Gene expression signatures of cigarette smoking in tumor and normal lung tissue from EAGLE

Lung cancer is the leading cause of cancer mortality in the US, and is largely due to tobacco smoking. To investigate molecular alterations in tobacco carcinogenesis, we studied the smoking-related gene expression changes in lung tumor and non-involved normal surrounding tissues from 20 never smoker, 26 former smoker, and 28 current smoker lung adenocarcinoma patients with similar age within the large population-based Environment And Genetics in Lung cancer Etiology (EAGLE) study. Total RNA was extracted from microscopically confirmed lung tissue samples frozen immediately after resection. Gene expression profiles were obtained using Affymetrix U133A GeneChip, and processed using the Bioconductor and Affymetrix Microarray Suite with Robust Multichip Analysis (RMA). Expression analyses were conducted using R (ANOVA model, adjusted by sex). We used Gene Set Enrichment Analysis (GSEA) to compare differentially expressed gene groups, and GoMiner to investigate molecular functions and biological processes. We selected genes with significant expression changes in stages I/II lung tumors and in normal tissues from all cases with p <0.001, fold difference >1.5, and False Discovery Rate (FDR) <12%. When compared to lung tumors of never smokers, 72 genes were significantly down-regulated and 38 genes were up-regulated in current smoker tumors. Among these genes, 62/72 and 11/38 were also significantly altered (p<0.001, GSEA analysis) in tumor tissue from former smokers, suggesting that many smoking-related gene alterations persist even after smoking cessation. Biologically, genes involved in mitosis and cell cycle (e.g., TPX2, TTK, NEK2, KIF2C, BRRN1, and CCNA2) were among those most up-regulated, while genes involved in the regulation of MAPK activity were down-regulated. In normal tissues from the same subjects, we identified 75 down- and 19 up-regulated genes, respectively, between current and never smokers. In contrast to the tumors, genes involved in immune and defense responses (e.g., CCL11, BST2, GZMA, CX3CR1, etc.) had the most prominent smoking-related changes in the normal lung tissues. These results suggest that smoking may increase susceptibility to lung cancer by altering host defense functions while disregulation of cell cycle control underlies smoking-related lung carcinogenesis. The most promising candidate genes are being confirmed by real-time PCR. Insights into the molecular pathogenesis of lung cancer resulting from these analyses may suggest new mechanisms relevant to therapy and prevention.