The mRNA expression levels of E2F family genes among the divers cancer types
To further evaluate the mRNA expression levels of E2F family genes, we compared the differential expression between the tumor and adjacent normal tissues across all the cancer types by utilizing the TIMER database. As shown in Figure 1, the elevated expression of E2F family members was observed in various kinds of cancers including LUAD and LUSC, which indicated the crucial role of E2F family in tumor progression. Especially, in the Skin Cutaneous Melanoma (SKMC), the mRNA expression of E2F3, E2F4, E2F5 and E2F8 in the metastatic lesions were significantly increased in contrast to the primary tumors. However, the expression levels of some ones were noticeably reduced in some cancers, such as the expression of E2F2 in rectum adenocarcinoma esophageal carcinoma (READ) and the expression of E2F6 in kidney chromophobe (KICH). In addition, the E2F7 expression was found to be significantly higher in almost all kinds of cancerous tissues compared with the adjacent normal tissues. Importantly, all the E2F family members were almost elevated in LUAD and LUSC with significant differences driving us to investigate the functions in the treatment for lung cancer. Similarly, in the lung cancer statistics of The Cancer Genome Atlas, E2F1 was reported to be overexpressed in LUAD and LUSC compared with the normal tissues evidenced by the fold change of 1.067 and 1.097, respectively (Table 1). The higher expression of E2F2 was found in LUAD (fold change, 1.039) versus normal samples. In the Gao’s study, E2F2 was also suggested as one elevated expression factor and one important prognostic factor in the NSCLC [26].
E2F3 expression level was noticeably upregulated in LUAD and LUSC with a fold change of 1.075 and 1.283 (Table 1). Based on the TCGA database, higher expressed E2F5/E2F6/E2F7 was found in the lung carcinomas. A similar trend of E2F5 expression is showed in the Stearman’s study [27]. Taken together, the expression of E2F family genes was increased in most of cancers.
Genetic alterations and mRNA expression of E2F family in LUAD and LUSC
To determine the genetic variations of E2F family members in human LUAD and LUSC, several studies from TCGA were included by utilizing the cBioPortal online tool. The mutations were more frequently observed in LUAD, whereas the amplifications appeared more in the LUSC (Figure 2A). This analysis revealed the varying degrees of genetic alteration among member genes of E2F family, of which E2F5 presented the highest incidence rate of genetic variations with the rate of 3% alteration (Figure 2A). The abovementioned statements suggested that the incidences of genetic alteration varied across the E2F family members.
To detect the transcriptomic expression of E2F family genes in LUAD or LUSC and normal lung tissues, transcript per million (TPM) and mRNA expression levels were analyzed by utilizing the Gepia database. The TPM levels were almost elevated in the cancer group, except that were decreased in the E2F4 (Figure 2B). In the LUAD, the expression levels of E2F1 and E2F8 were increased with the significant differences, as well as in the LUSC, we found the elevated expression of E2F1, E2F2, E2F7 and E2F8 (Figure 2B and 2C). Furthermore, the expression of E2F family genes in the disorder stages were also detected suggesting that the E2F2, E2F4, E2F5 and E2F8 groups significantly varied (Figure 3). Amongst them, E2F2 and E2F8 were significantly higher in the later stages of the disease, which indicated that these two factors could be considered as the key markers to label the tumor progression. Collectively, based on the above analyses, E2F8 actually plays a critical role in the lung cancer.
Having identified the mRNA expression levels of E2F1, E2F2 and E2F8, the protein levels of E2F expression were examined by immunohistochemistry in LUAD and LUSC tissues and their counterparts (Figure 4). As shown in Figure 4, we observed that E2F1, E2F2 and E2F8 proteins were expressed at the higher degrees in the cancer tissues than in the normal control.
The correlation between E2F family genes and survival in LUAD and LUSC
To explore the critical efficiency of E2F family genes in the outcomes of patients with LUAD and LUSC, survival plot module of Kaplan-Meier Plotter database was excavated to analyze the correlation between the mRNA levels of E2F family and the recurrence-free survival or the overall survival. For the recurrence-free survival, the overexpression of E2F3 and E2F6 was actually associated with the improved outcomes of LUAD patients. The overexpression of E2F2 impaired the survival of patients with LUSC (Figure 5). For the overall survival, the high level of E2F6 improved the outcome of LUAD patients. Notably, the results revealed that the increased levels of E2F1, E2F4 and E2F8 were significantly associated with the poorer prognosis of LUAD patients (Figure 5). All of the E2F family members produced no significances on the prognosis of LUSC patients. The prognostic potential of E2F family genes in non-small cell lung cancer was further verified by the RNA-seq data of TCGA database.
E2F family genes are correlated with immune infiltration levels in LUAD and LUSC.
Tumor microenvironment (TME) consists of tumor cells and the surrounding environmental ingredients, which presents the high heterogenous features. Several studies have revealed that genes expressed by tumor cells affect the immune cells infiltration levels, which can further play the important roles in the tumor progression. Herein, we explored the correlation between the levels of CD4+ and CD8+ T cells infiltration and the expression of E2F family genes in LUAD and LUSC by analyzing the TIMER database. As shown in Figure 6, we found that the mRNA expression levels of E2F family members were positively related to the degree of tumor purity in LUAD and LUSC. Tumor purity, as an important factor, can influence the analysis between mRNA expression of genes and immune infiltration in tumor samples. Most of E2F family members were obviously positive correlated with infiltrating level of CD4+ and CD8+ T cells. However, the overexpression of E2F2 and E2F5 negatively correlated to the infiltration of CD4+ and CD8+ T cells in both LUAD and LUSC. In the LUSC patient group, E2F6 and E2F7 were also found to impaired the infiltration of CD4+ and CD8+ T cells (Figure 6). These findings strongly suggested that E2F family members play a specific role in immune infiltration, especially those of CD4+ and CD8+ T cells.
The abovementioned results suggested that E2F8 might specifically affect the lung cancer progression. Herein, to further investigate the association between E2F8 expression and various immune infiltrating cells, we analyzed the correlations between mRNA expression and the marker sets of various immune cells, included CD8+ T cells, CD4+ T cells, Th1 and Th2 cells, Tfh cells, Th17 cells, Tregs cells, exhausted T cells, B cells, monocytes, TAMs, M1 and M2 macrophages, neutrophils and dendritic cells in LUAD and LUSC (Table 2). The results of correlation adjusted by tumor purity revealed that the E2F8 expression level was significantly correlated with most immune cells in LUAD and LUSC. Interestingly, the infiltration of anti-tumor T cells such as CD8+ and CD4+ cells were inhibited by E2F8. On the contrary, Treg cells, TAMs, or exhausted T cells were promoted to infiltrate in the TME. These results indicated that E2F8 may play a vital role in immune escape in the lung cancer microenvironment.
To evaluate the effects of immune cell infiltration on the prognosis of non-small cell lung cancer, the OS analysis of immune infiltration levels was performed including CD4+ T cells, CD8+ T cells, NK cells, Treg cells, B cells, macrophages and cancer-associated fibroblasts based on the TIMER database. The results showed that high levels of CD4+ T cells, Treg cells and macrophages would lead to the worse outcomes of patients (Figure 7).
The function of E2F family genes in LUAD and LUSC progression.
As previously reported, EGFR plays the crucial role in the lung cancer progression and Ki-67 works as an important mark to label the proliferation. Thus, we investigated the correlation of E2F family members with EGFR and Ki-67 expression using the Gepia online tool. As shown in Figure 8A and 8B, EGFR positively correlated with E2F2, E2F4, E2F6~8 and Ki-67 expression was significantly associated with all the E2F family members, which suggested that E2F family genes mostly contributed to the proliferation of tumor cells. To detect the connections with other functional molecules, the network was constructed by analyzing the E2Fs family genes and the 50 top frequently altered neighbor genes. We observed that the cell cycle-related specific genes (CCNE1, CCNE2, CCNF, CDK2 and CDC7), proliferation-related genes (PCNA), epigenetic alteration associated genes (EZH2 and FBXO5) were closely associated with E2F family clusters (Figure 8C).
To identify the signaling pathways associated with E2F family genes differentially activated in LUAD and LUSC, GO enrichment and Gene set enrichment analysis (GSEA) were conducted to obtain potential biological process enriched in non-small cell lung cancer with high expression of E2F family genes. Similar to the molecule networks, the GO enrichments indicated that the cellular growth associated pathways were significantly activated in LUAD and LUSC, such as Cell cycle, DNA replication, ATPase activity, Mitotic Nuclear Division and Catalytic Activity on RNA (Figure 8D). All gene sets that positively or negatively associated with E2F family genes were shown in Table 3. The result of GSEA revealed that immune progresses were positively correlated with E2F family gene expression in LUAD and LUSC, including the INFLAMMATORY_RESPONSE, ALLOGRAFT_REJECTION, IL6_JAK_STAT3_SIGNALING, and HALLMARK_INTERFERON_ALPHA_RESPONSE pathway. Therefore, these results further confirmed the findings that E2F family genes were specifically correlated with tumor cell proliferation and immune infiltration in LUAD and LUSC.
The scRNA expression of E2F family genes in non-small cell lung cancer
To analyze the molecular features of malignant and non-malignant cells in the NSCLC, we obtained 12,193 single cells (GSE143423) from NSCLC based on the TISCH database. To classify the major cell types in the tumor, we performed UMAP analysis and identified seven major clusters: malignant cells, CD8 T cells, Endothelial cells, Monocytes/macrophages, Oligodendrocytes, Pericytes and plasma (Figure 9A). To further explore the E2F family gene expression in each cluster, the used random forest6 analysis was used to identify that E2Fs were mainly expressed in the malignant cells, of which E2F2 and E2F4 stated at the highest levels (Figure 9B). By analyzing differentially expressed genes among the clusters, GO enrichment revealed that “E2F targets”, “G2M checkpoints” and “PI3K-AKT-MTOR”signaling pathways were activated in the malignant cells (Figure 9C), which suggested that E2F family genes in malignant cells significantly correlated to the cell proliferation to promote tumor progression.