E2F4 transcription factor is a prognostic biomarker related to immune infiltration of head and neck squamous cell carcinoma

Abstract

To investigate the relationship between the transcription factor, E2F4, and head and neck squamous cell carcinoma (HNSCC), and to preliminarily explore the signaling pathways and immunological role of E2F4. The mRNA expression of E2F4 in HNSCC was evaluated by searching Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. E2F4 protein expression was analyzed by immunohistochemistry using the CMU1h-ENT database. The association between E2F4 expression and tumor infiltration of immune cells was analyzed. Intracellular signaling by E2F4 was explored using KEGG and GO analysis. The correlation of E2F4 expression with clinical characteristics and its prognostic role were validated and analyzed in TCGA database. From the analysis of GEO and TCGA data, E2F4 expression was found to be up-regulated in HNSCC tumor tissues, and its level was associated with T, Grade, and M staging. Kaplan-Meier curve and Cox analyses indicated that the high expression of E2F4 was related to a poor prognosis. Thus, E2F4 was considered a potential prognostic factor for HNSCC. Immunohistochemical staining showed that E2F4 was mainly localized in the cell nucleus; it was highly expressed in HNSCC tissues, with a significant difference noted from that in pericancerous mucosa tissues. A correlation was observed between the differential expression of E2F4 and the immune infiltration of HNSCC. As revealed by KEGG and GO analysis, differential enrichment was found in the cell cycle, spliceosome, meiosis, microbial polysaccharide synthesis, and WNT signaling pathway, as well as in cyclic adenosine monophosphate, ERBB2, VEGF, GCNP and MYC pathways. E2F4 plays an important role in tumor progression and may be a critical biological prognostic factor for HNSCC. In addition, it functions in the nucleus as a transcription factor, regulates immune cells, and could be a promising molecular target for the diagnosis and treatment of HNSCC.

Figure 1

The workflow graph of this study.

Figure 2

Differential expression of E2F4 in GEO datasets.

Figure 3

Relationship between E2F4 expression and prognosis of HNSC patients based on the TCGA database. (A) Differential expression of E2F4 in HNSC tumor and normal tissues. (B) The survival curve for the differential expression of E2F4 was analyzed using GEPIA.

Figure 4

Correlation between E2F4 expression and clinicopathologic characteristics. (A) Subgroup analysis of T classification (T1, T2 and T3, T4). (B) Subgroup analysis of pathologic tumor grade (grades I, II and grades III, IV). (C) Subgroup analysis of M classifications (M0 and M1).

Figure 5

Bioinformatics analysis of E2F4 using the TCGA database (n = 544). (A) Univariate analysis of E2F4. (B) Multivariate analysis of E2F4. (red indicates positive correlation; green indicates negative correlation. P < 0.05).

Figure 6

E2F4 is expressed in HNSCC and normal pericancerous mucosal tissues. (A–C) Low expression of E2F4 in normal pericancerous tissues (×100, ×200, ×400). (D–F) High expression of E2F4 protein in well-differentiated HNSCC specimens (×100, ×200, ×400). (G–I) High expression of E2F4 protein in moderately differentiated HNSCC specimens (×100, ×200, ×400). (J–L) High expression of E2F4 protein in poorly differentiated HNSCC specimens (×100, ×200, ×400).

Figure 7

The main immune cells influenced by E2F4 expression included 17 subsets. The number of memory T cells and macrophages (R = 0.121, P = 0.00741) was increased, while CD8 + T and Treg cells numbers were decreased (R = − 0.185, P < 0.001; R = − 0.232, P < 0.001).

Figure 8

The E2F4 expression level demonstrated a significantly negative correlation with infiltration levels of CD4+ T (R = − 0.185, P = 1.47e−04), CD8+ T (R = − 0.185, P = 3.47e−05), Treg (R = − 0.232, P = 2.03e−07), and T cell follicular helper (R = − 0.208, P = 3.34e−06) cells, and M2 macrophages (R = 0.121, P = 7.41e−03) in HNSCC.