Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia

Evgeny A. Moskalev, Katrin Luckert, Ivan A. Vorobjev, Sergey E. Mastitsky, Aleena A. Gladkikh, Achim Stephan, Marita Schrenk, Kamil D. Kaplanov, Olga B. Kalashnikova, Oliver Pötz, Thomas O. Joos, Jörg D. Hoheisel

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Background: The Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples.Methods: Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.Results: For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2́-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2́-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.Conclusions: The results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

Original languageEnglish
Article number213
JournalBMC Cancer
Volume12
DOIs
Publication statusPublished - Jun 6 2012
Externally publishedYes

Fingerprint

Catenins
Wnt Signaling Pathway
B-Cell Chronic Lymphocytic Leukemia
Epigenomics
decitabine
Methylation
Genes
Cadherins
Cell Line
Neoplasms
DNA Methylation
Least-Squares Analysis
Microspheres
Immunoassay
Therapeutics
Gene Expression
Polymerase Chain Reaction
Control Groups

Keywords

  • β-catenin
  • B cell chronic lymphocytic leukaemia
  • DNA hypermethylation
  • Epigenetic silencing
  • Inhibitor genes
  • Wnt/β-catenin pathway

ASJC Scopus subject areas

  • Oncology
  • Genetics
  • Cancer Research

Cite this

Moskalev, E. A., Luckert, K., Vorobjev, I. A., Mastitsky, S. E., Gladkikh, A. A., Stephan, A., ... Hoheisel, J. D. (2012). Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia. BMC Cancer, 12, [213]. https://doi.org/10.1186/1471-2407-12-213

Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia. / Moskalev, Evgeny A.; Luckert, Katrin; Vorobjev, Ivan A.; Mastitsky, Sergey E.; Gladkikh, Aleena A.; Stephan, Achim; Schrenk, Marita; Kaplanov, Kamil D.; Kalashnikova, Olga B.; Pötz, Oliver; Joos, Thomas O.; Hoheisel, Jörg D.

In: BMC Cancer, Vol. 12, 213, 06.06.2012.

Research output: Contribution to journalArticle

Moskalev, EA, Luckert, K, Vorobjev, IA, Mastitsky, SE, Gladkikh, AA, Stephan, A, Schrenk, M, Kaplanov, KD, Kalashnikova, OB, Pötz, O, Joos, TO & Hoheisel, JD 2012, 'Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia', BMC Cancer, vol. 12, 213. https://doi.org/10.1186/1471-2407-12-213
Moskalev, Evgeny A. ; Luckert, Katrin ; Vorobjev, Ivan A. ; Mastitsky, Sergey E. ; Gladkikh, Aleena A. ; Stephan, Achim ; Schrenk, Marita ; Kaplanov, Kamil D. ; Kalashnikova, Olga B. ; Pötz, Oliver ; Joos, Thomas O. ; Hoheisel, Jörg D. / Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia. In: BMC Cancer. 2012 ; Vol. 12.
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title = "Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia",
abstract = "Background: The Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples.Methods: Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.Results: For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2́-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2́-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.Conclusions: The results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.",
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T1 - Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia

AU - Moskalev, Evgeny A.

AU - Luckert, Katrin

AU - Vorobjev, Ivan A.

AU - Mastitsky, Sergey E.

AU - Gladkikh, Aleena A.

AU - Stephan, Achim

AU - Schrenk, Marita

AU - Kaplanov, Kamil D.

AU - Kalashnikova, Olga B.

AU - Pötz, Oliver

AU - Joos, Thomas O.

AU - Hoheisel, Jörg D.

PY - 2012/6/6

Y1 - 2012/6/6

N2 - Background: The Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples.Methods: Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.Results: For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2́-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2́-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.Conclusions: The results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

AB - Background: The Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples.Methods: Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.Results: For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2́-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2́-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.Conclusions: The results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

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KW - B cell chronic lymphocytic leukaemia

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KW - Epigenetic silencing

KW - Inhibitor genes

KW - Wnt/β-catenin pathway

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