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

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

Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options

Aberrant activation of Wnt/β-catenin signaling plays a central role in the pathogenesis of a wide variety of malignancies and is typically caused by mutations in core Wnt pathway components driving constitutive, ligand-independent signaling. In multiple myelomas (MMs), however, these pathway intrinsic mutations are rare despite the fact that most tumors display aberrant Wnt pathway activity. Recent studies indicate that this activation is caused by genetic and epigenetic lesions of Wnt regulatory components, sensitizing MM cells to autocrine Wnt ligands and paracrine Wnts emanating from the bone marrow niche. These include deletion of the tumor suppressor CYLD, promotor methylation of the Wnt antagonists WIF1, DKK1, DKK3, and sFRP1, sFRP2, sFRP4, sFRP5, as well as overexpression of the co-transcriptional activator BCL9 and the R-spondin receptor LGR4. Furthermore, Wnt activity in MM is strongly promoted by interaction of both Wnts and R-spondins with syndecan-1 (CD138) on the MM cell-surface. Functionally, aberrant canonical Wnt signaling plays a dual role in the pathogenesis of MM: (I) it mediates proliferation, migration, and drug resistance of MM cells; (II) MM cells secrete Wnt antagonists that contribute to the development of osteolytic lesions by impairing osteoblast differentiation. As discussed in this review, these insights into the causes and consequences of aberrant Wnt signaling in MM will help to guide the development of targeting strategies. Importantly, since Wnt signaling in MM cells is largely ligand dependent, it can be targeted by drugs/antibodies that act upstream in the pathway, interfering with Wnt secretion, sequestering Wnts, or blocking Wnt (co)receptors

Syndecan-1 promotes Wnt/b-catenin signaling in multiple myeloma by presenting Wnts and R-spondins

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/b-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate (HS) proteoglycan syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its HS chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible short hairpin RNA–mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS chains decorating syndecan-1 mediate aberrant Wnt pathway activation in MM. HS-deficient MM cells exhibited strongly decreased autocrine Wnt/b-catenin pathway activity and reduced Wnt pathway–dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side chains of syndecan-1 and that this binding contributes to paracrine Wnt pathway activation through the Wnt receptor Frizzled (Fzd). Furthermore, in an HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Fzd degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Costimulation with R-spondin and its binding to HS chains decorating syndecan-1 are indispensable for optimal stimulation of Wnt signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/b-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets in the treatment of MM

Syndecan-1 promotes Wnt/β-catenin signaling in multiple myeloma by presenting Wnts and R-spondins

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/β-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt-pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate proteoglycan (HSPG) syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its heparan sulfate (HS) chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible shRNA-mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS-chains decorating syndecan-1 mediate aberrant Wnt-pathway activation in MM. HS-deficient MM cells exhibited a strongly decreased autocrine Wnt/β-catenin pathway activity and a reduced Wnt-pathway-dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side-chains of syndecan-1 and that this binding contributes to paracrine Wnt-pathway activation through the Wnt-receptor Frizzled. Furthermore, in a HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Frizzled degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Co-stimulation with R-spondin, and its binding to HS chains decorating syndecan-1, are indispensable for optimal stimulation of Wnt-signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt-signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/β-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets for the treatment of M

MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms

Background & Aims Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice.  Methods We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (AhCre/Metfl/fl/LacZ) or ISC-specific disruption of MET (Lgr5Creert2/Metfl/fl/LacZ) and control mice (AhCre/Met+/+/LacZ, Lgr5Creert2/Met+/+/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5Creert2/Metfl/fl/Apcfl/fl and Lgr5Creert2/Met+/+/Apcfl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in AhCre/Metfl/fl/Apcfl/+ mice compared with AhCre/Met+/+/Apcfl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44+/+, Cd44–/–, Cd44s/s, or Cd44v4–10/v4–10 mice).  Results Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in AhCre/Metfl/fl/LacZ mice. Lgr5Creert2/Metfl/fl/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5Creert2/Metfl/fl/Apcfl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (AhCre/Metfl/fl/Apcfl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44–/– mice did not expand to the same extent as crypts from Cd44+/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4–10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4–10.  Conclusions In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4–10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors

MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms

Background & Aims Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice.  Methods We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (AhCre/Metfl/fl/LacZ) or ISC-specific disruption of MET (Lgr5Creert2/Metfl/fl/LacZ) and control mice (AhCre/Met+/+/LacZ, Lgr5Creert2/Met+/+/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5Creert2/Metfl/fl/Apcfl/fl and Lgr5Creert2/Met+/+/Apcfl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in AhCre/Metfl/fl/Apcfl/+ mice compared with AhCre/Met+/+/Apcfl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44+/+, Cd44–/–, Cd44s/s, or Cd44v4–10/v4–10 mice).  Results Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in AhCre/Metfl/fl/LacZ mice. Lgr5Creert2/Metfl/fl/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5Creert2/Metfl/fl/Apcfl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (AhCre/Metfl/fl/Apcfl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44–/– mice did not expand to the same extent as crypts from Cd44+/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4–10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4–10.  Conclusions In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4–10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors

DKK1 expression represses Wnt pathway activation in MM.

<p>(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.</p

<i>DKK1</i> promoter methylation in MM cell lines.

<p>(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 <i>DKK1</i>, 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) <i>Upper panel</i>. Representation of bisulfite genomic sequencing results of 5 clones of the <i>DKK1</i> 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 <i>DKK1</i> 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. <i>Lower panel</i>. Electropherograms of bisulfite modified DNA from <i>DKK1</i> CpG island in HT-29 (U_DNA) and DLD-1 (M_DNA) cells. (D) Methylation specific PCR of the CpG island of the <i>DKK1</i> 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 <i>DKK1</i> 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.</p