Abstract
About 20% hepatocellular carcinomas (HCCs) display wild-type β-catenin, enhanced Wnt signaling, hepatocyte dedifferentiation and bad outcome, suggesting a specific impact of Wnt signals on HCC stem/progenitor cells. To study Wnt-specific molecular pathways, cell fates and clinical outcome, we fine-tuned Wnt/β-catenin signaling in liver progenitor cells, using the prototypical Wnt ligand Wnt3a. Cell biology assays and transcriptomic profiling were performed in HepaRG hepatic progenitors exposed to Wnt3a after β-catenin knockdown or Wnt inhibition with FZD8_CRD. Gene expression network, molecular pathology and survival analyses were performed on HCCs and matching non-tumor livers from 70 patients by real-time PCR and tissue micro-array-based immunohistochemistry. Wnt3a reprogrammed liver progenitors to replicating fibrogenic myofibroblast-like cells displaying stem and invasive features. Invasion was inhibited by 30 nM FZD7 and FZD8 CRDs. Translation of these data to human HCCs revealed two tight gene networks associating cell surface Wnt signaling, stem/progenitor markers and mesenchymal commitment. Both networks were linked by Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1), that appeared de novo in aggressive HCCs expressing cytoplasmic β-catenin and stem cell markers. HAPLN1 was independently associated with bad overall and disease-free outcome. In vitro, HAPLN1 was expressed de novo in EPCAM−/NCAM+ mesoderm-committed progenitors, upon spontaneous epithelial-mesenchymal transition and de-differentiation of hepatocyte-like cells to liver progenitors. In these cells, HAPLN1 knockdown downregulated key markers of mesenchymal cells, such as Snail, LGR5, collagen IV and α-SMA. In conclusion, HAPLN1 reflects a signaling network leading to stemness, mesenchymal commitment and HCC progression.
Highlights
20% of hepatocellular carcinoma (HCC) show activation of Wnt/β-catenin and TGFβ pathways, carry wild-type β-catenin, lose the hepatocyte phenotype and overexpress Frizzled (FZD) receptors [1, 2]
Wnt signals lead to myofibroblastic differentiation of liver progenitor cells As Wnt3a binds frizzled (FZD) receptors at the low nanomolar range [3], HepaRG hepatic progenitor cells were incubated with Wnt3a-conditioned medium, which contains
We show here that Wnt-specific signals foster fibrogenic, myofibroblast-like populations with stem cell features and impair hepatocyte and biliary lineage commitment of liver progenitors
Summary
20% of HCCs show activation of Wnt/β-catenin and TGFβ pathways, carry wild-type β-catenin, lose the hepatocyte phenotype and overexpress Frizzled (FZD) receptors [1, 2]. Wnt/β-catenin pathway activation involves interaction of Wnt ligands with cell surface FZD receptors and LRP5/6 co-receptors, followed by disruption of the Adenomatous polyposis coli (APC)-axin platform, halting GSK3B-dependent phosphorylation of β-catenin at key residues in exon 3. In the absence of interaction of Wnt ligands with their cell surface receptors, phosphorylated β-catenin undergoes proteasomal degradation. It is widely accepted that Wnt pathway activation in HCCs may be driven by upregulation of TGFB and tyrosine-kinase receptor pathways [2, 4, 5], Wnt ligands, their cell surface frizzled receptors and/or epigenetic silencing of a family of endogenous Wnt inhibitors, i.e., the Secreted Frizzled-Related Proteins (SFRPs) [6]. SFRPs are soluble decoy receptors composed of a cysteine-rich frizzled ligand-binding domain (FZD_CRD), thereby quenching Wnt activity at the cell surface [7]. Increasing the ratio of SFRP-like Wnt inhibitors over Wnt ligands or receptors controls tumor growth [8,9,10,11]
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