Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas

Abstract

Microarray analysis of promoter hypermethylation provides insight into the role and extent of DNA methylation in the development of colorectal cancer (CRC) and may be co-monitored with the appearance of driver mutations. Colonic biopsy samples were obtained endoscopically from 10 normal, 23 adenoma (17 low-grade (LGD) and 6 high-grade dysplasia (HGD)), and 8 ulcerative colitis (UC) patients (4 active and 4 inactive). CRC samples were obtained from 24 patients (17 primary, 7 metastatic (MCRC)), 7 of them with synchronous LGD. Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC. Tissue materials were studied for DNA methylation status using a 96 gene panel and for KRAS and BRAF mutations. Expression levels were assayed using whole genomic mRNA arrays. SFRP1 was further examined by immunohistochemistry. HT29 cells were treated with 5-aza-2’ deoxycytidine to analyze the reversal possibility of DNA methylation. More than 85% of tumor samples showed hypermethylation in 10 genes (SFRP1, SST, BNC1, MAL, SLIT2, SFRP2, SLIT3, ALDH1A3, TMEFF2, WIF1), whereas the frequency of examined mutations were below 25%. These genes distinguished precancerous and cancerous lesions from inflamed and healthy tissue. The mRNA alterations that might be caused by systematic methylation could be partly reversed by demethylation treatment. Systematic changes in methylation patterns were observed early in CRC carcinogenesis, occuring in precursor lesions and CRC. Thus we conclude that DNA hypermethylation is an early and systematic event in colorectal carcinogenesis, and it could be potentially reversed by systematic demethylation therapy, but it would need more in vitro and in vivo experiments to support this theory.

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer

“…Correspondingly to the previous findings, 11,17,[19][20] we found that another known inhibitor of canonical WNT pathway, DKK2, was significantly hypermethylated in CRC vs. NAT and AD vs. NAT comparisons. However, our promoter estimation considering the sequence in the region between 2000 bp upstream and 1000 bp downstream from the transcription start site (if the positive DNA strand was the coding strand) identified 'active promoter' in at least one of the 9 analyzed cell lines, according to the ENCODE ChromHMM results.…”

“…The most known WNT pathway alterations, such as APC and AXIN2 inactivation by mutations or loss of heterozygosity, affect the APC-GSK3-Axin complex leading to b-catenin stabilization, thereby resulting in the constitutive activation of canonical WNT signaling in CRC. 10 Promoter hypermethylation of negative regulators of canonical WNT pathway like secreted frizzledrelated proteins (SFRPs), [11][12][13][14][15][16][17][18] dickkopf family proteins (DKKs), 11,17,[19][20][21] and WIF1 WNT inhibitory factor 11,17,21 has also been described in malignancies, including CRC. Methylation patterns of CRC and normal adjacent tissue samples were compared in most of the previous global methylation studies focusing on WNT signaling pathway.…”

“…Methylation patterns of CRC and normal adjacent tissue samples were compared in most of the previous global methylation studies focusing on WNT signaling pathway. 20,22 Colorectal adenoma samples were involved in methylation analyses for selected WNT pathway genes 11,15,17,23,24 focusing mainly on promoter methylation level alterations. Serrated adenoma samples-representing another neoplastic pathway of CRC development-were also profiled in several previous methylation projects.…”

“…7 However, the DNA methylation changes also affect CIMP-negative (CIMP-zero or CIMP-low) CRCs, which represent the majority (approximately 75%) of sporadic CRCs. 7,17 In this study, we focused on CIMP-negative CRCs and their precancerous lesions in order to find common DNA methylation alterations of WNT pathway genes typical in development and progression of sporadic colorectal cancer.…”

A DNS-metiláció szerepe és megváltozása az öregedés és a daganatos betegségek kialakulása során

“…Régóta ismert, hogy bizonyos, vastagbéldaganatokban azonosított hipermetilált gének, például egyes tumorszuppresszor gének inaktivációja fokozza a sejt proliferációt, így szelektív előnyt jelenthet a tumorsejtek számára [69,70]. Az 1. táblázat a vastagbéldaganatok-ban legfontosabb hipermetilált tumorszuppresszor markereket foglalja össze [71][72][73][74][75][76][77][78][79][80][81][82][83][84].…”