Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

Chang Dong Yeo, In Kyoung Kim, Woo Ho Ban, Hye Seon Kang, Jin Woo Kim, Seung Joon Kim, Jong Y. Park, Sang Haak Lee

Abstract

Background: Smoking histories are independently associated with poor response to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. The aim of the present study was to determine the effect of nicotine exposure on programmed death-ligand 1 (PD-L1) expression in EGFR mutant lung cancer cells.
Methods: Human lung adenocarcinoma PC9 cells were exposed to 1 μM nicotine for 3 months designated as PC9/N, and cells were stimulated with gefitinib (0, 0.1, or 1 μM) for 48 hrs. Cell viability by the MTT assay and morphological changes by immunofluorescence staining were assessed. The protein expression of EGFR, mTOR, AKT, α1-nicotine acetylcholine receptor (nAchR) and PD-L1 were measured by Western blot. Gene expression of α1-nAchR and PD-L1 were examined by RT-PCR. Intratumoral levels of PD-L1 expression were compared according to the burden of smoking dosage in 54 EGFR mutant lung cancer patients.
Results: Cellular growth was inhibited by treatment with gefitinib, and PC9 cells were significantly more sensitive to gefitinib than PC9/N cells. Pleomorphic appearance with atypical nuclei and to be detached and shrunken with condensed nuclei in PC9 than PC9/N cells. The gene expression level of α1-nAchR and PD-L1 gene were higher in PC9/N cells compared to those in PC9 cells after treatment with gefitinib. Phosphorylation levels of EGFR, mTOR, AKT and PD-L1 level were decreased by gefitinib in PC9/N cells, which was to a lesser extent than that in PC9 cells. In tumors, heavy smokers (≥30 PY) showed 28.5% of ≥50% PD-L1 tumor proportion score (TPS) while light smoker and never smokers had 12.5% and 9.7% of ≥50% PD-L1 TPS, respectively. However, there was no statistical significance (P value =0.628).
Conclusions: Chronic nicotine exposure could increase PD-L1 expression related to intrinsic resistance to EGFR-TKI in NSCLC patients harboring activating EGFR mutation. Considering the clinical importance of inevitable EGFR resistance, further studies regarding the role of anti-PD-1/PD-L1 treatment are needed, especially in EGFR mutant smokers.