Research Article Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non–Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers David Chi-leung Lam,1,2 Luc Girard,4 Ruben Ramirez,4 Wing-shun Chau,3 Wai-sing Suen,3 Shelley Sheridan,4 Vicky P.C. Tin,2 Lap-ping Chung,2 Maria P. Wong,2 Jerry W. Shay,5 Adi F. Gazdar,4 Wah-kit Lam,1 and John D. Minna4 Departments of 1Medicine and 2Pathology, University of Hong Kong; 3Cardiothoracic Surgical Unit, The Grantham Hospital, HKSAR, China; and 4Hamon Center for Therapeutic Oncology Research and 5Department of Cell Biology, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas Abstract could also participate in lung cancer pathogenesis by activating Nicotine and its derivatives, by binding to nicotinic acetyl- signal transduction pathways such as the Akt pathway (1). One choline receptors (nAChR) on bronchial epithelial cells, can model could be that nicotine by stimulating nicotinic AChRs regulate cellular proliferation and apoptosis via activating the (nAChR) would activate Akt in lung epithelial cells and perhaps Akt pathway. Delineation of nAChR subtypes in non–small-cell stimulate cell proliferation and/or overcome apoptotic responses lung cancers (NSCLC) may provide information for prevention engendered by carcinogen exposure (1). If this model is true, then or therapeutic targeting. Expression of nAChR subunit genes one may ask whether lung tumors have different nAChR expression in 66 resected primary NSCLCs, 7 histologically non-involved patterns compared with normal lung tissues and whether lung lung tissues, 13 NSCLC cell lines, and 6 human bronchial cancers arising in smokers have different patterns compared with epithelial cell lines (HBEC) was analyzed with quantitative never smokers. Such differences would provide additional infor- PCR and microarray analysis. Five nonmalignant HBECs were mation that nicotine is playing a role via the nAChRs in lung cancer exposed to nicotine in vitro to study the variation of nAChR pathogenesis. In recent decades, there have been an increasing subunit gene expression with nicotine exposure and removal. proportion of female nonsmokers compared with male smokers in NSCLCs from nonsmokers showed higher expression of nAChR patients with lung cancer (2). It is possible that both gender and A6(P < 0.001) and B3(P = 0.007) subunit genes than those smoking, or an interaction of both factors, are playing roles in lung from smokers, adjusted for gender. In addition, nAChR A4 carcinogenesis. Differences between men and women may also (P < 0.001) and B4(P = 0.029) subunit gene expression showed make them respond to tobacco smoke in different ways, and lung significant difference between NSCLCs and normal lung. Using tumors derived from male smokers and female nonsmokers may Affymetrix GeneChip U133 Sets, 65 differentially expressed have adopted different carcinogenic pathways. Thus, it would be genes associated with NSCLC nonsmoking nAChR A6B3 important to analyze the role of nAChR expression in the context of phenotype were identified, which gave high sensitivity and gender as well. specificity of prediction. nAChR A1, A5, and A7 showed AChRs are divided into nicotinic (nAChR) and muscarinic significant reversible changes in expression levels in HBECs (mAChR) subtypes. nAChRs are further subdivided into neuronal or upon nicotine exposure. We conclude that between NSCLCs muscle subtypes, which could also be present in nonneuronal or from smokers and nonsmokers, different nAChR subunit gene non-muscle tissues. Neuronal nAChRs are composed of different h h h expression patterns were found, and a 65-gene expression subunits including a1, a2, a3, a4, a5, a6, a7, a9, a10, 1, 2, 3, or h h signature was associated with nonsmoking nAChR A6B3 4. In addition to these different a and subunits, the muscle type y q expression. Finally, nicotine exposure in HBECs resulted in nAChR may also contain , , and g subunits. Genes encoding for reversible differences in nAChR subunit gene expression. individual nAChR subunit are named CHRNA1, CHRNA2, CHRNA3, These results further implicate nicotine in bronchial carcino- CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA9, and CHRNA10 for genesis and suggest targeting nAChRs for prevention and the a subunits and CHRNB1, CHRNB2, CHRNB3, and CHRNB4 for h therapy in lung cancer. [Cancer Res 2007;67(10):4638–47] the subunits. nAChRs are found to be present throughout the central nervous system (CNS) and in nonneuronal tissues, such as Introduction a3, a5, and a7 in bronchial epithelium (3, 4); a4 in alveolar epithelial cells (4); and a3, a5, a7, h2, and h4 in pulmonary Tobacco smoking is the major cause of lung cancer, and nicotine neuroendocrine cells and human small cell lung cancer (SCLC) cell in tobacco smoke leads to both addiction and further metabolism lines (5–8), skin keratinocytes (9), vascular tissues (10), and human into potent carcinogen(s). In addition, recent discoveries of lymphocytes (11). nAChR holoreceptor is a pentamer consisting of functional acetylcholine receptors (AChR) on lung epithelial cells five homologous or different nAChR subunits surrounding a ligand- and lung tumors raise the question of whether exposure to nicotine gated channel (12) that responds to binding by ligands such as acetylcholine, nicotine, or its highly carcinogenic derivative 4(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK; ref. 13). Neu- Requests for reprints: John D. Minna, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center at Dallas, 6000 Harry ronal nAChR consists of only two types of subunits: either a Hines Boulevard, Dallas, TX 75390-8593. Phone: 214-648-4900; Fax: 214-648-4940; combination of a and h subunits or five copies of the same a E-mail: [email protected]. h I2007 American Association for Cancer Research. subunits (14). Both the a and subunits are thought to contribute doi:10.1158/0008-5472.CAN-06-4628 to the physiologic properties of nAChR, where a subunit contains Cancer Res 2007; 67: (10). May 15, 2007 4638 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Nicotinic Acetylcholine Receptors in Lung Cancer the principal sites for agonist binding, such as acetylcholine, and h (NSCLC) specimens and 14 normal lungs tissues. NSCLC tumor specimens subunits are believed to regulate the rate of binding and were collected from Hong Kong Chinese patients undergoing surgical dissociation by agonists (15). Functional receptors in the brain resection. Normal lung tissues used in this study were collected from are composed mainly of a4h2, a4a5h2, a6h2h3, a6h3, a4a6h2h3, patients with lung cancer undergoing surgical resection, and specimens were reviewed to show no tumor involvement. Written informed consent and a7 (16, 17). In fact, a4h2 in the brain is thought to be h for tumor and normal lung tissues collection were obtained from patients responsible for nicotine addiction (18, 19). a6 associated with 2 recruited before surgery, and ethics approval for study protocol was h and 3, a3ora4 is present in dopaminergic and adrenergic obtained from the local Institutional Review Board of the University of neurons in the brain (20); and a6h3, in particular, is a functional Hong Kong (HKU)/Hong Kong Hospital Authority Hong Kong West Cluster. nAChR (21). Receptor affinity for nicotine varies with different The demographic characteristics of these lung cancer patients, including composition of nAChR subunits (20). Transfection studies have their age, gender, smoking habits, and tumor-node-metastasis pathologic shown that the ratio of a/h subunits in nAChRs depends on the staging information, are summarized in Table 1. Nonsmokers were patients ratio of expression of the encoding nAChR subunit genes (22). who have never smoked for their lifetime. Smokers included patients who nAChRs were first implicated in the growth regulation of lung have been current active chronic smokers before surgery and patients who have been daily smoking for more than 6 months in the past but have quit cancer when nicotine was found to stimulate DNA synthesis in smoking at the time of surgery. All 66 NSCLC tumors collected were human SCLC cell lines (23), and this was supported by subsequent included in quantitative PCR, and 49 (all primary lung adenocarcinomas) of identification that the receptor involved was nAChR a7 (7, 24). We these 66 specimens were used for microarray studies; in addition, of the 14 have previously shown that lung cancer cells expressed nAChR, and normal lung tissue specimens, 9 were used for microarray analysis, whereas that nicotine, at concentrations found in smokers, blocked the 7 of them were included in quantitative PCR analysis (two normal lung induction of apoptosis in lung cancer cells (25); whereas West et al. tissue specimens were used in both microarray analysis and quantitative showed that activation of nAChR resulted in downstream PCR analysis). activation of the Akt pathway (1), protein kinase C pathway, and A panel of 13 NSCLC cell lines (9 NCI-H lung cancer cell lines and 4 the mitogen-activated protein kinase (MAPK) pathways, leading to HKULC lung cancer cell lines) were used in this study, and total RNA was extracted for quantitative PCR (Table 1). The NCI-H lung cancer cell lines inhibition of apoptosis and promotion of growth and proliferation (all were NSCLC lines: H1437, H1648, H1770, H1819, H1993, H2009, H2087, in human bronchial epithelial cells (HBEC; refs. 1, 25). H2122, and H2347) were maintained at the Hamon Center for Therapeutic a In the brain, nAChR 7 showed paradoxical up-regulation in Oncology Research, University of Texas Southwestern Medical Center at response to chronic exposure to nicotine, whereas other nAChR Dallas (31) and have been deposited in the American Type Culture subunits were down-regulated. It has also been shown that the Collection (ATCC) repository (32). The HKULC cell lines (HKULC1–4) were nAChR a7 in the lungs of monkeys is up-regulated by exposure to newly established lung adenocarcinomas from Hong Kong Chinese nicotine (26).