Abstract 4946: The nicotinic acetylcholine receptor genes CHRNA3 and CHRNB4 are epigenetically deregulated in lung tumorigenesis
Authors: David B. Scherf, Narek F. Sarkisyan, Hanna T. Jacobsson, Rainer Claus, Justo L. Bermejo, Thomas Muley, Michael Meister, Hendrik Dienemann, Christoph Plass, Angela Risch
Journal: Cancer Research (2010)

Abstract Background: Lung cancer, the leading cause of cancer-related death worldwide, is strongly connected with tobacco smoking. Genome-wide association studies have recently identified three lung cancer susceptibility regions in 15q25, 5p15.33 and 6p21.33 that strongly associate with lung cancer risk. Here we investigate whether these three susceptibility regions harbor genes with different DNA methylation patterns in tumor and adjacent normal lung tissue from the same individual. Methods: For DNA methylation analysis we used the MassARRAY (Sequenom), a MALDI-TOF based method, to quantify DNA methylation in tumor and normal lung tissue from 37 patients. We analysed 17 CpG islands situated in nine different genes situated in the three lung cancer susceptibility regions. Nonparametric and hurdle models were used to evaluate methylation differences. Additionally, reverse transcription PCR was performed with a LightCycler and expression analysis was conducted in 50 tumor/normal pairs. This included 25 adenocarcinoma (AdC) and 25 squamous cell carcinoma (SCC) (10xStage I, 10xStageII, 5xStageIII) matched pairs. In reexpression experiments we treated A549 and H1299 cells with 0.5 and 1 µM of 5-aza-deoxycitidine (5-Aza-dC). Results: Comparing tumor and normal tissue we obtained significant differences in DNA methylation for at least two CpG units situated in a putative promoter (p&amp;lt;0.001) for each of three genes, the alpha 3 and beta 4 subunit genes of the nicotinic acetylcholine receptor CHRNA3 and CHRNB4 and for the aminoglycoside phosphotransferase domain gene AGPHD. To assess RNA expression of these genes we analyzed 50 tumor/normal pairs by real-time quantitative RT-PCR. Matched pairs analysis showed a significant overall down regulation of CHRNA3 in SCC patient tumors (p=0.002). For CHRNB4 we detected increased transcription in lung tumors in both histologies (p&amp;lt;0.001). The expression analysis for AGPHD did not reveal a clear aberrant deregulation. After drug treatment increased expression was detected for CHRNA3 and CHRNB4 but not for AGPHD transcripts. Conclusion: In the present study we identified two genes situated in the nicotinic acetylcholine receptor cluster on chromosome 15q25, CHRNA3 and CHRNB4, as being differentially methylated in lung tumors compared to adjacent normal tissue, which is also reflected in gene expression. These results suggest CHRNA3 and CHRNB4 as strong candidate lung cancer susceptibility genes for further investigation in functional analyses to bring gene deregulation into a chronological context in lung tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4946.