TI - V3Nter is a cryptic inhibitor of Wnt/beta-catenin signaling . AB - SFRP1 , SFRP2 and SFRP5 suppress beta-catenin-T-cell factor ( TCF ) -regulated transcription ( CRT ) from a TCF/LEF responsive reporter in the CRC cell line HCT116 [15] . We asked whether V3Nter and V3FL could inhibit Wnt/beta-catenin signaling in cell lines carrying activating beta-catenin mutations , HCT116 (beta-catenin DeltaS45) and HepG2 ( HCC , beta-catenin Delta25-140 ) and used SFRP1 and SFRP5 as controls . Ectopic expression of SFRP1 , SFRP5 or V3Nter suppressed CRT in a dose -dependent manner ( Figure 2G ) . By contrast , V2Nter (Figure 2G) , V2FL and V3FL (Figure S1E) , increased CRT in HCT116 , but not in HepG2 cells . The increase in CRT by V2Nter , V2FL and V3FL was also observed in other cell lines . In the well-characterized human HCC cell line Huh-7 ( wild-type beta-catenin , baseline Wnt/beta-catenin signaling [20] ) Wnt1 , V2FL and V3FL increased CRT by more than 10 folds and V2Nter by 2.9 folds ( Figure S1F ) . Similar results were obtained in the mhAT3FS315 mouse HCC cell line ( not shown ) . C18 is a heparan sulfate proteoglycan ( HSPG ) , which contains heparan sulfate attachment sites common to all variants [8] . Thus , endogenous V2C18 and V3C18 , as well as their full-length and their amino terminal constructs could be HSPGs . Consistently , immunoblotting of proteoglycan-enriched fractions from a human HCC with anti-FZC18 , anti-DUF-959 and anti-Tsp-1C18 antibodies showed a high molecular weight smear typical of HSPGs . Important mobility shift and epitope unmasking were seen after digestion with heparin lyases , but not with chondroitinase ABC ( Figure S1 , A and G ) . Transfection of V2FL and V3FL in mouse hepatoma cells and detection of the most N-terminal (DUF-959) and C-terminal ( endostatin ) epitopes , as well as FZC18 , confirmed the high molecular weight smear typical of HSPG for V2FL and V3FL (Figure S1H) . Indeed , V2FL was detected in cell -conditioned medium ( Figure S1H ) . By contrast , V3FL was detected at high levels in cell layers but not in the conditioned medium ( Figure S1H ) , confirming its predominantly cell -surface location ( Figure S1B ) . Therefore , V2FL-HSPG and V3FL-HSPG might locally increase Wnt concentration , thereby increasing CRT activity , as has been shown for other HSPGs [21] . In addition , V2Nter and V3Nter share a C-terminal 47 amino-acid stretch ( Figure 2A ) from the heparan sulfate attachment site of C18 [8] , probably explaining the slight increase in CRT by V2Nter ( Figure 2G and S1F ) . Transient overexpression of V3Nter resulted in a reduced protein content of total beta-catenin , non PHOSphorylated beta-catenin , c-myc and cyclin D1 in HCT116 cells (Figure 2H) . Consistently with data on CRT , V2FL and V3FL did not reduce the protein content of total and non PHOSphorylated beta-catenin , cyclin D1 or c-myc (Figure 2H) . In addition , consistently with decreased cyclin D1 protein expression , reporter gene assays using the cyclin D1 promoter [22] upstream of luciferase cDNA confirmed that SFRP1 , SFRP5 and V3Nter decreased cyclin D1 promoter activity in HCT116 and HepG2 cells (Figure S1I) . Remarkably , overexpression of beta-catenin increased cyclin D1 promoter activity by 2.4 and 4.8 folds in HCT116 and HepG2 cell lines , respectively . Taken together , these data show that V3Nter can inhibit beta-catenin signaling in cancer cells carrying activating beta-catenin mutations and that the biological activity of the frizzled CRD is cryptic within full-length cell surface C18-HSPG .