We have previously demonstrated that the Wnt pathway, which is essential for normal T and B cell development 
and plays a key role in the development of several types of cancer 
, is aberrantly activated in MM and can promote MM tumor growth 
. Subsequent studies have confirmed the oncogenic potential of the Wnt pathway in MM by demonstrating that targeting the Wnt pathway by drugs or siRNAs leads to inhibition of MM growth 
. In addition, by knocking down β-catenin, Dutta-Simmons et al. revealed new potential Wnt/β-catenin transcriptional targets involved in various aspects of cell-cycle progression, such as CDC25 A/B, cyclins, cyclin-dependent kinases, and AurKA/B. Among the genes significantly downregulated upon β-catenin knock down, a significant proportion had LEF/TCF4 (GCTTTGT/A) binding sites in their promoters, identifying them as putative direct Wnt target genes 
Although the exact cause(s) of aberrant Wnt pathway activation in MM has not yet been established, the absence of detectable Wnt pathway mutations 
as well as the (over)expression of Wnt ligands in the BM microenvironment by both stromal cells and by the MM cells themselves 
, (and table S1), suggests a key role for autocrine and/or paracrine stimulation. As a consequence, loss of secreted Wnt pathway antagonists like DKKs and sFRPs could have a major impact on the pathogenesis of MM. Indeed, we observed that whereas the DKK1 protein is strongly expressed in most primary MMs, the expression of this Wnt antagonist is down-regulated or even completely absent in a subgroup of advanced (stage III) MMs (). In addition, the DKK1 protein was undetectable in MM cell lines, which represent the ultimate, microenvironment independent, phase of MM tumor progression and are almost invariably derived from extramedullary MMs ( and Figure S1
Interestingly, low or undetectable of DKK1 protein expression in BM samples of MM patients was correlated with an increased nuclear expression of β-catenin, a hallmark of canonical Wnt signaling (). DKK1 is a major Wnt pathway antagonist which acts by interfering with the binding of Wnt ligands to the LRP5/6 coreceptor 
. Importantly, the DKK1
gene itself is a direct target of β-catenin/TCF-mediated transcription 
, and DKK1 has been implicated in the feed-back regulation of Wnt signaling in several biological systems 
. Consistent with a tumor suppressor function, DKK1
silencing during tumor progression has been reported in several types of cancer 
. Our observation, that DKK1 levels can be low or undetectable in advanced MM and that restoration of its expression inhibits β-catenin/TCF transcriptional activity (), suggests that silencing of DKK1
may contribute to activation of the canonical Wnt pathway during MM progression.
Like loss of function mutations, aberrant methylation of the promoter of tumor suppressor genes can provide a selective advantage to neoplastic cells 
. We identified DKK1
promoter hypermethylation as a mechanism underlying the absence of DKK1 expression in MM (, , ). In four of the 6 MM cell lines tested, i.e.
, L363, LME-1, UM-1, and OPM-1, we showed hypermethylation of the DKK1
promoter (). The CpG island analyzed encompasses the first exon of the DKK1
gene, which encodes the transcriptional and translational start sites as well as a significant part of the region upstream of the coding sequence, an organization characteristic of genes targeted by epigenetic silencing 
. Indeed, this CpG island has previously been implicated in DKK1 silencing in several types of cancer, including colorectal cancer, gastric cancer, breast cancer, medulloblastoma and leukemia 
. Importantly, the promoter methylation was reduced and DKK1
expression was either restored and/or markedly increased by the DNA demethylating agent 5-aza-2-deoxycytidine (), confirming that the observed aberrant methylation indeed was instrumental in the silencing of DKK1
expression (). Interestingly, 5-azacytidine has been reported to have significant cytotoxic activity against MM cell lines as well as patient-derived malignant plasma cells, but not against peripheral blood mononuclear cells 
. Of the cell lines used in our study, OPM-1 and UM-1 display a very high sensitivity to 5-azacytidine and treatment of these cells with this compound result not only in promoter demethylation but also in rapid and extensive cell death, which explains the rather modest induction of DKK1 expression. Indeed, in the LME-1 cell line, which shows a lower sensitivity to 5-azacytidine-induced cell death, treatment results in a much stronger upregulation of DKK1 mRNA expression. In primary MM, we also observed dense methylation of the DKK1-associated CpG island ( and Figure S2
). Since methylation of the DKK1
promoter was not observed in normal bone marrow samples 
, this methylation can be considered aberrant and disease-related. In addition to silencing of DKK1
, silencing of other Wnt antagonists could also contribute to the enhanced Wnt signaling in advanced MM. Consistent with this notion, Chim et al.
, have reported that constitutive Wnt signaling in MM cell lines is associated with methylation dependent silencing of several Wnt inhibitors, including the sFRP1, 2, 4 and 5. Methylation of at least one of these soluble Wnt inhibitors was observed in most primary MM bone marrow samples 
Our finding that the DKK1
promoter is methylated and Wnt pathway is hyperactivated in advanced multiple myeloma, strongly suggests the presence of autocrine Wnt signaling in malignant plasma cells. In accordance with this hypothesis we observed inhibition of nuclear β-catenin levels and of Wnt reporter activity upon restoration of DKK1 in MM cells (). Importantly, MM cell lines used in this experiment have dense methylation of the DKK1
promoter around the transcription start region and lack detectable DKK1 transcript (, and Figure S1
). Taken together, these data suggest that activation of Wnt signaling in these cell lines is the consequence of DKK1
silencing and could reflect the progression-dependent Wnt pathway activation in patients with advanced MM.
Our current study, in conjunction with work of others, points to a multi-facetted role of DKK1 in the pathogenesis of MM. Studies by Shaughnessy and collegues have previously also reported that DKK1 is strongly expressed by the malignant plasma cells of most MM patients 
. It was shown that secretion of the DKK1 can contribute to MM bone disease by inhibiting Wnt signaling in osteoblasts, thereby interfering with their differentiation 
. Furthermore, in line with our current findings, which suggests that DKK1 may act as a feed-back tumor suppressor, these authors also reported loss of DKK1 protein expression in a subgroup of patients with advanced MM 
. In addition to causing bone disease, inhibition of osteoblast differentiation by DKK1 may also promote MM growth, since mature osteoblasts can suppress myeloma growth, whereas immature osteoblasts express high levels of IL-6, a central growth and survival factor for myeloma plasma cells 
. Furthermore, DKK1 enhances the expression of receptor activator of NF-kappa B ligand (RANKL) and downregulates the expression of osteoprotegerin (OPG) in immature osteoblast 
. The resulting increased RANKL/OPG ratio leads to osteoclast activation promoting osteolytic bone disease. Osteoclasts may also support the growth of myeloma cells through secretion of IL-6 and osteopontin, and by adhesive interactions, stimulating the proliferation of malignant plasma cells 
. Thus, like several other soluble factors expressed by MM cells, for example vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) 
, DKK1 can both, exercise paracrine effects on the BM microenvironment, and affect the MM cells in an autocrine fashion the net effect could be either enhanced or reduced tumor growth.
Consistent with this hypothesis, by employing a MM SCID/rab mouse model, Yaccoby et al. showed that treatment with anti-human DKK1-neutralizing antibody stimulates osteoblast activity, reduces osteoclastogenesis, and promotes bone formation in myelomatous and nonmyelomatous bones 
. MM burden was also reduced, but notably, not in all mice bearing human myeloma cells 
. Similar results were also obtained in a SCID/hu mouse model by Fulciniti et al 
. Together, these studies suggest that MM bone disease and tumor growth are interdependent, at least at the intramedullary stage, and that increased bone formation as a consequence of neutralization of DKK1, may also control MM growth 
.. However, in a 5T2MM murine myeloma model treatment with the anti-DKK1 antibody BHQ880 also caused a reduction of osteolytic bone lesions but did not have any effect on tumor burden 
. Give our current finding that DKK1 inhibits autocrine canonical Wnt signaling in MM cells, inhibition of DKK1 could hyperactivate the Wnt pathway and thereby promote tumor growth, especially at extramedullary sites. Indeed, stimulation of the Wnt signaling pathway in a 5TGM1 mouse myeloma model significantly increased subcutaneous tumor growth 
. In patients, extramedullary growth is associated with aggressive disease, occurring subsequent to the osteolytic bone disease, often resulting in plasma cell leukemia. Importantly, in a human-mouse xenograft MM model, Dutta-Simmons et al. demonstrated that the Wnt pathway not only controls the proliferation of MM plasma cells but also their metastatic potential 
. Taken together, these studies suggest a scenario in which DKK1 has a dual, stage depend, role: whereas high DKK1 expression in early MM contributes to a tumor permissive micronenviroment within the BM, advanced MMs that have acquired BM independence may benefit from DKK1 loss, which enhanced Wnt signaling and thereby promotes MM growth and dissemination. Although blocking DKK1 inhibits osteolytic bone disease in vivo
, targeting DKK1 in MM patients could enhance Wnt pathway activity in MM plasma cells, which might increase the metastatic potential and extramedullary growth of the tumor.
In conclusion, our study establishes for the first time a relation between low or absence of DKK1 expression and the presence Wnt pathway activation during MM progression. Moreover, we demonstrate the presence of a functional ligand-dependent Wnt signaling in MM cells and identify methylation of the DKK1 promoter as a mechanism underlying the absence of DKK1 expression in advanced stage MM. These data strongly suggest that epigenetic silencing of DKK1 unleashes Wnt signaling in a subset of advanced myelomas, promoting disease progression.