Abstract A27: Contribution of DNA methylation in development of lung cancer in former smokers

Abstract

Abstract A27 Background Lung cancer is the most common cause of cancer death worldwide with more than 1.2 million people dying of the disease each year. Half of newly diagnosed lung cancer patients are former smokers. Understanding why former smokers develop lung cancer is clearly important to the development of early detection, prevention and treatment strategies for these people. The effects of cigarette smoke on the epigenome are widespread as both global DNA methylation and local DNA methylation have been identified, associated with genomic instability and tumour suppressor gene (TSG) silencing, respectively. In a cancer-specific context, upregulation of oncogenes and silencing of tumour suppressor genes both occur as a result of tobacco smoke exposure. Therefore, molecular studies examining tumors at genomic and epigenomic levels will likely identify causal genetic events involved in cancer development in former smokers. Objective The objective of this study is to determine the contribution of DNA methylation as a mechanism to lung cancer development in former smokers. Hypothesis As smoking induces methylation changes in bronchioepithelial cells, we hypothesize that this constitutes the first hit to TSG inactivation. In order to inactivate both alleles, these methylation changes would be maintained in tumors where a second hit would be found at the same gene loci. Materials and Methods Epithelial cells from former smokers (those with >10 years of smoking cessation) were collected from peripheral airways during routine bronchoscopy. Half of the cells were fixed in Cytolyt and the other half in RNAlater for DNA and RNA extraction, respectively. Copy number profiling was performed by array comparative genomic hybridization (aCGH) using whole-genome tiling path SMRT v2 BAC array. Methylation analysis was performed by coupling affinity based enrichment of methylated sequences with hybridization to the same aCGH platform described above. Expression status of genes was determined by gene expression microarray analysis using Agilent 44K expression arrays. RESULTS: Preliminary MeDIP aCGH of bronchial brush cells from eight former smokers who had previous surgical removal of Stage I NSCLC, and eight former smokers without NSCLC, of similar age (68±7 versus 62±6), revealed distinct differences in frequency of DNA methylation between cancer and non-cancer groups. For example, at 11p13 the cancer group shows hypermethylation at the WT1 locus, a known TSG. Analysis in 62 NSCLC tumors for gene dosage, showed a high frequency of loss at the WT1 locus. To examine downstream effects of these events in tumours, expression of WT1 was assessed and found to be significantly underexpressed in the majority of NSCLC tumours compared to a normal lung reference. Collection of more samples and further integrative analysis is currently underway. Conclusion Differences in methylation between these two groups may explain why some former smokers develop cancer while others remain cancer free despite similar lifestyle changes. As methylation is a reversible DNA modification, this knowledge would prompt the development and application of DNA demethylation chemopreventative agents and unique therapeutic strategies. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A27.