Hypoxia-inducible factor-1α and nuclear factor-κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non-small-cell lung cancer cells

Abstract

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD-L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD-L1 expression regulation in non-small-cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD-L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho-IκBα (p-IκBα) and hypoxia-inducible factor-1α (HIF-1α). Flow cytometry results showed stronger membrane co-expression of EGFR and PD-L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF-1α indicated strong accordance among protein levels of PD-L1, p-IκBα, and HIF-1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p-IκBα, HIF-1α, and PD-L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p-IκBα, HIF-1α, and PD-L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non-small-cell lung cancer tissues with either p-IκBα or HIF-1α positive staining were more likely to possess elevated PD-L1 expression compared with tissues scored negative for both p-IκBα and HIF-1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF-κB and HIF-1α in PD-L1 expression regulation by EGFR mutants in NSCLC.

Keywords: Hypoxia-inducible factor-1α; NF-κB; epidermal growth factor receptor; non-small-cell lung cancer; programmed cell death ligand 1.

Figure 1

Activation of the epidermal growth factor receptor (EGFR) signaling pathway and programmed cell death ligand 1 (PD‐L1) expression in non‐small‐cell lung cancer (NSCLC) cells. A, Eight human NSCLC cell lines were cultured, and cellular proteins were extracted for western blot analysis of the protein levels of EGFR, phosphorylated (p‐)ERK1/2, ERK1/2, p‐AKT, AKT, p‐IκBα, hypoxia‐inducible factor‐1α (HIF‐1α), and PD‐L1 (top panel). Protein bands of p‐IκBα, HIF‐1α, and PD‐L1 were quantified and normalized to internal control Actin (bottom panel). M, EGFR mutant; W, WT EGFR. B, Flow cytometry analysis using specific Abs labeled with different fluorescent proteins (APC‐EGFR and PE‐PD‐L1) was used to examine the cell surface expression of EGFR and PD‐L1 of 8 NSCLC cell lines. APC, allophycocyanin; PE, phycoerythrin. The respective isotype control Abs, PE Mouse IgG1 and APC Mouse IgG2b, were used as controls

Figure 2

Effects of activation or inhibition of the epidermal growth factor receptor (EGFR) signaling pathway on programmed cell death ligand 1 (PD‐L1) expression in non‐small‐cell lung cancer cells. A, H661 cells transfected with WT EGFR (wt‐EGFR) or EGFR mutant expression vectors (e19del, e19del + T790M, L858R, and L858R + T790M) showed apparently elevated protein levels of phosphorylated (p‐)ERK, p‐AKT, p‐S6, p‐IκBα, hypoxia‐inducible factor‐1α (HIF‐1α), and PD‐L1. *P < .01 vs wt‐EGFR transfection. B,C Specific siRNA (si‐EGFR) transfection induced significantly downregulated EGFR expression, followed by obviously decreased protein levels of p‐ERK, p‐AKT, p‐S6, p‐IκBα, HIF‐1α, and PD‐L1 in H1975 (B) and H1299 (C) cells, respectively. D, H1975 cells received 2 dose treatments (1× and 3×) of 5 kinds of pathway inhibitor, with DMSO‐treated cells used as control. Actin was used as internal control and the graph indicates the relative protein levels of p‐IκBα, HIF‐1α, and PD‐L1. Mean ± SD values for continuous variables of 3 experiments. *P < .01 and **P < .05 vs DMSO group

Figure 3

Inhibitors of the epidermal growth factor receptor (EGFR) signaling pathway suppressed tumor growth and programmed cell death ligand 1 (PD‐L1) expression in a xenograft mouse model of non‐small‐cell lung cancer. Xenotransplanted tumor mouse model bearing H1975 cells was established and received treatments of 4 pathway inhibitors (U0126, LY294002, BAY11‐7082, and PX‐478, separately). A, All 4 experimental groups showed significantly decreased tumor burden compared with control group (DMSO). B,C. Western blot analysis (B) and immunohistochemistry analysis (C) showed that the protein levels of phosphorylated (p‐)IκBα, hypoxia‐inducible factor‐1α (HIF‐1α), and PD‐L1 were significantly downregulated to varying degrees in all inhibitor groups, with PX‐478 and BAY11‐7082 displaying the strongest inhibition action. Original magnification, 400×. *P < .01 and **P < .05 vs DMSO group

Figure 4

Immunohistochemistry staining of phosphorylated (p‐)IκBα, hypoxia‐inducible factor‐1α (HIF‐1α), and programmed cell death ligand 1 (PD‐L1) proteins in non‐small‐cell lung cancer tissues. Human non‐small‐cell lung cancer tissues (n = 149) were included for immunohistochemistry analysis of the expression of p‐IκBα, HIF‐1α, and PD‐L1 proteins. Positive expression of p‐IκBα (Anti‐p‐IκBα), HIF‐1α (Anti‐HIF‐1α), and PD‐L1 (Anti‐PD‐L1) is shown as brown staining in cytoplasm, cell nucleus, and cell membrane, respectively. Section without Ab treatment was used as negative control. Original magnification, 200 × (top panels) and 400 × (bottom panels)

Figure 5

Flowchart of programmed cell death ligand 1 (PD‐L1) expression regulation by epidermal growth factor receptor (EGFR) mutants through downstream signaling effectors phosphorylated (p‐)AKT, p‐ERK, nuclear factor‐κB (NF‐κB), and hypoxia‐inducible factor‐1α (HIF‐1α). IKK, IκB kinase