Yeo Chang Dong, Kim In Kyoung, Ban Woo Ho, Kang Hye Seon, Kim Jin Woo, Kim Seung Joon, Park Jong Y, Lee Sang Haak
Division of Pulmonology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea.
Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea.
Transl Cancer Res. 2019 Jul;8(Suppl 4):S378-S388. doi: 10.21037/tcr.2019.05.02.
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.
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.
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).
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.
吸烟史与携带表皮生长因子受体（EGFR）激活突变的非小细胞肺癌（NSCLC）患者对EGFR酪氨酸激酶抑制剂（TKIs）反应不佳独立相关。本研究的目的是确定尼古丁暴露对EGFR突变肺癌细胞中程序性死亡配体1（PD-L1）表达的影响。
将人肺腺癌PC9细胞暴露于1μM尼古丁中3个月，命名为PC9/N，然后用吉非替尼（0、0.1或1μM）刺激细胞48小时。通过MTT法评估细胞活力，通过免疫荧光染色评估形态变化。通过蛋白质印迹法检测EGFR、mTOR、AKT、α1-尼古丁乙酰胆碱受体（nAchR）和PD-L1的蛋白表达。通过RT-PCR检测α1-nAchR和PD-L1的基因表达。根据54例EGFR突变肺癌患者的吸烟剂量负担比较肿瘤内PD-L1表达水平。
吉非替尼处理可抑制细胞生长，且PC9细胞对吉非替尼的敏感性明显高于PC9/N细胞。与PC9/N细胞相比，PC9细胞出现具有非典型核的多形性外观，细胞核浓缩，细胞 detached 并萎缩。用吉非替尼处理后，PC9/N细胞中α1-nAchR和PD-L1基因的表达水平高于PC9细胞。吉非替尼可降低PC9/N细胞中EGFR、mTOR、AKT的磷酸化水平和PD-L1水平，但其降低程度小于PC9细胞。在肿瘤中，重度吸烟者（≥30包年）中PD-L1肿瘤比例评分（TPS）≥50%的比例为28.5%，而轻度吸烟者和从不吸烟者中该比例分别为12.5%和9.7%。然而，差异无统计学意义（P值 =0.628）。
长期尼古丁暴露可增加携带EGFR激活突变的NSCLC患者中与EGFR-TKI内在耐药相关的PD-L1表达。考虑到不可避免的EGFR耐药的临床重要性，需要进一步研究抗PD-1/PD-L1治疗的作用，尤其是在EGFR突变吸烟者中。
