Gene Expression Signature of Cigarette Smoking and Its Role in Lung Adenocarcinoma Development and Survival Maria Teresa Landi1*, Tatiana Dracheva2, Melissa Rotunno1, Jonine D. Figueroa1,3, Huaitian Liu4, Abhijit Dasgupta1, Felecia E. Mann2, Junya Fukuoka2, Megan Hames2, Andrew W. Bergen1, Sharon E. Murphy5, Ping Yang6, Angela C. Pesatori7, Dario Consonni7, Pier Alberto Bertazzi7, Sholom Wacholder1, Joanna H. Shih4, Neil E. Caporaso1., Jin Jen2. 1 Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Bethesda, Maryland, United States of America, 2 Center for Cancer Research, National Cancer Institute, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Bethesda, Maryland, United States of America, 3 Cancer Prevention Fellowship, National Cancer Institute, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Bethesda, Maryland, United States of America, 4 Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Bethesda, Maryland, United States of America, 5 Biochemistry, Molecular Biology and Biophysics, University of Minnesota Cancer Center, Minneapolis, Minnesota, United States of America, 6 Department of Health Science, Mayo Clinic, Rochester, Minnesota, United States of America, 7 Foundation OM Policlinic, University of Milan, Milan, Italy Abstract Background: Tobacco smoking is responsible for over 90% of lung cancer cases, and yet the precise molecular alterations induced by smoking in lung that develop into cancer and impact survival have remained obscure. Methodology/Principal Findings: We performed gene expression analysis using HG-U133A Affymetrix chips on 135 fresh frozen tissue samples of adenocarcinoma and paired noninvolved lung tissue from current, former and never smokers, with biochemically validated smoking information. ANOVA analysis adjusted for potential confounders, multiple testing procedure, Gene Set Enrichment Analysis, and GO-functional classification were conducted for gene selection. Results were confirmed in independent adenocarcinoma and non-tumor tissues from two studies. We identified a gene expression signature characteristic of smoking that includes cell cycle genes, particularly those involved in the mitotic spindle formation (e.g., NEK2, TTK, PRC1). Expression of these genes strongly differentiated both smokers from non-smokers in lung tumors and early stage tumor tissue from non-tumor tissue (p,0.001 and fold-change .1.5, for each comparison), consistent with an important role for this pathway in lung carcinogenesis induced by smoking. These changes persisted many years after smoking cessation. NEK2 (p,0.001) and TTK (p = 0.002) expression in the noninvolved lung tissue was also associated with a 3-fold increased risk of mortality from lung adenocarcinoma in smokers. Conclusions/Significance: Our work provides insight into the smoking-related mechanisms of lung neoplasia, and shows that the very mitotic genes known to be involved in cancer development are induced by smoking and affect survival. These genes are candidate targets for chemoprevention and treatment of lung cancer in smokers. Citation: Landi MT, Dracheva T, Rotunno M, Figueroa JD, Liu H, et al. (2008) Gene Expression Signature of Cigarette Smoking and Its Role in Lung Adenocarcinoma Development and Survival. PLoS ONE 3(2): e1651. doi:10.1371/journal.pone.0001651 Editor: Dawn Albertson, N, Minnesota State University Mankato, United States of America Received January 12, 2008; Accepted January 16, 2008; Published February 20, 2008 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This study was supported by the Intramural Research Program of NIH, National Cancer Institute, Division of Cancer Epidemiology and Genetics and the Center for Cancer Research, and from the NIH-R01-84354 grant to P.Y. J.F. was supported by the Cancer Prevention Fellowship Program, National Cancer Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *E-mail: landim@mail.nih.gov .These authors contributed equally to this work. Introduction Lung cancer is the leading cause of cancer death worldwide. Cigarette smoking is responsible for about 90% of lung cancers and decreases survival,[1] and yet the precise molecular alterations induced by smoking in lung that develop into cancer and impact survival have remained obscure. Using Affymetrix HG-U133A microarrays on 135 fresh frozen adenocarcinoma and paired non-tumor tissue samples from current, former and never smokers from the Environment And Genetics in Lung cancer Etiology (EAGLE) study (http://dceg.cancer.gov/eagle), we sought to identify the genes that are altered by smoking in lung, and those, within the smoking signature, that have a role in lung carcinogenesis and outcome from lung cancer. We chose adenocarcinoma, the predominant histological subtype of lung cancer, because it occurs in subjects with no history of smoking as well as in smokers, providing a range of exposures ideal for the study of smoking-induced carcinogenesis. Specifically, in early stage adenocarcinoma tissue we compared gene expression from current (C) and never (N) smokers and identified the major genes using stringent criteria for gene selection (p,0.001 and fold change .1.5), the Benjamini-Hochberg procedure[2] to calculate PLoS ONE | www.plosone.org 1 February 2008 | Volume 3 | Issue 2 | e1651