In this study, we first analyzed the expression levels of ZEB2 in pan-cancer based on the integrated data from TCGA and GETx databases. And the results indicate that the expression level of ZEB2 was significantly upregulated in 5 caner types and downregulated in 17 kinds of cancer (Fig. 1A, C). It is worth noting that the expression levels of ZEB2 were found to be downregulated in all gastroenteric tumor cell lines (Fig. 1B). The genomic alteration analysis results appeared to rule out the association between ZEB2 and genomic alterations in cancers but stopped short of definitively concluding why ZEB2 was differentially expressed between normal tissues and tumor tissues (Fig. 1D, E). A study of gut microbiota shows that ZEB2, a regulator of epithelial-mesenchymal transition, promotes the development of spontaneously invasive colorectal cancer in mice in a microbiota-dependent manner, and removal of the microbiota completely inhibited ZEB2-induced colorectal cancer16. Others also found that ZEB2 overexpression was highly associated with EMT which was implicated in the early stages of metastasis and/or cancer recurrence changes by disrupting the normal balance between differentiation and drug resistance of cancer cells29,30. However, there are several studies have reported that ZEB2 expression is essential for differentiation, maturation, and/or function of CD8+ cytotoxic T cells (CTLs) and NK cells, 2 types of immune cells involved in antitumor immune response31,32. This data suggests that the dysregulation of ZEB2 may contribute to COAD progression, it also raises a question that why ZEB2 was abnormally expressed in COAD and its role in the molecular mechanisms underlying COAD.
Therefore, we examined the relationship between ZEB2 expression and the clinical prognosis of COAD patients. First, we replicated the expression analysis of ZEB2 based on the integrated data from TCGA and GETx databases, and we also analyzed the ZEB2 expression level in TCGA database or microarray datasets (GSE87211 and GSE39582) separately. Consistent with above-mentioned results, we found that the ZEB2 expression levels were significantly downregulated in tumor tissues (Fig. 2A, B). Subsequently, we divided the TCGA datasets into high ZEB2 expression group (ZEB2_High Expression) and low ZEB2 expression group (ZEB2_Low Expression) according to the expression level of ZEB2, and performed survival analysis (Fig. 2D). As shown in the figures, the overall survival (OS) and progression-free survival (PFS) of the high ZEB2 expression group were significantly better than those of the low ZEB2 expression group, indicating that ZEB2 expression was positively related to a favorable prognosis of COAD patients. Our findings imply that ZEB2 may play an imperative part in predicting the prognosis of COAD patients and is expected to become a prognostic biomarker in COAD.
To illuminate the underlying molecular mechanism of how ZEB2 affects the prognosis of COAD patients, we further performed differential genes expression analysis between the high and low ZEB2 expression groups of COAD patients. We performed principal component analysis (PCA) on the grouping method before doing the variance, and the results showed that COAD patients could be well differentiated into two groups via this grouping method. We selected differentially expressed genes according to the criterion of P < 0.05, |logFC|>1. Subsequently, we performed pathway enrichment for selected genes. In GO enrichment analysis (Gene Ontology analysis), Biological Process (BP) found membrane potential regulation, Cellular Component (CC) found synaptic membrane structure, and Molecular Function (MF) found cation channel and other cellular components have statistical significance. KEGG pathway enrichment analysis found that pathways such as neutrophil extracellular trap pathway were closely related to ZEB2. Previous study has identified 23 genes that was released from human neutrophils as NETs characteristic by proteins enrichment. Therefore, we put above-mentioned proteins into the string to get PPI network, and the results showed that 23 proteins were found to be closely related to ZEB2, including MPO, LTF, ACTN1, ENO1, ACTB, LYZ, etc. The actin family was shown to play an essential role in enabling the release of nuclear DNA by neutrophils during NET formation33. Therefore, we hypothesized that ZEB2 may mediate the function of NETs by binding ACTB. From this point of view, consistent with previous studies, ZEB2 may be able to promote the proliferation, migration and invasion of COAD. Increasing evidences demonstrate that NETs are central elements of the innate immune response in cancers34–37, providing a direction for further exploration of the role of ZEB2 in tumors.
The expression of ZEB2 was positively associated with the infiltration of mast cells resting, B cells naive, macrophages M1, macrophages M2, and T cells CD4 naive. The relationships between the ZEB2 expression and immunosuppressive and immunostimulatory genes were also analyzed. We found that ZEB2 overexpression is closely associated with most kinds of cytokines, chemokines, and HLA histocompatibility antigen. The negative correlation between immunosuppressants and immunostimulants in ZEB2 high expression group further reflects the complexity of the tumor microenvironment.
However, we found that overexpression of ZEB2 was positively correlated with HLA class II histocompatibility antigen, which have been reported to act an important role in tumor associated antigen presentation and CD4 + T cell activation38. Since CD4 + T cells can play an antitumor role by directly inhibiting the cell cycle of tumor cells and indirectly pro-inflammatory/immune effects, we thought that may explain by how the ZEB2 overexpression was associated with better prognosis in patients with COAD39. In general, these results indicated a potential mechanism for ZEB2-associated immune activation