clinical management of HCC in the past five years (Llovet et al. 2022a). However, the widespread drug resistance of HCC has seriously hindered the long-term clinical benefits of existing treatments; it is urgent to find new treatments to overcome this drug resistance (Bao et al. 2021). High expression of EZH1 promotes the resistance of tumor cells to anti-HCC treatment. Exploring targeted inhibition of EZH1 is expected to improve the sensitivity of patients to drugs. In addition, autophagy and energy metabolism have been shown to be associated with tumor treatment resistance. In chronic myeloid leukemia, the drug 2-DG induces severe metabolic stress, including inhibition of glycolysis and mitochondrial oxygen consumption, leading to autophagic cell death in tumor cells, thereby solving the resistance of the TKI drug imatinib. Therefore, EZH1 inhibitors may contribute to overcoming HCC systemic drug resistance by regulating autophagy/energy metabolism, and the EZH1-ATG7 axis may be an effective targeting pathway.
This article has its limitations. First, there are few prognostic data included in this study, and the prognostic value of TMC5 may not be comprehensively evaluated. Second, there are not in vivo and in vitro experiments to verify the existence and mechanism of the EZH1-ATG7 regulatory axis in HCC development and treatment. The changes of autophagy and energy metabolism in this process also need further experiments to prove.