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

Patai ÁV, Valcz G, Hollósi P, Kalmár A, Péterfia B, Patai Á, Wichmann B, Spisák S, Barták BK, Leiszter K, Tóth K, Sipos F, Kovalszky I, Péter Z, Miheller P, Tulassay Z, Molnár B - PLoS ONE (2015)

Bottom Line: Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC.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.

View Article: PubMed Central - PubMed

Affiliation: 2nd Department of Medicine, Semmelweis University, Budapest, Hungary.

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.

No MeSH data available.


Related in: MedlinePlus

A. Gene expression of SFRP1 during ACS measured by mRNA expression microarray analysis. As indicated, transcript levels decreased across in LGD, HGD and CRC samples in 3 out of 4 SFRP1 transcripts. B. Protein expression of SFRP1 protein measured by immunohistochemistry.SFRP1 protein expression gradually decreased during ACS. The epithelial SFRP1 expression showed a continuous decrease during ACS. Epithelial SFRP1 expression was decreased in LGD compared to normal epithelium, but stromal SFRP1 protein expression was retained. In HGD and CRC samples, both epithelial and stromal SFRP1 protein expression was significantly reduced. Red staining: SFRP1, blue staining: Hoechst nuclear staining. N: normal sample, LGD: low-grade dysplasia, HGD: high-grade dysplasia, CRC: colorectal cancer
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pone.0133836.g003: A. Gene expression of SFRP1 during ACS measured by mRNA expression microarray analysis. As indicated, transcript levels decreased across in LGD, HGD and CRC samples in 3 out of 4 SFRP1 transcripts. B. Protein expression of SFRP1 protein measured by immunohistochemistry.SFRP1 protein expression gradually decreased during ACS. The epithelial SFRP1 expression showed a continuous decrease during ACS. Epithelial SFRP1 expression was decreased in LGD compared to normal epithelium, but stromal SFRP1 protein expression was retained. In HGD and CRC samples, both epithelial and stromal SFRP1 protein expression was significantly reduced. Red staining: SFRP1, blue staining: Hoechst nuclear staining. N: normal sample, LGD: low-grade dysplasia, HGD: high-grade dysplasia, CRC: colorectal cancer

Mentions: To analyze the effect of DNA hypermethylation, we examined mRNA and protein expression levels of SFRP1, a well-described antagonist of the Wnt pathway, frequently aberrantly activated in CRC. We confirmed the findings of previous studies, that DNA hypermethylation leads to subsequent underexpression of SFRP1 mRNA (Fig 3A) and lower protein levels (Fig 3B) in CRC, and also in precursor lesions. In normal samples we found strong, diffuse cytoplasmic SFRP1 protein expression in the epithelium and in the stroma, as well. In the stroma, SFRP1 positive cells were localized primarily in close proximity to epithelial cells. In LGD samples, epithelial SFRP1 protein expression decreased, but in the stroma, it was similar to that observed in normal samples. In HGD, reduction in epithelial SFRP1 protein expression was more pronounced than in LGD. In both HGD and CRC, there was no apparent stromal expression of SFRP1 proximal to the epithelia.


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

Patai ÁV, Valcz G, Hollósi P, Kalmár A, Péterfia B, Patai Á, Wichmann B, Spisák S, Barták BK, Leiszter K, Tóth K, Sipos F, Kovalszky I, Péter Z, Miheller P, Tulassay Z, Molnár B - PLoS ONE (2015)

A. Gene expression of SFRP1 during ACS measured by mRNA expression microarray analysis. As indicated, transcript levels decreased across in LGD, HGD and CRC samples in 3 out of 4 SFRP1 transcripts. B. Protein expression of SFRP1 protein measured by immunohistochemistry.SFRP1 protein expression gradually decreased during ACS. The epithelial SFRP1 expression showed a continuous decrease during ACS. Epithelial SFRP1 expression was decreased in LGD compared to normal epithelium, but stromal SFRP1 protein expression was retained. In HGD and CRC samples, both epithelial and stromal SFRP1 protein expression was significantly reduced. Red staining: SFRP1, blue staining: Hoechst nuclear staining. N: normal sample, LGD: low-grade dysplasia, HGD: high-grade dysplasia, CRC: colorectal cancer
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4546193&req=5

pone.0133836.g003: A. Gene expression of SFRP1 during ACS measured by mRNA expression microarray analysis. As indicated, transcript levels decreased across in LGD, HGD and CRC samples in 3 out of 4 SFRP1 transcripts. B. Protein expression of SFRP1 protein measured by immunohistochemistry.SFRP1 protein expression gradually decreased during ACS. The epithelial SFRP1 expression showed a continuous decrease during ACS. Epithelial SFRP1 expression was decreased in LGD compared to normal epithelium, but stromal SFRP1 protein expression was retained. In HGD and CRC samples, both epithelial and stromal SFRP1 protein expression was significantly reduced. Red staining: SFRP1, blue staining: Hoechst nuclear staining. N: normal sample, LGD: low-grade dysplasia, HGD: high-grade dysplasia, CRC: colorectal cancer
Mentions: To analyze the effect of DNA hypermethylation, we examined mRNA and protein expression levels of SFRP1, a well-described antagonist of the Wnt pathway, frequently aberrantly activated in CRC. We confirmed the findings of previous studies, that DNA hypermethylation leads to subsequent underexpression of SFRP1 mRNA (Fig 3A) and lower protein levels (Fig 3B) in CRC, and also in precursor lesions. In normal samples we found strong, diffuse cytoplasmic SFRP1 protein expression in the epithelium and in the stroma, as well. In the stroma, SFRP1 positive cells were localized primarily in close proximity to epithelial cells. In LGD samples, epithelial SFRP1 protein expression decreased, but in the stroma, it was similar to that observed in normal samples. In HGD, reduction in epithelial SFRP1 protein expression was more pronounced than in LGD. In both HGD and CRC, there was no apparent stromal expression of SFRP1 proximal to the epithelia.

Bottom Line: Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC.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.

View Article: PubMed Central - PubMed

Affiliation: 2nd Department of Medicine, Semmelweis University, Budapest, Hungary.

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.

No MeSH data available.


Related in: MedlinePlus