Induction of PD-L1 expression by epidermal growth factor receptor-mediated signaling in esophageal squamous cell carcinoma

Abstract

Purpose: The purpose of this study was to investigate the potential effect of activation of epidermal growth factor receptor (EGFR) signaling pathway on the expression of programmed death-ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC) cells with EGFR overexpression.

Methods: Flow cytometry and Western blot methods were used to assess PD-L1 expression on ESCC cells when EGFR signaling pathway was activated by epidermal growth factor (EGF) with or without EGFR-specific inhibitor AG-1478, and then EGFR signaling array was applied to analyze the potential signaling pathways involved.

Results: This study found that PD-L1 expression increased significantly in an EGFR-dependent manner by the activation of EGFR signaling and decreased sharply when EGFR signaling was blocked. The upregulated expression of PD-L1 was not associated with EGFR-STAT3 signaling pathway, but may be affected by EGFR-PI3K-AKT, EGFR-Ras-Raf-Erk, and EGR-PLC-γ signaling pathways.

Conclusion: The expression of PD-L1 can be regulated by EGFR signaling activation in ESCC, which indicates an important role for EGFR-mediated immune escape and potential molecular pathways for EGFR-targeted therapy and immunotherapy.

Keywords: epidermal growth factor receptor; esophageal squamous cell carcinoma; immune checkpoint; programmed death-ligand 1.

Figure 1

EGFR and PD-L1 expressions of ESCC cells in complete culture media. Notes: EGFR (A–C) and PD-L1 (D) expressions were tested by Western blot (A) and flow cytometry (B–D). (B and C) Cell surface and total EGFR expression. Abbreviations: EGFR, epidermal growth factor receptor; PD-L1, programmed death-ligand 1; ESCC, esophageal squamous cell carcinoma; MFI, median fluorescence intensity.

Figure 2

PD-L1 was induced by EGF. Notes: (A) PD-L1 expression after EGF treatment. ESCC cells were starved overnight and treated with EGF (20 ng/mL). After 24 h, PD-L1 expression was tested by flow cytometry. (B and C) AG1478 inhibited EGF-induced PD-L1 expression. Indicated concentration of AG1478 was added 30 min before EGF treatment. (D) Three repeated experiments for B and C. ^▲P<0.05 as significant difference. Abbreviations: PD-L1, programmed death-ligand 1; EGF, epidermal growth factor; ESCC, esophageal squamous cell carcinoma; FCS, fetal calf serum; DMSO, dimethyl sulphoxide.

Figure 2

PD-L1 was induced by EGF. Notes: (A) PD-L1 expression after EGF treatment. ESCC cells were starved overnight and treated with EGF (20 ng/mL). After 24 h, PD-L1 expression was tested by flow cytometry. (B and C) AG1478 inhibited EGF-induced PD-L1 expression. Indicated concentration of AG1478 was added 30 min before EGF treatment. (D) Three repeated experiments for B and C. ^▲P<0.05 as significant difference. Abbreviations: PD-L1, programmed death-ligand 1; EGF, epidermal growth factor; ESCC, esophageal squamous cell carcinoma; FCS, fetal calf serum; DMSO, dimethyl sulphoxide.

Figure 3

PD-L1 was not influenced by EGFR–STAT3 signaling pathway. Notes: ESCC cells were starved overnight and treated with EGF (20 ng/mL) for 30 min or pretreated with AG1478. Total protein was extracted and tested using Western blot. Abbreviations: EGFR, epidermal growth factor receptor; PD-L1, programmed death-ligand 1; EGF, epidermal growth factor; ESCC, esophageal squamous cell carcinoma; DMSO, dimethyl sulphoxide; STAT3, signal transducer and activator of transcription 3.

Figure 4

Potential EGFR signaling pathways involved in the regulation of PD-L1 expression. Notes: Starved ESCC cells were treated with EGF (20 ng/mL) for 30 min and analyzed using PathScan^® EGFR signaling antibody array kit. (A) Heatmap illustrator. (B) Increased phosphorylation in ESCC cells. Cutoff >1.5. ^▲P<0.05 as significant difference. Abbreviations: EGFR, epidermal growth factor receptor; PD-L1, programmed death-ligand 1; EGF, epidermal growth factor; ESCC, esophageal squamous cell carcinoma; FCS, fetal calf serum.