Protein tyrosine phosphatase receptor-like genes are frequently hypermethylated in sporadic colorectal cancer.

The activity of phosphatases could be influenced by genetic, as well as epigenetic alterations. In our study, we have investigated the methylation status of four PTPRs: PTPRM, PTPRT, PTPRR and PTPRZ1, which were pre-selected using microarray techniques as being alternatively methylated in sporadic colorectal cancer (CRC). The analyses were carried out on 131 surgical specimens obtained from sporadic CRC patients. The methylation status of the four genes was examined using methyl specific PCR (MSP). The analysis of promoter methylation using an Illumina 27K microarray revealed four protein tyrosine phosphatases PTPRM, PTPRT, PTPRR and PTPRZ1 as being hypermethylated with ß-value ≥0.2 and P≤0.05. Subsequent analysis using MSP confirmed these observations-the frequency of promoter methylation was significantly higher in tumor cells compared with matched normal tissue for each of the analyzed genes. There was no association observed between the methylation status of PTPRs and either CIMP, K-ras (codon 12) and BRAF (exon 15, V600E) mutations or tumor localization (proximal/distal). The results of our study show a statistically significant difference between promoter methylation in cancerous and healthy tissue. This result supports the hypothesis that the PTPR family has an important role in the etiology of CRC.

Assessment of three epigenotypes in colorectal cancer by combined bisulfite restriction analysis.

Recent investigations have demonstrated the clear heterogeneity of sporadic colorectal cancer (CRC) with regard to CpG island methylation. Two unsupervised cluster analyses revealed that CRCs form three distinct DNA methylation subsets, which are referred to as the high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME, respectively). A recent study by Yagi et al. found a fairly sensitive and specific identification of HME, IME, and LME using two marker panels analyzed by MALDI-TOF mass spectrometry (MassARRAY). However, the expensive equipment required for this method substantially increases the cost and complexity of the assay. In this article, we demonstrate the assessment of HME, IME, and LME in a group of 233 sporadic CRCs using seven markers proposed by Yagi et al. The DNA methylation of each marker was quantified using combined bisulfite restriction analysis (COBRA) and analyzed along with various genetic factors associated with CRC [the BRAF and KRAS mutations, MLH1 methylation and microsatellite instability (MSI)]. The baseline methylation of each marker was generated from pooled DNA isolated from 50 normal colon tissues. We demonstrate that the correlation of HME, IME, and LME epigenotyped by COBRA using different molecular classifiers is similar to that achieved by MassARRAY. Therefore, epigenotyping CRCs using COBRA is a simple, specific, and cost-effective method that has the potential to be widely used in CRC research.

Aberrant methylation of ERBB pathway genes in sporadic colorectal cancer.

The ErbB signalling network plays a crucial role in the growth and progression of several cancers, including colorectal cancer (CRC), and includes potentially drug-targetable genes. Oncogenic activation of the ErbB pathway by mutations and focal amplifications have emerged recently as an important predictive marker of the prognosis of CRC patients. However, in contrast to genetic events, little is known about epigenetic alternations of ErbB-associated genes and their impact on gene expression. Genome-wide methylation in sporadic CRCs (n = 12) paired with adjacent normal tissues have been previously analysed by Illumina Infinium HumanMethylation27 (HM27) at 27,578 CpG sites. For confirmation of our initial genome-wide analysis, we used a published HM27 dataset (GSE25062). Subsequently, CpG island methylation of selected ErbB pathway-associated genes was assessed on 233 CRC samples using methylation-sensitive polymerase chain reaction (MS-PCR) and analysed along with various genetic factors associated with CRC [epigenotype, BRAF and KRAS mutations, microsatellite instability (MSI)]. Methylation and expression integration was performed using published datasets including 25 pairs of CRC and normal colon tissues (GSE25062 and GSE25070), and confirmed with real-time PCR. Our previous microarray-based genome-wide DNA methylation analysis of 12 CRCs revealed that four ErbB-associated genes (PIK3CD, PKCΒ, ERBB4, ) were differentially methylated in CRCs. This was further confirmed by statistical re-analysis of an HM27 dataset (GSE25062). Frequent methylation at these loci in tumours was subsequently confirmed by MS-PCR (63%, 43%, 43% and 92%, respectively). Hypermethylation of PKCΒ associated with KRAS mutation (p = 0.04), whereas hypermethylation of ERBB4 associated with high-methylation epigenotypes (HME), BRAF mutation and MSI (p = 0.001, 0.002 and 0.0002, respectively). One of the four analysed genes (PKCΒ) was significantly downregulated in CRC tissue, as revealed by real-time PCR and re-analysis of the GSE25062 and GSE25070 datasets. After careful re-analysis of published methylation and expression data, we conclude that methylation of ERBB4, PAK7 and PIK3CD has no functional role in CRC carcinogenesis. In contrast, methylation seems to have a potential impact on the biology of colorectal tumours by negatively modulating the expression of PKCΒ. Importantly, the relationship between DNA methylation of PKCΒ and gene expression may warrant further attention in the context of colon cancer chemoprevention and anti-cancer therapy.

Copy number alterations of chromosomal regions enclosing protein tyrosine phosphatase receptor-like genes in colorectal cancer.

Protein tyrosine phosphatases that act in different cellular pathways are described most commonly as tumor suppressors, but also as oncogenes. Their role has previously been described in colorectal cancer, as well as in gastric, breast, thyroid, prostate, ovarian, pancreatic, glioma, liver, leukemia and many other cancers. In a previous study, we have described protein tyrosine phosphatase receptor type T, M, Z1 and Q genes (PTPRT, PTPRM, PTPRZ1 and PTPRQ) hypermethylated in sporadic colorectal cancer. Thus, in this study, we examined the relation of unbalanced chromosomal alterations within regions covering these four protein tyrosine phosphatase genes with this cancer. One hundred and two cancer tissues were molecularly characterized, including analysis of the BRAF and K-ras mutations and methylator phenotype. The analysis of chromosomal aberrations was performed using Comparative Genomic Hybridization. We observed amplification of three regions containing genes coding for PTPs, such as PTPRZ1 (7q31.3, amplified in 23.5% of cases), PTPRQ (12q21.2, amplified in 5.9% of cases), PTPRT (20q12, amplified in 29.4% of cases), along with deletions in the region of PTPRM (18p11.2, deleted in 21.6% of cases). These data may suggest that in sporadic colorectal cancer PTPRZ1, PTPRT, PTPRQ probably act as oncogenes, while PTPRM acts as a tumor suppressor gene. Our study also revealed that gains on chromosome 20q12 and losses on chromosome 18p11.2 are connected with the absence of the BRAF mutation and the conventional adenocarcinoma pathway.