DNA-methylation-dependent alterations of claudin-4 expression in human bladder carcinoma

Abstract

The expression pattern of tight junction (TJ) proteins is frequently disrupted in epithelial tumors. In particular, isoform- and organ-specific alterations of claudins have been detected in human cancers, highlighting them as interesting tools for the prognosis or treatment of various carcinomas. However, the molecular mechanisms responsible for these alterations are seldom identified. Here, we analyzed the expression and localization of claudins 1, 4, and 7 in human bladder carcinoma. Claudin-4 expression was significantly altered in 26/39 tumors, contrasting with the rare modifications detected in the expression of claudins 1 and 7. Overexpression of claudin-4 in differentiated carcinomas was followed by a strong downregulation in invasive/high-grade tumors, and this expression pattern was associated to the 1-year survival of bladder tumor patients. A CpG island was identified within the coding sequence of the CLDN4 gene, and treatment with a methyl-transferase inhibitor restored expression of the protein in primary cultures prepared from high-grade human bladder tumors. In addition, claudin-4 expression correlated with its gene methylation profile in healthy and tumoral bladders from 20 patients, and downregulation of claudin-4 expression was detected in the urothelium of mice overexpressing DNA methyl transferase 3a (Dnmt3a). Delocalization of claudins 1 and 4 from TJs was observed in most human bladder tumors and in the bladder tumor cell line HT-1376. Although the CLDN4 gene was unmethylated in these cells, pharmacological inhibition of methyl transferases re-addressed the two proteins to TJs, resulting in an increase of cell polarization and transepithelial resistance. These biological effects were prevented by expression of claudin-4-specific siRNAs, demonstrating the important role played by claudin-4 in maintaining a functional regulation of homeostasis in urothelial cells. Results of this study indicate that the TJ barrier is disrupted from early stages of urothelial tumorigenesis. In addition, we identified hypermethylation as the mechanism leading to the alteration of claudin-4 expression, and maybe also localization, in bladder carcinoma.

Claudin-4 overexpression is associated with epigenetic derepression in gastric carcinoma

“…Here, we demonstrate that upregulation of claudin-4 expression during multistep gastric tumor progression is associated with promoter DNA hypomethylation. Moreover, DNA hypomethylation was significantly correlated with high claudin-4 expression in gastric carcinoma, and decreased claudin-4 expression in advanced stages of gastric cancer was significantly associated with increased promoter DNA methylation, as has been similarly reported in ovarian 47 and bladder carcinoma, 21 suggesting that DNA methylation is an important mechanism regulating CLDN4 expression in gastric carcinogenesis (Supplementary Figure S3a).…”

“…31 Inactivation of several genes by DNA hypermethylation (for example, APC, E-cadherin, p14, p15, p16) has been reported in gastric cancer. 30 Similarly, alteration of claudin-4 expression in bladder carcinoma was found to be associated with DNA methylation, 21 and epigenetic modifications including histone acetylation and DNA demethylation have been reported to be involved in the increase of CLDN4 expression in ovarian cancer cells. 32,33 Interestingly, our recent study 33 revealed that claudin-4 overexpression during ovarian tumorigenesis is associated with loss of repressive histone methylations, suggesting that epigenetic derepression also contributes to tumorigenesis by activation of cancer-promoting genes.…”

“…17 Moreover, its expression was reported to be lower in poorly differentiated diffuse-type gastric cancer than in well-differentiated intestinal-type gastric cancer, suggesting its possible role in determining the diffuse phenotype. [17][18][19] In addition to its aberrant expression in several cancers, claudin-4 expression has been shown to be associated with prognosis in breast or bladder cancer, 20,21 and has a role in invasiveness and metastatic potential of pancreatic cancer cell, 22 as well as it induces angiogenic factors in ovarian cancer. 23 However, although there have been a few studies into the association of claudin-4 expression and prognosis, it remains unclear whether claudin-4 expression is associated with clinical outcomes in gastric cancer.…”

Expression Pattern and Prognostic Significance of Claudin 1, 4 and 7 in Pancreatic Cancer

“…Silencing of the expression of some claudins in several human cancers is correlated with promoter hypermethylation.These include claudin-7 in breast, claudin-4 in bladder and claudin-11 in gastric cancer (Kominsky et al, 2003;Boireau et al, 2007;Agarwal et al, 2009).…”

“…Claudin 1,4 and 7 are important members of this Claudin protein family and their expressions show alterations in many different malignancies such as oesophageal, gastric, biliary (Usami et al, 2006;Agarwal et al, 2009;Kwon et al, 2011), ovarian (Kwon et al, 2010), endometrial (Pan et al, 2007;Knoecny et al, 2008), bladder (Boireau et al, 2007;Szekely et al, 2011) renal (Lechpammer et al, 2008), prostate (Landers et al, 2008), breast (Kominsky et al, 2003), cholangiocarcinoma (Bunthot et al, 2012) and pancreatic cancer.…”