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

Abstract

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.

Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active β-catenin

“…Consistently with our recent report using transient expression experiments (Quelard et al, 2008), b-catenin-T-cell factor-regulated transcription (CRT) and cyclin D1 promoter activity were significantly decreased in HCT116 cells stably expressing V3Nter or SFRP-1, in contrast to cells expressing V2Nter or empty vector ( Supplementary Figures 1b and c). Similar effects on CRT were obtained with two different stable V3Nter cell batches (Supplementary Figure 1d).…”

“…As negative controls, we prepared cells expressing either the homologous C18 polypeptide lacking the frizzled module (V2Nter-HCT116) or empty pCDNA3.1 vector (Vector-HCT116, Figures 1a and b). In HCT116 cells, transient expression of V3Nter, SFRP-1 or SFRP-5 induces cell death (Suzuki et al, 2004;Quelard et al, 2008). Consistently, attempts to obtain clones by limiting dilution were unsuccessful, suggesting a negative selection pressure, as shown for SFRP-1 in breast cancer cells (Bafico et al, 2004).…”

“…In this context, we and others have shown that restoring expression of SFRP-1 or SFRP-5 inhibits Wnt/b-catenin stabilization and induces in vitro cell death in human CRC cells (Suzuki et al, 2004;Quelard et al, 2008). In addition to SFRPs, other endogenous molecules carrying frizzled CRDs bind to and inhibit Wnt signaling.…”

“…In addition to SFRPs, other endogenous molecules carrying frizzled CRDs bind to and inhibit Wnt signaling. We recently showed that a frizzled CRD derived from cell surface collagen XVIII (C18) functions in an SFRP-like fashion, binding to Wnt3a and inhibiting Wnt/b-catenin signaling in vitro (Quelard et al, 2008). C18 is expressed as three alternative tissue-specific variants, differing in their N-terminal (Nter) noncollagenous domains ( Figure 1a), which are generated by two alternative promoters and mRNA splicing (Muragaki et al, 1995;Rehn et al, 1996).…”

“…Variant 2 (V2) is a plasma protein produced mainly in the liver (Musso et al, 2001). Variant 3 (V3) is expressed at 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), and is not detected in metastatic CRCs (Musso et al, 2001). Proteolytic processing of V3 releases V3Nter, an aminoterminal glycoprotein containing a frizzled CRD, which locates at the cell surface in cancer cells and matches the three-dimensional structures of frizzled 8 and SFRP-3 CRDs (Quelard et al, 2008).…”