DNA Methylation Associated With DNMT1 Overexpression As A Probable Cause Of Esophagin Loss In Esophageal Cancer

NICOLAU-NETO P. ² , SOARES-LIMA S. ² , RIBEIRO- PINTO L. ^1,2 , SIMÃO T. ¹

Esophageal cancer (EC) is the sixth most frequent cancer and is the sixth leading cause of cancer-related deaths worldwide. Squamous cell carcinoma (ESCC) correspondes to 80% of the cases.  A previous study from our group showed a gradual loss of esophagin (SPRR3) expression in malignant transformation of the healthy esophagus into ESCC. However, molecular mechanisms studies involved in SPRR3 silencing are limited.  The aim of this study is to examine DNA methylation as regulatory mechanism of esophagin expression in ESCC.  For this, three CpG sites of esophagin gene were analyzed by pyrosequencing in esophageal cancer cell line (OE21) treated with the demethylating agent decitabine, and in tumor and matched normal surrounding mucosa from patients with ESCC. RT-qPCR was performed to evaluated esophagin and DNMT1 expression in the same samples. Our results demonstrate that decitabine treatment was able to induce SPRR3 demethylation in a time and dose-dependent manner in OE21, correlated with an increased mRNA expression.  The methylation of CpG sites analyzed was significantly higher in tumors in comparison with the adjacent tissues (A, p=0.0017; B, p=0.0002; C, p=0.0103). The ability of SPRR3 methylation to distinguish the adjacent mucosa from tumor samples using ROC curve analyses was significant for each of the CpG sites examined (A, sensitivity and specificity=84.62%, P=0.003; B. sensitivity=93.33% specificity=86.67%, P<0.0001; C, sensitivity and specificity=80.0%, P=0.0017).  We observed an inverse correlation between esophagin methylation and its mRNA expression for CpG sites evaluated (A, p<0.0001; B, p=0.0022; C, p=0.0006).  In order to investigate the mechanisms that could be involved in SPRR3 hypermethylation, we evaluated DNMT1 expression, which was higher in tumors with this profile (A, p=0,0028; B, p=0.0157; C, p=0.0080) and inversely correlated with SPRR3 expression (p=0.0009). Our data suggest DNA methylation induced by DNMT1 as a possible mechanism for esophagin silencing in ESCC. Funding Source: FAPERJ and MS/INCA.