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Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists.

Silva AL, Dawson SN, Arends MJ, Guttula K, Hall N, Cameron EA, Huang TH, Brenton JD, Tavaré S, Bienz M, Ibrahim AE - BMC Cancer (2014)

Bottom Line: We 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.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.Our 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. aeki2@cam.ac.uk.

ABSTRACT

Background: 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. 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.

Methods: We 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.

Results: We 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.

Conclusion: Our 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.

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Related in: MedlinePlus

Shown in the top row are the plots of the correlation between the expression fold-change along thex-axis and the corresponding average levels of methylation percentage along they-axis forSFRP1, SFRP2, SFRP5, WIF1andDKK2in a subset of tissue samples that included normal (n = 16, HRN), hyperplastic polyps (n = 3, HP), adenomas (n = 10, Ad) and adenocarcinomas (n = 12, pT). The bottom row shows the corresponding correlations in the CRC cell line HCT116 following 5′-azadeoxycytidine treatment as well as untreated controls. The dotted lines across the plots show the fitted linear model for the data from the corresponding gene. Note that the scale of the x-axis varies between the plots depending on the range of the expression fold-change for each of the genes.
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Fig2: Shown in the top row are the plots of the correlation between the expression fold-change along thex-axis and the corresponding average levels of methylation percentage along they-axis forSFRP1, SFRP2, SFRP5, WIF1andDKK2in a subset of tissue samples that included normal (n = 16, HRN), hyperplastic polyps (n = 3, HP), adenomas (n = 10, Ad) and adenocarcinomas (n = 12, pT). The bottom row shows the corresponding correlations in the CRC cell line HCT116 following 5′-azadeoxycytidine treatment as well as untreated controls. The dotted lines across the plots show the fitted linear model for the data from the corresponding gene. Note that the scale of the x-axis varies between the plots depending on the range of the expression fold-change for each of the genes.

Mentions: To establish whether the observed DNA hypermethylation is functionally relevant, we examined the levels of gene expression of five of the Wnt antagonists (SFRP1, SFRP2, SFRP5, DKK2 and WIF1) with progressive hypermethylation during neoplastic development of CRC. To do this, we used quantitative RT-PCR on total RNA extracted from a subset of matched normal, hyperplastic polyps/adenomas and adenocarcinoma tissue samples and determine the expression fold-change of these five genes. We observed a significant negative correlation between the expression fold-change and the DNA methylation levels for SFRP1 (r = −0.584, P < 0.001 Pearson correlation test), SFRP2 (r = −0.340, P = 0.032 Pearson correlation test), SFRP5 (r = −0.375, P = 0.038 Pearson correlation test) and WIF1 (r = −0.384, P = 0.048 Pearson correlation test) (Figure 2). This parallels previous results, which revealed an overall decrease in SFRP1, SFRP2 and SFRP5 expression correlating with promoter hypermethylation of these genes [12]. Only DKK2 (which is expressed at relatively low levels in the normal colorectal mucosa and whose methylation levels are low) showed no significant correlation between methylation and expression levels (r = −0.16, P = 0.36, Pearson correlation test). Either, our analysis was not sensitive enough to detect such a correlation, or the hypermethylation of DKK2 is simply a bystander effect, and functionally irrelevant.Figure 2


Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists.

Silva AL, Dawson SN, Arends MJ, Guttula K, Hall N, Cameron EA, Huang TH, Brenton JD, Tavaré S, Bienz M, Ibrahim AE - BMC Cancer (2014)

Shown in the top row are the plots of the correlation between the expression fold-change along thex-axis and the corresponding average levels of methylation percentage along they-axis forSFRP1, SFRP2, SFRP5, WIF1andDKK2in a subset of tissue samples that included normal (n = 16, HRN), hyperplastic polyps (n = 3, HP), adenomas (n = 10, Ad) and adenocarcinomas (n = 12, pT). The bottom row shows the corresponding correlations in the CRC cell line HCT116 following 5′-azadeoxycytidine treatment as well as untreated controls. The dotted lines across the plots show the fitted linear model for the data from the corresponding gene. Note that the scale of the x-axis varies between the plots depending on the range of the expression fold-change for each of the genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265460&req=5

Fig2: Shown in the top row are the plots of the correlation between the expression fold-change along thex-axis and the corresponding average levels of methylation percentage along they-axis forSFRP1, SFRP2, SFRP5, WIF1andDKK2in a subset of tissue samples that included normal (n = 16, HRN), hyperplastic polyps (n = 3, HP), adenomas (n = 10, Ad) and adenocarcinomas (n = 12, pT). The bottom row shows the corresponding correlations in the CRC cell line HCT116 following 5′-azadeoxycytidine treatment as well as untreated controls. The dotted lines across the plots show the fitted linear model for the data from the corresponding gene. Note that the scale of the x-axis varies between the plots depending on the range of the expression fold-change for each of the genes.
Mentions: To establish whether the observed DNA hypermethylation is functionally relevant, we examined the levels of gene expression of five of the Wnt antagonists (SFRP1, SFRP2, SFRP5, DKK2 and WIF1) with progressive hypermethylation during neoplastic development of CRC. To do this, we used quantitative RT-PCR on total RNA extracted from a subset of matched normal, hyperplastic polyps/adenomas and adenocarcinoma tissue samples and determine the expression fold-change of these five genes. We observed a significant negative correlation between the expression fold-change and the DNA methylation levels for SFRP1 (r = −0.584, P < 0.001 Pearson correlation test), SFRP2 (r = −0.340, P = 0.032 Pearson correlation test), SFRP5 (r = −0.375, P = 0.038 Pearson correlation test) and WIF1 (r = −0.384, P = 0.048 Pearson correlation test) (Figure 2). This parallels previous results, which revealed an overall decrease in SFRP1, SFRP2 and SFRP5 expression correlating with promoter hypermethylation of these genes [12]. Only DKK2 (which is expressed at relatively low levels in the normal colorectal mucosa and whose methylation levels are low) showed no significant correlation between methylation and expression levels (r = −0.16, P = 0.36, Pearson correlation test). Either, our analysis was not sensitive enough to detect such a correlation, or the hypermethylation of DKK2 is simply a bystander effect, and functionally irrelevant.Figure 2

Bottom Line: We 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.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.Our 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. aeki2@cam.ac.uk.

ABSTRACT

Background: 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. 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.

Methods: We 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.

Results: We 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.

Conclusion: Our 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.

Show MeSH
Related in: MedlinePlus