Epsin 3 potentiates the NF‑κB signaling pathway to regulate apoptosis in breast cancer

Abstract

Endocrine drug resistance is common in some patients with estrogen receptor (ER)‑positive breast cancer, so it is necessary to identify potential therapeutic targets. The aim of the present study was to investigate the regulatory effect and mechanism of epsin 3 (EPN3) expression level changes on the proliferation and apoptosis of ER‑positive breast cancer. Online GEPIA was used to analyze the expression level of EPN3 in breast cancer. The online Kaplan‑Meier plotter tool was used to analyze the relationship between EPN3 expression and the prognosis of patients with breast cancer. Reverse transcription‑quantitative PCR, immunohistochemistry and western blotting were performed to detect the mRNA and protein expression levels of EPN3 in breast cancer tissues and cells. A lentiviral infection system was used to knockdown the expression of EPN3 in breast cancer cell lines. Cell Counting Kit‑8 and flow cytometry assays were conducted to detect the effect of EPN3 knockdown on breast cancer cell proliferation and apoptosis. Western blotting was used to detect the regulation of EPN3 expression on NF‑κB, and immunofluorescence was performed to detect the effect of EPN3 expression on NF‑κB nuclear translocation. The results demonstrated that the expression level of EPN3 in breast cancer tissues was higher compared with that in adjacent tissues (P<0.05). The expression level of EPN3 in the ER‑positive breast cancer cell line, MCF7, was higher compared with that in the other cell lines (MCF10A, ZR75‑1, MDA‑MB‑231, BT549 and SK‑BR‑3). After knocking down the expression of EPN3 in MCF7 cells, the proliferative ability of the cells was decreased, and the apoptosis rate was increased (P<0.05). After EPN3 knockdown in MCF7 cells, the phosphorylation of NF‑κB was decreased (P<0.05), and the nuclear translocation signal was weakened. Thus, it was suggested that EPN3 promoted cell proliferation and inhibited cell apoptosis by regulating the NF‑κB signaling pathway in ER‑positive breast cancer.

Keywords: NF‑κB; apoptosis; epsin 3; estrogen receptor‑positive breast cancer.

Figure 1.

EPN3 is upregulated in breast cancer. (A) Analysis of EPN3 mRNA expression in normal breast tissues and breast cancer tissues in GEPIA. *P<0.05. Kaplan-Meier analysis of (B) OS and (C) RFS in breast cancer cases with EPN3 low or high expression in Kaplan-Meier Plotter analysis. (D) Reverse transcription-quantitative PCR was used to assessed EPN3 mRNA expression in in breast cancer tissues and paired normal adjacent tissues. (E and F) Immunohistochemistry was used to assessed EPN3 protein expression in different tissues. Magnification, ×10 and ×40. **P<0.01. OS, overall survival; RFS, release-free survival; HR, hazard ratio; EPN3, epsin 3.

Figure 2.

EPN3 is associated with estrogen receptor-positive breast cancer. (A) mRNA and (B) protein expression levels of EPN3 in breast cancer cell lines, as tested via reverse transcription-quantitative PCR and western blotting, respectively. (C) EPN3-related genes were enriched in pathways as determined via KEGG analysis. (D-G) Gene set enrichment analysis was used to analyze the signaling pathways enrichment in different groups. EPN3, epsin 3; FDR, false discover rate; NES, normalized enrichment score.

Figure 3.

EPN3 promotes cell proliferation and inhibits apoptosis in breast cancer. (A and B) Western blotting and (C) reverse transcription-quantitative PCR analyses of EPN3 protein and mRNA expression in MCF7 cell transfected with shRNA1 and shRNA2 compared with NC. (D) Cell Counting Kit-8 assay was performed to examined cell proliferation. EPN3 knockdown suppressed the proliferation of MCF7 cells. (E and F) Flow cytometry was performed to assess the difference of cell cycle between shEPN3-MCF7 and NC-MCF7 cells. (G and H) Flow cytometry was performed to assess the difference in the apoptosis rate between shEPN3-MCF7 and NC-MCF7 cells. Data were represented as Mean ± SD. *P<0.05, **P<0.01. n.s., not significant; NC, negative control; shRNA/sh, short hairpin RNA; EPN3, epsin 3.

Figure 4.

EPN3 facilitates NF-κB activation in breast cancer cells. Effects of EPN3 knockdown on the (A and B) caspase family and (C and D) Bcl-2 family were assessed via western blotting. (E and F) Effects of EPN3 knockdown on phosphorylation of NF-κB (p65) were assessed via western blotting. (G) Immunofluorescent staining revealed that the nuclear translocation of NF-κB (p65) was decreased by EPN3 knockdown. Scale bar, 50 µm. (H) Gene set enrichment analysis was used to analyze the NF-κB pathway enrichment in high groups. **P<0.01. n.s., not significant; NC, negative control; shRNA/sh, short hairpin RNA; EPN3, epsin 3; p-, phosphorylated; FDR, false discover rate; NES, normalized enrichment score.

Figure 5.

EPN3 knockdown inhibits tumor growth in vivo. (A) Immunohistochemistry was used to (B) assessed EPN3 protein expression between the two groups. Magnification, ×10 and ×40. (C) Tumor volume and (D) weight were compared between the two groups. **P<0.01. NC, negative control; shRNA/sh, short hairpin RNA; EPN3, epsin 3.