Abstract
BackgroundThere is emerging evidence that Wnt pathway activity may increase during the progression from colorectal adenoma to carcinoma and that this increase is potentially an important step towards the invasive stage. Here, we investigated whether epigenetic silencing of Wnt antagonists is the biological driver for this increased Wnt activity in human tissues and how these methylation changes correlate with MSI (Microsatelite Instability) and CIMP (CpG Island Methylator Phenotype) statuses as well as known mutations in genes driving colorectal neoplasia.MethodsWe conducted a systematic analysis by pyrosequencing, to determine the promoter methylation of CpG islands associated with 17 Wnt signaling component genes. Methylation levels were correlated with MSI and CIMP statuses and known mutations within the APC, BRAF and KRAS genes in 264 matched samples representing the progression from normal to pre-invasive adenoma to colorectal carcinoma.ResultsWe discovered widespread hypermethylation of the Wnt antagonists SFRP1, SFRP2, SFRP5, DKK2, WIF1 and SOX17 in the transition from normal to adenoma with only the Wnt antagonists SFRP1, SFRP2, DKK2 and WIF1 showing further significant increase in methylation from adenoma to carcinoma. We show this to be accompanied by loss of expression of these Wnt antagonists, and by an increase in nuclear Wnt pathway activity. Mixed effects models revealed that mutations in APC, BRAF and KRAS occur at the transition from normal to adenoma stages whilst the hypermethylation of the Wnt antagonists continued to accumulate during the transitions from adenoma to carcinoma stages.ConclusionOur study provides strong evidence for a correlation between progressive hypermethylation and silencing of several Wnt antagonists with stepping-up in Wnt pathway activity beyond the APC loss associated tumour-initiating Wnt signalling levels.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-891) contains supplementary material, which is available to authorized users.
Highlights
There is emerging evidence that Wnt pathway activity may increase during the progression from colorectal adenoma to carcinoma and that this increase is potentially an important step towards the invasive stage
We examined the DNA methylation of 134 CpG dinucleotides within promoter-CpG islands associated with these genes in 264 matched normal, adenomatous or hyperplastic polyps and adenocarcinoma tissue samples obtained from 126 patients
This criterion revealed two main clusters with significantly different CpG patterns (P < 0.05, Fisher’s exact test) (Additional file 6), dependent on whether the genes encode Wnt antagonists or agonists (Figure 1). The first of these clusters contains seven genes (SFRP1, SFRP2, SFRP5, DKK2, WIF1, WNT3A and SOX17) whose CpG methylation increased significantly from normal to adenoma (P < 0.001, Wilcoxon signed rank test or paired t-test depending on data distribution and false discovery rate adjusted for multiple testing (Additional file 7)): of these, six encode Wnt antagonists (SFRP1, SFRP2, SFRP5, DKK2, WIF1 and SOX17) with only the Wnt antagonists SFRP1, SFRP2, WIF1 and DKK2 showing further significant increase in methylation from adenoma to carcinoma (P < 0.05, Wilcoxon signed rank test or paired t-test depending on data distribution (Additional file 7))
Summary
There is emerging evidence that Wnt pathway activity may increase during the progression from colorectal adenoma to carcinoma and that this increase is potentially an important step towards the invasive stage. The role of Wnt signaling in initiating CRC is well documented It is less clear whether hyperactive Wnt signaling is required for the progression from adenoma to carcinoma. Epigenetic inactivation of extracellular Wnt signaling antagonists has been observed in colorectal carcinomas, which could boost Wnt signaling to levels above those caused by the initial mutational inactivation of APC [12]. All these are indications that the level of Wnt signaling increases from the adenoma to the carcinoma stage, implying that the sustained (or increased) activity of β-catenin could be critical throughout CRC progression
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