HIF-1α and NF-κB play important roles in regulating PD-L1 expression by EGFR mutants in non-small cell lung cancer cells.

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-IB (p-IB) and hypoxia-inducible factor-1 (HIF-1). 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, IB, and HIF-1 indicated strong accordance among protein levels of PD-L1, p-IB, and HIF-1 in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p-IB, HIF-1, and PD-L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p-IB, HIF-1, and PD-L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non-small-cell lung cancer tissues with either p-IB or HIF-1 positive staining were more likely to possess elevated PD-L1 expression compared with tissues scored negative for both p-IB and HIF-1. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF-B and HIF-1 in PD-L1 expression regulation by EGFR mutants in NSCLC.