The presence of aberrant DNA methylation in noncancerous esophageal mucosae in association with smoking history

Abstract

BACKGROUND: Esophageal squamous cell carcinomas (ESCCs) tend to have multiple primary lesions, and it is believed that they arise from background mucosae with accumulation of genetic/epigenetic alterations. In this study, the objective was to elucidate the effects of smoking and drinking on the accumulation of epigenetic alterations in background mucosae. METHODS: Genes that are silenced in human ESCCs were searched for by treating 3 ESCC cell lines with the demethylating agent, 5‐aza‐2′‐deoxycytidine and performing oligonucleotide microarrays. Methylation levels were analyzed by quantitative methylation‐specific polymerase chain reaction analysis of 60 ESCCs and their corresponding background mucosae. RESULTS: Forty‐seven genes were identified as methylation‐silenced in at least 1 of the 3 ESCC cell lines, and 14 of those genes (claudin 6 [CLDN6]; G protein‐coupled receptor 158 [GPR158]; homeobox A9 [HOXA9]; metallothionein 1M [MT1M]; neurofilament, heavy polypeptide 200 kDa [NEFH]; plakophilin 1 [PKP1]; protein phosphatase 1, regulatory [inhibitor] subunit 14A [PPP1R14A]; pyrin domain and caspase recruitment domain containing [PYCARD]; R‐spondin family, member 4 [RSPO4]; testis‐specific protein, Y‐encoded–like 5 [TSPYL5]; ubiquitin carboxyl‐terminal esterase L1 [UCHL1]; zinc‐finger protein 42 homolog [ZFP42]; zinc‐finger protein interacting with K protein 1 homolog [ZIK1]; and zinc‐finger and SCAN domain containing 18 [ZSCAN18]) were used as markers. In the background mucosae, methylation levels of 5 genes (HOXA9, MT1M, NEFH, RSPO4, and UCHL1) had significant correlations with smoking duration (ρ = .268; P = .044; ρ = .405; P = .002; ρ = .285; P = .032; ρ = .300; P = .024; and ρ = .437; P = .001, respectively). In contrast, an inverse correlation between PYCARD methylation levels and alcohol intake was observed (ρ = −.334, P = .025) among individuals with the inactive aldehyde dehydrogenase 2 (ALDH2) genotype. CONCLUSIONS: The current results suggested that ESCCs developed from an epigenetic field for cancerization, which was induced by exposure to carcinogenic factors, such as tobacco smoking. The epigenetic field defect will be a novel target for risk diagnosis and prevention of ESCCs. Cancer 2009. © 2009 American Cancer Society.

Revisit of Field Cancerization in Squamous Cell Carcinoma of Upper Aerodigestive Tract: Better Risk Assessment with Epigenetic Markers

“…In contrast, the performance of our 4-gene panel may effectively identify subjects who will benefit from endoscopic surveillance. Our panel's superiority may be due to our meticulous search for candidate genes by oligonucleotide microarray, which decreased bias in choosing targets (30), and to using qMSP to extract full information without truncating the methylation value (23). Imaging-enhanced endoscopies minimized the risk of contamination during sample collection (28,29).…”

“…The DNA methylation of the epigenetic markers was quantified with the qMSP. The laboratory work was similar to the details previously reported that used an independent test set of 60 tumor samples derived from Japanese individuals (30). In brief, 14 genes whose promoter CpG islands were methylated in esophageal SCCs were isolated by a genome-wide screening of genes reexpressed after esophageal SCC cell lines were treated with the demethylating agent 5-aza-2 0 -deoxycytidine.…”

“…Laboratory analyses were done by laboratory technicians who were blinded to the clinical status of the samples analyzed. As in our previous report (30), DNA was digested by BamHI, denatured, and treated by 15 cycles denaturing for 30 seconds at 95 C and incubating for 15 minutes at 50 C in 3.1 N sodium bisulfite (pH 5.0) and 0.5 mmol/L hydroquinone. The samples were desalted and then desulfonated in 0.3 N NaOH.…”

“…The sample size of this prospective study was planned previously based on our previous study (30). That study found that methylation levels in the normal esophageal mucosae of cancer patients (i.e., group 3) were 2.7% AE 2.5% (average AE SD; median: 2.4%; range: 0%-16.3%) for HOXA9, 7.2% AE 5.1% (6.2%; 0.4%-20.3%) for NEFH, 1.8% AE 3.6% (0.9%; 0%-22.4%) for UCHL1, and 1.9% AE 1.7% (1.3%; 0%-6.8%) for MT1M.…”

Epigenetics in Head and Neck Squamous Cell Carcinoma

“…The last ten years brought evidence that the genetic changes in the field are frequently accompanied by epigenetic aberrations. The epigenetic field defect was observed for oesophageal (Oka et al, 2009), lung (Guo et al, 2004) or stomach (Ushijima, 2007) cancers. Similarly, epigenetic www.intechopen.com changes in normal mucosa cells derived from surgical margins were detected in head and neck carcinomas (Martone et al, 2007).…”