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

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

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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). Up-regulation of functional nAChR a7 subunits has patients (33) being maintained and stored at the University of Hong been shown in normal human bronchial epithelial cells (NHBE) Kong, HKSAR, China. The demographic characteristics of patients from upon exposure to nicotine (10). In SCLC cell lines, up-regulation of whom the cell lines were established, including their gender, smoking nAChR a7 has been shown in response to NNK stimulation, with habits, and tumor cell types, were known. The normal bronchial epithelial cell lines included in this study were one cell line derived from peripheral overexpression and phosphorylation of serine-threonine protein alveolar space, the small airway epithelial cell (Clonetics), and other kinase Raf-1 and extracellular signal regulated kinases 1 and 2 and bronchial epithelial cell lines, including NHBE (Clonetics), BEAS-2B (ATCC activation of c-myc (27), leading to increased proliferation (1, 28). CRL-9609), and five NHBE cell lines of the HBEC-KT series (Dr. John There is also speculation that chronic tobacco smoking may induce Minna’s Laboratory; ref. 34). a positive feedback loop that amplifies nicotine response in NHBE Complementary DNA synthesis. Total RNA was extracted from tissue cells (10, 29). On the other hand, tolerance to the adverse effects of specimens and cell lines. RNA samples (1 Ag) were reverse transcribed in nicotine could reflect desensitization of nAChR. Thus, it would be 20 AL reaction mix [5Â First-Strand Buffer (Invitrogen), 100 mmol/L DTT important to know if other nAChR subunit genes respond to (Promega), 1 mmol/L deoxynucleotide triphosphate (Amersham Bioscien- nicotine exposure in a similar way, and these nicotine stimulation ces), oligo-dT12-18 primers (Invitrogen) and random hexamer (Promega), responses may give insight into their potential roles in nicotine RNaseOUT Recombinant RNase Inhibitor (Invitrogen), and Superscript II Reverse Transcriptase (Invitrogen)] with 1-h reaction at 42jC. addiction or bronchial carcinogenesis. h Quantitative PCR reactions. Twelve pairs of primers spanning across Recent reports of clinical trials of the nAChR a4 2 antagonist, intron-exon junctions were designed for quantitative PCR targeting nAChR h which target a4 2 receptors in the brain, have shown its clinical subunit genes a1, a3, a4, a5, a6, a7, a9, a10, h2, h3, and h4 (Table 2), with efficacy in smoking cessation (30). It is possible that similar 18S as the reference gene. Reaction conditions were validated separately for nAChR antagonists could block the effect on lung epithelial cells each pair of primers, with single peak of dissociation curves produced in or tumor cells. each run of reaction. All of these observations led to our current study of the Quantitative PCR reactions were carried out in triplicates. SYBR Green I quantitative mRNA expression analysis of various nAChR subunit (SYBR Green JumpStart Taq ReadyMix, Sigma) was used as the detection genes in lung cancers, normal lung, and lung epithelial cells and dye, and ribosomal 18S was used as the reference gene. Final reaction A A their variation with the smoking history of the patients. In fact, we volume was 10 L, with 1.25 L of one-tenth TE-diluted cDNA from reverse transcription reaction, 0.1 Amol/L of each specific primer, and 5 AL SYBR found significant differences in nAChR receptor subunit expression Green Jumpstart Taq ReadyMix. Quantitative PCR cycles were set at 10-min patterns in comparisons of tumor and normal tissue and also denaturation followed by 40 cycles of 95jC for 15 s, 60jC for 5 s, and 72jC differences between lung adenocarcinomas, depending on smoking for 20 s and 72jC for 10 min as the final extension step. Dissociation curves (nicotine) exposure. were inspected for each pair of primers, and only one dissociation peak must be present for each run of reaction before the results were considered to be valid. A 5-fold serial dilution of a reference sample was used for Materials and Methods construction of standard curves with respect to each pair of primers. Clinical characteristics of tumor and cell line samples. Total RNA Quantitative PCR was run for each tumor or cell line cDNA sample, and the was extracted from frozen tissue of 66 surgically resected non-SCLC Ct for a particular sample was obtained from the standard curves for a www.aacrjournals.org 4639 Cancer Res 2007; 67: (10). 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Table 1. Summary of demographic characteristics of NSCLCs, normal lung tissue samples, NSCLC cell lines, and HBEC lines

NSCLCs (resected specimens) Normal lung tissues (resected specimens) NSCLC cell lines HBEC lines

Total no. samples 66 7 13 6 Age range 38-81 49-64 38-69 18-71 Mean age 62.0 55.6 54.8 58.0 Males/females 27:39 2:5 5:8 3:3 Histology (AD/SCC/LC) 54/6/5, adenosquamous = 1 Normal lung tissues AD = 12, NSCLC = 1 NHBE Never smokers/smokers 37:29 3:4 4:9 2:4

NOTE: The tumor-node-metastasis stages for resected lung cancer specimens were 36/12/17/1 (pathologic stages I/II/III/IV). Abbreviations: AD, adenocarcinoma; SCC, squamous cell carcinoma; LC, large cell carcinoma.

specific pair of primers. The Ct for unknown samples was compared with RNA was checked with denaturing formamide gel electrophoresis, which the Ct of reference samples to obtain the normalized relative amount for showed two sharp and distinct bands of 18S and 28S. Quality check was also unknown samples. The normalized relative amount was then log 2 done by the Agilent Bioanalyzer with graphical analysis showing two transformed, and approximation to normal distribution was estimated. distinct peaks of 18S and 28S without additional peaks of degradation. The Excel RGB (red-green-blue color coding macro) coding was done with a total RNA was then hybridized onto Affymetrix GeneChip HG-U133 A and B black-blue-white scale representing highest to lowest level of nAChR gene sets according to standard protocols (35). expression detected in this study for expression pattern inspection. For Absolute signals from an individual chip were captured and processed quantitative PCR data, the normalized relative amount for all samples was with the MicroArray Suite 5.0 software (Affymetrix). Captured signals were multiplied by a common factor of 1,024 and then log 2 transformed to give further scaled and normalized to median expression level with an in-house values in the same range as the microarray data so that the same color scale Visual Basic software ‘‘MATRIX’’ (Microarray Transformation in Excel) could be used for both microarray and transformed quantitative PCR data version 1.31 written by Luc Girard at the University of Texas Southwestern for direct visual inspection and comparison. Medical Center at Dallas. The MATRIX program allowed input of multiple Microarray GeneChip expression analysis. Total RNA (5 Ag) was CHP files from MicroArray Suite 5.0 into an Excel spreadsheet where extracted with RNeasy Miniprep (Qiagen) protocol. The quality of the total normalization, t tests, and color display were done and further statistical analysis could be done with raw, normalized, and transformed probeset signal intensities for each sample in Excel spreadsheet format. Table 2. A list of the 12 pairs of primers designed for The BRB ArrayTool 3.4.0 program (developed by Amy Peng Lam and quantitative PCR with SYBR green I dye and their Richard Simon at the Biometric Research Branch, National Cancer Institute) corresponding sequences was used for significance analysis of microarray (SAM) to analyze for differentially expressed genes between different group phenotypes and for class prediction to identify signature genes that predict group phenotypes nAChR subunit and Quantitative PCR primer sequences by computer algorithm such as support vector machine (SVM). All lung reference genes cancer samples were designated as either having a higher than the mean level of expression or a lower than the mean level of expression for CHRNA1 5¶-GCTCTGTCGTGGCCATCAA-3¶ individual subunit gene. Comparisons using SAM were made between 5¶-CACTCCCCGCTCTCCATG-3¶ samples above and below their respective mean levels of expression for CHRNA3 5¶-AACCTGTGGCTCAAGCAAATCT-3¶ individual nAChR subunit genes a1, a3, a4, a5, a6, a7, a9, a10, h2, h3, and 5¶-CATGAACTCTGCCCCACCAT-3¶ h4 or different combinations of the expression levels of these nAChR CHRNA4 5¶-GTGGATGAGAAGAACCAGATGATG-3¶ subunit genes. 5¶-CAGCGCAGCTTGTAGTCGTG-3¶ Response to nicotine exposure in NHBE cell lines. Five NHBE cell CHRNA5 5¶-AGATGGAACCCTGATGACTATGGT-3¶ lines (HBEC-KT 1–5; ref. 34) were cultured, and an equal number of 5¶-AAACGTCCATCTGCATTATCAAAC-3¶ passages for each cell line were randomized into a nicotine group and CHRNA6 5¶-TGGCCAACGTGGATGAAGTAA-3¶ control group. The HBECs were seeded and incubated for 24 h before 5¶-TCTCAATGCCATCATATTCCATTG-3¶ addition of nicotine. At time 0 h, nicotine (100 nmol/L) was added to the CHRNA7 5¶-GCTGCTCGTGGCTGAGATC-3¶ nicotine group, and the same volume of culture medium without nicotine 5¶-TGGCGAAGTACTGGGCTATCA-3¶ was added to the control group. Both groups were incubated for 72 h, after CHRNA9 5¶-AAAGATGAACTGGTCCCATTCCT-3¶ which medium from both groups was removed and replaced with the same 5¶-AAGGTCATTAAACAACTTCTGAGCATAT-3¶ volume of fresh medium without nicotine. Both groups were further CHRNA10 5¶-TGCCTATGGTGGCCTGGAT-3¶ incubated for 72 h until time 144 h was reached. Cells were harvested at 5¶-GGCGGAACCTGGAGGCT-3¶ time 0, 72, and 144 h. Total RNA was extracted for reverse transcription and CHRNB2 5¶-CTGGATCCTTCCCGCTACAAC-3¶ quantitative PCR as described above. 5¶-TGGGTCAGCCAGACATTGGT-3¶ Statistical analysis. Microarray analysis was done by the in-house CHRNB3 5¶-AACAGTTCCGTTTGATTTCACGAT-3¶ program MATRIX 1.31, an Excel-based visual basic program written for 5¶-CAGCCAGGTAGTACAAGACTGGAAAT-3¶ analysis of microarray data. Student’s t tests, with assumptions of two tails CHRNB4 5¶-TCACAGCTCATCTCCATCAAGCT-3¶ and unequal variance, were used for comparison of expression level of 5¶-CCTGTTTCAGCCAGACATTGGT-3¶ different subunit genes between groups of samples with different gender 18S 5¶-AGGAATTGACGGAAGGGCAC-3¶ and smoking history. m2 tests were used for comparing the distribution of 5¶-GGACATCTAAGGGCATCACA-3¶ smokers and nonsmokers with high or low mean expression of individual or combinations of nAChR subunit genes. SAM and class prediction with SVM

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Figure 1. Results from quantitative PCR analysis: a color-scale display of the log 2 normalized relative amount of the different nAChR subunit gene expression in all samples with different gender and smoking habits. A, resected NSCLC tumors and normal lung tissues. SCC, squamous cell carcinoma; LC, large cell carcinoma. B, NSCLC cell lines and NHBE cell lines. The expression level of the nAChR subunits are color coded from dark blue to white for highest to lowest expression levels, with the color scale attached. C, summary of comparison of different nAChR subunit gene expression in different NSCLC tumors, normal lung tissues, NSCLC cell lines, and NHBE cell lines. *, P < 0.05, Student’s t tests, two sided with unequal variance assumed. were done with the BRB ArrayTools program version 3.4.0; t tests and m2 primary lung cancer specimens and normal lung tissue (Fig. 1A). tests were carried out with SPSS for Windows version 11.5. Likewise, some but not all of these nAChR subunit genes were expressed in the NSCLC lines (with the exception of a4; whereas Results a3, a6, a9, and h2 were only expressed in a few of the tumor lines). Quantitative PCR analysis of nAChR subunit gene expres- HBECs only expressed a1, a5, a10, and h4(Fig.1B). Significant sion. Many of the nAChR subunit genes were expressed in the difference was found in the levels of different CHRN gene www.aacrjournals.org 4641 Cancer Res 2007; 67: (10). May 15, 2007

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Research expression when NSCLC tumors, normal lung tissues, NSCLC cell compared. This difference remained after adjusting for gender (for lines, and NHBE cell lines were compared (Fig. 1C). When resected men: CHRNA6, P = 0.003; CHRNB3, P = 0.031 and for women: NSCLC were compared with normal lung tissues, there were CHRNA6, P = 0.001; CHRNB3, P = 0.027; Fig. 2C and D). Among the statistically significant differences in the expression levels of samples with low nAChR a6h3 expression level, 8 of 34 (23.5%) CHRNA4 (mean NSCLC/mean normal lung = 3.10/6.32, P < 0.001) were smokers, and 3 of 24 (8.8%) were nonsmokers; and among the and B4 (mean NSCLC/mean normal lung = 7.56/5.96, P = 0.029). samples with high nAChR a6h3 expression levels, 3 of 24 (8.8%) A subgroup analysis with separate comparisons between the were smokers, and 20 of 34 (58.8%) were nonsmokers. The squamous cell carcinoma and adenocarcinoma against normal lung proportion of smokers and nonsmokers among samples with low tissue was done. For the comparison between squamous or high nAChR a6h3 gene expression was significantly different cell carcinoma (n = 6) and normal lung tissues (n = 7), no (m2 distribution, P < 0.001). statistically significant difference in the expression levels of any Expression analysis with Affymetrix GeneChips. No signifi- nAChR subunit gene was observed. When adenocarcinomas cant differential gene expression from the Affymetrix arrays was (n = 54) were compared with normal lung tissues (n = 7), significant found that identified tumor groups with high or low expression by differences were found in the expression of nAChR a4 (mean quantitative PCR for individual nAChR subunit genes a1, a3, a4, adenocarcinoma/mean normal lung = 1.92/6.32, P < 0.001) and b4 a5, a6, a7, a9, a10, h2, h3, and h4 as well as for combinations of (mean adenocarcinoma/mean normal lung = 7.56/5.96, P = 0.029). low a4 and high h4, or with low a4 and low h2. However, with However, CHRNA4 and CHRNB4 did not show statistically combined nAChR a6h3 expression levels, 173 genes were found to significant difference when NSCLC cell lines were compared with be differentially expressed between 34 samples with nAChR a6h3 HBEC lines. Instead, the expression levels of CHRNA5 (mean NSCLC low (n = 11) compared with samples with nAChR a6h3 high cell lines/mean HBECs = 6.83/5.00, P = 0.022), CHRNA7 (mean (n = 23), when the false discovery rate was controlled at 0.05 (i.e., NSCLC cell lines/mean HBECs = 6.20/À0.05, P = 0.023), CHRNA9 allowed 5% chance of false discovery among the list of significant (mean NSCLC cell lines/mean HBECs = 2.33/À1.46, P = 0.001), differentially expressed genes identified). Class prediction with and CHRNB2 (mean NSCLC cell lines/mean HBECs = 1.84/À1.78, leave-one-out analysis and SVM was done using these 173 genes. P = 0.037) were found to be significantly higher in NSCLC cell lines Repetitive training and testing with division of samples into compared with NHBE cell lines. training set (random two third) and test set (random one third) Significant differences for the expression levels of the CHRNA6 resulted in 65 signature genes (Table 3) predictive of the ‘‘nAChR (mean smokers/mean nonsmokers = 4.41/6.81, P < 0.001) and a6h3 phenotype’’ with 100% sensitivity and 90% specificity. When CHRNB3 (mean smokers/mean nonsmokers = 6.51/7.16, P = 0.007) these 65 genes were used for prediction of all the 49 samples, the subunit genes (Fig. 2A and B) were found when primary lung prediction sensitivity and specificity were 87% and 61%, respec- adenocarcinomas arising in smokers versus nonsmokers were tively, for smoking habits.

Figure 2. Box plots to show expression levels and comparison between smokers and nonsmokers of (A) CHRNA6 and (B) CHRNB3 genes in primary lung adenocarcinomas. C and D, statistically significant differences in expression levels between smokers and nonsmokers persisted when analyses were stratified according to gender.

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Table 3. List of differentially expressed genes between primary lung adenocarcinomas with both CHRNA6 and CHRNB3 gene expression levels above mean level compared with lung cancers showing both CHRNA6 and CHRNB3 gene expression levels below mean level

Gene symbol Description Probeset Mean intensities Mean intensities Parametric P in samples with both in samples with both A6 and B3 down A6 and B3 up

RABGAP1L RAB GTPase activating 203020_at 357.1 792.6 <0.000001 protein 1-like B3GALT4 UDP-Ga1:hGlcNac 210205_at 57.4 148.8 <0.000001 h-1,3-galactosyltransferase, polypeptide 4 MRMA full-length insert cDNA 232151_at 384.7 1111.7 0.000002 clone EUROIMAGE 2344436 RUTBC3 RUN and TBC1 domain 203014_x_at 107.9 198.9 0.000002 containing 3 GGT1 g-Glutamyltransferase 1 207131_x_at 190.9 530.2 0.000002 PHYH Phytanoy I-CoA hydroxylase 203335_at 298.8 632.6 0.000002 (Refsum disease) TPM1 Tropomyosin 1 (a) 206116_s_al 378.2 933.3 0.000002 MGC14839 Similar to RIKEN cDNA 238805_at 160.7 540.5 0.000003 2310030G06 gene P8 p8 protein (candidate of 209230_s_at 340.7 1150.7 0.000004 metastasis 1) USP18 Ubiquitin-specific peptidase 18 219211_at 105.3 276.3 0.000004 HLA-DQB1 MHC class II, DQ h 1 209823_x_at 557.2 2023.4 0.000004 PPFIBP2 PTPRFinteracting protein, 212841_s_at 103.3 355.7 0.000004 binding protein 2 (liprin h 2) MGC17330 HGFL gene 221756_at 125.8 331.6 0.000005 SCUBE2 Signal peptide, CUB domain, 219197_s_at 83.8 325.7 0.000005 EGF-like 2 GPR160 G protein–coupled 223423_at 878.8 2477.8 0.000006 receptor 160 PIP5K1B Phosphatidylinositol-4-phosphate 205632_s_at 103.6 212.5 0.000007 5-kinase, type I, h MGC17299 Hypothetical protein MGC17299 225822_at 1238 3335.8 0.000012 LOC440441 230388_s_at 313.6 496.2 0.000013 PLN Phospholamban 204939_s_at 36.7 146.3 0.000013 GPNMB Glycoprotein (transmembrane) nmb 201141_at 1141.9 2958.6 0.000013 COMP Cartilage oligomeric matrix protein 205713_s_at 31.8 199.6 0.000013 13CDNA73 Hypothetical protein CGOO3 214319_at 41.8 116 0.000015 HLA-E MHC class I, E 200904_at 575.2 1384.8 0.000017 NDRG2 NDRG family member 2 206453_s_at 184.9 432.4 0.000022 HNMT Histamine N-methyltransferase 211732_x_at 115 263.1 0.000031 MRVI1 Murine retrovirus integration site 1 226047_at 190.5 640.5 0.000031 homologue PAPSS2 3¶-Phosphoadenosine 5¶-phosphosulfate 203058_s_at 162.2 304.9 0.000034 synthase 2 CEBPA CCAAT/enhancer binding protein 204039_at 127.5 418.2 0.000034 (C/EBP), a DCN Decorin 211813_x_at 697.7 2510.1 0.000038 PIGR Polymeric immunoglobulin receptor 226147_s_at 1972.9 20996.8 0.00004 EGFR Epidermal growth factor receptor 201984_s_at 200.3 507 0.00004 (erythroblastic leukemia viral (v-erb-b) oncogene homologue, a vian) HSD17B6 Hydroxysteroid (17-h) dehydrogenase 6 205700_at 50.4 407.4 0.000041 INSIG1 Insulin-induced gene 1 201626_at 200.6 348.1 0.000051 DPT Dermatopontin 213071_at 31.1 132.2 0.000052

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Gene symbol Description Probeset Mean intensities Mean intensities Parametric P in samples with both in samples with both A6 and B3 down A6 and B3 up

MYH11 Myosin, heavy polypeptide 11, 201497_x_at 88.6 620.7 0.000057 smooth muscle CLEC7A C-type lectin domain family 7, 221698_s_at 119.6 307.8 0.000064 member A GGT2 g-Glutamyltransferase 2 215603_x_at 201.3 432.1 0.00007 TMEM51 Transmembrane protein 51 218815_s_at 155.8 285.4 0.000072 FBP1 Fructose-1,6-bisphosphatase 1 209696_at 413.8 1069.1 0.000077 GSDML Gasdermin-like 219233_s_at 112.1 274.8 0.00008 TNFSF13/ Tumor necrosis factor (ligand) 210314_x_at 384 941.7 0.000094 TNFSF12-TNFSF13 superfamily, member 13/tumor necrosis factor (ligand) superfamily, member 12-member 13 C18orf1 Chromosome 18 open reading 207996_s_at 65.5 146.7 0.000115 frame 1 SFRP4 Secreted frizzled-related 204052_s_at 69.5 192.8 0.000119 protein 4 ACOT2 Acyl-CoA thioesterase 2 202982_s_at 196.8 304.4 0.000119 HMOX2 Heme oxygenase (decycling) 2 218120_s_at 194.9 383.3 0.000131 C22orf13 Chromosome 22 open reading 223039_at 963.6 1573.3 0.000134 frame 13 TINF2 TERF1 (TRF1)-interacting nuclear 220052_s_at 154.1 253.5 0.000157 factor 2 CLUL1 Clusterin-like 1 (retinal) 206556_at 34.1 61.9 0.000179 APOC1 Apolipoprotein C-I 213553_x_at 643.4 1334.1 0.000196 RNF128 Ring finger protein 128 219263_at 104.3 365.2 0.000207 DOCK8 Dedicator of cytokinesis 8 232843_s_at 211.6 541.1 0.000233 DPP4 Dipeptidylpeptidase 4 (CD26, 203716_s_at 47.4 347.5 0.00027 adenosine deaminase completing protein 2) PLUNC Palate, lung, and nasal epithelium 220542_s_at 453.2 104.6 0.00016 carcinoma–associated phosphoribosyl amino imidazole carboxylase, phosphoribosyl amino imidazole succinocarboxamide synthetase PAICS 201013_s_at 1069.7 486.3 0.000054 FXR1 Fragile X mental retardation, autosomal 201637_s_at 1205.3 697.6 0.000051 homologue 1 STC1 Stanniocalcin 1 204595_s_at 337.2 150.4 0.000051 NME1 Non-metastatic cells 1, protein (NM23A) 201577_at 1670.8 980.2 0.00005 expressed in MXI1 MAX interactor 1 202364_at 681.9 389.5 0.000032 MGC14289 Similar to RIKEN cDNA 228280_at 829.4 428.2 0.000028 1200014N16 gene CCT7 Chaperonin containing TCP1, 200812_at 1033.6 656.8 0.000025 subunit 7 (eta) PFN2 Profilin 2 204992_s_at 1311.6 493.7 0.000012 FLJ13089 Hypothetical protein FLJ13089 227245_at 602.9 342.7 0.000009 MYC v-myc myelocytomatosis viral 202431_s_at 294.5 69.5 0.000008 oncogene homologue (avian) TOP1 Topoisomerase (DMA) I 208901_s_at 1246 667.9 0.000007 WDR12 WD repeat domain 12 218512_at 554.9 322 0.000005

NOTE: Parametric P values referred to significance level in class prediction.

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Exposure of NHBE cell lines to nicotine. We wished to know if subunit gene expression levels allowed for quantitative comparison brief exposure to nicotine would alter the expression level of any of between groups of samples with respect to gender and smoking the nAChR subunit genes in bronchial epithelial tissue. We took habits. We have shown overall statistically significant differences in advantage of our new immortalized HBEC to study this. When the expression levels of CHRNA4 and CHRNB4 between NSCLC HBEC lines were exposed to 100 nmol/L nicotine and then RNA tumor and normal lung tissues as well as CHRNA6 and CHRNB3 was harvested at 72 and 144 h, a significant increase in the between NSCLC tumors from smokers and nonsmokers (Fig. 2) and expression levels of CHRNA1, CHRNA5, and CHRNA7 were found at CHRNA1 and CHRNA7 with respect to gender. The expression 72 h, with return to baseline levels of expression upon nicotine levels of CHRNA6 and CHRNB3 were statistically different even removal (Fig. 3). No significant increase or decrease was found in after adjusting for the effect of gender. the levels of expression of other nAChR subunit genes analyzed. CHRNA4 is the major nAChR subtype found throughout the CNS and was thought to be responsible for nicotine addiction (19). The expression level of CHRNA4 was found to be significantly lower in Discussion NSCLC tumor compared with normal lung tissues. A modest The distribution, function, and ligand-binding affinity of nAChR elevation of CHRNB4 subunit gene expression in NSCLC compared depends on the composition of nAChR subunits, although the exact with normal lung tissues was detected in this study. Such function and physiologic roles of individual nAChR subtypes and differences in the levels of expression of CHRNA4 and CHRNB4 its subunits are not completely understood. To our knowledge, the were not found when comparing NSCLC cell lines or NHBE cells. pattern of expression of these nAChR subunits has not been There have been previous reports of functional polymorphism of reported in NSCLC. The quantitative PCR analysis of nAChR nAChR a4h4 leading to variable nicotine-induced transmembrane conductance in vitro (36). The relatively lower level of CHRNA4 expression in NSCLC tumor might imply that CHRNA4 expression was down-regulated in the process of lung carcinogenesis. One possible mechanism for this to happen could be desensitization of nAChR subunit gene expression on chronic exposure to stimulation by its agonists such as nicotine. The differences in the levels of expression of CHRNA4 and CHRNB4 subunit genes might imply that they were involved in the process of lung carcinogenesis, but the potential mechanism deserve further evaluation. The NSCLC cell lines showed other nAChR subunit gene expression differences when compared with NHBE cell lines (i.e., CHRNA5, CHRNA7, CHRNA9, and CHRNB2). The nAChR a5 subunit has been reported to be present in the brain and usually co- assembled with a3, a4, h2, or h4 to form various nAChR subtypes such as a4a5h2 and a3a5h4 (16). The presence of a5 subunit in nAChR has been shown to alter the calcium permeability and nicotine sensitivity in vitro (37). The nAChR a7 subunit was found to be important in the control of nicotine-induced calcium influx in SCLC (38) and was thus thought to be important in growth signal transduction induced by nicotine binding to nAChR. nAChR a9 was present mainly in inner ear hair cells and has been found to form nAChR with a10 subunits (39), but its function has not been fully characterized. nAChR h2 formed the nAChR a4h2 subtype that was thought to mediate nicotine addiction in the brain (20, 22). The fact that CHRNA5, CHRNA7, CHRNA9, and CHRNB2 subunit genes showed elevated levels of expression in NSCLC cell lines could indicate their involvement in constitutive cellular processes in cancer cell lines. CHRNA6 and CHRNB3 showed differences in expression level between NSCLCs from smokers (relatively lower expression levels) and nonsmokers (relatively higher expression levels), even after adjustment for the effect of gender. The pattern of CHRNA6 and CHRNB3 gene expression in our study of NSCLCs is similar to the observations that CHRNA6 and CHRNB3 are coexpressed in the CNS (16), and they form functional nAChR in the brain (21). In fact, CHRNA6 and CHRNB3 were located closely on chromosome 8(8p11.21 and 8p11.2, respectively), and it was possible that they share a common regulatory mechanism. The fact that both CHRNA6 and CHRNB3 subunit genes showed lower expression in Figure 3. Effects of nicotine stimulation on the expression levels of different NSCLCs from smokers when compared with nonsmokers could nAChR subunit genes in NHBE cell lines. HBEC(1-5)-KT, human bronchial epithelial cells stimulated by nicotine (nicotine groups); HBEC(1-5)-KTc, cells imply desensitization with chronic exposure to tobacco smoke. In without nicotine exposure (control groups). NSCLC cell lines, no significant difference in the expression of www.aacrjournals.org 4645 Cancer Res 2007; 67: (10). May 15, 2007

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Research nAChR subunit genes was found between cell lines derived from hyperplastic foci indicative of increased proliferative activity in the smokers and cell lines derived from nonsmokers. This could be lungs of smokers with COPD and other chronic pulmonary diseases attributed to the small number of NSCLC cell lines (n = 13) being (54). It was possible that, in addition to smoking status or nicotine studied, or that the effects of tobacco smoking on nAChR gene exposure, the presence of nonneoplastic pulmonary disease that expression had ceased with in vitro culture. impairs lung oxygenation may influence the expression of CHRNA7. A list of 65 differentially expressed genes was found to be CHRNA1, CHRNA5, and CHRNA7 showed significant reversible predictors of nAChR a6h3 phenotypes, whereas no significant induction of expression on in vitro exposure to nicotine, providing differentially expressed genes were found between samples with direct evidence that these subunit genes respond to acute nicotine different levels of expression of individual nAChR subunit genes or exposure and could mediate the immediate or short term effects of combinations, including a4h4, a4h2, a6h2, etc. Some of these genes nicotine. Little is known about the functional significance of the are involved in the regulation of apoptosis (p8; ref. 40), potential expression these specific CHRNA1, CHRNA5, and CHRNA7 in the tumor suppressor genes in lung cancer [CEBPA (41) and NME1 CNS or in lung cancer, although CHRNA7 had been reported to be (42)], or in other cancers [NDRG2 (43) and MXI1 (44)] and genes expressed in SCLC cell lines (5) and was thought to be related to involved in tumor progression and metastasis [DPP4 (45), PLUNC smoking in schizophrenic patients (50), as discussed above. Up- (46), and SFRP4 (47)]. Both EGFR and MYC were found to be on the regulation of functional CHRNA7 has also been shown in NHBE cells list of differentially expressed genes with respect to the a6h3 on exposure to nicotine (10). The return of the expression levels of phenotypes. Binding of nAChR by nicotine was thought to activate those three subunits to baseline upon cessation of nicotine exposure downstream signaling pathway via Akt phosphorylation (1). It has may reflect that continued or chronic exposure to nicotine (usually been reported that nAChR may trigger the MAPK pathway with taken to be more than 10 days of continuous exposure to nicotine; which EGFR and MYC were involved (reference: KEGG pathway ref. 55) was required for nicotine addiction or other cellular effects of hsa04010: classic MAPK pathway), eventually leading to promotion nicotine mediated by specific nAChR subunits. The effects of chronic of cell growth and proliferation. EGFR expression was higher in the exposure to nicotine on nAChR subunit gene expression in these high a6h3 tumors. Inhibitors of EGFR prevented nicotine-induced NHBE cells and whether the same response is maintained, or other Akt phosphorylation in mouse pheochromocytoma cell line (PC12; nAChR subunit genes would be involved, in chronic nicotine ref. 48). Thus, cross-talk between signaling downstream of EGFR exposure warrant further evaluation. As the baseline expression and nAChR activation via the AKT and MAPK pathways may level of CHRNA6 and CHRNB3 are low, we cannot determine whether together promote carcinogenesis in this group of tumors (48). On they are down-regulated on nicotine stimulation. the other hand, the low a6h3 tumors showed high MYC and low In summary, we have determined the pattern of expression of CEBPA expression. MYC overexpression had been found to lead to CHRN subunit genes in NSCLCs, normal lung, normal bronchial reduction in CEPBA-mediated cytochrome activation in the epithelial cells, and tumor cell lines. The combination of CHRNA6 presence of EGF stimulation in proliferating cells (49). Together, and CHRNB3 high expression correlated with NSCLCs in non- the data suggest involvement of distinctive pathways mediated by smokers, whereas the combination of low expression correlated EGFR or MYC in tumors that express different combination with NSCLCs from smokers. This leads to the identification of patterns of A6 and B3 nAChR subunits. Further investigation into differential gene expression with class predictive significance. With their relation with nicotine exposure and verification with short-term exposure to nicotine, there were elevated level of functional studies would be warranted. expression for CHRNA1, CHRNA5, and CHRNA7 subunit genes. There were gender differences detected in the expression level of These nAChR subunit genes could be playing a role in the CHRNA7 in lung cancer cell lines. CHRNA7 had been most well pathogenesis of bronchogenic carcinoma and may mediate the studied in patients with schizophrenia (50). There were suggestions effects of nicotine addiction in lung cancer patients of different of CHRNA7 correlating with gender and nicotine dependence in sexes. Further evaluation of the functions and roles played by these patients with schizophrenia (51). CHRNA7 has been reported to be nAChR subunit genes in nicotine addiction and lung carcinogenesis expressed in SCLC cell lines (5, 6). There was previous reports of are warranted to allow for opportunities in development of sensitization of the nAChR a7 subunit in human SCLC cell lines by chemoprevention strategy. elevated carbon dioxide level at the expense of oxygen level (52). It was thus proposed that chronic pulmonary condition such as chronic obstructive pulmonary disease (COPD) may promote the Acknowledgments growth of SCLC. This was further supported by demonstration of Received 12/21/2006; revised 2/21/2007; accepted 3/8/2007. up-regulation of the nAChR a7 subunit of endothelial cells by Grant support: Lung Cancer Specialized Programs of Research Excellence grant second-hand smoke and hypoxia (53). In addition, the growth of P50CA70907 and the Hong Kong Special Administrative Region RGC grant 7468/04M. The costs of publication of this article were defrayed in part by the payment of page pulmonary neuroendocrine cells was shown to be under the charges. This article must therefore be hereby marked advertisement in accordance control of the nAChR a7 subunit (27), with formation of multiple with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non−Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers

David Chi-leung Lam, Luc Girard, Ruben Ramirez, et al.

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