DNA methylation profiles at precancerous stages associated with recurrence of lung adenocarcinoma

PLoS One. 2013;8(3):e59444. doi: 10.1371/journal.pone.0059444. Epub 2013 Mar 27.

Abstract

The aim of this study was to clarify the significance of DNA methylation alterations at precancerous stages of lung adenocarcinoma. Using single-CpG resolution Infinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue obtained from patients without any primary lung tumor, 145 samples of non-cancerous lung tissue (N) obtained from patients with lung adenocarcinomas, and 145 samples of tumorous tissue (T). Stepwise progression of DNA methylation alterations from normal lung tissue to non-cancerous lung tissue obtained from patients with lung adenocarcinomas, and then tumorous tissue samples, was observed at 3,270 CpG sites, suggesting that non-cancerous lung tissue obtained from patients with lung adenocarcinomas was at precancerous stages with DNA methylation alterations. At CpG sites of 2,083 genes, DNA methylation status in samples of non-cancerous lung tissue obtained from patients with lung adenocarcinomas was significantly correlated with recurrence after establishment of lung adenocarcinomas. Among such recurrence-related genes, 28 genes are normally unmethylated (average β-values based on Infinium assay in normal lung tissue samples was less than 0.2) and their DNA hypermethylation at precancerous stages was strengthened during progression to lung adenocarcinomas (Δβ(T-N)>0.1). Among these 28 genes, we focused on 6 for which implications in transcription regulation, apoptosis or cell adhesion had been reported. DNA hypermethylation of the ADCY5, EVX1, GFRA1, PDE9A, and TBX20 genes resulted in reduced mRNA expression in tumorous tissue samples. 5-Aza-2'-deoxycytidine treatment of lung cancer cell lines restored the mRNA expression levels of these 5 genes. Reduced mRNA expression in tumorous tissue samples was significantly correlated with tumor aggressiveness. These data suggest that DNA methylation alterations at precancerous stages determine tumor aggressiveness and outcome through silencing of specific genes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / genetics*
  • Adenocarcinoma / pathology*
  • Adenocarcinoma of Lung
  • Adult
  • Aged
  • Aged, 80 and over
  • Azacitidine / pharmacology
  • DNA Methylation / drug effects
  • DNA Methylation / genetics*
  • DNA Probes / metabolism
  • Female
  • Gene Expression Regulation, Neoplastic / drug effects
  • Gene Silencing
  • Genes, Neoplasm / genetics
  • Humans
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / pathology*
  • Male
  • Middle Aged
  • Neoplasm Recurrence, Local / genetics*
  • Neoplasm Recurrence, Local / pathology*
  • Neoplasm Staging
  • Precancerous Conditions / genetics*
  • Precancerous Conditions / pathology*
  • Proportional Hazards Models
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism

Substances

  • DNA Probes
  • RNA, Messenger
  • Azacitidine

Grant support

This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NiBio, 10–42, http://www.nibio.go.jp/part/promote/fundamental/doc/index.html) and partially supported by a Grant-in-Aid for the Third Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan (2, http://www.mhlw.go.jp/bunya/kenkyuujigyou/hojokin-koubo14/09.html), National Cancer Center Research and Development Fund (23-A-11, http://www.ncc.go.jp/jp/about/rinri/kaihatsu/files/h24_ncc_research_list.pdf), and Grants-in-Aid for Scientific Research (B, 233900 90, http://www.jsps.go.jp/j-grantsinaid/) and for Young Scientists (B, 23790456, http://www.jsps.go.jp/j-grantsinaid/) from the Japan Society for the Promotion of Science (JSPS). National Cancer Center Biobank is supported by the National Cancer Center Research and Development Fund (23-A-1, http://www.ncc.go.jp/jp/about/rinri/kaihatsu/files/h24_ncc_research_list.pdf), Japan. T. Sato is an awardee of a research resident fellowship from the Foundation for Promotion of Cancer Research in Japan (http://www.fpcr.or.jp/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.