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Tyrosine phosphorylation is one of the basic mechanisms for signal transduction in the cell. Receptors exhibiting tyrosine kinase activity are widely involved in carcinogenesis and are negatively regulated by receptor protein tyrosine phosphatases (RPTP). Genes encoding different RPTPs are affected by aberrant epigenetic regulation in cancer. PTPRH (SAP-1) has been previously described to be overexpressed in colorectal cancer (CRC) and classified as an oncogenic factor. Previous microarray-based mRNA expression comparison of colorectal adenomas (AD), CRC and normal mucosa samples (NM) demonstrated that PTPRH tumor expression is the most reduced of all RPTP genes. qRT-PCR validation revealed gene downregulation for CRC (7.6-fold-change; P<0.0001) and AD (3.4-fold-change; P<0.0001) compared to NM. This was confirmed by immunohistochemical staining of tumor and NM sections as pronounced decrease of protein expression was observed in CRCs compared to the corresponding normal tissue. DNA methylation of two PTPRH promoter fragments was analyzed by pyrosequencing in a group of CRC, and AD patients as well as NM samples and CRC cell lines. The mean DNA methylation levels of these two regions were significantly higher in CRC than in NM. Both regions were highly methylated in SW480 and HCT116 cell lines contrary to unmethylated HT29 and COLO205. Cell lines with highly methylated promoters notably showed lower PTPRH expression levels, lower RNA II polymerase concentrations and higher levels of H3K27 trimethylation in the promoter and gene body, measured by chromatin immunoprecipitation. Cells were cultured with 5-aza-deoxycitidine and an increase in PTPRH expression was observed in SW480 and HCT116, whereas this was unchanged in the unmethylated cell lines. The results indicate that PTPRH is downregulated in colorectal tumors and its expression is epigenetically regulated via DNA methylation and chromatin modifications.

Altered epigenetic mechanisms, similar to gene mutations, contribute to the pathogenesis and molecular heterogeneity of neoplasms, including colorectal cancer (CRC). Enhancer of zeste 2 (EZH2) is a histone methyltransferase, which is involved in epigenetic gene silencing and is aberrantly expressed in CRC. Therefore, the identification of the genes regulated by EZH2 in CRC is important to improve current understanding of its role in cancer epigenetics. The present study used chromatin immunoprecipitation (ChIP) followed by deep sequencing to assess genome-wide EZH2‑DNA interactions in healthy or CRC mucosa samples. In total, 86.9/61.6 and 92.5/62.6 million tags were sequenced/mapped in healthy and CRC mucosa samples, respectively. The EZH2-binding densities were correlated with transcriptomic datasets and this demonstrated that the claudin-23 (CLDN23) gene, which encodes a component of cell-cell adhesion structures, was occupied by EZH2 and significantly silenced in CRC tissue. The measurement of DNA methylation at the CLDN23 promoter using pyrosequencing excluded the possibility that silencing of this gene in CRC patient samples was a result of DNA hypermethylation. Following treatment of the Colo205 and HT-29 CRC cell lines, with the EZH2 inhibitor, GSK126, the level of histone H3 lysine 27 trimethylation (H3K27me3) was reduced and the mRNA and protein expression levels of CLDN23 were increased. ChIP analysis confirmed that the level of H3K27m3 along the CLDN23 gene was decreased in the GSK126-treated cell lines. Furthermore, ChIP analysis of these samples detected histone H3 lysine 4 trimethylation (H3K4me3) at the CLDN23 promoter, demonstrating that the balance between H3K27me3 and H3K4me3 may underlie the regulation of the expression of CLDN23. The present study demonstrated an epigenetic link between the activity of the EZH2 methyltransferase at the CLDN23 locus and the expression of CLDN23 in CRC tissue.

DNA methylation is an epigenetic event that plays a role in gene expression regulation. Alterations in DNA methylation contribute to cancer development and progression. The aim of this study was to identify gene promoters aberrantly methylated in colorectal tumor tissue in comparison to normal colonic mucosa. Analyses were performed on two pooled DNA samples: from normal and cancerous tissue obtained from CRC patients. DNA was fractionated according to methylation degree with the use of affinity column containing methyl-CpG binding domain. To identify novel hypermethylated gene promoters, methylated DNA from normal and from cancerous tissues were analyzed with the use of promoter microarrays. We identified nine novel genes hypermethylated in colorectal cancer. The frequency of their promoter methylation was assessed in the larger group of patients (n = 77): KCNK12 (methylated in 41% of CRC patients), GPR101 (40%), CDH2 (45%), BARX1 (56%), CNTFR (22%), SYT6 (64%), SMO (21%), EPHA5 (43%), and GSPT2 (21%). The results of gene expression level analysis suggest the role of promoter methylation in downregulation of six out of nine genes examined. We did not find correlation between gene methylation and age, gender, tumor grade or stage. Importantly, in stage IV CRC methylation of GPR101 correlated with longer time to progression (P = 0.0042; HR = 2.5468; 95% CI 1.5391-10.0708)., (Copyright © 2011 Wiley Periodicals, Inc.)