TEAD4 is overexpressed in glioma cancer and correlated with malignancy grades and the 1p/19q codeletion status
According to a previous study, TEAD4 is overexpressed in cancer, and it has emerged as a novel prognostic marker for a variety of cancers, including gastric cancer, breast cancer, colorectal cancer, melanoma, and head-neck squamous cell carcinoma. To identify the novel correlation between TEAD4 expression and clinical outcome, we first assayed the relative TEAD4 mRNA expression level in LGG specimens and compared the level with that in normal brain tissues using the CGGA database. As shown in Figure 1A, the TEAD4 level was significantly up-regulated in glioma tissues compared with respective normal tissues. To investigate the relationship between TEAD4 expression and glioma grade, we examined the TEAD4 mRNA level from grade WHO II and III glioma tissues using data from both CGGA and TCGA databases. We found that the TEAD4 mRNA level was significantly increased in WHO III glioma tissues, indicating increased TEAD4 expression was closely correlated with a higher WHO grade (Figure 1B, C).
In addition, we investigated whether TEAD4 expression was associated with classic genetic alterations in glioma, including the IDH mutation and the 1p/19q codeletion. As there were few LGGs with both the IDH wildtype and the 1p/19q codeletion, we mainly focused on three molecular subgroups: (1) the IDH mutation and the 1p/19q codeletion (muIDH + Codel); (2) the IDH mutation and the 1p/19q non-codeletion (muIDH + Non-codel); and (3) the IDH wildtype and the 1p/19q non-codeletion (wtIDH + Non-codel). We observed that TEAD4 expression was decreased in muIDH + Codel LGG specimens compared with muIDH + Non-codel LGG specimens in CGGA and TCGA datasets (Figure 1D, E). However, we did not observe significant differences in the TEAD4 mRNA level between muIDH + Non-codel and wtIDH + Non-codel gliomas in both databases. This analysis indicated that the 1p/19q codeletion, but not the IDH mutation, can contribute to the down-regulation of the TEAD4 level.
To verify the data from CGGA and TCGA databases, we collected glioma specimens from the Human Protein Atlas and examined the TEAD4 protein level in glioma and normal tissues using immunohistochemistry data. We found that the TEAD4 level was higher in gliomas compared with respective normal tissues. In addition, TEAD4 displayed significantly stronger staining in high-grade (++) tumor specimens compared with low-grade (+) counterparts in immunohistochemistry assays (Figure 1F). These results indicated that the elevated TEAD4 level is highly associated with the statistically significant probability of an increased glioma malignancy grade.
TEAD4 up-regulation is correlated with decreased life span
To confirm our conclusion, we graphed the survival curve of LGG patients from CGGA and TCGA databases according to the differential expression of TEAD4. The statistical analysis of TEAD4 expression with regard to life span was performed using Kaplan–Meier curves. These results showed that LGG patients with higher TEAD4 expression exhibited a significantly poorer outcome and a shorter median overall survival (Figure 2A, B). Furthermore, as a survival predicting factor, ROC curve analysis provided a good predictive value of TEAD4 expression in LGG specimens. The AUC values of TEAD4, 0.613 and 0.660, respectively, in two cohorts (Figure 2C, D), also confirmed that high TEAD4 expression was associated with a statistically significant probability of increased malignancy and decreased life span.
TEAD4 overexpression promotes glioma proliferation, colony formation, and migration in vitro
To elucidate the role of TEAD4 in glioma cells, we overexpressed TEAD4 in U87 and U251 cell lines and compared the findings with those of the negative control (NC) group (Figure 3A). We carried out transwell assays to analyze the effects of TEAD4 overexpression on glioma cell migration. The migratory ability was significantly increased upon TEAD4 overexpression. These results showed that cell migration was enhanced in TEAD4-OE cells (Figure 3B, C).
Next, CCK-8 assay results showed that TEAD4 overexpression promoted glioma cell proliferation (Figure 3D). In addition, colony formation assay results demonstrated that TEAD4 overexpression significantly increased the number of glioma cell colonies (Figure 3E). In conclusion, these results indicated that TEAD4 can promote glioma cell migration, proliferation, and colony formation in vitro.
GSEA and PPI of the TEAD4 expression phenotype
In light of the aforementioned findings, we confirmed that TEAD4 plays an oncogenic role in LGGs. To gain insight into the underlying mechanisms, we utilized GSEA to identify GO items and KEGG pathways enriched in TEAD4 high and low expression phenotypes. A NOM p-value < 0.05 and a FDR q-value < 0.05 were taken as the thresholds. We presented the five most enriched GO terms and KEGG pathways in the TEAD4 high expression group (Table 2) and ordered the data according to the NES. As shown in Figure 4A, five KEGG pathways, including those involved in viral myocarditis, glycan biosynthesis, antigen processing and presentation, autoimmune thyroid disease, and the intestinal immune network for IgA production, were significantly enriched in the TEAD4 high expression group. Five GO terms, including antigen processing and presentation, tumor necrosis factor-mediated signaling, antigen processing and presentation of peptide antigens, lymphocyte apoptosis, and response to tumor necrosis factor, exhibited the strongest positive correlation with high TEAD4 expression (Figure 4B). Meanwhile, no gene sets showed significantly differential enrichment in the TEAD4 low expression phenotype.
To further understand the connection between TEAD4 low and high expression phenotypes, we constructed a PPI network of DEGs. “MCODE” (Figure 4C) and “cytoHubba” (Figure 4D) were applied to identify the key cluster and hub genes in the network. The cluster and hub genes in the network included immune checkpoint genes (PDCD1, IDO1, ICOS), cytokines (IL-10, CXCL11), cytokine receptors (CCR2, CXCR3, CXCR6, IL2RA), and some other mediators of immune function (CD19, CD80, CD40LG, GATA3, SLAMF1). We then annotated the biological functions of the top six hub genes and observed that multiple immune processes were significantly enriched (Figure 4E).
Taken together, GSEA showed nearly all the top enriched gene sets associated with immune processes (Table 2) and PPI results revealed the hub genes were highly immune-related.
Association of glioma purity with TEAD4 expression and multivariate analysis
Given the immune phenotype of TEAD4 in LGG specimens, we further investigated the role of TEAD4 in immune cell infiltration. We used the “ESTIMATE” package in R to infer the tumor purity as well as stromal and immune scores of LGG specimens. Stromal and immune scores were positively correlated with the infiltration level of stromal cells and immune cells. The increased TEAD4 level correlated with elevated stromal and immune scores, but reduced tumor purity in LGG patients (Figure 5A), indicating the negative association of glioma purity with TEAD4 expression in LGG.
Next, we investigated whether tumor purity or TEAD4 expression was an independent prognostic biomarker among other factors. We performed correlation univariate analysis using Cox regression. These results revealed the relationship between overall survival and age, grade, tumor purity, and TEAD4 expression (Table 3). Specifically, clinical outcome showed a negative correlation with age, tumor grade, and TEAD4 expression but a positive correlation with tumor purity. Subsequently, multivariate Cox regression analysis revealed that tumor purity independently correlated with a better survival consequence (HR = 0.076, P = 0.034). Age (HR = 1.059, P < 0.001), grade (HR = 2.422, P < 0.01), and TEAD4 expression (HR = 1.171, P = 0.015) were independent prognostic indicators of LGG (Table 3, Figure 5B).
TEAD4 overexpression affects TIICs in LGG
According to the analysis of glioma purity, we further aimed to explore the connection between TEAD4 expression and the specific types of TIIC by TIMER and CIBERSORT assays. Consistent with the results observed from the ESTIMATE algorithm assay, TEAD4 expression was negatively correlated with glioma purity (r = −0.039, P = 2.93e-01) (Figure 5A). As immune cell infiltration indicated glioma progression and a higher malignancy grade, we tested the correlation between TEAD4 expression and immune cell infiltration by the TIMER assay. These results showed that TEAD4 expression was positively correlated with the infiltration level of multiple immune cells, including B cells (r = 0.326, P = 2.76e-13), CD8+ T cells (r = 0.138, P = 2.48e-03), CD4+ T cells (r = 0.445, P = 1.79e-24), macrophages (r = 0.369, P = 1.14e-16), neutrophils (r = 0.474, P = 5.14e-28), and dendritic cells (r = 0.501, P = 1.41e-31) in LGG (Figure 5A). Meanwhile, an elevated infiltration level of these six types of immune cells was associated with a worse cumulative survival rate in LGG (Figure 5B).
To further confirm our conclusion, we employed CIBERSORT to access the proportions of 22 different types of TIICs in LGG specimens. As presented in Figure 5C and D, plasma cells, CD8+ T cells, and macrophages M1 and M2 exhibited higher proportions with elevated TEAD4 expression compared with counterparts in both CGGA and TCGA databases. Furthermore, the proportion of naive CD4+ T cells was significantly decreased in the TEAD4 high expression group. These results indicated a largely triggered immune cell infiltration in LGG patients with higher TEAD4 expression, especially in those with high malignancy grade tumors, resulting in decreased overall survival.
TEAD4 is positively associated with immune checkpoint genes
Recently, the immune checkpoint blockade has shed light on the novel immune-based therapeutic management of many cancers, including glioma. Stimulated immune checkpoint genes can lead to T cell exhaustion and escape from immune surveillance . Clinical trials targeting CTLA-4 or PD-1/PD-L1 are currently underway, although initial results have been unpromising . Moreover, some immune checkpoint genes were found to be essential components of the hub gene network, as discussed in the “PPI” section. We explored the association between the expression of TEAD4 and multiple immune checkpoint genes, including CTLA4, PDCD1, PD-L1, CD39, TIM-3, and B7-H3 in LGG, and TEAD4 expression was positively correlated with these six genes. These results indicated that LGGs with an elevated TEAD4 level exhibited a higher expression of immune checkpoint genes, suggesting an evasion of immune surveillance. Thus, it would be beneficial for LGGs with an elevated TEAD4 level to block immune checkpoint genes, and we suppose that a combinatorial approach of immunotherapy and targeted therapy can optimize efficacy and reduce chemoresistance.