Smoking Signature in Lung Figure 1. Comparison of gene expression differentiating current from never smokers (C/N) and gene expression differentiating former from never smokers (F/N) in early stage tumor tissue (T) using Gene Set Enrichment Analysis (GSEA). Left: Running Enrichment Score (y axis) is calculated by walking down the entire list of probes from Affymetrix HG-U133A chip (numbered from 1 to 22,283 in the x axis) ordered by the ANOVA coefficients divided by the standard error values from the Former/Never (F/N) smoking comparison. This running-sum statistic increases when a given probe is in the Current/Never (C/N) Gene Set of interest and decreases when the probe is not in the C/N Gene Set, with the magnitude of increment depending on the strength of the correlation between the probe and the F/N comparison. The Enrichment Score (ES) is the maximum deviation of the Running Enrichment Score from zero encountered in the random walk and reflects the degree to which the Gene Set is overrepresented at the extremes (top or bottom) of the entire ranked probe list. We report results for two different C/N Gene Sets: on the top, the 64 up-regulated probes, with ES = 0.87 and, on the bottom, the 98 down-regulated probes, with ES = 20.90. A leading edge subset of the Gene Set is defined as those probes in the Gene Set that appear in the probes ranked list at, or before, the point where the running sum reaches its maximum deviation from zero. The leading edge for the Gene Set of the C/N up-regulated probes contains 58 probes over 64 and the leading edge for the Gene Set of down-regulated probes contains 90 over 98 probes. This confirms that among the 64 up-regulated probes from the C/N comparison, 58 are also found in the F/N comparison; and among the 98 down-regulated probes from the C/N comparison, 90 are also found in the F/N comparison. Right: distributions of ES values created using a permutation procedure for (top) the Gene Set of up-regulated probes in C/N and (bottom) the Gene Set of down-regulated probes in C/N. These distributions are used to calculate the statistical significance (nominal p-value) of the observed ES values (p-value,0.002 in both cases). doi:10.1371/journal.pone.0001651.g001 Validation was based on 68 samples, including 43 T and 25 NT, also used for the microarray analysis. All 19 genes were up- regulated in the C/N comparison in these samples (Table 4). Confirmation was based on 40 independent samples (19 T and 21 NT) from EAGLE (samples not used for microarray analysis) and the Mayo Clinic, Rochester, MN. All the 14 genes in T and 4 of 5 genes in NT were up-regulated by smoking also in the independent samples (Table 4). Discussion In a population-based study with fresh frozen tissue samples of adenocarcinoma and noninvolved lung tissue (mostly paired samples), we identified a smoking signature that persists years after smoking cessation and is related to lung cancer development and survival. Aneuploidy and chromosome instability are two of the most common abnormalities in cancer cells that arise through unequal segregation of chromosomes between daughter cells during mitosis. Thus, mitotic alterations are highly relevant for carcino- genesis. We found that smoking induces deregulation of this very mitotic process proceeding from lung tissue changes through cancer development to cancer death or survival. In fact, the smoking signature we identified comprises genes that regulate the mitotic spindle formation. These genes, such as NEK2[11,12] and CENPF[11] (both on 1q32-q41), TPX2[13,14] and STK6 (or AURKA)[15] (related to the Aurora-A activation pathway important in tumor progression[16]), TTK (linked to cell mitosis through EGFR,[17] a critical drug target for lung adenocarcino- ma[18]), and BIRC5 (Survivin),[19] have all been found over- expressed in smoking-related tumors. While previous studies have proposed these genes as targets for therapeutic interven- tions,[16,18–21] our work suggests that they may be targets for chemoprevention in smokers as well. In fact, they were strongly induced by smoking in the early stage tumor tissue and some, e.g., NEK2 and TTK, were also associated with increased mortality PLoS ONE | www.plosone.org 5 February 2008 | Volume 3 | Issue 2 | e1651