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

Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, 06800, Ankara, Turkey.
Molecular Cancer (Impact Factor: 4.26). 10/2009; 8(1):90. DOI: 10.1186/1476-4598-8-90
Source: PubMed


beta-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.
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 beta-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 beta-catenin in HCC cell lines.
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.

Download full-text


Available from: Kamil Can Akcali,
  • Source
    • "Certain types of Fzs are upregulated in HCC. Fz3, 6, and 7 are upregulated in the tumorous tissues of HCC (15) and Fz2 is expressed in Hep3B (16). In the present study, quantitative PCR demonstrated that Fz2 was expressed to a greater degree in the HCC and HB cell lines than in the adult liver. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In the present study, Frizzled-2 (Fz2), a receptor of the Wnt ligand, was investigated as a potential target of molecular therapy for hepatocellular carcinoma (HCC). Quantitative polymerase chain reaction (PCR) was performed to determine the expression levels of Fz2. A surgical specimen of HCC was immunostained with an Fz2 antibody. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed on HCC cell lines, including HLF and Hep3B, 72 h after the transfection of the short hairpin (sh)RNA of Fz2 (shRNA-Fz2). RNA was isolated from the Hep3B and HLF cells 48 h after transfection and subjected to quantitative PCR. All cell lines had elevated levels of Fz2 compared with those in an adult liver. The highest and lowest expression levels of Fz2 were 246.9±15.7 in the HLF cells and 5.8±1.4 in the Hep3B cells, respectively. Fz2 was expressed in the tumorous HCC tissue, but not in the surrounding non-tumorous tissue. Cell proliferation was suppressed to 28.6±6.4% in the HLF cells and to 29.8±4.3% in the Hep3B cells at 100 ng shRNA-Fz2 per well. Levels of cyclin D1 expression decreased to 65.2±5.9% in the HLF cells and to 60.8±14.6% in the Hep3B cells at 2.5 μg per well. In conclusion, Fz2 was upregulated in the HCC cells. shRNA-Fz2 suppressed the proliferation of the Hep3B and HLF cells, decreasing Fz2 expression. As it was not expressed in the surrounding non-tumorous tissue, Fz2 may be an ideal molecular therapeutic target for HCC.
    Oncology letters 10/2014; 8(4):1519-1522. DOI:10.3892/ol.2014.2408 · 1.55 Impact Factor
  • Source
    • "Additionally, crosstalk between canonical and noncanonical Wnt signaling has been shown previously. It was found that Wnt/Ca2+ signaling could activate the TAK1-NLK MAPK pathway, which might reduce the transcription of TCF/LEF 1 without influencing the accumulation of β-catenin, thus, inhibiting canonical Wnt signaling [29], [30]. However, some recent studies suggested that noncanonical Wnt signaling also could stimulate the canonical Wnt pathway and the effect of noncanonical Wnt ligands on the activation of canonical Wnt signaling might be related to the kind of FZD receptors on the target cells [31], [32]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells is critical for reepithelization and recovery in acute respiratory distress syndrome (ARDS), and Wnt signaling was considered to be the underlying mechanisms. In our previous study, we found that canonical Wnt pathway promoted the differentiation of MSCs into AT II cells, however the role of the noncanonical Wnt pathway in this process is unclear. It was disclosed in this study that noncanonical Wnt signaling in mouse bone marrow-derived MSCs (mMSCs) was activated during the differentiation of mMSCs into AT II cells in a modified co-culture system with murine lung epithelial-12 cells and small airway growth media. The levels of surfactant protein (SP) C, SPB and SPD, the specific markers of AT II cells, increased in mMSCs when Wnt5a was added to activate noncanonical Wnt signaling, while pretreatment with JNK or PKC inhibitors reversed the promotion of Wnt5a. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. We found that the Wnt5a supplement promoted the vertical and horizontal migration of mMSCs, ameliorated the cell death and the reduction of Bcl-2/Bax induced by H2O2. The effect of Wnt5a on the migration of mMSCs and their survival after H2O2 exposure were partially inhibited with PKC or JNK blockers. In conclusion, Wnt5a through Wnt/JNK signaling alone or both Wnt/JNK and Wnt/PKC signaling promoted the differentiation of mMSCs into AT II cells and the migration of mMSCs; through Wnt/PKC signaling, Wnt5a increased the survival of mMSCs after H2O2 exposure in vitro.
    PLoS ONE 03/2014; 9(3):e90229. DOI:10.1371/journal.pone.0090229 · 3.23 Impact Factor
  • Source
    • "In addition to the high prevalence of CTNNB1 mutations, genetic alterations of other components, such AXIN1, AXIN2, and APC genes that encode proteins containing the destruction box, also occur at a lower frequency. Wnt5a, which is up-regulated in poorly differentiated and highly motile mesenchymal-like HCC cells, has been suggested to play a role in tumor progression by inducing epithelial mesenchymal transition [28]. In line with this, Wnt5a up-regulation was significantly shown to enhance migration, proliferation, and invasiveness in pancreatic cancer cells in vitro[29]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: To gain biological insights into lung metastases from hepatocellular carcinoma (HCC), we compared the whole-genome sequencing profiles of primary HCC and paired lung metastases. We used whole-genome sequencing at 33X-43X coverage to profile somatic mutations in primary HCC (HBV+) and metachronous lung metastases (> 2 years interval). In total, 5,027-13,961 and 5,275-12,624 somatic single-nucleotide variants (SNVs) were detected in primary HCC and lung metastases, respectively. Generally, 38.88-78.49% of SNVs detected in metastases were present in primary tumors. We identified 65-221 structural variations (SVs) in primary tumors and 60-232 SVs in metastases. Comparison of these SVs shows very similar and largely overlapped mutated segments between primary and metastatic tumors. Copy number alterations between primary and metastatic pairs were also found to be closely related. Together, these preservations in genomic profiles from liver primary tumors to metachronous lung metastases indicate that the genomic features during tumorigenesis may be retained during metastasis. We found very similar genomic alterations between primary and metastatic tumors, with a few mutations found specifically in lung metastases, which may explain the clinical observation that both primary and metastatic tumors are usually sensitive or resistant to the same systemic treatments.
    BMC Medical Genomics 01/2014; 7(1):2. DOI:10.1186/1755-8794-7-2 · 2.87 Impact Factor
Show more

Similar Publications