Metformin suppresses NFE2L1 pathway activation to inhibit gap junction beta protein expression in NSCLC

Abstract

Objective: Non-small-cell lung cancer (NSCLC) is a deadly form of cancer that exhibits extensive intercellular communication which contributed to chemoradiotherapy resistance. Recent evidence suggests that arrange of key proteins are involved in lung cancer progression, including gap junction proteins (GJPs).

Methods and results: In this study, we examined the expression patterns of GJPs in NSCLC, uncovering that both gap junction protein, beta 2 (GJB2) and gap junction protein, beta 2 (GJB3) are increased in LUAD and LUSC. We observed a correlation between the upregulation of GJB2, GJB3 in clinical samples and a worse prognosis in patients with NSCLC. By examining the mechanics, we additionally discovered that nuclear factor erythroid-2-related factor 1 (NFE2L1) had the capability to enhance the expression of connexin26 and connexin 31 in the NSCLC cell line A549. In addition, the use of metformin was discovered to cause significant downregulation of gap junction protein, betas (GJBs) by limiting the presence of NFE2L1 in the cytoplasm.

Conclusion: This emphasizes the potential of targeting GJBs as a viable treatment approach for NSCLC patients receiving metformin.

Keywords: NFE2L1; NSCLC; gap junction proteins; metformin; prognosis.

FIGURE 1

mRNA expression of GJBs and GJB2 and GJB3 survival analysis in the TCGA database. The mRNA expression level of GJB1 (A), GJB2 (B), GJB3 (C), GJB4 (D), and GJB5 (E) in LUAD and LUSC compared with normal lung tissue in the TCGA database; (F) K‐M OS curves based on the expression levels of GJB2 in LUAD; (G) K‐M OS curves based on the expression levels of GJB3 in LUAD; (H) K‐M OS curves based on both high expression levels GJB2 and GJB3 and other cohorts in LUAD; (I) K‐M OS curves based on the expression levels of GJB2 in LUSC; (J) K‐M OS curves based on the expression levels of GJB3 in LUSC; (K) K‐M OS curves based on both high expression levels GJB2 and GJB3 and other cohorts in LUSC, *p<0.05.

FIGURE 2

Clinical specimen data analysis. Over‐survival curve analyses curves based on the expression levels of GJB2(A), GJB3(B), and both high expression (GJB2 and GJB3) cohorts compared with other cohorts(C) of NSCLC patients using the log‐rank test. (D) mRNA expression level in NSCLC tissue and paired adjacent non‐tumors tissues. Immunohistochemistry of GJB2 (E) in normal lung tissue, LUAD, and LUSC. Immunohistochemistry of GJB3 (F) in normal lung tissue, LUAD, and LUSC (the scale bars of E F pictures are 100 μm).

FIGURE 3

MET influenced GJB2 and GJB3 expression in the A549 cell line. (A) Viability of A549 after exposure with different concentrations of MET after 24H or 48H. (B) mRNA level of GJB2 and GJB3 after exposure with different concentrations of MET after 24H in the A549 cell line. The protein level of connexin 26 (C) and connexin 31 (D) after exposure with different concentrations of MET after 24H in the A549 cell line (the above protein images were all repeated for three experiments). The coregulation analysis between NFE2L1 and GJB2 (E) or GJB3 (F) in NSCLC tissue is based on TCGA data. (G) mRNA level of NFE2L1 after being infected with different siRNA sequences. (H) The protein level of NFE2L1 after being infected with different siRNA sequences.

FIGURE 4

The mechanism of NFE2L1 regulating GJB2 and GJB3 expression under MET exposure. (A–D) Luciferase activity assay showed an increase in transcription activity of GJB2 and GJB3 promoter in NFE2L1 over‐expressed A549 and 95D cells (n = 3, *p < 0.05, **p < 0.01, with one‐way ANOVA followed by Dunnett's post‐test). Western blot for GJB2 (E) and GJB3 (F) expression in A549 cells infected with control or siNFE2L1 lentivirus, with or without MET exposure. β‐actin served as a control (the above protein images were all repeated for three experiments). (G) qRT‐PCR for NFE2L1 expression in A549 cells infected with control or siNFE2L1 lentivirus (n = 3, *p < 0.05, **p < 0.01, with t‐test). (H) Immunofluorescence analysis of NFE2L1 location in A549 cells after exposure with different concentrations of MET (the scale bar of H picture is 10 μm). (I) Protein immunoblotting experiments demonstrated that metformin treatment inhibited the nuclear entry of NFE2L1 and its retention in the cytoplasm.