Aberrant methylation of the MSH3 promoter and distal enhancer in esophageal cancer patients exposed to first-hand tobacco smoke

Abstract

Purpose: Polymorphisms in MSH3 gene confer risk of esophageal cancer when in combination with tobacco smoke exposure. The purpose of this study was to investigate the methylation status of MSH3 gene in esophageal cancer patients in order to further elucidate possible role of MSH3 in esophageal tumorigenesis.

Methods: We applied nested methylation-specific polymerase chain reaction to investigate the methylation status of the MSH3 promoter in tumors and matching adjacent normal-looking tissues of 84 esophageal cancer patients from a high-risk South African population. The Cancer Genome Atlas data were used to examine DNA methylation profiles at 17 CpG sites located in the MSH3 locus.

Results: Overall, promoter methylation was detected in 91.9 % of tumors, which was significantly higher compared to 76.0 % in adjacent normal-looking esophageal tissues (P = 0.008). When samples were grouped according to different demographics (including age, gender and ethnicity) and smoking status of patients, methylation frequencies were found to be significantly higher in tumor tissues of Black subjects (P = 0.024), patients of 55-65 years of age (P = 0.032), males (P = 0.037) and tobacco smokers (P = 0.015). Furthermore, methylation of the MSH3 promoter was significantly more frequent in tumor samples from smokers compared to tumor samples from non-smokers [odds ratio (OR) = 31.9, P = 0.031]. The TCGA data confirmed significantly higher DNA methylation level at the MSH3 promoter region in tumors (P = 0.0024). In addition, we found evidence of an aberrantly methylated putative MSH3-associated distal enhancer element.

Conclusion: Our results suggest that methylation of MSH3 together with exposure to tobacco smoke is involved in esophageal carcinogenesis. Due to the active role of the MSH3 protein in modulating chemosensitivity of cells, methylation of MSH3 should further be examined in association with the outcome of esophageal cancer treatment using anticancer drugs.

Fig. 1

Representative methylation-specific PCR (MSP). PCR products in U lanes represent unmethylated MSH3 promoter, whereas PCR products in M lanes indicate the presence of methylated MSH3 promoter. L marks 100-bp DNA ladder

Fig. 2

MSH3 promoter methylation frequency. Comparison of methylation frequency in tumors (closed bars) and adjacent normal-looking tissues (open bars) from MSP analysis. All bars are divided into semi-methylated (lower part) and completely methylated (upper part) samples. The number of methylated samples versus total number of samples is indicated above each bar. Chi-square test was used (*P < 0.05; **P < 0.01)

Fig. 3

DNA methylation at MSH3 gene region on chromosome 5. a Graphical representation of MSH3 gene region using UCSC Genome Browser (http://genome.ucsc.edu). Chr5:79949332-80217274 and chr5:79949332-79959924 (zoom in) genomic regions are aligned to February 2009 (GRCh37/hg19) assembly of the human genome. Genomic locations of investigated CpG sites (blue), position of MSP region examined in this study (red) and linkage disequilibrium pattern (r ² > 0.80) of rs26279 (green) are shown. b DNA methylation levels of CpG sites in tumors versus matching normal tissues are compared (box plots). Only significant differences (P < 0.05) are shown