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Open Access Highly Accessed Research article

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

Evgeny A Moskalev17*, Katrin Luckert2, Ivan A Vorobjev34, Sergey E Mastitsky5, Aleena A Gladkikh3, Achim Stephan1, Marita Schrenk1, Kamil D Kaplanov6, Olga B Kalashnikova6, Oliver Pötz2, Thomas O Joos2 and Jörg D Hoheisel1

Author Affiliations

1 Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany

2 Biochemistry Department, NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, 72770, Reutlingen, Germany

3 Functional Morphology of Hemablastoses, National Hematology Research Centre of Russian Academy of Medical Sciences, Novy Zykovsky passage 4a, 125167, Moscow, Russia

4 A.N. Belozersky Institute and Biological Faculty, Moscow State University, Leninskie Gory 1, 119991, Moscow, Russia

5 Theoretical Bioinformatics, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany

6 Department of Haematology, Volgograd Regional Clinical Oncological Dispensary No.1, Zemlyachki str. 78, 400138, Volgograd, Russia

7 Diagnostic Molecular Pathology, Institute of Pathology, University of Erlangen-Nürnberg, Krankenhausstr. 8-10, 91054, Erlangen, Germany

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BMC Cancer 2012, 12:213  doi:10.1186/1471-2407-12-213

Published: 6 June 2012

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-2´-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-2´-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.

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