Expression of claudin 1, claudin 4, and claudin 7 in colorectal cancer and its relation with CLDN DNA methylation patterns

Abstract

IntroductionTargeting tumor suppressor genes by epigenetic silencing especially in the promotor region is one of the probable mechanisms of carcinogenesis. 1 However, differential methylation may also occur outside of the promoter region and previous studies have shown that gene expression and tissue differentiation are powerfully affected by such methylation sites. 1,2 So far, there are limited data available on claudin (CLDN), epigenetics, and the relation to claudin protein expression as well as its role in the development of colorectal carcinoma (CRC). While DNA methylation of claudin 6 has been shown to be involved in the invasion of breast cancer cells 3 Abstract Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p ≤ 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Methylatedclaudin-11associated with metastasis and poor survival of colorectal cancer

“…Interestingly, claudin expression is in a tissue specific manner [ 29 ]. Victoria and his colleagues have demonstrated that one of the principal reasons of increased relatively increased expression of CLDN1 in CRC is the hypomethylation of its promoter region [ 30 ], which is also identified in our methylation array data ( Supplementary Figure 2 ). Whereas, the different phenomenon was observed in breast cancer, hypermethylation and reduced expression of CLDN1, suggesting that the expression of CLDN1 varies in different tissues [ 31 ].…”

Emerging clinical significance of claudin-7 in colorectal cancer: a review

“…Claudin-7 expression in CRC also has a dual role. IHC showed significantly lower claudin-7 staining intensity in CRC tissue than adjacent non-tumor tissue, 73 – 78 and abnormal cytoplasmic localization was observed. 73 There was an important association between claudin-7 expression and lymphovascular invasion, neurotropic invasion, lymphocyte status, and tumor grade.…”

“…IHC showed significantly lower claudin-7 staining intensity in CRC tissue than adjacent non-tumor tissue, 73 – 78 and abnormal cytoplasmic localization was observed. 73 There was an important association between claudin-7 expression and lymphovascular invasion, neurotropic invasion, lymphocyte status, and tumor grade. 74 – 76 , 79 A decrease in claudin-7 mRNA levels is an early event in the development of CRC that may contribute to the damage of the epithelial barrier in adenomas.…”

“…Claudin-7 mRNA and protein expression increased after applying the methylation inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC); however, the claudin-7 expression was significantly decreased by DNA methyltransferases 3a (DNMT3a), which further suggested that hypermethylation inhibited claudin-7 expression in CRC. Hahn-Strömberg et al 73 used bisulfite pyrosequencing to analyze DNA methylation of claudin-1, -4, and -7 in tumors and the adjacent non-neoplastic mucosa and found that claudin-1 was significantly hypomethylated in the tumor tissues, while claudin-7 methylation levels showed no significant difference between tumors and paracancerous tissues. Claudin gene hypomethylation seems to be related to the abnormal compartmentalization of TJ proteins.…”

Methylation of the claudin‑3 promoter predicts the prognosis of advanced gastric adenocarcinoma

“…There is accumulating evidence that alterations in DNA methylation contribute to the deregulation of claudin expression in cancer. For example, colorectal cancer tissue showed DNA hypomethylation of the claudin-1 gene as well as reduced membrane expression and increased cytoplasmic expression of claudin-1, as compared with adjacent non-neoplastic mucosa ( 18 ). Ovarian cancer cells that exhibit high expression of claudin-3 showed increased DNA methylation and enhanced histone H3 acetylation of the promoter region ( 19 ).…”