Smoking is associated with hypermethylation of the APC 1A promoter in colorectal cancer: the ColoCare Study

Abstract

Smoking tobacco is a known risk factor for the development of colorectal cancer and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome-wide analysis of DNA methylation in colorectal tumours from 36 never-smokers, 47 former smokers, and 13 active smokers, and in adjacent mucosa from 49 never-smokers, 64 former smokers, and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS < 1 × 10-5 ) in the tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never-smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, ρ = 0.26, p = 0.03) and was confined to tumours, with hypermethylation never being observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking-associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: APC; DNA methylation; colorectal cancer; epigenetics; smoking; tobacco.

Conflict of interest statement

The authors report no conflicts of interest.

Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Figures

Figure 1

Overview of analyses by smoking behaviours in tumours and adjacent mucosa. Differentially methylated sites between smokers and never smokers were identified in tumour tissue and in adjacent mucosa. Further analyses were performed to identify sites displaying smoking-specific differential methylation between tumours and adjacent mucosa.

Figure 2

Manhattan plots showing differentially methylated sites between never and active smokers. Results of the analyses between tumours from never and active smokers (A) and differential methylation between tumours and adjacent mucosa unique to active smokers (B). Gene symbols of the genes associated with the most significantly different sites are provided. The threshold (line) represents statistical significance (pLIS−5)

Figure 3

Methylation of the APC promoter 1A in tumours and matched adjacent mucosa. Mean methylation levels (beta values) for each patient were calculated across the 15 CpG sites mapping to the 1A promoter that were identified as differentially methylated by smoking status (Figure 2). (A) promoter methylation in tumours by patient smoking status. Mean values by smoking status are indicated by horizontal lines. (B) promoter methylation in tumours by AJCC stage in all patients. Mean values by stage are indicated by horizontal lines. (C) promoter methylation in matched samples of tumours and adjacent mucosa from 89 patients (33 never smokers, 43 former smokers, and 13 active smokers). Lines indicate matched samples from the same patient.