Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia

Abstract

DKK-3: is a newly characterised mortalisation-related gene and an antagonist of the Wnt oncogenic signalling pathway whose expression is decreased in a variety of cancer cell lines, suggesting that the Dkk-3 gene, located at chromosome 11p15.1, functions as a tumour suppressor gene. Although 11p15 is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Dkk-3 abnormalities has never been evaluated in this disease. We analysed CpG island methylation of the Dkk-3 promoter in six ALL cell lines and 183 ALL patients. We observed Dkk-3 hypermethylation in all cell lines and in cells from 33% (60/183) of ALL patients. Moreover, Dkk-3 methylation was associated with decreased Dkk-3 mRNA expression and this expression was restored after exposure to the demethylating agent 5-AzaC. Clinical features did not differ between hypermethylated and unmethylated patients. Estimated disease-free survival (DFS) and overall survival at 10 and 11 years, respectively, were 49.8 and 45.6% for normal patients and 10.5 and 15.1% for hypermethylated patients (P=0.001 and 0.09). Multivariate analysis demonstrated that Dkk-3 methylation was an independent prognostic factor predicting DFS (P=0.0009). Our data suggest that Dkk-3 methylation occurs at an early stage in ALL pathogenesis and probably influences the clinical behaviour of the disease.

Figure 1

Methylation status and expression levels of Dkk-3 in ALL cell lines and patients. (A) MSP analysis of CpG island within Dkk-3 promoter in five ALL cell lines. Pos-C indicates methylated control; Neg-C indicates unmethylated control (marrow mononuclear cells from a healthy donor); UM=unmethylated alleles; M=methylated alleles. Promoter hypermethylation is observed in all cell lines. (B) RT–PCR analysis with the Dkk-3 and GAPDH (as control for mRNA integrity) primers. Neg-C indicates unmethylated control. Lack of Dkk-3 expression in observed in all cell lines. (C) MSP analysis of CpG island within Dkk-3 promoter in 12 ALL patients. (D) Analysis of Dkk-3 protein expression by Western blot. The levels of beta-tubulin were also analysed as loading control. 1: ALL-derived MY cell line; 2–4: MY cell line treated with 2, 4 or 6 mM of 5-Aza-2′-deoxycytidine. The demethylating agent restores Dkk-3 expression.

Figure 2

Kaplan–Meier survivor function for ALL patients. DFS curves for ALL patients enrolled in this study according to methylation status of the Dkk-3 gene. Solid lines, unmethylated patients; dashed lines, methylated patients.