Malignant mesothelioma is a rare primary cancer with a survival of less than 1 year[1]. A trimodality treatment approach combining surgery, chemotherapy, and sequential radiation therapy (RT) represents the mainstream of current mesothelioma treatment options[7]. But survival is still not improved, and prognostic biomarkers are urgently needed to guide clinical decision-making. While the TCGA database is able to obtain a large number of data from mesothelioma patients, we searched for potential new prognostic biomarkers from them based on the TCGA database.
EZH2 is the catalytic subunit of polycomb repressive complex 2 (PRC2) that represses target genes by H3K27me3[8]. In addition, highly activated EZH2 mutations are frequently found in the early stages of cancer, suggesting a role for activated PRC2 cancer drivers. Excessive EZH2 expression, which alters histone modifications, leads to an aberrant epigenetic landscape that may contribute to cancer progression[9]. Based on this, we performed the correlation between EZH2 mRNA levels and the prognosis of mesothelioma patients and immune infiltration analysis based on the TCGA database.
We collated the data obtained from the TCGA database and performed univariate and multivariate analysis of general data including age, gender, stage, pathological type, and whether they had received radiotherapy, and the results showed that high expression of EZH2 was associated with poor prognosis in mesothelioma patients. The results of survival analysis of EZH2 expression group showed that the survival time of low expression group was 26.3(23-32.7) months; the survival time of high expression group was 13.7(9.9-17.9) months. The EZH2 expression was an independent prognostic factor for the survival of mesothelioma patients (HR=2.63, 95% CI: 1.02-6.79, P=0.046) (Fig.1B). As shown in Fig.1A, ROC curves showed that EZH2 expression predicted 1-year survival with an AUC of 0.740, 2-year survival with an AUC of 0.756, and 3-year survival with an AUC of 0.692 (Fig.1D), which indicated that EZH2 expression had a good predictive effect on prognosis. To further investigate the mechanism of EZH2 action we performed further analysis and performed KEGG pathway analysis by GSEA software to investigate the potential biological function of EZH2. KEGG pathway analysis showed that there were five pathways with the strongest positive correlation with EZH2 expression: Cell cycle, DNA replication, Cell adhesion molecule, primary immunodeficiency, and taste conduction; and five pathways with the strongest negative correlation with EZH2 expression: glycolytic gluconeogenesis pathway, drug metabolism cytochrome P450 pathway, retinol metabolism pathway, fatty acid metabolism pathway, and ribosome pathway (Fig.2). These results suggest that pathways regulating cell cycle control and fatty acid metabolism, glycolysis, and gluconeogenesis are essential in mesothelioma patients, and they are closely associated with EZH2 expression.
The complex interplay between tumors and their microenvironment remains to be elucidated. Recently, it has been found that immune infiltrating components change at every tumor stage, and specific cells have an important impact on survival. As tumors progress, the density of TH cells and NK cell increases, while the density of most T cells decreases. B cells are a key role in the core immune network and are associated with prolonged survival, but high expression in advanced patients has a dual role in promoting tumor recurrence and progression, so they have a dual role in tumor development. Our results showed that EZH2 was closely related to the survival of mesothelioma patients. In order to explore the relationship between EZH2 and tumor immune microenvironment, we analyzed the correlation between EZH2 expression in mesothelioma tissues and immune infiltration level using TIMER. These results indicate that EZH2 expression plays an important role in immune infiltration. In addition, we also sought to determine whether there is a difference in the tumor immune microenvironment between mesothelioma patients with high EZH2 levels and mesothelioma patients with low EZH2 levels. Among them, in NK cells, High expression group was lower than low expression group (P=0.023). In Mast cells, high expression group was lower than low expression group (P=0.040). In TH cells, high expression group was higher than low expression group (P=0.021). In Th17 cells, high expression group was lower than low expression group (P=0.005). In Th2 cells, high expression group was higher than low expression group (P<0.001).
There is increasing evidence that EZH2 can not only inhibit tumor genes, but also participate in regulating collective immune homeostasis and regulating immune-related cells, especially in the development, differentiation and function of T cells[10]. EZH2 has some regulatory effects on T cell differentiation and epigenetic inheritance of Treg function. Pharmacological inhibition of EZH2 in human T cells using CPI-1205 caused phenotypic and functional alterations in Tregs and enhanced the cytotoxic activity of Teffs. Regulating EZH2 expression in T cells could improve the antitumor response elicited by anti-CTLA-4 treatment[11]. EZH2 inhibition was found to restore the cytotoxic response of CD8 T cells in patients with systemic lupus erythematosus (SLE), reducing the incidence of infection and thus reducing death due to infection[12]. Shane et al showed that the phosphorylation status of EZH2 determines its ability to maintain anti-tumor immunity in CD8 + T memory precursor cells, and Akt-mediated EZH2 phosphorylation is a key target for enhancing anti-tumor immunotherapy strategies[13].
In addition, it has been shown that EZH2 is associated with tumor resistance and its inhibitors can overcome resistance to immunotherapy. EZH2 is a major driving force in cancer cellimmunoediting. It mediates immune escape by down-regulating immune recognition and activation, up-regulating immune checkpoints and generating an immunosuppressive tumor microenvironment[14]. Knockdown or inhibition of EZH2 upregulated MET expression and phosphorylation and improved cell proliferation and EGFR-TKI resistance in vitro. Inhibition of MET or PI3K/AKT elevated EZH2 levels and restored sensitivity to EGFR-TKIs. These findings suggest that the “MET-AKT-EZH2” feedback loop regulates EGFR-TKI resistance[15]. EZH2 was also highly expressed in lung cancer with positive KRAS expression, showing a positive correlation. The expression of EZH2 was positively correlated with the expression of BRAF, especially in lung squamous cell carcinoma. High expression of EZH2 and its possible synergy with KRAS and BRAF mutations[3].
EZH2 can regulate the metabolic activity of tumor cells through epigenetic regulation, which in turn affects tumor progression[16], the famous Warburg Effect indicates that cancer cells can remodel their glucose metabolism even under hypoxia[17] As can be seen from our results, the expression of EZH2 is closely related to the glycolytic gluconeogenesis pathway. In a study, Brookes et al. found that up-regulation of EZH2 can increase intracellular deoxyglucose levels and induce a slight increase in mitochondrial oxidative capacity. However, cells overexpressing EZH2 may also rely on enhanced glycolysis as their main energy source. EZH2 can promote tumorigenesis and malignant progression by activating glycolysis through EAF2-HIF1α signaling axis[18]. Accumulating evidence demonstrates that EZH2, as the catalytic subunit of PRC2, also plays a key role directly or indirectly in the metabolic process of cancer cells[16]. Recent studies have shown that EZH2 expression is significantly downregulated in response to glucose deprivation in glucose-sensitive colorectal cancer cell lines, and that EZH2 knockout cells are more resistant to glucose deprivation. EZH2 deficiency upregulates glutaminase (GLS) expression and promotes glutamate production, which in turn leads to increased intracellular glutathione (GSH) synthesis and ultimately attenuates glucose deprivation-induced reactive oxygen species (ROS)-mediated cell death[19].