Methylome profiling reveals functions and genes which are differentially methylated in serrated compared to conventional colorectal carcinoma

Abstract

BackgroundSerrated adenocarcinoma (SAC) is a recently recognized colorectal cancer (CRC) subtype accounting for 7.5–8.7 % of CRCs. It has been shown that SAC has a worse prognosis and different histological and molecular features compared to conventional carcinoma (CC) but, to date, there is no study analysing its methylome profile.ResultsThe methylation status of 450,000 CpG sites using the Infinium Human Methylation 450 BeadChip array was investigated in 103 colorectal specimens, including 39 SACs and 34 matched CCs, from Spanish and Finnish patients. Microarray data showed a higher representation of morphogenesis-, neurogenesis-, cytoskeleton- and vesicle transport-related functions and also significant differential methylation of 15 genes, including the iodothyronine deiodinase DIO3 and the forkhead family transcription factor FOXD2 genes which were validated at the CpG, mRNA and protein level using pyrosequencing, methylation-specific PCR, quantitative polymerase chain reaction (qPCR) and immunohistochemistry. A quantification study of the methylation status of CpG sequences in FOXD2 demonstrated a novel region controlling gene expression. Moreover, differences in these markers were also evident when comparing SAC with CRC showing molecular and histological features of high-level microsatellite instability.ConclusionsThis methylome study demonstrates distinct epigenetic regulation patterns in SAC which are consistent to previous expression profile studies and that DIO3 and FOXD2 might be molecular targets for a specific histology-oriented treatment of CRC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0128-7) contains supplementary material, which is available to authorized users.

Hyper-Methylated Loci Persisting from Sessile Serrated Polyps to Serrated Cancers

“…The resulting loss of mismatch repair capacity leads to microsatellite instability (MSI) or further dysregulation of methylation control over cell cycle (CIMP-positive), allowing uncontrolled cell proliferation [16]. Compared to conventional carcinomas, serrated adenocarcinomas from the Garcia-Solano et al [15] study are more likely to be CIMP high (9% vs. 30%, respectively p = 0.0035) [17]. Recent data show that SSA/Ps in the proximal region (right-side) of the colon are associated with a three to four-fold increased risk of concurrent advanced neoplasia and colorectal cancer [18].…”

“…These lesions included:

Left-sided SSA/P ( n = 16) and HP specimens ( n = 34: 55% left-sided, 45% right-sided) from the NH Colonoscopy Registry cohort.

Colorectal cancers described in Garcia-Solano et al GEO (GSE68060) [15] ( n = 17 serrated adenocarcinomas vs. n = 11 adjacent normal mucosal samples from the Spanish cohort and n = 17 serrated adenocarcinomas vs. n = 4 adjacent normal mucosal samples from the Finnish cohort). Features used to diagnose serrated adenocarcinoma included “epithelial serrations, clear or eosinophilic cytoplasm, abundant cytoplasm, vesicular nuclei, absence of, or less than 10% necrosis of the total surface area, mucin production and cell balls and papillary rods in mucinous areas of a tumour” [17,37,38].

Additional colorectal cancer samples that showed high microsatellite instability (MSI-H) ( n = 9), as well as n = 6 normal samples from Garcia-Solano et al [15].

…”

“…Features used to diagnose serrated adenocarcinoma included “epithelial serrations, clear or eosinophilic cytoplasm, abundant cytoplasm, vesicular nuclei, absence of, or less than 10% necrosis of the total surface area, mucin production and cell balls and papillary rods in mucinous areas of a tumour” [17,37,38]. …”

Biology and Therapeutic Targets of Colorectal Serrated Adenocarcinoma; Clues for a Histologically Based Treatment against an Aggressive Tumor

“…Consequently, the loss of E-cadherin expression and the increase in mesenchymal markers are more evident in SAC than in CC [19]. These histological and immunohistochemical manifestations of the invasive activity of SAC tumor cells were further confirmed by analyzing the molecular signatures of SAC compared to CC, where functions associated with cytoskeleton rearrangement and small GTPases' activity were frequently enriched in SAC [12,20]. Intriguingly, the epithelial mesenchymal transition (EMT) in SAC does not seem to involve the canonical Wnt/β-catenin, as the nuclear expression of β-catenin was lower in SAC than in CC.…”

Differences in gene expression profiling and biomarkers between histological colorectal carcinoma subsets from the serrated pathway

“…Previous studies comparing SAC and CC molecular signatures have highlighted that antiapoptosis-, neural differentiation-, GTPases-, calcium signalling-and cytoskeleton-related functions, are characteristic of SAC. 15,27,28 Given its role as part of a receptor complex for a small neuropeptide, and its participation in the regulation of GTPase function, in calcium signalling, and in granulocytic differentiation, [29][30][31] CRCP was chosen to validate the microarray result of it being up-regulated in SAC as compared with hmMSI-H. In agreement with the array, CRCP was overexpressed in SAC as assessed by qPCR, although we could not validate this finding at the protein level.…”