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Epigenetic screen identifies genotype-specific promoter DNA methylation and oncogenic potential of CHRNB4

Oncogene volume 32, pages 3329–3338 (2013)Cite this article

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Abstract

Genome-wide association studies have highlighted three major lung cancer susceptibility regions at 15q25.1, 5p15.33 and 6p21.33. To gain insight into the possible mechanistic relevance of the genes in these regions, we investigated the regulation of candidate susceptibility gene expression by epigenetic alterations in healthy and lung tumor tissues. For genes up or downregulated in lung tumors, the influence of genetic variants on DNA methylation was investigated and in vitro studies were performed. We analyzed 394 CpG units within 19 CpG islands in the susceptibility regions in a screening set of 34 patients. Significant findings were validated in an independent patient set (n=50) with available DNA and RNA. The most consistent overall DNA methylation difference between tumor and adjacent normal tissue on 15q25 was tumor hypomethylation in the promoter region of CHRNB4 with a median difference of 8% (P<0.001), which resulted in overexpression of the transcript in tumors (P<0.001). Confirming previous studies, we also found hypermethylation in CHRNA3 and telomerase reverse transcriptase (TERT) with significant expression changes. Decitabine treatment of H1299 cells resulted in reduced methylation levels in gene promoters, elevated transcript levels of CHRNB4 and CHRNA3, and a slight downregulation of TERT demonstrating epigenetic regulation of lung cancer cells. Single-nucleotide polymorphisms rs421629 on 5p15.33 and rs1948, rs660652, rs8040868 and rs2036527 on 15q25.1, previously identified as lung cancer risk or nicotine-addiction modifiers, were associated with tumor DNA methylation levels in the promoters of TERT and CHRNB4 (P<0.001), respectively, in two independent sample sets (n=82; n=150). In addition, CHRNB4 knockdown in two different cell lines (A549 and H1299) resulted in reduced proliferation (PA549<0.05;PH1299<0.001) and propensity to form colonies in H1299 cells. These results suggest epigenetic deregulation of nicotinic acetylcholine receptor subunit (nAChR) genes which in the case of CHRNB4 is strongly associated with genetic lung cancer susceptibility variants and a functional impact on tumorigenic potential.

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Acknowledgements

We thank Frau Heinzmann-Groth, Birgit Jäger and all other members of the clinical and laboratory teams for help with sample and/or data collection and archiving for the Heidelberg lung study. We are grateful to all patients at the Thoraxklinik Heidelberg, who participated in the study. We thank Ruprecht Kuner for the cell lines, Oliver Mücke for competent technical assistance, Chris Amos for helpful comments on the manuscript and the members of the Plass Laboratory for thoughtful discussions. The scientific development and funding of this project were in part supported by the Genetic Associations and Mechanisms in Oncology (GAME-ON): a NCI Cancer Post-GWAS Initiative. This work was in part supported by the National Institute of Health (USA; grant number CA148127) and earlier sample collection by the Deutsche Krebshilfe (grant number 70–2387). JLB was in part supported by a grant of the Deutsche Forschungsgemeinschaft (DFG, SFB/TRR77, project Z2).

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Author notes

  1. T Muley, M Meister, H Dienemann and A Risch: Translational Lung Research Centre Heidelberg (TLRC-H), Member of the German Center for Lung Research.

Authors and Affiliations

  1. Division of Epigenomics and Cancer Risk Factors (C010), German Cancer Research Center (DKFZ), Heidelberg, Germany

    D B Scherf, N Sarkisyan, H Jacobsson, R Claus, L Gu, C Plass & A Risch

  2. Division of Molecular Genetic Epidemiology, Institute of Medical Biometry and Informatics, University Hospital Heidelberg, German Cancer Research Center, Heidelberg, Germany

    J L Bermejo & B Peil

  3. Translational Research Unit, Thoraxklinik-Heidelberg gGmbH, University of Heidelberg, Germany

    T Muley & M Meister

  4. Department of Thoracic Surgery, Thoraxklinik-Heidelberg gGmbH, University of Heidelberg, Germany

    H Dienemann

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Scherf, D., Sarkisyan, N., Jacobsson, H. et al. Epigenetic screen identifies genotype-specific promoter DNA methylation and oncogenic potential of CHRNB4. Oncogene 32, 3329–3338 (2013). https://doi.org/10.1038/onc.2012.344

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