Chronic nicotine exposure mediates resistance to EGFR-TKI in EGFR-mutated lung cancer via an EGFR signal
Introduction
Lung cancer is the leading cause of cancer-related death in the developed world [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancers, and the prognosis of advanced NSCLC remains very poor despite advances in treatment [2].
Epidermal growth factor receptor (EGFR) is recognized as an important molecular target in cancer therapy [3], and the somatic activating mutation of the EGFR gene (EGFR mutation) is an oncogenic driver mutation in NSCLC [4]. Patients with NSCLC harboring EGFR mutations generally respond to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) [5], [6], [7], [8], [9], [10]. Although these patients respond dramatically to such treatment, some of them show poor response and the majority eventually undergo disease progression [11]. Many studies have revealed several resistance mechanisms and candidates, including EGFR secondary mutations, MET gene amplification, and epithelial-mesenchymal transition (EMT) [12], [13], [14].
Cigarette smoking is the strongest documented risk factor for the development of lung cancer. Tobacco-derived carcinogens are known to form DNA adducts, mutate vital growth regulatory genes such as p53 and Ras, and initiate oncogenesis [15], [16]. Nicotine, while not carcinogenic by itself, has been shown to induce proliferation, angiogenesis, and EMT in several experimental models [17], [18], [19]. Previous preclinical studies have shown that cigarette smoke exposure renders EGFR-TKIs ineffective in EGFR-mutated cell lines [20], [21]. In addition, another study has demonstrated that short-term nicotine exposure activates an EGFR signal and induces EGFR-TKI resistance [22], but the influence of chronic exposure has remained unclear. In the present study, the association between chronic nicotine exposure and EGFR-TKI resistance was investigated experimentally. Furthermore, we investigated whether the smoking status was a predictive and prognostic factor in patients with EGFR-mutated NSCLC who were treated with the EGFR-TKI gefitinib.
Section snippets
Cell culture and reagents
The PC-9 (EGFR exon 19 deletion) and 11_18 (EGFR exon 21 L858R) cell lines (human NSCLC cell lines) were maintained in RPMI1640 medium with 10% FBS (Sigma-Aldrich, St. Louis, MO). All the cell lines were maintained in a 5% CO2-humidified atmosphere at 37 °C. The cell lines that were maintained in the presence of 1 μM nicotine (Sigma-Aldrich) for 3 months were designated as PC-9/N and 11_18/N, respectively.
Gefitinib and hexamethonium (nicotinic acetylcholine receptor [nAChR] inhibitor) was
Resistance of PC-9/N and 11_18/N cell lines to EGFR-TKI
In order to evaluate the influence of chronic nicotine exposure on EGFR-TKI resistance, the PC-9 and 11_18 cell lines were cultured with 1 μM nicotine for 3 months (PC-9/N and 11_18/N, respectively). Both PC-9/N and 11_18/N cell lines were resistant to gefitinib, compared with the controls and the 50% inhibitory concentrations (IC50) of PC-9/N and 11_18/N cell lines became about 3 times higher than the controls (Fig. 1A and B). In contrast, no significant difference in the sensitivity to
Discussion
EGFR mutations occur in 30% of patients with NSCLC who are of East Asian ethnicity, compared with 8% of patients of other ethnicities [28]. In the IRESSA Pan-Asia Study, EGFR mutation was a stronger predictor of a response to gefitinib, compared with smoking status [5], [29]. However, whether smoking status can serve as a predictor of a response to EGFR-TKI treatment among patients with EGFR mutations remained unclear. Our clinical data showed that smoking status tends to be associated with the
Conclusion
Our experiments suggest that chronic nicotine exposure induced by cigarette smoking mediates resistance to EGFR-TKIs via an EGFR signal through the α subunits of nAChRs. Smoking cessation is of great importance, and resistance may be overcome by high-dose EGFR-TKI treatment. To confirm this hypothesis, further research is needed.
Financial support
This study was supported by the Third-Term Comprehensive 10-Year Strategy for Cancer Control (Kazuto Nishio; 13801892) and Grant-in Aid for Japan Society for Promotion of Science Fellows (Yosuke Togashi; 26-12493).
Conflict of interest statement
None declared.
Acknowledgments
We thank Ms. Tomoko Kitayama and Ms. Ayaka Kurumatani for their technical assistance. This research was partially supported by the Third-Term Comprehensive 10-Year Strategy for Cancer Control (Kazuto Nishio; 13801892) and Grant-in Aid for Japan Society for Promotion of Science Fellows (Yosuke Togashi; 26-12493).
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