Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma

Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
PLoS ONE (Impact Factor: 3.23). 02/2012; 7(2):e30359. DOI: 10.1371/journal.pone.0030359
Source: PubMed


The Wnt/β-catenin pathway plays a crucial role in the pathogenesis of various human cancers. In multiple myeloma (MM), aberrant auto-and/or paracrine activation of canonical Wnt signaling promotes proliferation and dissemination, while overexpression of the Wnt inhibitor Dickkopf1 (DKK1) by MM cells contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggest that DKK1 is part of a negative feedback loop in MM and may act as a tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is low or undetectable in a subset of patients with advanced MM as well as in MM cell lines. This absence of DKK1 is correlated with enhanced Wnt pathway activation, evidenced by nuclear accumulation of β-catenin, which in turn can be antagonized by restoring DKK1 expression. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling.

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Available from: Richard W J Groen
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    • "DKK1 is highly expressed in primary MM, and its expression is downregulated or completely suppressed in the advanced stage (Luo et al. 2012; Kocemba et al. 2012). Aberrant methylation of DKK1 is a reason for DKK1 silencing in the advanced stage, which unleashes the expression of β-catenin/ TCF and activates the Wnt signaling pathway (Kocemba et al. 2012). Increased activity of Wnt signaling plays an important role in MM pathogenesis and progression through inhibition of its antagonist during methylation. "
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    ABSTRACT: Multiple myeloma (MM) is a B cell malignancy characterized by accumulation of malignant plasma cells in the bone marrow. Pathogenesis of MM involves a complex pattern of structural and numerical chromosomal aberrations. In addition, epigenetic changes such as DNA methylation and histone modifications may play a role in this disease by affecting the expression of different genes. This article reviews recent findings on the role of epigenetic alterations in MM pathogenesis, which affect the expression of cell cycle regulatory molecules, apoptosis, DNA repair system, CD markers, cell signaling pathways as well as tumor suppressor miRNAs. Given these results, it can be stated that these epigenetic changes play an important role in the initiation and progression of MM. Therefore, understanding the impact of epigenetics in MM pathogenesis in each stage of disease progression can help develop therapeutic targets to increase survival and reduce drug resistance.
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    • "Both canonical and non-canonical signaling pathways participate in many physiological and pathological processes [1-4]. Several lines of evidence suggest that Wnt/β-catenin signaling is essential for bone formation and resorption [4-8]. Activating mutations in lipoprotein receptor-related protein 5 (LRP5), a Wnt co-receptor, induce high bone mass phenotype, whereas inactivating mutations cause osteoporosis-pseudoglioma syndrome, characterized by osteoporosis and blindness [9,10]. "
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    ABSTRACT: The Wnt/β-catenin signaling pathway is essential for controlling bone mass; however, little is known about the variable effects of the constitutive activation of β-catenin (CA-β-catenin) on bone growth and remodeling at different postnatal stages. The goal of the present study was to observe the effects of CA-β-catenin on vertebral bone growth and remodeling in mice at different postnatal stages. In particular, special attention was paid to whether CA-β-catenin has detrimental effects on these processes. Catnblox(ex 3) mice were crossed with mice expressing the TM-inducible Cre fusion protein, which could be activated at designated time points via injection of tamoxifen. β-catenin was stabilized by tamoxifen injection 3 days, and 2, 4, 5, and 7 months after birth, and the effects lasted for one month. Radiographic imaging, micro-computed tomography, immunohistochemistry, and safranin O and tartrate-resistant acid phosphatase staining were employed to observe the effects of CA-β-catenin on vertebral bone growth and remodeling. CA-β-catenin in both early (3 days after birth) and late stages (2, 4, 5, and 7 months after birth) increased bone formation and decreased bone resorption, which together increased vertebral bone volume. However, when β-catenin was stabilized in the early stage, vertebral linear growth was retarded, and the mice demonstrated shorter statures. In addition, the newly formed bone was mainly immature and located close to the growth plate. In contrast, when β-catenin was stabilized in the late stage, vertebral linear growth was unaffected, and the newly formed bone was mainly mature and evenly distributed throughout the vertebral body. CA-β-catenin in both early and late stages of growth can increase vertebral bone volume, but β-catenin has differential effects on vertebral growth and remodeling when activated at different postnatal stages.
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    ABSTRACT: Our previous studies had shown that DAZAP2 was profoundly downregulated in bone marrow mononuclear cells from multiple myeloma patients. In this report, we analyzed epigenetic changes in multiple myeloma cell lines to understand the molecular mechanisms underlying the downregulation of DAZAP2. Four multiple myeloma cell lines, KM3, MM.1S, OPM-2 and ARH-77, were studied. The results of methylation specific PCR (MSP) showed that the promoter of DAZAP2 was methylated for KM3, MM.1S, OPM-2 and unmethylated for ARH-77. The DAZAP2 promoter region was amplified to obtain a series of different length sequences. All of the amplified sequences were inserted to luciferase reporter vector. The constructs were transfected into COS-7 cells and the luciferase activities were measured to search for the core region of DAZAP2 promoter. Two CpG islands were found in DAZAP2 promoter region. The results of luciferase assay showed that CpG island 1 displayed weak transcriptional activity, whereas CpG island 2 exhibited strong transcriptional activity (273 folds) compared to the control. The sequence that covered both CpG islands 1 and 2 showed higher activity (1,734 folds) compared to the control, suggesting that the two islands had synergistic effect on regulating DAZAP2 expression. We also found that M. Sss I methylase could inhibit the luciferase activity, whereas demethylation using 5-aza-2'-deoxycytidine treatment rescued the expression of DAZAP2 for multiple myeloma cell lines. These data revealed that methylation of DAZAP2 promoter was involved in downregulation of DAZAP2 in multiple myeloma cells.
    Preview · Article · Jul 2012 · PLoS ONE
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