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. 2014 May 14;5(6):457-64.
doi: 10.7150/jca.9145. eCollection 2014.

Regulation of Desmocollin3 Expression by Promoter Hypermethylation is Associated with Advanced Esophageal Adenocarcinomas

Qinggang Wang  1 , DunFa Peng  2 , Shoumin Zhu  2 , Zheng Chen  3 , TianLing Hu  2 , Mohammed Soutto  2 , Rama Saad  4 , Shutian Zhang  5 , Wael Ei-Rifai  6
Affiliations

Regulation of Desmocollin3 Expression by Promoter Hypermethylation is Associated with Advanced Esophageal Adenocarcinomas

Qinggang Wang et al. J Cancer. .

Abstract

Background: Desmocollin3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and plays an important role in tumor invasion and metastasis. In this study, we investigated the epigenetic mechanism that regulates DSC3 expression in esophageal adenocarcinomas (EACs).

Methods: Expression of DSC3 was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The promoter DNA methylation level of DSC3 was examined using quantitative bisulfite pyrosequencing.

Results: The qRT-PCR analysis demonstrated significant down-regulation of the DSC3 mRNA levels in human EAC cell lines and tissue samples (P<.001). In addition, the EAC cell lines and tumor samples have aberrant promoter hypermethylation as compared to normal esophageal samples (P<.001). DSC3 promoter hypermethylation (>10% methylation level) was detected in 97.5% (39/40) of EAC samples whereas none of the normal tissue samples showed hypermethylation (P<.0001). There was a significant inverse correlation between promoter DNA methylation levels and mRNA expression folds for DSC3 (coefficient r=-0.685, P<.0001). Treatment of FLO-1 and SKGT4 EAC cells with 5-Aza-deoxytidine led to a significant reduction in the promoter DNA methylation levels with restoration of the DSC3 expression, suggesting that promoter DNA methylation is a key epigenetic mechanism regulating DSC3 expression. High DSC3 promoter DNA methylation levels were significantly correlated with advanced tumor stage (P<.001) and lymph node metastasis (P<.001).

Conclusion: Taken together, our results demonstrate that epigenetic silencing of DSC3 is a frequent finding in EAC that is possibly associated with advanced stages.

Keywords: DSC3; cancer; epigenetics; esophageal; metastasis..

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Conflict of interest statement

Competing Interests: All the authors declared no conflict of interest for the purpose of this study.

Figures

Figure 1
Figure 1
DSC3 promoter CpG island and Pyrosequencing. A) A schematic chart shows DSC3 genomic structure. DSC3 has 16 exons as shown in black boxes. A CpG island with dense CpG sites is present from -400 to +200 bp relative to the transcription start site (TSS); each vertical bar represents one CpG site. Forward and reverse primers for Pyrosequencing assay PCR locate in the gap of CpG site and the 7 CpG sites sequenced is shown within green box. B) and C) Representative Pyrosequencing profile of the 7 CpG sites in a matched normal (B) and tumor (C) sample pair, showing DNA methylation level for each individual CpG site.
Figure 2
Figure 2
DSC3 mRNA expression and DNA methylation level in non-neoplastic esophageal cell lines and EAC cell lines. A) The expression of DSC3 in EAC cell lines were significantly down-regulated compared with non-neoplastic esophageal cell lines. B) DSC3 promoter DNA methylation levels in EAC cell lines were significantly higher than in non-neoplastic esophageal cell lines. *** P<.001, compared with the average value of normal samples.
Figure 3
Figure 3
DSC3 mRNA expression and DNA methylation level in human normal and EAC tissues. A) DSC3 mRNA expression was significantly higher in normal tissues, while down-regulated in tumor tissues (relative fold was 9.36 vs 0.24; P<.001). B) DSC3 promoter methylation level was low in normal samples but aberrant hypermethylation was found in EAC tissues (% average DNA methylation level was 5.86 vs 38.6 in normal and tumor respectively; P<.001). A line on 10% level displays the cutoff threshold for hypermethylation. C) The Spearman rank correlation analysis between DNA methylation level and mRNA expression fold of DSC3. Significant inverse correlation was found (r=-0.685, P<.0001).
Figure 4
Figure 4
DSC3 mRNA expression and promoter DNA methylation status in matched human normal tissues and EAC tissues. A) DSC3 gene expression was significantly down-regulated in tumors. B) Tumor tissues had aberrant hypermethylation levels when compared with matched normal tissues. C) Methylation levels of 8 representative matching normal and tumor samples.
Figure 5
Figure 5
Treatment with 5-Aza and TSA reverses DNA methylation and gene expression patterns of DSC3 in esophageal adenocarcinoma cell lines. A) Treatment restores gene expression in FLO-1 cell line. 5-Aza treatment alone led to significantly induction of relative mRNA expression from 1-180 fold. B) DSC3 promoter methylation level decreased in FLO-1 after treatments. C) Treatment restores gene expression in SKGT4 cell line. 5-Aza treatment alone led to significant induction of relative expression from 1-150 fold. D) DSC3 promoter methylation level significantly decreased in SKGT4 after treatments.
Figure 6
Figure 6
DSC3 methylation is associated with advanced tumor stage and lymph node metastasis. A) Increased DNA methylation of DSC3 promoter correlates with advanced tumor staging (P<.001). B) Increased DNA methylation of DSC3 promoter correlates with lymph node metastasis (P<.001).
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