E2F3 renders an immunosuppressive tumor microenvironment in nasopharyngeal carcinoma: Involvements of the transcription activation of PRC1 and BIRC5

Abstract

Background: E2F transcription factors are well-recognized oncogenic molecules, and their correlation with immune cell infiltration has recently been reported. This work studies the impacts and mechanism of E2F transcription factor 3 (E2F3) in the growth and tumor microenvironment (TME) of nasopharyngeal carcinoma (NPC).

Methods: Aberrantly expressed transcription factors in NPC were screened by abundant bioinformatics analyses. Gene expression in NPC cells was analyzed by reverse transcription-quantitative polymerase chain reaction and Western blot analyses. Malignant behaviors of NPC cells were analyzed by cell counting kit-8, 5-ethynyl-2'-deoxyuridine labeling, Transwell assays, and xenograft tumor models. TPA-induced THP-1 cells (macrophages) were cultured in the conditioned medium of NPC cells to mimic tumor-associated macrophages (TAMs) in vivo, and these TAMs were cocultured with CD8⁺ T cells. Regulation of E2F3 on protein regulator of cytokinesis 1 (PRC1) and baculoviral IAP repeat containing 5 (BIRC5) was validated by chromatin immunoprecipitation and luciferase reporter assays.

Results: E2F3 was highly expressed in NPC cells, and its knockdown suppressed malignant behavior and tumorigenic ability of the cells. The E2F3 knockdown condition downregulated M2 cytokines CD163 and interleukin-10 in TAMs, which further enhanced proliferation and activation of the cocultured CD8⁺ T cells. E2F3 promoted transcription of PRC1 and BRIC5. Furthermore, PRC1 or BRIC5 upregulation in NPC cells restored the malignant properties of NPC cells, reprogrammed the TAMs to M2 phenotype, and suppressed the CD8⁺ T cell proliferation and activation.

Conclusion: This work suggests that E2F3 renders an immunosuppressive TME in NPC by activating PRC1 and BIRC5. Suppression of any member involved might favor tumor elimination.

Keywords: E2F transcription factor 3; baculoviral IAP repeat containing 5; macrophages; nasopharyngeal carcinoma; protein regulator of cytokinesis 1; tumor microenvironment.

Figure 1

E2F3 is abundantly expressed in NPC cells. (A) Volcano plots for DE genes between NPC and control in the GSE12452, GSE13597, GSE53819, and GSE64634 datasets; (B) a Venn diagram for the intersecting factors among the DE genes and human transcription factors; (C) expression profiles of PAX5, LHX2, POU2F2, E2F3, KLF15, and HOXC6 in head and neck squamous cell carcinoma in ULACAN system; (D) E2F3 mRNA, and protein expression in NP69, HNE3, C666‐1 and HK1 cells detected by RT‐qPCR and WB analysis (n = 3, one‐way ANOVA). ANOVA, analysis of variance; DE, differentially expressed; NPC, nasopharyngeal carcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; WB, Western blot. *p < .05.

Figure 2

E2F3 knockdown blocks malignant properties of NPC cells. (A) E2F3 mRNA and protein expression in HNE3 and C666‐1 cells after shRNA administration determined by RT‐qPCR and WB analysis (n = 3, two‐way ANOVA); (B, C) viability (B) and DNA replication (C) of NPC cells determined by CCK‐8 and EdU labeling assays (n = 3, two‐way ANOVA); (D, E) migration (D) and invasion (E) of NPC cells tested by Transwell assays (n = 3, two‐way ANOVA). ANOVA, analysis of variance; NPC, nasopharyngeal carcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; WB, Western blot. *p < .05.

Figure 3

E2F3 knockdown in NPC cells reprograms the phenotype of TAMs and promotes CD8⁺ T cell activation. (A) Expression of M2 cytokines CD163 and IL‐10 in the conditioned medium (of NPCs)‐stimulated TAMs examined by RT‐qPCR (n = 3, two‐way ANOVA); (B, C) proliferation of (B) and the expression of IFN‐γ in (C) CD8⁺ T cells after coculture with TAMs examined by flow cytometry (n = 3, two‐way ANOVA); (D) growth rate of xenograft tumors formed by HNE3 cells (n = 5, two‐way ANOVA); (E) tumor weight on Day 30 (n = 5, unpaired t test); (F, G) infiltration of M2 TAMs (F) and CD8⁺ T cells (G) in tumor tissues analyzed by flow cytometry (n = 5, unpaired t test). ANOVA, analysis of variance; NPC, nasopharyngeal carcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; TAM, tumor‐associated macrophage. *p < .05.

Figure 4

E2F3 downregulation suppresses transcription of PRC1 and BIRC5. (A) Intersections of downstream targets of E2F3 and the DE genes in the GSE12452, GSE13597, GSE53819, and GSE64634 data sets; (B) a protein–protein interaction network of the 10 intersecting factors; (C) expression profiles of PRC1 and BIRC5 in the UALCAN system; (D) mRNA expression of PRC1 and BIRC5 in sh‐E2F3‐introduced NPC cells examined by RT‐qPCR (n = 3, two‐way ANOVA); (E) protein levels of PRC1 and BIRC5 in sh‐E2F3‐introduced NPC cells examined by WB analysis; (F) putative binding sites of E2F3 with the promoters of PRC1 and BIRC5 predicted in the hTFtarget system; (G) PRC1 and BIRC5 promoter fragments enriched by E2F3 protein examined by the ChIP‐qPCR assay (n = 3, two‐way ANOVA); (H) transcriptional regulation of E2F3 on PRC1/BIRC5 examine by dual‐luciferase reporter gene assay (n = 3, two‐way ANOVA). ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; DE, differentially expressed; NPC, nasopharyngeal carcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; WB, Western blot. *p < .05.

Figure 5

PRC1 or BIRC5 overexpression rescues the malignant behavior of NPC cells. A, mRNA and protein levels of PRC1 (A) and BIRC5 (B) in NPC cell lines examined by RT‐qPCR and WB analysis (n = 3, two‐way ANOVA); (C, D) viability (C) and DNA replication (D) of NPC cells determined by CCK‐8 and EdU labeling assays (n = 3, two‐way ANOVA); (E, F) migration (E) and invasion (F) of NPC cells tested by Transwell assays (n = 3, two‐way ANOVA). ANOVA, analysis of variance; CCK‐8, cell counting kit‐8; EdU, 5‐ethynyl‐2′‐deoxyuridine; NPC, nasopharyngeal carcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; WB, Western blot. *p < .05.

Figure 6

PRC1 or BIRC5 overexpression blocks the E2F3 silencing‐mediated immune activation. (A) Expression of M2 cytokines CD163 and IL‐10 in the conditioned medium (of NPCs)‐stimulated TAMs examined by RT‐qPCR (n = 3, two‐way ANOVA); (B, C) proliferation of (B) and the expression of IFN‐γ in (C) CD8⁺ T cells after coculture with TAMs examined by flow cytometry (n = 3, two‐way ANOVA); (D) growth rate of xenograft tumors formed by HNE3 cells (n = 5, two‐way ANOVA); (E) tumor weight on Day 30 (n = 5, one‐way ANOVA); (F, G) infiltration of M2 TAMs (F) and CD8⁺ T cells (G) in tumor tissues analyzed by flow cytometry (n = 5, one‐way ANOVA). ANOVA, analysis of variance; NPC, nasopharyngeal carcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; TAM, tumor‐associated macrophage. *p < .05.