Transcriptional silencing of the Wnt-antagonist DKK1 by promoter methylation is associated with enhanced Wnt signaling in advanced multiple myeloma - PubMed (original) (raw)

Transcriptional silencing of the Wnt-antagonist DKK1 by promoter methylation is associated with enhanced Wnt signaling in advanced multiple myeloma

Kinga A Kocemba et al. PLoS One. 2012.

Abstract

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The relation between the Wnt pathway activation and the loss of DKK1 expression during MM progression.

(A) Representative pictures of immunohistochemical staining of a multiple myeloma patient displaying either high DKK-1 and low β-catenin expression (upper panel) or with DKK-1 loss and increased nuclear β-catenin localization (lower panel). Immunohistochemical stainings are shown for CD138 (left column), β-catenin (middle column) and DKK1 (right column). (B) Nuclear β-catenin expression in relation to multiple myeloma progression (n = 48, p<0.05). (C) DKK-1 expression in relation to multiple myeloma progression (p<0.05). (D) Representative pictures of immunocytochemical staining of multiple myeloma cell lines with goat polyclonal anti-DKK1 antibody (magnification: 400×). Prostate cancer cell line (PC-3) was used as positive control (PC) for the DKK-1 staining. (E) Relation between the loss of DKK-1 expression and nuclear localization of β-catenin (p>0.05). A significant correlation (p<0.05) between expression of nuclear β-catenin and DKK-1 was observed in the two extreme groups identified based on β-catenin expression. * indicates p value<0.05.

Figure 2

Figure 2. DKK1 expression represses Wnt pathway activation in MM.

(A) MM cell lines OPM-1 and UM-1 were transduced with either the LZRS-pBMN-IRES-eGFP (control) or the LZRS-pBMN-DKK1-IRES-eGFP (DKK1) virus. Conditioned medium of sorted, transduced cells was harvested and immunoblotted using a goat polyclonal antibody against DKK1. Representative immunoblot confirms the expression of DKK1 in the conditioned medium of LZRS-pBMN-DKK1-IRES-eGFP transduced cells. β-actin is shown as internal control for equal cell number. (B) Cytoplasmic and nuclear proteins were prepared from the LZRS-pBMN-IRES-eGFP (control) or the LZRS-pBMN-DKK1-IRES-eGFP (DKK1) MM cells, stimulated for 24h with Wnt3a conditioned medium (+). As a control, L-cells conditioned medium was applied (−).To assess β-catenin accumulation, nuclear and cytoplasmic cells lysate was immunoblotted by using a monoclonal anti-β-catenin antibody. The bottom part of the blot was stained with β-tubulin and Histone H2B as controls for cytoplasmic and nuclear proteins, respectively. (C) LZRS-pBMN-IRES-eGFP (control) or the LZRS-pBMN-DKK1-IRES-eGFP (DKK1) cells were transfected with TOPFLASH reporter and renilla contruct. 24 hours upon transfection cells were treated with L-cells conditioned medium (−) or Wnt3a conditioned medium (+).The relative light units value of LZRS-pBMN-IRES-eGFP cells treated with L-cells conditioned medium was normalized to 1. The mean ± SD of representative experiment performed in triplicate is shown. * indicates p value<0.05 *** indicates p value<0.001. by student's t test.

Figure 3

Figure 3. DKK1 promoter methylation in MM cell lines.

(A) Total RNA was isolated and RT–PCR analyses were performed with the specific primers indicated. Complementary DNA from prostate cancer cell line (PC-3) was used as positive control (PC) for DKK1 expression. The β-actin expression was used as a loading control. (B) Schematic representation of the promoter area analyzed for DKK1, containing a CpG island. White arrows indicate the positions of primers used for bisulfite sequencing, and black arrows indicate the positions of primers used for methylation specific PCR. Each of the CpG dinucleotides is presented as open circle. (C) Upper panel. Representation of bisulfite genomic sequencing results of 5 clones of the DKK1 promoter region in HT-29 and DLD-1 colon cell lines used as unmethylated (U_DNA) and methylated (M_DNA) control, respectively. The amplified 326 bp product corresponds to the DKK1 promoter region from −193 to +122. In total, 18 CpG dinucleotides (CpGs) within the CpG island were analyzed and are represented as open and closed circles, which indicate unmethylated and methylated CpG sites, respectively. Lower panel. Electropherograms of bisulfite modified DNA from DKK1 CpG island in HT-29 (U_DNA) and DLD-1 (M_DNA) cells. (D) Methylation specific PCR of the CpG island of the DKK1 promoter region in MM cell lines. DNA bands in lanes labeled with U indicate PCR products amplified with primers recognizing unmethylated promoter sequences, whereas DNA bands in lanes labeled with M represent amplified products with primers designed for the methylated template. (E) Bisulfite sequencing analysis of the the DKK1 promoter region in multiple myeloma cell lines, open circles indicating unmethylated CpG sites, and closed circles representing methylated CpG sites. Percentages indicate the fraction of methylated CpG dinucleotides of the total CpG sites analyzed.

Figure 4

Figure 4. Analysis of DKK1 promoter methylation in MM bone marrow samples.

Methylation specific PCR of the CpG island of the DKK1 promoter region in the bone marrow samples of twelve patients with multiple myeloma (P1–P12), HT-29 and DLD-1 colon cell lines were used as unmethylated (U_DNA) and methylated (M_DNA) control respectively. DNA bands in lanes labeled with U and M indicate PCR products amplified with primers recognizing unmethylated and methylated promoter sequences respectively.

Figure 5

Figure 5. Restoration of DKK1 expression in MM cell lines by 5-aza-2-deoxycytidine treatment.

(A) DKK-1 promoter methylation analyzed by bisulfite genomic sequencing of 10 clones, on DNA isolated from 5-aza-2-deoxycytidine treated (5-aza-CdR) and untreated (PBS) MM cell lines UM-1 and OPM-1. Frequency of methylation was calculated by dividing the number of methylated CpG sites by the total number of analyzed CpG sites. (B) Reverse transcriptase-PCR analysis for DKK1 gene expression in multiple myeloma cell lines in the absence and presence of the demethylating agent 5-aza-2-deoxycytidine. β-actin expression is shown as an input control.

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References

    1. Podar K, Chauhan D, Anderson KC. Bone marrow microenvironment and the identification of new targets for myeloma therapy. Leukemia. 2009;23:10–24. - PMC - PubMed
    1. Raab MS, Podar K, Breitkreutz I, Richardson PG, Anderson KC. Multiple myeloma. Lancet. 2009;374:324–329. - PubMed
    1. Edwards CM, Zhuang J, Mundy GR. The pathogenesis of the bone disease of multiple myeloma. Bone. 2008;42:1007–1013. - PMC - PubMed
    1. Giuliani N, Morandi F, Tagliaferri S, Lazzaretti M, Donofrio G, et al. Production of Wnt inhibitors by myeloma cells: potential effects on canonical Wnt pathway in the bone microenvironment. Cancer Res. 2007;67:7665–7674. - PubMed
    1. Oshima T, Abe M, Asano J, Hara T, Kitazoe K, et al. Myeloma cells suppress bone formation by secreting a soluble Wnt inhibitor, sFRP-2. Blood. 2005;106:3160–3165. - PubMed

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