WNT/β-catenin signaling and hepatocellular carcinoma

Authors

  • Jack R. Wands,

    Corresponding author
    1. Liver Research Center, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI
    • Address reprint requests to: Jack R. Wands, M.D., Liver Research Center, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI 02905. E-mail: jack_wands_md@brown.edu.

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  • Miran Kim

    1. Liver Research Center, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI
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  • Potential conflict of interest: Nothing to report.

  • See Article on Page 576

Abbreviations
FZD

Frizzled

HCC

hepatocellular carcinoma

HSPG

heparin sulfate proteoglycans

IHS

immunohistochemical staining

LRP

lipoprotein receptor-related protein

PKC

protein kinase-C

TCF/LEF

T-cell factor/lymphoid enhancer-binding factor

Hepatocellular carcinoma (HCC) is the third leading cause of death by cancer worldwide.[1] There are approximately 1 million deaths per year on a global basis and there are limited therapeutic options.[2] The growing incidence of HCC has generated intense research to understand cellular, genetic, and molecular mechanisms in the hope of developing more effective therapies.

The pathogenesis of HCC involves a complex multistep process, which develops from a normal hepatocyte or precursor cell type to a transformed phenotype due to the accumulation of aberrant genetic and epigenetic changes and the activation of growth factor signaling pathways.[3-9] The extensive genetic heterogeneity of HCC is understandable based on its diverse etiology and there have been few common genetic abnormalities found between tumors, as described in numerous studies that attempt to define a general sequence of genetic events involved in transformation, as has been observed in colon cancer, for example.[10] However, gene expression and micro-RNA profiling have been useful in the identification of patient subgroups with different prognosis and clarification of potential driver genes important in hepatic oncogenesis.[9, 11-13] Because the genetic heterogeneity of HCC is quite complex, other investigations have focused on identifying common signaling pathways that may link these tumors with diverse etiologies, to common and better defined molecular mechanisms. In this regard, signaling pathways that are commonly up-regulated in >90% of HCCs irrespective of etiology include insulin/IGF1/IRS1/Raf/Raf/MAPK/Erk and insulin/IGF1/IRS1/PI3K/AKT and WNT/β-catenin signaling cascade; however, there are undoubtedly many others that are activated.[6, 14]

Increasing evidence suggests that activation of the WNT/β-catenin-mediated signaling cascade could play a key role in hepatic oncogenesis. This signaling networks involves the binding of one or more of 19 known extracellular soluble secreted WNT ligands to one or more of the 10 Frizzled (FZD) cell surface transmembrane receptors on tumor cells that promote activation of two well-described pathways called the canonical (involves β-catenin) and the noncanonical or c-Jun NH2-terminal kinase (JNK) and protein kinase-C (PKC) cascades that are principally active during embryogenesis but are important later during adult tissue homeostasis; however, such pathways become reactivated during the transformation of normal hepatocytes to the malignant phenotype. The level of β-catenin in the hepatocyte is low due to the activity of a destruction complex comprised of APC, axin, and GSK-3 which binds β-catenin, phosphorylates it, followed by degradation in the proteasome. As shown in Fig. 1, activation of the canonical WNT/β-catenin is initiated by the binding of the WNT extracellular ligands to the transmembrane FZD receptors and the low-density lipoprotein receptor-related protein (LRP) which subsequently dissolves the formation of the β-catenin destruction complex and leads to the accumulation of β-catenin in the cytoplasm. The β-catenin protein can then translocate to the nucleus where it serves as a transcription activator to form a transcriptional complex with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins followed by the activation of target genes that up-regulate cell-proliferation, migration, invasion, cell cycle progression, and metastasis (Fig. 1A). Therefore, constitutive activation of this pathway may be important to establish and maintain the hepatic malignant phenotype. This deregulated WNT/β-catenin signaling cascade has been observed in ∼95% of HCCs.[15] Furthermore, this pathway can also be activated by mutations and deletions in the β-catenin gene that renders the protein not degradable by the destruction complex, allowing it to accumulate in the cytoplasm, translocate to the nucleus, and activate downstream growth-related genes.[5] Therefore, overexpression of upstream of FZD receptors and WNT ligands drive this pathway towards activation as the principle mechanism contributing to the accumulation of β-catenin in the cytoplasm, where it can translocate to the nucleus and be detected by immunohistochemical staining (IHS). One of the most important ligands involved in this process is WNT3, which is often overexpressed in HCC and, following binding to its FZD7 partner, activates canonical signaling in hepatitis B virus (HBV) and HCV-related tumors.[17, 18] In this context, interrupting the interaction between WNT ligands and FZD receptors has been directly proposed as a means of inhibiting WNT/β-catenin signaling to potentially reduce tumor cell migration and invasion.[18] Recently, there is accumulating evidence to indicate that WNT ligands may be concentrated on the cell surface by binding to heparan sulfate proteoglycans (HSPG), as shown in Fig. 1A; subsequently, WNTs are released to interact with the FZD receptors to activate the β-catenin signaling cascade.

Figure 1.

Activation of WNT/β-catenin signaling cascade and modulation of WNT binding to the heparin sulfate containing proteoglycan GPC3 by the human monoclonal antibody HS20. (A) WNT binds to FZD and LRP receptors which activates the canonical β-catenin signaling cascade. GPC3 increases the binding of WNTs to the FZDs receptors by facilitating the formation of a complex. Activation of the subsequent downstream signal results in the accumulation of β-catenin in the cytoplasm where it translates to the nucleus and up-regulates growth-promoting genes by forming a transcriptional complex with LEF/TLF protein. (B) Binding of HS20 to the heparin sulfate side chains of GPC3 to "block" activation of WNT/β-catenin signaling in HCC cells.

The article by Gao et al.[19] in this issue has put forth a novel approach of how to interrupt the WNT3/β-catenin signaling cascade. This carefully performed study presents convincing evidence that one of the HSPGs of particular interest is glypican-3 (GPC3), which is expressed on HCC tumors but not normal human tissues or liver. In this regard, GPC3 may attract and concentrate WNT ligands to the HCC cell surface and subsequently activate the WNT/β-catenin pathway. Their hypothesis was to block the binding and accumulation WNT3a to the GPC3 molecules by using a human monoclonal antibody (HS20) that recognized the heparan sulfate side chains of the GPC3. The HS20 antibody could then disrupt the WNT3/β-catenin signaling cascade through inhibiting the interaction of GPC3 with WNT3a and reduced access of the ligand to the FZD receptors as shown in Fig. 1B. Gao et al. demonstrate that HS20 probably binds to the heparan sulfate side chains linked to the GPC3 core. The authors reveal that HS20 disrupts this interaction between WNT3a and GPC3 since it inhibits WNT3a-dependent HCC cell proliferation in vitro. More important, results are presented to demonstrate that HCC development and growth, in a xenograph nude mouse model, was inhibited by intravenous administration of HS20 without apparent toxicity to the animals.

This study raises numerous additional questions that will stimulate further research such as mapping of the exact binding site of HS20 to GPC3. Moreover, will this approach inhibit other growth signaling pathways like insulin/IGF1 which crosstalk with the WNT/β-catenin signaling cascade and appear important in hepatic oncogenesis?[6] Furthermore, tumor cell migration, invasion, proliferation, and metastasis are probably regulated by other WNT/FZD combinations and it is not clear whether HS20 will interfere with these activities as well. Nevertheless, this research presents a new idea of how to manipulate a molecular pathway known to be important in human HCC pathogenesis. There is evidence in the preclinical animal model study to suggest that it has antitumor effects on HCC progression. Since HS20 is an entirely human antibody, it should be further studied to evaluate its potential for treatment of patients with this devastating disease.

  • Jack R. Wands, M.D. and Miran Kim, Ph.D.

  • Liver Research Center Rhode Island Hospital and the Warren Alpert Medical School of Brown University Providence, RI

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