Liver Cancer Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

An Introduction to Liver Cancer

Hepatocellular carcinoma (HCC) also known as liver cancer, is the sixth most common type of cancer in the world, and is also the third most common cause of cancer related mortality. Over the past decade, the incidence of HCC has risen in many countries. Main risk factors of the HCC include hepatitis B and C virus infection, excessive alcohol consumption, non alcoholic steatohepatitis, exposure to environmental toxins such as aflatoxin B, hemochromatosis, cirrhosis, diabetes and obesity. Generally, the standard treatments for early stage HCC include surgical resection and liver transplantation, which can cure certain population of patients. However, due to the asymptomatic nature of early HCC and the lack of effective screening strategies, 80% of patients developed advanced HCC at the time of diagnosis. Moreover, conventional chemotherapy using cytotoxic agents is not very effective in changing the progress of the tumor growth. Thus, the mortality rate is quite high of advanced stage HCC, and 5-year survival rate is only 7% in patients with HCC. In recent decades, great progress has been made in understanding the molecular mechanism of carcinogenesis. Many cell signaling pathways have been found that are associated with tumor pathogenesis, which renders the new identification of molecular targets for therapeutic development. Targeted therapy acts directly and specifically on the components that regulate tumorigenesis compared with conventional chemotherapy. The new therapy strategy has present clinical benefits in various tumor types, such as breast, colorectal and lung cancers.

1 Main Signaling Pathways in Liver Cancer Therapy

1.1 Wnt/β-catenin signaling cascade

Wnt signaling plays important roles in various normal liver functions and in the liver development and postnatal liver homeostasis. A large number of evidences showed that aberrant Wnt signaling involves in HCC. The activation of Wnt signaling pathway is frequently found in liver carcinoma. Previous studies have shown that the subcellular localization of β-catenin and β-catenin-associated related cell adhesion molecules has changed significantly in hepatocellular carcinoma. Although it is no doubt that abnormal Wnt signal transduction is related to HCC, the exact role of activated Wnt pathway in the pathogenesis of liver tumor is still unclear. Research found that pharmacologic inhibition of β-catenin can significantly decreases survival of hepatoma cells. All studies results suggest that the role of the Wnt pathway in the development of liver cancer is highly context-dependent and involves cross-talk with other pathways. Nonetheless, components of the Wnt pathway may be potential therapeutic intervention targets for treating HCC.

1.2 VEGF signaling cascade

HCC is a hypervascular tumor and many pro-angiogenic factors are over-expressed in HCC cells such as VEGF receptor. VEGF ligand binding leads to VEGF receptors dimerization and autophosphorylation. The phosphorylated tyrosine residues in receptors act as docking sites for various signal transduction proteins, and ultimately activate the cellular processes involved in angiogenesis. VEGF also can induce activation of Rho GTPase, which plays an important role during angiogenesis processes. The VEGF pathway is significantly associated with HCC pathogenesis, such as the expression of VEGF mRNA has been found in a majority of HCC patients with liver tumors. The major route of HCC dissemination and metastasis is through the portal vein in the liver. VEGF mRNA level is closely related to the formation of portal vein tumor thrombus (PVTT), suggesting that VEGF may play an important role in the invasion and metastasis of HCC. The increase level of serum VEGF before operation can also predict the high incidence of postoperative tumor recurrence. In addition, increased VEGF expression has also been detected in cirrhotic and dysplastic livers that can lead to liver cancer.

1.3 FGF signaling cascade

Aberrant FGF/FGFR signaling is clearly associated with tumorigenesis. In HCC, studies have shown that plasma FGF2 level was significantly increased, and overexpression of FGFR1 in hepatocytes accelerated the growth of HCC induced by carcinogen diethylnitrosamine (DEN). FGF19 is another member in FGF family associated with the development of HCC. FGF19 can induce hepatocellular proliferation which eventually leads to development of HCC. FGF19 is also able to activate the Wnt pathway in hepatocytes. Due to the abnormal Wnt signal correlates closely with HCC tumorigenesis, which may be one of the mechanisms of liver tumor formation induced by FGF19. FGFR4 is the main FGF receptor expressed in the liver to mediate FGF19 induced tumorigensis. Many studies revealed that FGFR4 involves in HCC and is frequently overexpressed in patients with hepatocellular carcinoma.

1.4 MAPK signaling cascade

Mitogen-activated protein kinase (MAPK) is serine-threonine kinase associated with a variety of cellular activities. There are three members in the mammalian MAPK family including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. ERK signaling involves in cell proliferation, migration and survival in many tumors. During the ERK pathway, some genes such as HRAS, KRAS, and CRAF, are often upregulated in HCC. For example, the CRAF is overexpresed in patients with HCC and phosphorylated ERK level is higher in HCC tissues. JNK is another main MAPK signaling pathway, which is closely related to HCC. One example, JNK1 is over-activated in most of HCC patients. The activation of p38 is triggered by MKK3, 4, and 6, and autophosphorylation. Interestingly, p38 seems to play a suppressive role in HCC unlike ERK and JNK pathways.

1.5 PI3k/AKT/mTOR signaling cascade

mTOR regulates several important cellular processes such as the regulation of protein translation. Abberent protein translation often causes abnormal cell growth and tumorigenesis. The PI3K/Akt/mTOR signaling pathway can be up-regulated in various carcinoma cells. For example, phospho-mTOR is overexpressed in HCC. The high expression of Akt phosphorylation was also found in HCC and early HCC recurrence and poor prognosis. There is a high frequency of somatic PI3K mutations in HCC specimens. PTEN plays a negative regulator role in the PI3K/Akt/mTOR, and its expression is reduced in half of all HCC tumors, leading to the over activation of the pathway. Decreased PTEN expression often causes advanced tumor stage, high recurrences rate and poor survival outcome in HCC patients.

1.6 Other miscellaneous signaling cascade

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase. The EGFR signaling pathway is one of the most important pathways associated with the regulate growth, survival, proliferation, and differentiation in mammalian cells. Aberrant EGFR signaling plays a critical role in tumor angiogenesis and proliferation such as the HCC. EGFR overexpression has been found in HCC. Besides, deregulation of the insulin-like growth factor (IGF) pathway has also been found involved in the development of HCC. Obvious alterations in the expression of components of the IGF pathway have been found during hepatocarcinogenesis, such as the IGF-2 is overexpressed in HCC. The transforming growth factor-beta (TGF-β) pathway also involves in the regulation of tumor progression in HCC, including angiogenesis, production of the extracellular matrix and immune suppression. Increased expression of TGF-β in HCC patients' sera and urine are associated with disease progression.

Liver Cancer Diagnosis

2.1 Molecular Markers for Liver Cancer

APC, CTNNB1, AXIN1 and Secreted Frizzled related protein 1 (SFRP1) genes are commonly altered and are detected in HCC. Aberrant DNA methylation of the APC gene is the most frequent alteration found in HCC. Another most frequently detected DNA marker is the epigenetic alteration of the SFRP1 gene. SFRP1 protein forms an inhibitory complex with the frizzled receptor and acts as a Wnt pathway inhibitor, which can act as a negative regulator of cell invasion and predict the metastatic potential of a tumor. Hypermethylation of the SFRP1 promoter is observed in more than 50% of HCC.

Other frequently identified DNA modifications in the cell cycle regulation pathway are RASSF1A gene and methylation of CDKN2A (pl6) in HCC. Besides, mutations of the TP53 gene are associated with chromosomal instability, occur in 20-50% of HCC. IRF2 is an upstream gene of TP53 recently found to be mutated in approximately 5% of HCC. mRASSF1A and TP53 genes mutations in the serum and urine of HCC patients are detected at rates up to 90 and 50% respectively, indicating these genes are promising biomarkers for non invasive HCC screening.

ARID1A, ARID1B and ARID2 involve in the chromatin remodeling pathway and are the most frequently detected in HCC. PTEN gene mutations and methylation are the most frequent recurrent modifications in the PI3K/AKT/mTOR signaling pathway pathway, which can be used as DNA markers for HCC diagnosis. Other molecular markers include circulating nucleic acids (mRNAs), alpha-Fetoprotein mRNA (AFP mRNA), gamma-Glutamyl Transferase mRNA (GGT mRNA), insulin-Like Growth Factor II (IGF-II) mRNA, albumin mRNA and microRNAs (miRNAs).

2.2 Protein Markers for Liver Cancer

One of protein markers is oncofetal and glycoprotein antigens, such as Alpha-Fetoprotein (AFP) and glypican-3 (GPC3). Alpha-fetoprotein (AFP) was the first serologic assay for detection and clinical followup of patients with hepatocellular carcinoma, which has been the standard tumor biomarker for HCC for many years. Glypican-3 is a membrane-anchored heparin sulfate proteoglycan. GPC3 mRNA is upregulated significantly in tumor tissues of HCC, and the expression of GPC3 in the serum of HCC patients was significantly higher than that in the serum of healthy adults or patients.

Another type of protein marker is enzymes and isoenzymes, including Des-gamma-carboxy (abnormal) prothrombin (DCP), gamma-glutamyl transferase (GGT), serum alpha-1-fucosidase (AFU), human carbonyl reductase 2 and golgi phosphoprotein 2 (GOLPH2). DCP is produced by the malignant hepatocyte, which is closely related to HCC and used as a marker in the HCC diagnosis. GGT is mainly secreted by hepatic Kupffer cell and endothelial cell of bile duct, and its activity increases in HCC tissues. AFU is a lysosomal enzyme present in all mammalian cells playing a role in hydrolyzing fucose glycosidic linkages of glycoprotein and glycolipids, which displays increased activity in HCC patients thus can be used as detection marker. Human carbonyl reductase 2 is expressed in the human liver and kidney, which have been shown to be inversely correlated to the pathological grading of HCC. GOLPH2 is a Golgi-apparatus-associated protein and has been shown to have a higher sensitivity than AFP in the detection of HCC.

Other protein markers include growth factors and their receptors, such as transforming growth factor-beta (TGF-Beta), tumor-specific growth factor (TSGF), epidermal growth factor receptor family, hepatocyte growth factor/scatter factor and basic fibroblast growth factor.

3 Targeted Therapy for Liver Cancer

The Wnt/β-catenin, VEGF, FGF, MAPK, PI3k/AKT/mTOR signaling pathway plays important roles in malignant transformation, prevention of apoptosis, drug resistance and metastasis. The overexpression of oncogenes and tumor suppressor genes include VEGFR, PDGFR, Raf, FGFR-1, mTOR and c-MET has been used as target for targeted therapy in liver cancer process.

We have focused on recently discovered therapeutic targets for liver cancer, such as the VEGF receptor signaling pathway, RAF/MEK/ERK signaling pathway, mTOR pathway, Wnt/β-catenin signaling pathway, and c-MET inhibitors, as well as a few immunotherapies that have recently been developed for the treatment of liver cancer based on these targets along with their progress in clinical trials (Table 1-6).

3.1 Liver cancer therapy for VEGF pathway

VEGF is the most well-known growth factor in angiogenesis. It functions its effect by binding to its receptors, VEGF receptor 1 (VEGF-R1), VEGF-R2, and VEGF-R3, present on endothelial cells. VEGF secreted by tumour cells binding to its receptors and causing activation of signal transduction pathways promoting cell migration, proliferation, and survival of cancer cells lead to angiogenesis. Sorafenib is the only drug approved for treatment of advanced HCC patients currently, and it is an orally active anti-angiogenic multi-kinase inhibitor. Bevacizumab, an anti-VEGF monoclonal antibody has shown improved efficacy in patients with unresectable HCC. Ramucirumab is another monoclonal antibody targeting VEGFR-2, which blocks the receptor. Sunitinib is an orally administered multi-kinase inhibitor with activity used in various kinases including VEGFR and PDGFR. Other VEGF pathway inhibitors include Linifanib and Regorafenib.

Table 1 Clinical trials of multi-kinase inhibitor Sorafenib

According to statistics, a total of 72 Sorafenib projects targeting liver cancer multi-kinase are currently in clinical stage, of which 60 are recruiting and 12 are not recruiting.

Table 2 Clinical trials of VEGF inhibitor Bevacizumab

According to statistics, a total of 29 Bevacizumab projects targeting liver cancer VEGF are currently in clinical stage, of which 16 are recruiting and 13 are not recruiting.

Table 3 Clinical trials of VEGFR-2 inhibitor Ramucirumab

According to statistics, a total of 8 Ramucirumab projects targeting liver cancer VEGFR-2 are currently in clinical stage, of which 3 are recruiting and 5 are not recruiting.

Table 4 Clinical trials of VEGF inhibitor Regorafenib

According to statistics, a total of 10 Regorafenib projects targeting liver cancer VEGF are currently in clinical stage, of which 7 are recruiting and 3 are not recruiting.

3.2 Liver cancer therapy for RAF/MEK/ERK pathway

The mitogen-activated protein kinase (MAPK) cascade contains serine/threonine kinases, which converts extracellular molecules such as growth factors, hormones, and differentiation factors, into intracellular signals for regulating several cellular processes including proliferation, apoptosis and migration. As an oral MEK inhibitor, Selumetinib is evaluated in a small phase II study (NCT00604721) in patients with advanced HCC. Another MEK inhibitor, refametinib is evaluated in a phase II study (NCT01204177) in patients with unresectable HCC.

3.3 Liver cancer therapy for mTOR pathway

This pathway acts as a critical regulator of numerous physiological processes and involves in cell proliferation and metastasis of transformed human cancers including HCC. Everolimus inhibitor inhibits tumour growth in patient-derived xenograft models of advanced HCC. Other mTOR inhibitors include temsirolimus and sirolimus.

Table 5 Clinical trials of mTOR inhibitor Everolimus

According to statistics, a total of 3 Everolimus projects targeting liver cancer mTOR are currently in clinical stage, of which 1 is recruiting and 2 are not recruiting.

Table 6 Clinical trials of mTOR inhibitor temsirolimus

According to statistics, a total of 3 temsirolimus projects targeting liver cancer mTOR are currently in clinical stage, and not recruiting.

3.4 c-MET inhibitors for liver cancer therapy

MET is a receptor encoded by the c-MET proto-oncogene, which is the ligand of hepatocyte growth factor (HGF). MET serves as tyrosine kinase receptor regulating metastatic progression. Binding of MET to HGF activates the RAS-MAPK and PI3K-AKT signaling pathways resulting in tumour development and metastasis in HCC. Foretinib is the first c-MET inhibitor of broad spectrum, including c-Met and VEGFR, to be tested in clinical trials. Other c-MET inhibitors include Tivantinib and Cabozantinib.

3.5 Liver cancer therapy for Wnt/β-catenin pathway

The Wnt/β-catenin signalling plays a critical role in a host of physiological and pathophysiological processes such as embryonic development, cell proliferation, regeneration, angiogenesis and cancer. It also involves in maintaining liver health, which is dysregulated in HCC with mutation in β-catenin in most HCC patients and to be a potential important target of therapy. Pimozide, an antipsychotic drug can inhibit cell proliferation and apoptosis in HCC cell lines. OMP-18R5 is a monoclonal antibody targeting the wnt/β-catenin signaling evaluated in clinical phase I trials (NCT01345201) for the treatment of solid tumours and myeloid malignancies, suggesting potential use for HCC treatment.