Abstract
The downregulation of tight junction protein CLDN6 promotes breast cancer cell migration and invasion; however, the exact mechanism underlying CLDN6 downregulation remains unclear. CLDN6 silence is associated with DNA methyltransferase 1 (DNMT1) mediated DNA methylation, and DNMT1 is regulated by the transforming growth factor beta (TGFβ)/SMAD pathway. Therefore, we hypothesized that TGFβ/SMAD pathway, specifically SMAD2, may play a critical role for CLDN6 downregulation through DNA methyltransferase 1 (DNMT1) mediated DNA methylation. To test this hypothesis, we blocked the SMAD2 pathway with SB431542 in two human breast cancer cell lines (MCF-7 and SKBR-3). Our results showed that treatment with SB431542 led to a decrease of DNMT1 expression and the binding activity for CLDN6 promoter. The methylation level of CLDN6 promoter was decreased, and simultaneously CLDN6 protein expression increased. Upregulation of CLDN6 inhibited epithelial to mesenchymal transition (EMT) and reduced the migration and invasion ability of both MCF-7 and SKBR-3 cells. Furthermore, knocked down of CLDN6 abolished SB431542 effects on suppression of EMT associated gene expression and inhibition of migration and invasion. Thus, we demonstrated that the downregulation of CLDN6 is regulated through promoter methylation by DNMT1, which depends on the SMAD2 pathway, and that CLDN6 is a key regulator in the SMAD2/DNMT1/CLDN6 pathway to inhibit EMT, migration and invasion of breast cancer cells.
Highlights
Claudins (CLDNs) are small transmembrane proteins, and 27 members have been identified for this protein family [1,2,3]
SMAD2 Signaling Suppresses Claudin 6 (CLDN6) in MCF-7 and SKBR-3 Cells
These results suggest that SMAD pathways regulate the expression of CLDN6, and inactivation of SMAD2/3 proteins restore CLDN6 expression in breast cancer cells
Summary
Claudins (CLDNs) are small transmembrane proteins, and 27 members have been identified for this protein family [1,2,3]. Claudin 6 (CLDN6) is a component of tight junctions (TJs), which maintain cell–cell junctions in epithelial cell sheets. Silencing of CLDN6 enhanced migration ability of the human breast epithelium cell line HBL-100 [5]. Epithelial to mesenchymal transition (EMT) is one of the mechanisms of tumor migration and invasion [6,7,8]. During the initial stage of EMT, the expression of epithelial genes is suppressed, whereas mesenchymal marker expression is increased [9]. We believe that CLDN6 may inhibit migration and invasion of cancer cells via EMT suppression. The exact mechanism underlying CLDN6 downregulation remains unclear
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