**6.1. Wnt/β-catenin signalling**

The Wnt/β-catenin signalling plays a pivotal role in a host of physiological and pathophysiological processes such as embryonic development, cell proliferation, regeneration, angiogenesis and cancer [48]. It is also an important player in maintaining liver health, but it is found to be dysregulated in HCC with mutation in β-catenin observed in about 40–70% of HCC cases, proving to be a potential important target of therapy.

At physiological levels β-catenin is regulated by a destruction complex consisting of adenomatous polyposis coli (APC)/Axin/glycogen synthase kinase 3b (GSK3β), and casein kinase 1 (CK1) which phosphorylates β-catenin at Ser33, Ser37, Thr41, and Ser45 residues located in exon 3. The phosphorylated β-catenin is polyubiquitinated by β-transducin repeat containing protein (β-TrCP) and degraded by the proteasome. However, wnt signalling is activated upon binding of the wnt to one of the frizzled (FZD) family members and to low- density lipoprotein receptor-related protein 5 (LRP5) or LRP6, resulting in the inhibition of β-catenin degradation. The accumulated cytoplasmic β-catenin translocates to the nucleus where it forms a complex with T-cell factor (TCF)/lymphoid, displacing the transcriptional inhibitor Groucho, and the β-catenin-TCF complex enhances transcription of target genes that are implicated in cancer development for example, c-Myc and cyclin D1.

Nuclear β-catenin accumulation has been found to be associated to tumour progression and poor prognosis. Cytoplasmic β-catenin accumulation has been reported in HCCs larger than 5 cm in diameter and with reduced disease-free survival. Dysregulation of the wnt/β-catenin signalling has also shown to regulate angiogenesis and metastasis [49]. Aberrant activation of wnt signalling has also resulted from deregulation of other components of the pathway e.g. up regulation of wnt genes (Wnt3, Wnt4 and Wnt5A) and FZD (FZD3, FZD6 and FZD7) in about 60–90% of HCCs with more than 5% occurring in peritumours, implying that their expression could be an early event in hepatocarcinogenesis [50].

Disruption of β-catenin and TCF association in the nucleus by two fungal-derived compounds, PKF115-584 and CGC049090, has shown dose-dependent cytotoxicity against HCC cells and 10 times reduced toxicity in normal hepatocytes [51]. The disruption reduced expression of wnt/β-catenin target genes (c-Myc, cyclin D1, survivin) and inhibited *in vivo* tumour growth [52]. For reasons yet to be delineated, the presence of EpCAM, hepatic stem cell marker and a direct target of the wnt/β-catenin pathway, sensitised HCC cells to these antagonists [53]. Together these results suggest that EpCAM expression may facilitate HCC prognosis by effective stratification of HCC patients responsive to wnt/β-catenin signalling antagonists.

in OS and TTP in HCC patients with c-MET protein expression between tivantinib and placebo. These results suggest the potential of c-met protein expression to select HCC patients who may benefit from tivantinib [45]. However, surprisingly, two large randomised double-blind placebo-controlled phase III trials i.e. METIV-HCC (NCT01755767) and JET-HCC (NCT02029157), have both failed to demonstrate improved OS in advanced HCC patients

Cabozantinib is also an oral inhibitor of c-MET, VEGFR and PDGFR. *In vitro* and *in vivo* studies have demonstrated its reduced invasive and migratory properties in HCC. A phase II randomised trial is on-going to investigate the efficacy to cabozantinib in solid tumours. A phase III, randomised, double-blind, controlled trial is underway to evaluate the efficacy of cabozantinib *versus* placebo as a second-line treatment for advanced HCC who have received

The Wnt/β-catenin signalling plays a pivotal role in a host of physiological and pathophysiological processes such as embryonic development, cell proliferation, regeneration, angiogenesis and cancer [48]. It is also an important player in maintaining liver health, but it is found to be dysregulated in HCC with mutation in β-catenin observed in about 40–70% of HCC cases,

At physiological levels β-catenin is regulated by a destruction complex consisting of adenomatous polyposis coli (APC)/Axin/glycogen synthase kinase 3b (GSK3β), and casein kinase 1 (CK1) which phosphorylates β-catenin at Ser33, Ser37, Thr41, and Ser45 residues located in exon 3. The phosphorylated β-catenin is polyubiquitinated by β-transducin repeat containing protein (β-TrCP) and degraded by the proteasome. However, wnt signalling is activated upon binding of the wnt to one of the frizzled (FZD) family members and to low- density lipoprotein receptor-related protein 5 (LRP5) or LRP6, resulting in the inhibition of β-catenin degradation. The accumulated cytoplasmic β-catenin translocates to the nucleus where it forms a complex with T-cell factor (TCF)/lymphoid, displacing the transcriptional inhibitor Groucho, and the β-catenin-TCF complex enhances transcription of target genes that are implicated in

Nuclear β-catenin accumulation has been found to be associated to tumour progression and poor prognosis. Cytoplasmic β-catenin accumulation has been reported in HCCs larger than 5 cm in diameter and with reduced disease-free survival. Dysregulation of the wnt/β-catenin signalling has also shown to regulate angiogenesis and metastasis [49]. Aberrant activation of wnt signalling has also resulted from deregulation of other components of the pathway e.g. up regulation of wnt genes (Wnt3, Wnt4 and Wnt5A) and FZD (FZD3, FZD6 and FZD7) in about 60–90% of HCCs with more than 5% occurring in peritumours, implying that their

with high c-met protein expression [46].

180 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

**6.1. Wnt/β-catenin signalling**

prior sorafenib (CELESTIAL, NCT01908426) [47].

**6. Other potential therapeutic targets in HCC**

proving to be a potential important target of therapy.

cancer development for example, c-Myc and cyclin D1.

expression could be an early event in hepatocarcinogenesis [50].

Recently, two FDA-approved drugs have been identified to antagonise wnt/β-catenin pathway by differing mechanisms. First, pyrvinium was identified in a chemical screen for small molecules. It binds to CK1 potentiating its activity and leading to stabilisation of the destruction complex resulting in degradation of cytoplasmic and nuclear levels of β-catenin [54]. Recently, Pimozide, an antipsychotic drug, has been shown to inhibit cell proliferation and apoptosis in HCC cell lines by reducing EpCAM and β-catenin [55]. The specific role of these inhibitors has yet to be completely elucidated.

Another class of compounds regulate the wnt/β-catenin pathway by inhibiting tankyrases (TNK1 and TNK2). TNKs destabilise Axin leading to β-catenin stabilisation. Thus, inhibition of TNKs prolongs half-life of Axin preventing β-catenin accumulation. These compounds include XAV939 and WXL-8 and also reduce tumourigenicity *in vivo* [56].

Another therapeutic strategy to regulate the wnt/β-catenin signalling is to block the interaction between wnt ligands and FZD receptors. This has been achieved with monoclonal antibodies or using recombinant soluble fragment of FZD (sFZD). A monoclonal antibody, OMP-18R5, developed using the extracellular domain of FZD7, binds to five FZD receptors and blocks wnt signalling. It inhibits *in vivo* tumour growth and acts synergistically with chemotherapeutic drugs including taxol, irinotecan and gemcitabine [57]. OMP-18RS, is the only potential compound targeting the wnt pathway to make it to clinical phase I trials (NCT01345201) for the treatment of solid tumours and myeloid malignancies, suggesting potential use for HCC treatment.

Sorafenib has also been proposed as a potential wnt modulator, decreasing β-catenin and also expression of liver-specific wnt targets (GLUL, LGR5, and TBX3) in several HCC cell lines accompanied by reduced tumour volume *in vivo* using HepG2 xenografts in nude mice [58].

Several studies have also evaluated the significance of combination therapy for targeting the wnt pathway. A small molecular target, FH535 inhibits proliferation of HCC cell lines by inhibiting recruitment of β-catenin coactivators and also suppresses peroxisome proliferatoractivated receptor (PPAR) signalling. Galuppo et al. [59] reported FH535 and sorafenib synergistically inhibited HCC cell line and liver cancer stem cells by targeting the RAS/RAF/ MAPK and WNT/β-catenin pathways. Western blot demonstrated cleaved increased poly (ADP-ribose) polymerase (PARP) and reduced cyclin D1 and c-Myc.

Identification of pharmacological inhibitors of the wnt/βcatenin pathway is still underway. In the complex network of wnt ligands, receptors and β-catenin, preclinical studies have yielded promising results but wnt inhibitors targeting HCC have not yet reached clinical trials.
