Blocking Wnt Signaling by SFRP-Like Molecules Inhibits in Vivo Cell Proliferation and Tumor Growth in Cells Carrying Active B-Catenin

Oncogene (2011) 30, 423–433 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active b-catenin E Lavergne1,2, I Hendaoui1,2, C Coulouarn1,2, C Ribault1,2, J Leseur1,2,3, P-A Eliat2,4, S Mebarki1,2, A Corlu1,2, B Cle´ment1,2 and O Musso1,2 1INSERM, UMR991, Liver Metabolisms and Cancer, Rennes, France; 2Universite´ de Rennes 1, Rennes, France; 3Centre Re´gional de Lutte contre le Cancer, Rennes, France and 4PRISM, IFR 140, Biogenouest, Rennes, France Constitutive activation of Wnt/b-catenin signaling in cancer Introduction results from mutations in pathway components, which frequently coexist with autocrine Wnt signaling or epige- The Wnt/b-catenin pathway is a major regulator of cell netic silencing of extracellular Wnt antagonists. Among the proliferation, migration, differentiation and metabo- extracellular Wnt inhibitors, the secreted frizzled-related lism, controlling tissue homeostasis and tumor progres- proteins (SFRPs) are decoy receptors that contain soluble sion (MacDonald et al., 2009). In the majority of Wnt-binding frizzled domains. In addition to SFRPs, other colorectal cancers (CRC), the coexistence of several endogenous molecules harboring frizzled motifs bind to and mechanisms such as genetic defects in pathway compo- inhibit Wnt signaling. One of such molecules is V3Nter, a nents, autocrine Wnt signaling and epigenetic silencing soluble SFRP-like frizzled polypeptide that binds to Wnt3a of extracellular Wnt antagonists results in activation of and inhibits Wnt signaling and expression of the b-catenin Wnt/b-catenin signaling and provides a selective ad- target genes cyclin D1 and c-myc. V3Nter is derived from vantage to tumor cells (Polakis, 2007). Pathway activa- the cell surface extracellular matrix component collagen tion involves interaction of Wnt ligands with cell surface XVIII. Here, we used HCT116 human colon cancer cells Frizzled receptors and LRP5/6 co-receptors. This carrying the DS45 activating mutation in one of the alleles disrupts the Adenomatous polyposis coli (APC)–axin of b-catenin to show that V3Nter and SFRP-1 decrease complex, thus halting proteasomal degradation of baseline and Wnt3a-induced b-catenin stabilization. Con- b-catenin, which is stabilized and interacts with T-cell sequently, V3Nter reduces the growth of human colorectal factor transcription factors, displacing repressors and cancer xenografts by specifically controlling cell prolifera- recruiting activators of target gene expression. tion and cell cycle progression, without affecting angiogen- The activity of the Wnt/b-catenin pathway can be esis or apoptosis, as shown by decreased [3H]-thymidine antagonized by several families of secreted proteins. (in vitro)orBrdU(in vivo) incorporation, clonogenesis Interaction of Wnt ligands with the Frizzled receptors assays, cell cycle analysis and magnetic resonance imaging is enhanced by heparan sulfate glycosaminoglycans in living mice. Additionally, V3Nter switches off the controlling Wnt diffusion at the cell surface (Mikels b-catenin target gene expression signature in vivo.More- and Nusse, 2006) or inhibited by several families of over, experiments with b-catenin allele-targeted cells extracellular antagonists (Kawano and Kypta, 2003; showed that the DS45 b-catenin allele hampers, but does Bovolenta et al., 2008). Members of the Dickkopf not abrogate, inhibition of Wnt signaling by SFRP-1 or by (DKK) family antagonize canonical signaling by bind- the SFRP-like frizzled domain. Finally, neither SFRP-1 ing to LRP5/6, thus disrupting the Wnt-induced nor V3Nter affect b-catenin signaling in SW480 cells Frizzled-LRP5/6 complex (MacDonald et al., 2009). carrying nonfunctional Adenomatous polyposis coli.Thus, Wnt inhibitory factor-1 binds directly to Wnts, altering SFRP-1 and the SFRP-like molecule V3Nter can inhibit their ability to interact with the receptors. Members of tumor growth of b-catenin-activated tumor cells in vivo. the family of extracellular decoy receptors known as Oncogene (2011) 30, 423–433; doi:10.1038/onc.2010.432; secreted frizzled-related proteins (SFRPs) possess a published online 20 September 2010 frizzled cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled Keywords: frizzled; collagen XVIII; Wnt; b-catenin; receptors. Frizzled CRDs contain 10 cysteines at colorectal cancer; secreted frizzled-related proteins conserved positions, which form a highly conserved three-dimensional structure through a precise pattern of disulfide bridges. Frizzled CRDs bind Wnts and form homodimers or heterodimers (Dann et al., 2001). Thus, SFRPs can modulate Wnt signaling by sequestering Correspondence: Dr O Musso, INSERM, UMR 991, Liver Metabo- Wnts through the CRD or by acting as dominant- lisms and Cancer, Hoˆpital Pontchaillou, Universite´de Rennes, rue negative inhibitors, forming inactive complexes with the Henri le Guilloux, RENNES Cedex F-35033, France. E-mail: [email protected] frizzled receptors (Bovolenta et al., 2008). In addition, Received 1 March 2010; revised 10 August 2010; accepted 11 August engineered SFRP-like proteins such as the soluble CRD 2010; published online 20 September 2010 of the receptor Frizzled 8 potently inhibit autocrine Wnt An SFRP-like frizzled motif blocks tumor growth E Lavergne et al 424 signaling and tumor growth in mice carrying teratomas tion pressure, as shown for SFRP-1 in breast cancer (DeAlmeida et al., 2007). cells (Bafico et al., 2004). However, after seeding three It has been suggested that SFRPs may function as cells per well, several antibiotic-resistant cell popula- tumor suppressors in CRC because allelic loss or tions expressing the relevant epitopes (Supplementary epigenetic inactivation of SFRP genes contributes to Figure 1a) were expanded. By immunocytochemi- constitutive activation of the Wnt/b-catenin pathway stry, they were composed of B50% of cells expressing (Caldwell et al., 2004; Suzuki et al., 2004). In this high levels of V3Nter or SFRP-1 and low b-catenin levels, context, we and others have shown that restoring and B50% of cells showing low V3Nter or SFRP-1 expression of SFRP-1 or SFRP-5 inhibits Wnt/b-catenin content and high b-catenin levels (Figure 1c). In V3Nter stabilization and induces in vitro cell death in human (–) cells neighboring V3Nter ( þ )foci,b-catenin was CRC cells (Suzuki et al., 2004; Quelard et al., 2008). In restricted to cell membranes and particularly to cell addition to SFRPs, other endogenous molecules carry- contacts (Figure 1c, close-up), suggesting paracrine inhibi- ing frizzled CRDs bind to and inhibit Wnt signaling. We tion of Wnt/b-catenin signaling. Controls, that is, Vector- recently showed that a frizzled CRD derived from cell and V2Nter-HCT116 cells, showed widespread high surface collagen XVIII (C18) functions in an SFRP-like b-catenin levels (Figure 1c). These features were stably fashion, binding to Wnt3a and inhibiting Wnt/b-catenin maintained at least throughout 15 passages. signaling in vitro (Quelard et al., 2008). C18 is expressed Consistently with our recent report using transient as three alternative tissue-specific variants, differing in expression experiments (Quelard et al., 2008), b-catenin- their N-terminal (Nter) noncollagenous domains T-cell factor-regulated transcription (CRT) and cyclin (Figure 1a), which are generated by two alternative D1 promoter activity were significantly decreased in promoters and mRNA splicing (Muragaki et al., 1995; HCT116 cells stably expressing V3Nter or SFRP-1, in Rehn et al., 1996). Variant 1 is a ubiquitous structural contrast to cells expressing V2Nter or empty vector basement membrane component (Saarela et al., 1998). (Supplementary Figures 1b and c). Similar effects on Variant 2 (V2) is a plasma protein produced mainly in the CRT were obtained with two different stable V3Nter liver (Musso et al., 2001). Variant 3 (V3) is expressed at cell batches (Supplementary Figure 1d). very low levels during Xenopus embryogenesis (Elamaa et al., 2002), in human fetal (Elamaa et al., 2003), normal adult and tumor liver tissues (Quelard et al.,2008),andis Paracrine inhibition of Wnt3a-induced b-catenin not detected in metastatic CRCs (Musso et al.,2001). stabilization in HCT116 cells Proteolytic processing of V3 releases V3Nter, an amino- V3Nter attenuated CRT in a dose–response assay to terminal glycoprotein containing a frizzled CRD, which soluble Wnt3a (Figure 2a). In contrast, V2Nter en- locates at the cell surface in cancer cells and matches the hanced CRT in this context (Figure 2a). Indeed, three-dimensional structures of frizzled 8 and SFRP-3 heparan sulfates attached to the 47-aa stretch common CRDs (Quelard et al.,2008). to the three C18 variants (Figure 1b) may locally These findings led us to explore the potential inhibitory increase Wnt concentration and thus CRT, as shown for effects of the frizzled module of C18 on tumor growth. We C18 (Quelard et al., 2008) and for other heparan sulfate show here that V3Nter and SFRP-1 attenuate baseline proteoglycans (Mikels and Nusse, 2006). Accordingly, and Wnt3a-induced b-catenin stabilization in HCT116 the slope of the Wnt3a dose–response curve for V2Nter- cells, reducing in vitro and in vivo tumor growth through HCT116 cells was steeper than that of the other cells, slowed cell cycle progression. Moreover, V3Nter inhibits including Vector-HCT116 cells. In contrast,

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