Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non–Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers

David Chi-Leung Lam,Lap-Ping Chung,Wah-Kit Lam,Jerry W Shay,John D Minna,Wing-Shun Chau,Vicky P.C Tin,Maria P Wong,Shelley Sheridan,Adi F Gazdar,Luc Girard,Ruben Ramirez,Wai-Sing Suen

doi:10.1158/0008-5472.can-06-4628

Abstract

Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChR) on bronchial epithelial cells, can regulate cellular proliferation and apoptosis via activating the Akt pathway. Delineation of nAChR subtypes in non-small-cell lung cancers (NSCLC) may provide information for prevention or therapeutic targeting. Expression of nAChR subunit genes in 66 resected primary NSCLCs, 7 histologically non-involved lung tissues, 13 NSCLC cell lines, and 6 human bronchial epithelial cell lines (HBEC) was analyzed with quantitative PCR and microarray analysis. Five nonmalignant HBECs were exposed to nicotine in vitro to study the variation of nAChR subunit gene expression with nicotine exposure and removal. NSCLCs from nonsmokers showed higher expression of nAChR alpha6 (P < 0.001) and beta3 (P = 0.007) subunit genes than those from smokers, adjusted for gender. In addition, nAChR alpha4 (P < 0.001) and beta4 (P = 0.029) subunit gene expression showed significant difference between NSCLCs and normal lung. Using Affymetrix GeneChip U133 Sets, 65 differentially expressed genes associated with NSCLC nonsmoking nAChR alpha6beta3 phenotype were identified, which gave high sensitivity and specificity of prediction. nAChR alpha1, alpha5, and alpha7 showed significant reversible changes in expression levels in HBECs upon nicotine exposure. We conclude that between NSCLCs from smokers and nonsmokers, different nAChR subunit gene expression patterns were found, and a 65-gene expression signature was associated with nonsmoking nAChR alpha6beta3 expression. Finally, nicotine exposure in HBECs resulted in reversible differences in nAChR subunit gene expression. These results further implicate nicotine in bronchial carcinogenesis and suggest targeting nAChRs for prevention and therapy in lung cancer.

Highlights

Tobacco smoking is the major cause of lung cancer, and nicotine in tobacco smoke leads to both addiction and further metabolism into potent carcinogen(s)
We have previously shown that lung cancer cells expressed nicotinic acetylcholine receptors (AChR) (nAChR), and that nicotine, at concentrations found in smokers, blocked the induction of apoptosis in lung cancer cells [25]; whereas West et al showed that activation of nAChR resulted in downstream activation of the Akt pathway [1], protein kinase C pathway, and the mitogen-activated protein kinase (MAPK) pathways, leading to inhibition of apoptosis and promotion of growth and proliferation in human bronchial epithelial cells (HBEC; refs. 1, 25)
All 66 non–small-cell lung cancers (NSCLC) tumors collected were included in quantitative PCR, and 49 of these 66 specimens were used for microarray studies; in addition, of the 14 normal lung tissue specimens, 9 were used for microarray analysis, whereas 7 of them were included in quantitative PCR analysis

Summary

Introduction

Tobacco smoking is the major cause of lung cancer, and nicotine in tobacco smoke leads to both addiction and further metabolism into potent carcinogen(s). All of these observations led to our current study of the quantitative mRNA expression analysis of various nAChR subunit genes in lung cancers, normal lung, and lung epithelial cells and their variation with the smoking history of the patients. We found significant differences in nAChR receptor subunit expression patterns in comparisons of tumor and normal tissue and differences between lung adenocarcinomas, depending on smoking (nicotine) exposure.

Results

Conclusion