Inhibition of Wnt/β-Catenin Signaling by a Soluble Collagen-Derived Frizzled Domain Interacting with Wnt3a and the Receptors Frizzled 1 and 8

The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth

A Cryptic Frizzled Module in Cell Surface Collagen 18 Inhibits Wnt/β−Catenin Signaling

Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries structural identity with the extracellular cysteine-rich domain of the frizzled receptors. We show that V3C18 is a cell surface heparan sulfate proteoglycan, its topology being mediated by the FZC18 module. V3C18 mRNA was expressed at low levels in 21 normal adult human tissues. Its expression was up-regulated in fibrogenesis and in small well-differentiated liver tumors, but decreased in advanced human liver cancers. Low FZC18 immunostaining in liver cancer nodules correlated with markers of high Wnt/β−catenin activity. V3C18 (Mr = 170 kD) was proteolytically processed into a cell surface FZC18-containing 50 kD glycoprotein precursor that bound Wnt3a in vitro through FZC18 and suppressed Wnt3a-induced stabilization of β−catenin. Ectopic expression of either FZC18 (35 kD) or its 50 kD precursor inhibited Wnt/β−catenin signaling in colorectal and liver cancer cell lines, thus downregulating major cell cycle checkpoint gatekeepers cyclin D1 and c-myc and reducing tumor cell growth. By contrast, full-length V3C18 was unable to inhibit Wnt signaling. In summary, we identified a cell-surface signaling pathway whereby FZC18 inhibits Wnt/β−catenin signaling. The signal, encrypted within cell-surface C18, is released by enzymatic processing as an active frizzled cysteine-rich domain (CRD) that reduces cancer cell growth. Thus, extracellular matrix controls Wnt signaling through a collagen-embedded CRD behaving as a cell-surface sensor of proteolysis, conveying feedback cues to control cancer cell fate

Régulation de la voie de signalisation Wnt/b-caténine par le microenvironnement : rôle du domaine Frizzled du collagène XVIII (FZC18)

Le cancer colorectal et le carcinome hépatocellulaire font partie des cancers les plus fréquents au monde. Les traitements disponibles à ce jour pour les formes avancées de ces cancers ne sont que palliatifs et ont une efficacité relativement faible. Les biothérapies ciblant les mécanismes moléculaires impliqués dans la croissance ou la différenciation des cellules tumorales permettent de proposer des traitements nouveaux ayant une forte spécificité et une faible toxicité, prolongeant ainsi les rémissions avec une meilleure qualité de vie. Nous nous sommes intéressés au collagène XVIII qui est un composant majeur des membranes basales. Un des variants de ce collagène possède un domaine Frizzled que nous avons nommé FZC18. Celui-ci contient un motif CRD (Cysteine-rich Domain) homologue à celui présent dans la portion extracellulaire des récepteurs Frizzled et à celui des SFRPs (Secreted Frizzled-related Proteins), deux acteurs majeurs de la voie de signalisation Wnt/b-caténine.Colorectal cancer and hepatocellular carcinoma are among the most common cancers in the world. Currently available treatments for advanced froms of these cancers are only palliative and have a relatively low efficacy. Biotherapy targeting the molecular mechanisms involved in growth or differentiation of tumor cells provide treatments with high specificity and low toxicity, prolonging remissions with a better quality of life. A major issue in the use of biomolecules is teir ability to enter in the tumor cells, which can be adressed by using biomolecules that target cell surface receptors. We focused on collagen XVIII, which is a major basement membrane component. One of the variants of this collagene has a FZC18 domain, which contains a CRD motif (Cysteine-rich Domain) homologous to the CRD of the extracellular Wnt-binding domain of the Frizzled receptors and the SFRPs (Secreted Frizzled-related Proteins). Both of them are major actors of the Wnt/b-catenin signaling pathway.RENNES1-BU Santé (352382103) / SudocSudocFranceF

Tenascin-C is required for normal Wnt/β-catenin signaling in the whisker follicle stem cell niche.

Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/β-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed β-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear β-catenin in wild-type mice reflecting active Wnt/β-catenin signaling, whereas β-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/β-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted β-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/β-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/β-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells

Tenascin-W Is a Novel Stromal Marker in Biliary Tract Cancers

Extrahepatic cancers of the biliary system are typically asymptomatic until after metastasis, which contributes to their poor prognosis. Here we examined intrahepatic cholangiocarcinomas (n = 8), carcinomas of perihilar bile ducts (n = 7), carcinomas of the gallbladder (n = 11) and hepatic metastasis from carcinomas of the gallbladder (n = 4) for the expression of the extracellular matrix glycoproteins tenascin-C and tenascin-W. Anti-tenascin-C and anti-tenascin-W immunoreactivity was found in all biliary tract tumors examined. Unlike tenascin-C, tenascin-W was not detected in normal hepatobiliary tissue. Tenascin-W was also expressed by the cholangiocarcinoma-derived cell line Huh-28. However, co-culture of Huh-28 cells with immortalized bone marrow-derived stromal cells was necessary for the formation and organization of tenascin-W fibrils in vitro. Our results indicate that tenascin-W may be a novel marker of hepatobiliary tumor stroma, and its absence from many normal tissues suggests that it may be a potential target for biotherapies

Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active β-catenin.: An SFRP-like frizzled motif blocks tumor growth

International audienceConstitutive activation of Wnt/β-catenin signaling in cancer results from mutations in pathway components, which frequently coexist with autocrine Wnt signaling or epigenetic silencing of extracellular Wnt antagonists. Among the extracellular Wnt inhibitors, the secreted frizzled-related proteins (SFRPs) are decoy receptors that contain soluble Wnt-binding frizzled domains. In addition to SFRPs, other endogenous molecules harboring frizzled motifs bind to and inhibit Wnt signaling. One of such molecules is V3Nter, a soluble SFRP-like frizzled polypeptide that binds to Wnt3a and inhibits Wnt signaling and expression of the β-catenin target genes cyclin D1 and c-myc. V3Nter is derived from the cell surface extracellular matrix component collagen XVIII. Here, we used HCT116 human colon cancer cells carrying the ΔS45 activating mutation in one of the alleles of β-catenin to show that V3Nter and SFRP-1 decrease baseline and Wnt3a-induced β-catenin stabilization. Consequently, V3Nter reduces the growth of human colorectal cancer xenografts by specifically controlling cell proliferation and cell cycle progression, without affecting angiogenesis or apoptosis, as shown by decreased [(3)H]-thymidine (in vitro) or BrdU (in vivo) incorporation, clonogenesis assays, cell cycle analysis and magnetic resonance imaging in living mice. Additionally, V3Nter switches off the β-catenin target gene expression signature in vivo. Moreover, experiments with β-catenin allele-targeted cells showed that the ΔS45 β-catenin allele hampers, but does not abrogate, inhibition of Wnt signaling by SFRP-1 or by the SFRP-like frizzled domain. Finally, neither SFRP-1 nor V3Nter affect β-catenin signaling in SW480 cells carrying nonfunctional Adenomatous polyposis coli. Thus, SFRP-1 and the SFRP-like molecule V3Nter can inhibit tumor growth of β-catenin-activated tumor cells in vivo

FZC18 reduces cell sensitivity to soluble Wnt3a.

<p>HEK293T cell batches stably expressing FZC18 (1; 4; 5) or empty vector (V) were incubated with either 50% control or Wnt3a conditioned medium (CM) for 16 hr before lysis. CRT (β-catenin-T-Cell factor Regulated Transcription) assays using Super8•Topflash or the negative control Super8•Fopflash reporters are representative of three independent experiments performed in triplicate and normalized to Renilla luciferase activity (mean±SD).</p

Frizzled 1 and 8 receptors partially rescue the inhibition of Wnt3a-induced CRT by FZC18.

<p>CRT reporter gene assays using the β-catenin-TCF responsive reporter Super8•Topflash in HEK293T cells stably expressing FZC18 (A, B and C) and vector (A, B). Twenty-four hours after transfection with the CRT reporter and increasing amounts of either FZD1 receptor (A), FZD8 receptor (B), FZD8 receptor, FZD8_CRD or FZC8_CRD-GPI (C) cDNAs, cells were incubated either with 50% control CM (L) or with 50% Wnt3a CM for 16 hr. Results are representative of three independent experiments performed in triplicate and normalized to Renilla luciferase activity. Error bars represent standard deviations.</p