Oxidative DNA damage induces hypomethylation in a compromised base excision repair colorectal tumourigenesis.

BACKGROUND: A compromised base excision repair (BER) promotes carcinogenesis by accumulating oxidative DNA-damaged products as observed in MUTYH-associated polyposis, a hereditary colorectal cancer syndrome marked by adenomas and cancers with an accumulation of 8-oxoguanine. Remarkably, DNA global demethylation has been shown to be mediated by BER, suggesting a relevant interplay with early colorectal tumourigenesis. To check this hypothesis, we investigated a cohort of 49 adenomas and 10 carcinomas, derived from 17 MUTYH-associated polyposis patients; as adenoma controls, we used a set of 36 familial adenomatous polyposis and 24 sporadic polyps. METHODS: Samples were analysed for their mutational and epigenetic status, measured as global LINE-1 (long interspersed nuclear element) and gene-specific LINE-1 MET methylation by mass spectrometry and pyrosequencing. RESULTS: MUTYH-associated polyposis adenomas were strikingly more hypomethylated than familial adenomatous and sporadic polyps for both DNA demethylation markers (P=0.032 and P=0.007 for LINE-1; P=0.004 and P<0.0001 for LINE-1 MET, respectively) with levels comparable to those of the carcinomas derived from the same patients. They also had mutations due mainly to KRAS/NRAS p.G12C, which was absent in the controls (P<0.0001 for both sets). CONCLUSIONS: Our results show that DNA demethylation, together with specific KRAS/NRAS mutations, drives the early steps of oxidative damage colorectal tumourigenesis.

Small-bowel carcinomas associated with celiac disease: transcriptomic profiling shows predominance of microsatellite instability-immune and mesenchymal subtypes.

Celiac disease (CD) is a risk factor for developing small-bowel carcinoma with a 14-fold higher risk compared with general population. As small-bowel carcinomas associated with CD (CD-SBCs) are extremely rare, very few molecular data are available about their pathogenesis, and information about their transcriptomic profiling is lacking. We generated RNA-seq data on 13 CD-SBCs, taken from the largest well-characterized series published so far, collected by the Small Bowel Cancer Italian Consortium, and compared the tumor transcriptional signatures with the four Consensus Molecular Subtypes (CMS) of colorectal carcinoma by applying the "CMS classifier." CpG Island Methylator Phenotype (CIMP) was evaluated using methylation-sensitive multiple ligation-dependent probe amplification. Up to 12 of 13 cancers fell within the two main subtypes exhibiting high immune and inflammatory signatures, i.e., subtypes 1 and 4. The first and predominant subset was commonly microsatellite unstable, and exhibited CIMP and high CD3+ and CD8+ T cell infiltration. Moreover, it showed increased expression of genes associated with T helper 1 and natural killer cell infiltration, as well as upregulation of apoptosis, cell cycle progression, and proteasome pathways. By contrast, cancers falling in subtype 4 showed prominent transforming growth factor-ß activation and were characterized by complement-associated inflammation, matrix remodeling, cancer-associated stroma production, and angiogenesis. Parallel histologic and histochemical analysis confirmed such tumor subtyping. In conclusion, two molecular subtypes have been consistently identified in our series of CD-SBCs, a microsatellite instability-immune and a mesenchymal subtype, the former likely associated with an indolent and the latter with a worse tumor behavior.

The polymorphic variant rs1800734 influences methylation acquisition and allele-specific TFAP4 binding in the MLH1 promoter leading to differential mRNA expression.

Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele.

Methylation-associated Silencing of microRNA-126 and its Host Gene EGFL7 in Malignant Pleural Mesothelioma.

BACKGROUND/AIM: We recently reported that miR-126 is down-regulated in malignant pleural mesothelioma (MPM) and can be combined into a 4-microRNA-classifier that can accurately diagnose MPM with high sensitivity and specificity. Herein we analyzed the epigenetic regulation of miR-126 and its host gene EGF-like domain, multiple 7 (EGFL7). MATERIALS AND METHODS: Resected formalin-fixed paraffin-embedded MPM tissues from 29 patients, 14 patient-matched non-neoplastic pleura (NNP) specimens, 5 MPM diagnostic biopsies (DB), and 5 samples of pneumothorax-induced benign reactive mesothelial proliferation (PTHX) were analyzed. miR-126 and EGFL7 mRNA were quantified by RT-qPCR. CpG-islands' methylation in the EGFL7 promoter was analyzed using methylation-specific PCR and in the MIR126-containing intron 7 was quantified by pyrosequencing. RESULTS: Relative to NNP, EGFL7 was under-expressed more than 4-fold in MPM (p<0.001). EGFL7 mRNA and miR-126 levels correlated in MPM (p<0.01) and NNP (p<0.001). The EGFL7 promoter region was hypermethylated in 69% of MPM and 80% of DB samples, but not in NNP and PTHX samples. EGFL7 promoter hypermethylation was associated with epithelioid histology (p<0.05) and reduced patient-survival (p<0.05). CONCLUSION: In MPM, DNA-hypermethylation down-regulates miR-126 and its host gene EGFL7, therefore is a poor prognostic factor, and may represent a future therapeutic target for de-methylating strategies re-establishing EGFL7 and miR-126 expression.