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.

Tight junction is composed of transmembrane proteins important for maintaining cell polarity and regulating ion flow. Among these proteins are the tissue-specific claudins, proteins that have recently been suggested as tumor markers for several different types of cancer. An altered claudin expression has been observed in colon, prostatic, ovarian, and breast carcinoma. The aim of this study was to analyze the allele frequencies of three common single nucleotide polymorphisms (SNPs) in the genes for claudin 1 and claudin 7 in colon cancer (CC) patients and in a control population of healthy blood donors. Pyrosequencing was used to genotype the CLDN1 SNP rs9869263 (c.369C>T), and the CLDN7 SNPs rs4562 (c.590C>T) and rs374400 (c.606T>G) in DNA from 102 formalin fixed paraffin embedded (FFPE) colon cancer tissue, and 111 blood leukocyte DNA from blood/plasma donors. These results were correlated with clinical parameters such as TNM stage, tumor localization, tumor differentiation, complexity index, sex, and age. We found that there was a significant association between the CLDN1 genotype CC in tumor samples and a higher risk of colon cancer development (OR 3.0, p < 0.001). We also found that the CLDN7 rs4562 (c.590C>T) genotype CT had a higher risk of lymph node involvement (p = 0.031) and a lower degree of tumor differentiation (p = 0.028). In the control population, the allele frequencies were very similar to those in the HapMap cohort for CLDN7. The CLDN1 rs9869263 genotype (c.369C>T) was related to increased risk of colon cancer, and the CLDN7 rs4562 genotype (c.590C>T) was related to tumor differentiation and lymph node involvement in colon carcinoma. Further studies are warranted to ascertain their potential uses as biomarkers predicting tumor development, proliferation, and outcome in this disease.

Background and objective: Claudins (CLDNs) are members of a large family of tissue specific adherent proteins which have been suggested as tumor markers. In this study we analyzed the protein expression of CLDN 1, 3, 4, 5 and 7, their clinical significance and association with tumor growth pattern in colon carcinoma.Methods: Immunohistochemical staining was used to detect the expression of CLDN 1, 3, 4, 5 and 7 in samples diagnosed with colon carcinoma as well as the adjacent normal mucosa. Complexity Index (CI) was calculated using images of cytokeratin-8 stained slides of the tumours using Photoshop CS, Fovea Pro, and Image J computer programs. The results from the Immunohistochemistry (IHC) and CI were correlated to clinicopathological parameters as well as 5-years survival of the patients diagnosed with colon carcinoma.Results: Significantly high staining intensity was observed in normal mucosa as compared to colon cancer tissue for CLDN 4 (p=0.031) and 7 (p=0.011) and number of stained cells for CLDN 4 (p=0.001). A significant association was also observed between weaker expression at the invasive front of the tumor tissue and heterogenous expression of CLDN 1 (p=0.000), CLDN 3 (p=0.003), CLDN 5 (p=0.001) and CLDN 7 (p=0.000). There was no significant correlation between the expression of CLDNs, CI and clinicopathological parameters. Similarly, no significant association was found between CLDN expression and 5-years survival of the patients diagnosed with colon carcinoma. Conclusion:Altered expression of CLDN 1, 3, 4, 5 and 7 in colon carcinoma cells may play a promoting role in colon carcinoma development and is inversely proprtional to higher expression at invasive front of the tumor. CLDN protein expression can be used as a tumor marker since the expression generally is weaker in tumor tissue compared to the normal mucosa.

There have been numerous studies on the gene expression of connective tissue growth factor (CTGF) in colorectal cancer, however very few have investigated polymorphisms in this gene. The present study aimed to determine whether single nucleotide polymorphisms (SNPs) in the CTGF gene are associated with a higher susceptibility to colon cancer and/or an invasive tumor growth pattern. The CTGF gene was genotyped for seven SNPs (rs6918698, rs1931002, rs9493150, rs12526196, rs12527705, rs9399005 and rs12527379) by pyrosequencing. Formalin-fixed paraffin-embedded tissue samples (n=112) from patients diagnosed with colon carcinoma, and an equal number of blood samples from healthy controls, were selected for genomic DNA extraction. The complexity index was measured using images of tumor samples (n=64) stained for cytokeratin-8. The images were analyzed and correlated with the identified CTGF SNPs and clinicopathological parameters of the patients, including age, gender, tumor penetration, lymph node metastasis, systemic metastasis, differentiation and localization of tumor. It was demonstrated that the frequency of the SNP rs6918698 GG genotype was significantly associated (P=0.05) with an increased risk of colon cancer, as compared with the GC and CC genotypes. The other six SNPs (rs1931002, rs9493150, rs12526196, rs12527705, rs9399005 and rs12527379) exhibited no significant difference in the genotype and allele frequencies between patients diagnosed with colon carcinoma and the normal healthy population. A trend was observed between genotype variation at rs6918698 and the complexity index (P=0.052). The complexity index and genotypes for any of the studied SNPs were not significantly correlated with clinical or pathological parameters of the patients. These results indicate that the rs6918698 GG genotype is associated with an increased risk of developing colon carcinoma, and genetic variations at the rs6918698 are associated with the growth pattern of the tumor. The present results may facilitate the identification of potential biomarkers of the disease in addition to drug targets.