Smoking Signature in Lung Table 2. Cell cycle genes differentiating current from never smokers (C/N) in the early stage tumor (T) tissue samples, and corresponding values in the former/never smoker (F/N) and in the smokers’ paired tumor/non-tumor tissue (T/NT) comparisons. Chromosomal Current/Never{ N = 30 Former/Never N = 23 Tumor/Non-Tumor N = 36 Fold-change Fold-change p-value Fold-change Probe ID 204641_at 204822_at 218009_s_at 207828_s_at 202095_s_at 203362_s_at 219918_s_at 210559_s_at 201897_s_at 204170_s_at 222077_s_at 203214_x_at 219306_at 209642_at 210052_s_at 203418_at 212020_s_at 201088_at 211519_s_at 218252_at 204887_s_at 211080_s_at 214894_x_at 208634_s_at 202284_s_at 208893_s_at RACGAP1* Gene Symbol NEK2* TTK* PRC1* CENPF* BIRC5* MAD2L1 ASPM CDC2 CKS1B CKS2 CDC2 KIF15* BUB1* TPX2* CCNA2 MKI67 KPNA2 KIF2C* CKAP2 PLK4 NEK2* MACF1 MACF1 CDKN1A DUSP6 Location 1q32.2–q41 6q13–q21 15q26.1 1q32–q41 17q25 4q27 1q31 10q21.1 1q21.2 9q22 12q13.12 10q21.1 3p21.31 2q14 20q11.2 4q25–q31 10q25-qter 17q23.1–q23.3 1p34.1 13q14 4q27–q28 1q32.2–q41 1p32–p31 1p32–p31 6p21.2 12q22–q23 3.45 3.27 2.99 2.88 2.72 2.67 2.59 2.54 2.36 2.36 2.35 2.29 2.22 2.17 2.06 1.99 1.95 1.82 1.78 1.75 1.74 1.57 0.65 0.60 0.54 0.34 p-value 0.0001 ,0.0001 0.0007 ,0.0001 0.0002 0.0003 0.0008 0.0009 0.0002 0.0006 0.0003 0.0006 0.0002 0.0009 0.0006 ,0.0001 ,0.0001 ,0.0001 0.0004 0.0008 0.0001 0.0001 0.0003 0.0001 0.0003 0.0003 2.84 2.08 2.61 2.28 2.10 1.93 2.12 2.02 1.89 2.02 1.91 1.98 2.00 1.68 1.87 1.85 1.71 1.53 1.67 1.52 1.55 1.50 0.64 0.58 0.70 0.32 0.0036 0.0123 0.0109 0.0034 0.0145 0.0309 0.0218 0.0298 0.0152 0.0148 0.0178 0.0150 0.0047 0.0507 0.0100 0.0012 0.0016 0.0079 0.0062 0.0292 0.0066 0.0019 0.0016 0.0004 0.0668 0.0012 3.14 2.22 2.60 2.77 2.55 2.74 2.87 2.37 2.47 1.69 2.13 2.12 1.90 2.02 2.07 1.82 1.41 2.34 1.51 1.47 1.48 1.36 0.52 0.42 0.65 0.84 p-value ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 0.0015 ,0.0001 ,0.0001 0?0001 0.0001 ,0.0001 ,0.0001 0.0006 ,0.0001 0.0002 0.0001 ,0.0001 0.0002 ,0.0001 ,.0.0001 0.0082 0.3102 {Probe selection restricted to estimates with p,0.001 and fold-change .1.5 or ,0.6667, and within the most inclusive category of genes with p#0.001 in the GoMiner analysis (GO ID 7049, Appendix S2D). *Genes involved in the mitotic spindle formation. The double line separates up-regulated and down-regulated probes. doi:10.1371/journal.pone.0001651.t002 genes with the stringent selection criteria (Table 1, and Appendix S3A, S3B). As expected, the CYP1B1 gene, known to be induced by smoking[9,10] was strongly up-regulated. The GoMiner results showed that the most smoking-altered genes were involved in cellular defense response (5 of 90 cellular defense genes on the chip, p,0.001), and more generally in immune response (Appendix S3C). MACF1, UBE21, and CBX7 (p,0.001), and C16orf30 (p = 0.001) were shared between T and NT C/N comparisons. C16orf30 and UBE21, both on chromosome 16p13.3, are located within 246kb, but they do not appear to share specific transcriptional regulation mechanisms (Appendix S4A). The GSEA analysis revealed some similarities between T and NT in the overall pattern of smoking-induced alteration (p = 0.08 and 0.04, for up- and down-regulated genes, respectively, Appendix S4B, S4C, and S4D). Notably, NEK2 and TTK were among those similarly altered in both T and NT in the GSEA analysis. In contrast, the F/N comparison in NT showed no statistically significant genes (Table 1), and was not further explored. Smoking-associated gene expression signature and survival from lung cancer We studied the overall gene expression signature of smoking in T and NT (98+64 C/N in T, 75+28 C/N in NT, minus 3 overlapping probes between T and NT, for a total of 262 probe- sets representing 230 genes) in relation to survival from adenocarcinoma in smokers (n = 54, Appendix S5A). Since only 262 probe-sets were included in this analysis, we used a less stringent criterion of p,0.01 for gene selection (Table 3). Altered expression in NT of genes involved in the mitotic spindle formation, e.g., NEK2 (p,0.001) and TTK (p = 0.001) were associated with a 3-fold increased mortality risk (Table 3, analysis adjusted for stage, sex, and smoking). Validation and confirmation of gene expression smoking signature We selected 19 genes (20 probes) for validation by QRTPCR, including 14 genes for T and 5 for NT tissue, based on fold change (.2) and cancer relevance. PLoS ONE | www.plosone.org 4 February 2008 | Volume 3 | Issue 2 | e1651