Volume 127, Issue 4, Pages 1110-1122 (October 2004) Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma  Philippe Merle, Suzanne de la Monte, Miran Kim, Marc Herrmann, Shinji Tanaka, Annette Von Dem Bussche, Michael C. Kew, Christian Trepo, Jack R. Wands  Gastroenterology  Volume 127, Issue 4, Pages 1110-1122 (October 2004) DOI: 10.1053/j.gastro.2004.07.009 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Real-time PCR assay for frizzled-7 gene. ♦, Standard curves for (A) 18SrRNA and (B) FZD7. ♢, Mean values of unknown HCC tumor samples performed in duplicate. The standard curves show a 5 order of magnitude linear dynamic range. (C) The FZD7 mRNA steady-state levels in HCC cell lines expressed as relative abundance of FZD7 mRNA. The value represents the mean ± SD from 3 separate experiments. Gastroenterology 2004 127, 1110-1122DOI: (10.1053/j.gastro.2004.07.009) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Measurement of HCC cell motility. (A) The percent of nonmigrated and total migrated (migrated-adherent, and migrated nonadherent) cells were evaluated by a luminescent-based assay. The values (percent of cells) are expressed as mean ± SD from 6 separate measurements. t test, *P < 0.01, **P < 0.001 as compared with values obtained with HepG2 cells. (B) Correlation between FZD7 mRNA steady-state levels and the percent of total migrated cells (both migrated-adherent and migrated-nonadherent). Z test of correlation, P = 0.02. Gastroenterology 2004 127, 1110-1122DOI: (10.1053/j.gastro.2004.07.009) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Western blot analysis of total cellular β-catenin and Tcf reporter assay. (A) There is no correlation of proliferating cell nuclear antigen with FZD7 mRNA. (B) However, high levels of FZD7 gene expression are associated with nuclear accumulation of β-catenin; lower FZD7 expression levels were associated with both nuclear and cytoplasmic accumulation in HepG2 and Hep3B cells. (C) Measurement of Tcf transcriptional activity. These HCC cell lines were cotransfected with either TOPflash (■) or FOPflash (□) and β-galactosidase-expressing plasmid. Note the high level of Tcf-mediated transcription activity in all 3 cell lines. There was a general correlation between FZD7 levels and Tcf transcriptional activity. Gastroenterology 2004 127, 1110-1122DOI: (10.1053/j.gastro.2004.07.009) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Construction and expression of the FZD7 mutant proteins. (A) FZD7-FL, wild-type full-length FZD7 protein; FZD7-▵C, FZD7 protein truncated in the intracellular domain; FZD7-▵T▵C, secreted FZD7 protein truncated in both the transmembrane and intracellular domains; CRD, cystein-rich domain. The Wnt-ligand interacts with the CRD of FZD7 and with the epidermal growth factor repeat (EGFR) regions 1 and 2 of LRP5/6 as represented by grey boxes. (B) Expression in a heterogenous population of Huh7 cells stably transfected with pcDNA3/FZD7-▵T▵C or pcDNA3/empty vector as a control. Expression was assessed by quantitative real-time RT-PCR with either a first set of primers located in the extracellular domain, or a second set located in the intracellular domain of FZD7 receptor. Expression levels are normalized to the values obtained with pcDNA3. (C) Expression of FZD7-▵T▵C as assessed by Western blot analysis with the monoclonal anti-M2 flag tag antibody in a heterogenous population of Huh7 cells stably transfected with FZD7-▵T▵C or pcDNA3/empty vector. (D) Expression of various forms of FZD7 in Huh7 cells. Cells were stably transfected with pLenti6/V5-GFP, clone-4 (GFP-C4) served as a negative control; Huh7 cells were stably transfected with pLenti6/V5-FZD7-▵C, clones 1 to 8 (▵C-C1–C8); Huh7 cells transiently transduced with pLenti6/V5-FZD7-▵C (▵C-Tr). Huh7 cells stably transduced with pLenti6/V5-FZD7-FL, clone-1 (FL-C1) served as a positive control for RT-PCR assessment with sets of primers targeting either the extracellular domain or the intracellular domain of FZD7 receptor. Expression levels are normalized to the values observed with pLenti6/V5-GFP-C4. (E) Expression of FZD7-▵C as assessed by Western blot analysis with rabbit polyclonal anti-human FZD7 antibody with or without immunoprecipitation with goat anti-human V5 antibodies coupled to agarose beads. Experiments were performed in Huh7 cells transduced with pLenti6/V5-FZD7-▵C and pLenti6/V5-GFP-C4 as a negative control. Gastroenterology 2004 127, 1110-1122DOI: (10.1053/j.gastro.2004.07.009) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 β-catenin protein levels in HCC cells after ectopic expression of a dominant-negative FZD7-▵T▵C mutant receptor-expressing construct. (A) Levels of β-catenin protein in Huh7, Focus, Hep3B, and HepG2 HCC cell lines transfected with the secreted form of the FZD7-▵T▵C receptor. (B) Level of β-catenin protein in Huh7 cells after transduction with the transmembrane FZD7-▵C mutant receptor cloned into pLenti6/V5. (C) Motility of Huh7 cells toward soluble collagen-I under conditions of transient transfection with pLenti6/V5-FZD7-▵T▵C, pLenti6/V5-FZD7-▵C, or pLenti6/V5-GFP as a control. (D) Motility measurements of representative clones under conditions of stable integration and expression of: C1 = FZD7-▵T▵C, C5 = FZD7-▵C, and C6 = FZD7-▵C. The percent of nonmigrated and total migrated (migrated-adherent and migrated nonadherent) cells were evaluated by a luminescent-based assay after 3 hours at 37°C. The values (percent of cells), expressed as mean ± SD, are from 6 separate measurements. t test, *P < 0.05, **P < 0.01 when compared with GFP-negative control. Gastroenterology 2004 127, 1110-1122DOI: (10.1053/j.gastro.2004.07.009) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Schematic representation of β-catenin constructs and cell motility assay. (A) Approximate location of functional domains: black, protein instability; right-leaning hatch, N-terminal transactivation domain; left-leaning hatch, C-terminal transactivation domain; grey, armadillo repeats, protein-protein interaction. (B) Motility measurements of FZD7-▵C-C6 and GFP-C4 blasticidin-selected clonal Huh7 cell populations initially transduced with pLenti6/V5-D-TOPO lentiviral vectors expressing either the FZD7-▵C negative-dominant mutant or GFP as control, and cotransduced once again with a pLenti6/V5-D-TOPO lentiviral vector expressing a biologically active ▵N/▵C β-catenin mutant or GFP to keep constant the total amounts of plasmid DNA. The percent of nonmigrated and total migrated (migrated-adherent and migrated nonadherent) cells were evaluated by a luminescent-based assay after 3 hours at 37°C. The values (percent of cells) expressed as mean ± SD were derived from 6 separate measurements. Note the restoration of cell motility by the ▵N/▵C mutant β-catenin in the setting of inhibition of the signal transduction of the receptor level with stable expression of the dominant-negative FZD7 ▵C-C6 receptor mutant protein. Gastroenterology 2004 127, 1110-1122DOI: (10.1053/j.gastro.2004.07.009) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 (A) Quantitative real-time RT-PCR assessment of FZD7 mRNA levels in human HCC tumors (T) and the corresponding peritumorous liver parenchyma (PT), derived from Taiwan and South Africa. Nonparametric paired test, *P < 0.0001; paired t test, P = 0.0187, when comparing levels in tumor with peritumoral areas. (B) Western blot analysis of FZD7 receptor protein expression in HCC tumors (T) and the corresponding peritumorous areas (PT), as well as in Huh7 and HepG2 human hepatoma cell lines. (C) Western blot analysis of β-catenin protein accumulation in cytosolic (C) or nuclear (N) enriched fractions from 2 HCC tumors and their corresponding peritumoral areas compared with 2 normal liver samples. Both peritumoral area and tumor overexpress FZD7 mRNA as shown by values listed below the Western blots and expressed as relative abundance of FZD7 mRNA. Each tumor and peritumor region had a wild-type β-catenin exon-3 as assessed by PCR and sequencing. Gastroenterology 2004 127, 1110-1122DOI: (10.1053/j.gastro.2004.07.009) Copyright © 2004 American Gastroenterological Association Terms and Conditions