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Cited by 217Wnt/β-catenin signaling in development and cancerCancer Cells and MetastasisSignaling Pathways in Disease

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Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells

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Yuzugullu, Haluk, Benhaj, Khemais, Ozturk, Nuri, Senturk, Serif, Celik, Emine, Toylu, Asli, Tasdemir, Nilgun, Yilmaz, Mustafa, Erdal, Esra, Akcali, Kamil Can, Atabey, Nese, Ozturk, Mehmet·2009·Molecular Cancer
DOI 10.1186/1476-4598-8-90PMC PMC2770486
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Abstract

Background: β-catenin mutations that constitutively activate the canonical Wnt signaling have been observed in a subset of hepatocellular carcinomas (HCCs). These mutations are associated with chromosomal stability, low histological grade, low tumor invasion and better patient survival. We hypothesized that canonical Wnt signaling is selectively activated in well-differentiated, but repressed in poorly differentiated HCCs. To this aim, we characterized differentiation status of HCC cell lines and compared their expression status of Wnt pathway genes, and explored their activity of canonical Wnt signaling. Results: We classified human HCC cell lines into "well-differentiated" and "poorly differentiated" subtypes, based on the expression of hepatocyte lineage, epithelial and mesenchymal markers. Poorly differentiated cell lines lost epithelial and hepatocyte lineage markers, and overexpressed mesenchymal markers. Also, they were highly motile and invasive. We compared the expression of 45 Wnt pathway genes between two subtypes. TCF1 and TCF4 factors, and LRP5 and LRP6 co-receptors were ubiquitously expressed. Likewise, six Frizzled receptors, and canonical Wnt3 ligand were expressed in both subtypes. In contrast, canonical ligand Wnt8b and noncanonical ligands Wnt4, Wnt5a, Wnt5b and Wnt7b were expressed selectively in well- and poorly differentiated cell lines, respectively. Canonical Wnt signaling activity, as tested by a TCF reporter assay was detected in 80% of well-differentiated, contrary to 14% of poorly differentiated cell lines. TCF activity generated by ectopic mutant β-catenin was weak in poorly differentiated SNU449 cell line, suggesting a repressive mechanism. We tested Wnt5a as a candidate antagonist. It strongly inhibited canonical Wnt signaling that is activated by mutant β-catenin in HCC cell lines. Conclusion: Differential expression of Wnt ligands in HCC cells is associated with selective activation of canonical Wnt signaling in well-differentiated, and its repression in poorly differentiated cell lines. One potential mechanism of repression involved Wnt5a, acting as an antagonist of canonical Wnt signaling. Our observations support the hypothesis that Wnt pathway is selectively activated or repressed depending on differentiation status of HCC cells. We propose that canonical and noncanonical Wnt pathways have complementary roles in HCC, where the canonical signaling contributes to tumor initiation, and noncanonical signaling to tumor progression.

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Figure 1 Expression analysis of a-fetoprotein (AFP), E-cadherin and five mesenchymal cell markers in HCC cell lines. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. GAPDH was used as a control for expression analyses shown here and in figures 4 to 7.
Figure 1 Expression analysis of a-fetoprotein (AFP), E-cadherin and five mesenchymal cell markers in HCC cell lines. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. GAPDH was used as a control for expression analyses shown here and in figures 4 to 7.CC BY · PubMed Central
Figure 2 Immunocytochemical analysis of mesenchymal marker vimentin protein in AFP+ and AFP- HCC cell lines. Cells grown on coverslips were subjected to immunoperoxidase assay using anti-vimentin antibody (brown), and counterstained with hematoxylin (blue).
Figure 2 Immunocytochemical analysis of mesenchymal marker vimentin protein in AFP+ and AFP- HCC cell lines. Cells grown on coverslips were subjected to immunoperoxidase assay using anti-vimentin antibody (brown), and counterstained with hematoxylin (blue).CC BY · PubMed Central
Figure 3 Expression of hepatocyte lineage markers HNF-4α and HNF-1α in HCC cell line correlate with low motility. (a)Selective expression of HNF-4α and HNF-1α in AFP+ HCC cell lines. Total RNAs were extracted from cells and used for RT-PCR analysis of HNF-4α and HNF-1α expression. GAPDH RT-PCR was used as a loading control. (b. c) Differential motility of AFP+ and AFP- HCC cell lines. Cells were cultured in six-well culture plates, and a single linear wound was made with a pipette tip in confluent monolayer cells. The distances between wound edges were measured at fixed points in each dish according to standardized template. After 24 hours migration, cell migration into the wound was visualized using phase contrast microscopy at ×20 magnification (b). The number of cells migrating beyond the wound edge was counted (c). Assays in six replicates, error bars; SD. SNU475 cells are larger cells giving rise to visual overestimation of migrating cell number in the picture shown in b.
Figure 3 Expression of hepatocyte lineage markers HNF-4α and HNF-1α in HCC cell line correlate with low motility. (a)Selective expression of HNF-4α and HNF-1α in AFP+ HCC cell lines. Total RNAs were extracted from cells and used for RT-PCR analysis of HNF-4α and HNF-1α expression. GAPDH RT-PCR was used as a loading control. (b. c) Differential motility of AFP+ and AFP- HCC cell lines. Cells were cultured in six-well culture plates, and a single linear wound was made with a pipette tip in confluent monolayer cells. The distances between wound edges were measured at fixed points in each dish according to standardized template. After 24 hours migration, cell migration into the wound was visualized using phase contrast microscopy at ×20 magnification (b). The number of cells migrating beyond the wound edge was counted (c). Assays in six replicates, error bars; SD. SNU475 cells are larger cells giving rise to visual overestimation of migrating cell number in the picture shown in b.CC BY · PubMed Central
Figure 4 Comparative analysis of TCF/LEF transcription factors in hepatocellular carcinoma cell lines. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay of four members of TCF/LEF family. See Figure 1 for GAPDH loading control.
Figure 4 Comparative analysis of TCF/LEF transcription factors in hepatocellular carcinoma cell lines. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay of four members of TCF/LEF family. See Figure 1 for GAPDH loading control.CC BY · PubMed Central
Figure 5 Comparative analysis of Frizzled receptors and LRP co-receptors in hepatocellular carcinoma cell lines. Frizzled receptors involved in canonical and noncanonical Wnt signaling were tested for expression by RT-PCR assay (Top). The expression of LRP co-receptors was analyzed similarly (bottom). Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. See Figure 1 for GAPDH loading control.
Figure 5 Comparative analysis of Frizzled receptors and LRP co-receptors in hepatocellular carcinoma cell lines. Frizzled receptors involved in canonical and noncanonical Wnt signaling were tested for expression by RT-PCR assay (Top). The expression of LRP co-receptors was analyzed similarly (bottom). Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. See Figure 1 for GAPDH loading control.CC BY · PubMed Central
Figure 6 Comparative analysis of canonical Wnt ligands in hepatocellular carcinoma cell lines. Canonical Wnt ligands were tested for expression by RT-PCR assay. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. See Figure 1 for GAPDH loading control.
Figure 6 Comparative analysis of canonical Wnt ligands in hepatocellular carcinoma cell lines. Canonical Wnt ligands were tested for expression by RT-PCR assay. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. See Figure 1 for GAPDH loading control.CC BY · PubMed Central
Figure 7 Comparative analysis of noncanonical (top) and unclassified (bottom) Wnt ligands in hepatocellular carcinoma cell lines. Noncanonical and unclassified Wnt ligands were tested for expression by RT-PCR assay. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. See Figure 1 for GAPDH loading control.
Figure 7 Comparative analysis of noncanonical (top) and unclassified (bottom) Wnt ligands in hepatocellular carcinoma cell lines. Noncanonical and unclassified Wnt ligands were tested for expression by RT-PCR assay. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. See Figure 1 for GAPDH loading control.CC BY · PubMed Central
Figure 8 Frequent constitutive activation of canonical Wnt signaling in well-differentiated, but not in poorly differentiated hepatocellular carcinoma cell lines. (a) Comparative analysis of the canonical Wnt signaling in hepatoma cell lines with known mutations of β-catenin or Axin-1 genes. TCF reporter assay shows that well-differentiated HepG2 cells display high signaling activity. In contrast, canonical Wnt signaling is attenuated in poorly differentiated SNU398, and undetectable in poorly differentiated SNU475 cell line. Assays in triplicate, error bars; SD. (b) Comparative analysis of the canonical Wnt signaling in HCC cell lines with wild-type β-catenin and Axin-1 genes. Huh7 and Hep3B cell lines (both well-differentiated) display weak but significantly increased TCF reporter activity. Other cell lines (all poorly differentiated, except Hep40) display no detectable TCF reporter activity. TCF activity denotes the ratio of signals detected with pGL3-OT (OT) and pGL3-OF (OF) plasmids, respectively. Assays in triplicate, error bars; S. D. Cells were transfected with the reporter gene pGL3-OT (OT) harboring LEF-1/TCF binding sites for β-catenin and the corresponding pGL3-OF (OF) without these sites.
Figure 8 Frequent constitutive activation of canonical Wnt signaling in well-differentiated, but not in poorly differentiated hepatocellular carcinoma cell lines. (a) Comparative analysis of the canonical Wnt signaling in hepatoma cell lines with known mutations of β-catenin or Axin-1 genes. TCF reporter assay shows that well-differentiated HepG2 cells display high signaling activity. In contrast, canonical Wnt signaling is attenuated in poorly differentiated SNU398, and undetectable in poorly differentiated SNU475 cell line. Assays in triplicate, error bars; SD. (b) Comparative analysis of the canonical Wnt signaling in HCC cell lines with wild-type β-catenin and Axin-1 genes. Huh7 and Hep3B cell lines (both well-differentiated) display weak but significantly increased TCF reporter activity. Other cell lines (all poorly differentiated, except Hep40) display no detectable TCF reporter activity. TCF activity denotes the ratio of signals detected with pGL3-OT (OT) and pGL3-OF (OF) plasmids, respectively. Assays in triplicate, error bars; S. D. Cells were transfected with the reporter gene pGL3-OT (OT) harboring LEF-1/TCF binding sites for β-catenin and the corresponding pGL3-OF (OF) without these sites.CC BY · PubMed Central
Figure 9 Expression analysis of genes inhibiting canonical Wnt signaling downstream to β-catenin in HCC cells. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. GAPDH RT-PCR was used as a loading control.
Figure 9 Expression analysis of genes inhibiting canonical Wnt signaling downstream to β-catenin in HCC cells. Total RNAs were extracted from cell lines and used to detect gene expression by RT-PCR assay. GAPDH RT-PCR was used as a loading control.CC BY · PubMed Central
Figure 10 Ectopic expression of mutant β-catenin induces high canonical Wnt activity in well-differentiated, but not in poorly differentiated hepatocellular carcinoma cells. (a) Well-differentiated Huh7, and poorly differentiated SNU449 and SNU182 cell lines have been co-transfected with either pCI-neo-mutant β-catenin (S33Y) plasmid (S33Y-β-catenin +) or empty pCI-neo plasmid (S33Y-β-catenin -), and cellular β-catenin levels at post-transfection 48 h were tested by immunoblotting. Calnexin was used as a loading control. (b) Cell lines were treated as described, then pCI-neo-mutant β-catenin (S33Y)-transfected cells were subjected to TCF reporter assay. TCF activity denotes the ratio of signals detected with pGL3-OT (OT) and pGL3-OF (OF) plasmids, respectively. Assays in triplicate, error bars; SD. Co-transfections included pGL-OT or pGL-OF, in addition to pCI-neo plasmids in both (a) and (b).
Figure 10 Ectopic expression of mutant β-catenin induces high canonical Wnt activity in well-differentiated, but not in poorly differentiated hepatocellular carcinoma cells. (a) Well-differentiated Huh7, and poorly differentiated SNU449 and SNU182 cell lines have been co-transfected with either pCI-neo-mutant β-catenin (S33Y) plasmid (S33Y-β-catenin +) or empty pCI-neo plasmid (S33Y-β-catenin -), and cellular β-catenin levels at post-transfection 48 h were tested by immunoblotting. Calnexin was used as a loading control. (b) Cell lines were treated as described, then pCI-neo-mutant β-catenin (S33Y)-transfected cells were subjected to TCF reporter assay. TCF activity denotes the ratio of signals detected with pGL3-OT (OT) and pGL3-OF (OF) plasmids, respectively. Assays in triplicate, error bars; SD. Co-transfections included pGL-OT or pGL-OF, in addition to pCI-neo plasmids in both (a) and (b).CC BY · PubMed Central
Figure 11 Minimal TCF reporter activity and lack of nuclear accumulation of mutant β-catenin in poorly differentiated SNU449.cl8 cells. SNU449 cells were stably transfected with Tet-responsive ΔN-β-catenin expression vector to obtain SNU448.cl8 cells. (a) Induced expression of N-terminally truncated ΔN-β-catenin protein in the Tet-Off conditions, as tested by western blot assay. Total cell lysates were extracted from cells and subjected to western blot assay using anti-β-catenin antibody. (b) TCF activity is only weakly induced in Tet-off conditions, as tested by duplicate experiments. (c) SNU449.cl8 cells at Tet-On state express wild-type endogenous β-catenin protein principally located at cell membrane. Under Tet-Off conditions the staining pattern remains almost identical despite ΔN-β-catenin expression. Note lack of nuclear accumulation. SW480 cells used as positive control display strong nuclear β-catenin staining. Cells were grown on coverslips, subjected to indirect immunofluorescence assay using anti-β-catenin antibody (red), counterstained with DAPI (blue) and examined by confocal microscopy.
Figure 11 Minimal TCF reporter activity and lack of nuclear accumulation of mutant β-catenin in poorly differentiated SNU449.cl8 cells. SNU449 cells were stably transfected with Tet-responsive ΔN-β-catenin expression vector to obtain SNU448.cl8 cells. (a) Induced expression of N-terminally truncated ΔN-β-catenin protein in the Tet-Off conditions, as tested by western blot assay. Total cell lysates were extracted from cells and subjected to western blot assay using anti-β-catenin antibody. (b) TCF activity is only weakly induced in Tet-off conditions, as tested by duplicate experiments. (c) SNU449.cl8 cells at Tet-On state express wild-type endogenous β-catenin protein principally located at cell membrane. Under Tet-Off conditions the staining pattern remains almost identical despite ΔN-β-catenin expression. Note lack of nuclear accumulation. SW480 cells used as positive control display strong nuclear β-catenin staining. Cells were grown on coverslips, subjected to indirect immunofluorescence assay using anti-β-catenin antibody (red), counterstained with DAPI (blue) and examined by confocal microscopy.CC BY · PubMed Central
Figure 12 Wnt5a inhibits canonical Wnt signaling activity in Huh7 and HepG2 cells. (a) Huh7 cells were co-transfected with either pCI-neo-mutant β-catenin (S33Y) plasmid (S33Y-β-catenin +) along with pShuttle-IRES-WNT5a or empty pShuttle-IRES vector. 48 hours post transfection; cells were subjected to TCF reporter assay. TCF activity denotes the ratio of signals detected with pGL3-OT (OT) and pGL3-OF (OF) plasmids, respectively. Assays in triplicate, error bars; SD. Co-transfections included pGL-OT or pGL-OF, in addition to pCI-neo-S33Y-β-catenin and pIRES plasmids. (b) HepG2 experiments were performed under similar conditions, except that pCI-neo-mutant β-catenin (S33Y) plasmid was omitted.
Figure 12 Wnt5a inhibits canonical Wnt signaling activity in Huh7 and HepG2 cells. (a) Huh7 cells were co-transfected with either pCI-neo-mutant β-catenin (S33Y) plasmid (S33Y-β-catenin +) along with pShuttle-IRES-WNT5a or empty pShuttle-IRES vector. 48 hours post transfection; cells were subjected to TCF reporter assay. TCF activity denotes the ratio of signals detected with pGL3-OT (OT) and pGL3-OF (OF) plasmids, respectively. Assays in triplicate, error bars; SD. Co-transfections included pGL-OT or pGL-OF, in addition to pCI-neo-S33Y-β-catenin and pIRES plasmids. (b) HepG2 experiments were performed under similar conditions, except that pCI-neo-mutant β-catenin (S33Y) plasmid was omitted.CC BY · PubMed Central

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Authors
Yuzugullu, H., Benhaj, K., Ozturk, N., Senturk, S., Celik, E., Toylu, A., Tasdemir, N., Yilmaz, M., Erdal, E., Akcali, K. C., Atabey, N., & Ozturk, M.
Affiliations
  • Bilkent University (TR)
  • Dokuz Eylül University (TR)
ORCID
Venue
Molecular Cancer
Publisher
Springer Science and Business Media LLC
Published
2009-10-22
Volume
8
Issue
1
Pages
90-90
ISSN
1476-4598 (ISSN-L)
DOI
10.1186/1476-4598-8-90
PMC
PMC2770486
OpenAlex
W2152347253
Cited by
215
References
76
Citations by year
2026: 5 · 2025: 12 · 2024: 9 · 2023: 16 · 2022: 14 · 2021: 21 · 2020: 19 · 2019: 9 · 2018: 13 · 2017: 9 · 2016: 17 · 2015: 21 · 2014: 13 · 2013: 9 · 2012: 13
FWCI
3.76
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Molecular Cancer (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Bilkent University Institutional Repository (Bilkent University) (submittedVersion)
  • Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion)
  • HAL (submittedVersion, other-oa)
  • Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • UNC Libraries
Funders
  • Kræftens Bekæmpelse
  • Türkiye Bilimsel ve Teknolojik Araştırma Kurumu
  • Türkiye Bilimler Akademisi
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Signaling Pathways in Disease
Keywords
hepatocellular carcinoma, Wnt/β-catenin signaling, non-canonical Wnt signaling, Wnt5a, CTNNB1 mutation, tumor differentiation, TCF transcription factors, Frizzled receptors, tumor invasion, tumor progression
MeSH terms
Wnt-5a Protein, Cell Differentiation, Cell Nucleus, Carcinoma, Hepatocellular, Humans, Immunohistochemistry, Ligands, Liver Neoplasms, Mutation, Neoplasm Proteins, Proto-Oncogene Proteins, Receptors, LDL, Biomarkers, Tumor, Signal Transduction, Gene Expression Regulation, Neoplastic, Genes, Reporter, Autocrine Communication, Cell Line, Tumor, Wnt Proteins, Frizzled Receptors, TCF Transcription Factors

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