**5. c-MET inhibitors**

suppressor p53 [31]. HCV infection has led to anti-apoptotic effect also following activation

Selumetinib is an oral MEK inhibitor. In a small phase II study (NCT00604721) involving 19 HCC patients with advanced HCC, selumetinib was given at a dose of 100 mg twice per day but the study was terminated at interim analysis because there was no response and the TTP was only 8 weeks. However western blot of biopsy samples taken pre and post treatment showed phosphorylation of MEK1/2, and ERK1/2, suggesting failure of selumetinib was not due to lack of target inhibition [33]. A recent phase I study (NCT01029418) looked into the safety, maximum tolerated dose (MTD), and tolerability of selumetinib in combination with sorafenib in 27 Asian patients with advanced HCC. The MTD of selumetinib was at 75 mg daily with sorafenib at 400 mg twice daily. Common treatment-related adverse events included diarrhoea, rash, and hypertension, fatigue, anorexia and hand-foot and mouth disease. Seven patients had a PR and stable disease for more than 6 months. The OS was 14.4 months. Due to the acceptable adverse events, this combination of selumetinib and sorafenib deserves further

Another MEK inhibitor, refametinib, was evaluated in a phase II study (NCT01204177) in combination with sorafenib in 95 patients with unresectable HCC. Patients received twicedaily refametinib at 50 mg plus twice-daily sorafenib at 200 mg (morning)/400 mg (evening), with dose escalation to sorafenib 400 mg twice daily after cycle 2. The TTP was 122 days and OS was 290 days. Interestingly, the best responders to the combination treatment were those harbouring Ras mutation. A recently completed proof of concept phase II trial (NCT01915602) of refametinib in combination with sorafenib in Ras mutant HCC has recently been completed with results expected soon. Given that Ras mutations are only observed in 3–5% of HCC patients, this study raises questions about feasibility and costs of screening large cohort of

patients to identify a small sub-group with particular mutations.

**4. Mammalian target of rapamycin (mTOR) signalling pathway**

This pathway is a critical regulator of numerous physiological processes and also plays a pivotal role in cell proliferation and metastasis of transformed human cancers including HCC. It is upregulated in around 40% of HCC and has been associated to poor prognosis and early recurrence independent of underlying liver aetiology [35]. Two mTOR inhibitors have been

Preclinical studies have shown everolimus (taken orally) to dose-dependently inhibit tumour growth in patient-derived xenograft models of advanced HCC [36]. In a phase I/II study (NCT00516165) in advanced HCC patients, Zhu et al. [37] reported daily dose of 10 mg per day to be well tolerated in 28 patients producing a medium PFS and OS of 3.8 months and 8.4 months respectively. The subsequent phase II study involving 28 patients with prior systemic therapy with daily dose of 10 mg could not be completed as two patients remained progression free for 24 weeks. Although everolimus was well tolerated this study had some limitations including small sample size and lack of randomised control. The efficacy of

by Ras/Raf/Mek/Erk signalling [32].

178 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

evaluation [34].

studied in clinical trials.

c-Met is a proto-oncogene that encodes the receptor, MET, for the ligand of hepatocyte growth factor (HGF). MET is a tyrosine kinase receptor regulating metastatic progression. Binding of MET to HGF activates the RAS-MAPK and PI3K-AKT signalling pathways leading to tumour development and metastasis. In HCC, c-MET protein is overexpressed in 70% of HCC and has been associated to poor prognosis [43].

Foretinib was the first c-MET inhibitor of broad spectrum, including c-Met and VEGFR, to be tested in clinical trials. In a phase I/II study (NCT00920192) involving patients with advanced HCC, the median TTP was 4.2 months and the OS was 15.7 months. Its toxicity profile was also acceptable with the most adverse events including hypertension and anorexia. Baseline plasma levels of Interleukin 6 (IL6) and Interleukin 8 (IL8) were identified as independent predictors of OS by multivariate analysis. A larger randomised study is needed to warrant the effects of foretinib [44].

Tivantinib is a selective oral inhibitor of c-MET. In a randomised, placebo controlled phase II study (NCT00988741), advanced HCC patients were administered 240 mg daily resulting in a small improvement in TTP (1.4 months *versus* 1.6 months) compared to the placebo group. Additionally, HCC tumours expressing high levels of c-MET protein, as judged by immunohistochemical analysis, demonstrated an improved OS (7.2 months *versus* 3.8 months) and longer TTP (2.7 months *versus* 1.4 months) compared to placebo. There was no difference 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 with high c-met protein expression [46].

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 effec-

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tive stratification of HCC patients responsive to wnt/β-catenin signalling antagonists.

inhibitors has yet to be completely elucidated.

potential use for HCC treatment.

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

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

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

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

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.

include XAV939 and WXL-8 and also reduce tumourigenicity *in vivo* [56].

(ADP-ribose) polymerase (PARP) and reduced cyclin D1 and c-Myc.

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 prior sorafenib (CELESTIAL, NCT01908426) [47].
