PD-L1 overexpression is partially regulated by EGFR/HER2 signaling and associated with poor prognosis in patients with non-small-cell lung cancer

Abstract

Immunocheckpoint inhibitors targeting the programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) axis have shown promising results in patients with non-small-cell lung cancer (NSCLC). Recent research has shown that epidermal growth factor receptor (EGFR) signaling affects PD-L1 expression in NSCLC cells; however, the mechanism regulating PD-L1 expression in tumor cells remains unclear. Using immunohistochemistry, we evaluated the impact of expression of PD-L1 and EGF family receptors EGFR and human epidermal growth factor receptor 2 (HER2) in tumor cells from 91 patients with pathological Stage IA-IIIA NSCLC. Overexpression of PD-L1 was observed in 14% of the resected tumors, and associated with poor recurrence-free survival (p = 0.021) and overall survival (p = 0.033). PD-L1 expression is positively correlated with EGFR expression and inversely correlated with HER2. NSCLC cell lines were treated in vitro with the EGFR ligand EGF with or without inhibition of EGFR or HER2, after which PD-L1 expression was evaluated using flow cytometry. Consistent with previous reports, PD-L1 expression was clearly enhanced by EGF. EGFR-tyrosine kinase inhibitors or EGFR small interfering RNA (siRNA) blocked EGF-induced PD-L1 overexpression in NSCLC cell lines, but HER2 siRNA did not. Moreover, our findings suggest that PD-L1 expression could be partially regulated via the PI3K/AKT and JAK/STAT pathways. We conclude that PD-L1 overexpression is associated with poor prognosis and is positively correlated with EGFR expression but inversely correlated with HER2 expression in NSCLC. We also showed that EGFR and HER2 have different effects on EGF-induced PD-L1 expression in NSCLC cell lines.

Keywords: EGFR; HER2; Non-small-cell lung cancer (NSCLC); PD-L1; Prognostic factor.

Fig. 1

Recurrence-free survival and overall survival in patients with NSCLC. Kaplan–Meier (a): recurrence-free survival (RFS) or (b): overall survival (OS) curves in patients with expression status of EGFR, HER2, or PD-L1. Significantly better overall survival was noted for the group with high HER2 expression than that with low HER2 expression. Significantly worse RFS and OS were noted for the group with high PD-L1 expression than that with low PD-L1 expression. Data were analyzed using log-rank test

Fig. 2

PD-L1 expression in NSCLC tissue positively correlates with EGFR expression and inversely correlates with HER2 expression. a H score of PD-L1 in 91 NSCLC tissues positively correlates with the H score of EGFR. b H score of PD-L1 inversely correlates with the H score of HER2 in resected NSCLC tissues. Data were analyzed using Spearman’s rank correlation test. *p < 0.05

Fig. 3

Silencing EGFR blocks EGF-induced PD-L1 but silencing HER2 does not. a PD-L1 expression in cells treated with small interfering RNA (siRNA) targeting each EGF family receptor (siEGFR or siHER2) or control siRNA (siCtr) for 48 h, after which expression of PD-L1 was assessed by flow cytometry. b PD-L1 expression in cells treated with control siRNA for 24 h followed by EGF for 24 h, after which PD-L1 expression was evaluated. c PD-L1 expression in cells treated with siRNA targeting EGFR or HER2 or control for 24 h followed by 100 ng/mL of EGF for 24 h, after which expression of PD-L1 was evaluated. Representative data from at least three independent experiments are shown

Fig. 4

Inhibition of PI3K/AKT or JAK/STAT pathways attenuates EGF-induced PD-L1 expression in NSCLC cells. a NSCLC cell lines were cultured in the presence or absence of LY294002 (LY), PD98059 (PD), or tofacitinib (Tof) for 24 h and then the expression of PD-L1 was assessed by flow cytometry. b NSCLC cell lines were cultured in the presence or absence of LY294002, PD98059, or tofacitinib for 24 h followed by 100 ng/mL of EGF for 24 h, and then the expression of PD-L1 was assessed by flow cytometry. Representative data from three independent experiments are shown