Hypomethylation of CLDN4 Gene Promoter Is Associated with Malignant Phenotype in Urinary Bladder Cancer

Abstract

The tight junction (TJ) protein claudin-4 (CLDN4) is overexpressed in bladder urothelial carcinoma (BUC) and correlates with cancer progression. However, the mechanism of CLDN4 upregulation and promotion of malignant phenotype is not clear. Here, we analyzed 157 cases of BUC and investigated the hypomethylation of CpG island in the CLDN4 promoter DNA and its correlation with cancer progression. In hypomethylated cases, CLDN4 expression, cell proliferation, stemness, and epithelial-mesenchymal transition were increased. Treatment of three human BUC cell lines with the demethylating agent aza-2′-deoxycytidine (AZA) led to excessive CLDN4 expression, and, specifically, to an increase in CLDN4 monomer that is not integrated into the TJ. The TJ-unintegrated CLDN4 was found to bind integrin β1 and increase stemness, drug resistance, and metastatic ability of the cells as well as show an anti-apoptosis effect likely via FAK phosphorylation, which reduces upon knockdown of CLDN4. Thus, CLDN4 is overexpressed in BUC by an epigenetic mechanism and the high expression enhances the malignant phenotype of BUC via increased levels of TJ-unintegrated CLDN4. CLDN4 promoter DNA methylation is expected to be a novel indicator of BUC malignant phenotype and a new therapeutic target.

Claudin-4: A New Molecular Target for Epithelial Cancer Therapy

“…Overexpression of CLDN4 has been reported in various cancers, such as gastric cancer [ 6 , 7 , 8 ], pancreatic cancer [ 9 , 10 , 11 ], colorectal cancer [ 12 , 13 ], breast cancer [ 14 , 15 ] (especially triple-negative breast cancer [ 16 , 17 ]), oral squamous cell carcinoma [ 18 ], ovarian cancer [ 19 ], bladder cancer [ 20 , 21 ], non-small cell lung cancer [ 22 ], and cholangiocarcinoma [ 23 ]. In all of these cases, CLDN4 expression correlated with disease progression and poor prognosis.…”

“…Hypermethylation of CpG islands in the CLDN4 promoter region reduces CLDN4 expression in gastric, bladder, and colon cancers [ 32 , 33 , 34 ]. In contrast, CLDN4 hypomethylation and CLDN4 overexpression have been reported in gastric, breast, ovarian, and bladder cancers [ 21 , 35 , 36 , 37 ]. In bladder cancer, CLDN4 promoter DNA hypomethylation was shown to correlate with CLDN4 overexpression, high-grade tumors, and invasion, and the increase in CLDN4 promoted anti-apoptosis, stemness, and epithelial-mesenchymal transition (EMT) [ 21 ].…”

“…In contrast, CLDN4 hypomethylation and CLDN4 overexpression have been reported in gastric, breast, ovarian, and bladder cancers [ 21 , 35 , 36 , 37 ]. In bladder cancer, CLDN4 promoter DNA hypomethylation was shown to correlate with CLDN4 overexpression, high-grade tumors, and invasion, and the increase in CLDN4 promoted anti-apoptosis, stemness, and epithelial-mesenchymal transition (EMT) [ 21 ]. Several studies have suggested that the depletion of methyl donors by the upregulation of methyltransferases may be a likely cause of hypomethylation [ 38 , 39 , 40 ].…”

“…CLDN4 suppresses cell death caused by apoptosis [ 15 , 89 ] and endoplasmic reticulum stress [ 90 ]. It is believed that non-TJ CLDN4 activates integrin β1 as a binding partner and suppresses apoptosis via FAK signaling [ 8 , 21 ]. In addition, CLDN1 [ 91 , 92 ] increases resistance to anoikis, whereas CLDN6 decreases it [ 93 ].…”

“…CLDN4 is overexpressed in bladder cancer due to promoter DNA hypomethylation [ 21 ]. Further demethylation via aza-2′-deoxycytidine (AZA) treatment induces expression of CLDN4 to levels above that necessary for TJ function.…”