**2.2 VEGF pathway**

Angiogenesis is critical for cancer development. Tumor cells require oxygen and nutrients for survival and proliferation and they need to be located within 100 to 200 m from blood vessels to obtain an adequate supply of oxygen (Carmeliet and Jain, 2000). Solid tumors smaller than 1 to 2 cubic millimeters are not vascularized (Hawkins, 1995). However, beyond the critical volume of 2 cubic millimeters, new blood vessels need to be recruited to supply oxygen and nutrients and to remove metabolic wastes (Hawkins, 1995). Neovascularization also facilitates the dissemination of cancer cells throughout the entire body eventually leading to metastasis formation.

HCC is a hypervascular tumor and many pro-angiogenic factors are over-expressed in HCC cells and in the surrounding microenvironment (Shen et al., 2010). Among them, VEGF receptor signaling is one of the most well studied. The major VEGF that mediates tumor angiogenesis is VEGF-A, and it has several splicing variants that can be produced simultaneously. The most predominant forms are VEGF-A121 and VEGF-A165. Other members in the VEGF family include Placenta growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D (Kaseb et al., 2009; Roskoski, 2007). VEGF signals through VEGF receptor (tyrosine kinase receptor) on the cell surface. There are three main subtypes of VEGF receptors,

Although there is little doubt about the strong correlation between aberrant Wnt signaling and HCC, the precise role of activated Wnt pathway in the pathogenesis of liver tumor is less well understood. It has been shown that pharmacologic inhibition of -catenin decreases survival of hepatoma cells (Behari et al., 2007). Inactivation of -catenin suppressor APC led to spontaneous development of HCC in a mice model, suggesting the direct contribution from activated Wnt signaling to hepatocarcinogenesis (Colnot et al., 2004). However mice overexpressing a gain-of-function -catenin mutant (exon 3 deletion) only showed increased susceptibility to developing HCC after exposure to carcinogen diethylnitrosamine (DEN), instead of developing spontaneous liver tumor (Harada et al., 2002). These 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 represent potential therapeutic

Several approaches could be envisioned to target Wnt pathways. Extracellularly, it is possible to design molecules to disrupt Wnt ligand-receptor complexes, preventing initiation of the signaling events. For this approach, Wnt ligands, extracellular Wnt regulators such as DKKs, SFRPs proteins, and members of the receptor complexes can all be targeted. These proteins themselves or variants could be directly considered as candidates for drug developments. Alternatively, antibodies or other modalities that could block ligand receptor interactions may also be explored for therapeutic development. Intracellular components could also be targeted. Small molecule ligands toward kinases of the signaling pathway could also be screened that may regulate Wnt signaling. In addition, small molecule antagonists have been identified to interfere with the binding between -catenin and TCF/LEF proteins or its coactivator CEBP, blocking downstream gene activation (Dahmani et al., 2011). However, due to the inherent complexity of Wnt signaling in the liver, further research is needed to fully understand the implications of therapeutic

Angiogenesis is critical for cancer development. Tumor cells require oxygen and nutrients for survival and proliferation and they need to be located within 100 to 200 m from blood vessels to obtain an adequate supply of oxygen (Carmeliet and Jain, 2000). Solid tumors smaller than 1 to 2 cubic millimeters are not vascularized (Hawkins, 1995). However, beyond the critical volume of 2 cubic millimeters, new blood vessels need to be recruited to supply oxygen and nutrients and to remove metabolic wastes (Hawkins, 1995). Neovascularization also facilitates the dissemination of cancer cells throughout the entire

HCC is a hypervascular tumor and many pro-angiogenic factors are over-expressed in HCC cells and in the surrounding microenvironment (Shen et al., 2010). Among them, VEGF receptor signaling is one of the most well studied. The major VEGF that mediates tumor angiogenesis is VEGF-A, and it has several splicing variants that can be produced simultaneously. The most predominant forms are VEGF-A121 and VEGF-A165. Other members in the VEGF family include Placenta growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D (Kaseb et al., 2009; Roskoski, 2007). VEGF signals through VEGF receptor (tyrosine kinase receptor) on the cell surface. There are three main subtypes of VEGF receptors,

intervention points for treating HCC.

inhibition of the pathway in HCC.

body eventually leading to metastasis formation.

**2.2 VEGF pathway** 

numbered 1, 2 and 3 (Kaseb et al., 2009; Roskoski, 2007). They all have an extracellular region consisting of 7 immunoglobulin-like domains, a single transmembrane domain, and an intracellular portion containing a split tyrosine-kinase domain. VEGF ligand binding induces dimerization and autophosphorylation of VEGF receptors. Phosphorylated tyrosine residues in the receptor serve as a docking site for various signal transduction proteins that can eventually activate cellular processes involved in angiogenesis. For example, VEGFR-2 phosphorylation activates PLC- which in turn leads to protein kinase C activation (PKC). PKC can activate MAP kinase signaling and promote cell proliferation as well as increase vascular permeability through activation of endothelial nitric oxide synthase (Kaseb et al., 2009; Roskoski, 2007). VEGF also induce activation of Rho GTPase, which plays a crucial role during angiogenesis processes such as vascular permeability, extra cellular matrix degradation, cellular migration and invasion (van der Meel et al., 2011).

Among the three VEGF receptors, VEGFR-2 appears to mediate almost all of the known cellular responses to VEGFs. The activation of VEGFR-2 in endothelial cells results in their proliferation, migration, and increased survival and promotes vascular permeability (Kaseb et al., 2009; Roskoski, 2007). The function of VEGFR-1 is less known. Although it has higher affinity for VEGF than VEFGR-2, it has weak tyrosine kinase phosphorylation activity following ligand stimulation. Activation of VEGFR-1 has no direct proliferative or cytoskeletal effects. It is possibly involved in modulating VEGFR-2 activity. VEGF-C and VEGF-D, but not VEGF-A, are ligands for a third receptor (VEGFR-3), which is important for lymphangiogenesis (Kaseb et al., 2009; Roskoski, 2007).

The VEGF pathway is clearly important for HCC pathogenesis. Expression of VEGF mRNA in liver tumors was found in a majority of HCC patients. And the expression of VEGF steadily increases with the progression of the hepatocarcinogenic process from a normal liver, to a dysplastic nodule, to HCC (Yamaguchi et al., 1998). The levels of VEGF mRNA expression in tumors with tumorous emboli and in poor-encapsulated tumors were higher than those without tumorous emboli and in well-encapsulated tumors. The principle route of HCC dissemination and metastasis is through the portal vein in the liver and VEGF mRNA level correlated well with portal vein tumor thrombus (PVTT) formation of HCC, suggesting VEGF may play an important role in HCC invasion and metastasis (Zhou et al., 2000). Immunohistochemical staining also detected very high VEGF expression in welldifferentiated HCC as well as areas surrounding the HCC tissues, where inflammatory cell filtration was apparent. High serum VEGF levels have been shown to correlate with poor response to chemotherapy and poor survival among HCC patients. Increased preoperative serum VEGF may also predict high incidence of tumor recurrence after surgical resection (Whittaker et al., 2010). Furthermore, increased VEGF expression has also been detected in cirrhotic and dysplastic livers, which often lead to liver cancer (El-Assal et al., 1998).

The most direct evidence supporting the role of the VEGF signaling pathway in HCC came from recent progress in molecular targeted therapy inhibiting this pathway. Bevacizumab, an anti–VEGF monoclonal antibody was tested in patients with unresectable HCC and resulted in significant disease-stabilizing effect (Siegel et al., 2008). Clinical effects were assessed by tumor regression and progression-free survival (PFS). Of the 46 patients in the study, 13% had objective response and 65% were progression free at 6 months. The mean PFS time was 6.9 months and median overall survival time is 12.4 months. Overall survival rates were 53% at 1 year, 28% at 2 years and 23% at 3 years. Treatment was associated with significant reductions in tumor arterial enhancement and circulating VEGF-A level. Besides VEGF antibody, small molecule VEGF inhibitors have also been developed and tested in clinic. Sorafenib, an inhibitor targeting the VEGF pathway has been shown to prolong overall survival in patient with advanced HCC (Llovet et al., 2008). In a randomized, placebo-controlled phase III trial, sorafenib prolonged median survival time of advanced HCC patients by 2.8 months, from 7.9 months in the placebo group to 10.7 months in the sorafenib treatment group. Time to radiologic progression was delayed by 2.7 months, from 2.8 months to 5.5 months. This result is quite significant since no effective systemic therapy ever existed for patients with advanced hepatocellular carcinoma before the sorafenib trial. There are several other VEGF small molecule inhibitors that are currently being tested in the clinic for HCC treatment including, sunitinib, vatalanib, cediranib, brivanib, and linifanib (Shen et al., 2010; Whittaker et al., 2010). Studies have also been carried out to assess the benefit of the combined therapy using those compounds.

### **2.3 FGF pathway**

Supported by various mouse genetic models as well as human genetic studies, aberrant FGF/FGFR signaling is clearly associated with tumorigenesis (Beenken and Mohammadi, 2009; Knights and Cook, 2010; Krejci et al., 2009; Turner and Grose, 2010). Signaling is activated upon FGF ligands binding to the FGF receptors on the cell surface. There are more than 20 different FGFs in the FGF family, making it the largest family of growth factors. Fibroblast growth factor receptors consist of an extracellular ligand binding domain, a single transmembrane domain, and an intracellular domain with tyrosine kinase activity. The extracellular domain of the FGF receptor is composed of three immunoglobin-like domains. Alternative splicing of four FGF receptors genes (FGFR1-4) results in over 48 different isoforms of FGFRs, and they have different affinity towards different FGF ligands and have distinct expression patterns (Ornitz and Itoh, 2001; Ornitz et al., 1996). FGFRs are tyrosine kinase receptors that upon ligand binding induce dimerization and kinase activation in the presence of the co-factor, heparan sulfate (Plotnikov et al., 1999). Phosphorylation of the tyrosine residues on the receptor provides docking sites for downstream adaptor proteins, which can couple to the activation of different intracellular signaling pathways. One of the key adaptor proteins of FGF receptors is FGF substrate 2 (FRS2) which can be phosphorylated by FGFR receptors and recruit more adaptor proteins such as son of sevenless (SOS) and growth factor receptor-bound 2 (GRB2) to activate RAS GTPase (Eswarakumar et al., 2005). RAS GTPase promotes several downstream signaling, such as Wnt, MAPK, and PI3K/Akt pathways (Knights and Cook, 2010). These FGF downstream signaling pathways have all been implicated in several aspects of tumorigenesis, such as proliferation, survival, cell migration and invasion, as well as angiogenesis (Balmanno and Cook, 2009; Dailey et al., 2005; Presta et al., 2005; Xian et al., 2005). In HCC, it has been shown that plasma FGF2 level was significantly increased (Hsu et al., 1997) and overexpression of FGFR1 in hepatocytes accelerated the growth of HCC chemically induced by DEN in a mouse model (Huang et al., 2006).

Recently, attention has focused on a unique FGF family member, FGF19, and its involvement in the development of HCC (Nicholes et al., 2002; Wu and Li, 2009). FGF19 belongs to a unique FGF subfamily that has weakened affinity towards heparan sulfate. The reduced affinity liberates FGF19 from tissues where it is expressed, allowing it to act as an endocrine hormone. Furthermore, FGF19 requires Klotho as a co-receptor in activating FGF

significant reductions in tumor arterial enhancement and circulating VEGF-A level. Besides VEGF antibody, small molecule VEGF inhibitors have also been developed and tested in clinic. Sorafenib, an inhibitor targeting the VEGF pathway has been shown to prolong overall survival in patient with advanced HCC (Llovet et al., 2008). In a randomized, placebo-controlled phase III trial, sorafenib prolonged median survival time of advanced HCC patients by 2.8 months, from 7.9 months in the placebo group to 10.7 months in the sorafenib treatment group. Time to radiologic progression was delayed by 2.7 months, from 2.8 months to 5.5 months. This result is quite significant since no effective systemic therapy ever existed for patients with advanced hepatocellular carcinoma before the sorafenib trial. There are several other VEGF small molecule inhibitors that are currently being tested in the clinic for HCC treatment including, sunitinib, vatalanib, cediranib, brivanib, and linifanib (Shen et al., 2010; Whittaker et al., 2010). Studies have also been carried out to assess the

Supported by various mouse genetic models as well as human genetic studies, aberrant FGF/FGFR signaling is clearly associated with tumorigenesis (Beenken and Mohammadi, 2009; Knights and Cook, 2010; Krejci et al., 2009; Turner and Grose, 2010). Signaling is activated upon FGF ligands binding to the FGF receptors on the cell surface. There are more than 20 different FGFs in the FGF family, making it the largest family of growth factors. Fibroblast growth factor receptors consist of an extracellular ligand binding domain, a single transmembrane domain, and an intracellular domain with tyrosine kinase activity. The extracellular domain of the FGF receptor is composed of three immunoglobin-like domains. Alternative splicing of four FGF receptors genes (FGFR1-4) results in over 48 different isoforms of FGFRs, and they have different affinity towards different FGF ligands and have distinct expression patterns (Ornitz and Itoh, 2001; Ornitz et al., 1996). FGFRs are tyrosine kinase receptors that upon ligand binding induce dimerization and kinase activation in the presence of the co-factor, heparan sulfate (Plotnikov et al., 1999). Phosphorylation of the tyrosine residues on the receptor provides docking sites for downstream adaptor proteins, which can couple to the activation of different intracellular signaling pathways. One of the key adaptor proteins of FGF receptors is FGF substrate 2 (FRS2) which can be phosphorylated by FGFR receptors and recruit more adaptor proteins such as son of sevenless (SOS) and growth factor receptor-bound 2 (GRB2) to activate RAS GTPase (Eswarakumar et al., 2005). RAS GTPase promotes several downstream signaling, such as Wnt, MAPK, and PI3K/Akt pathways (Knights and Cook, 2010). These FGF downstream signaling pathways have all been implicated in several aspects of tumorigenesis, such as proliferation, survival, cell migration and invasion, as well as angiogenesis (Balmanno and Cook, 2009; Dailey et al., 2005; Presta et al., 2005; Xian et al., 2005). In HCC, it has been shown that plasma FGF2 level was significantly increased (Hsu et al., 1997) and overexpression of FGFR1 in hepatocytes accelerated the growth of HCC chemically induced

Recently, attention has focused on a unique FGF family member, FGF19, and its involvement in the development of HCC (Nicholes et al., 2002; Wu and Li, 2009). FGF19 belongs to a unique FGF subfamily that has weakened affinity towards heparan sulfate. The reduced affinity liberates FGF19 from tissues where it is expressed, allowing it to act as an endocrine hormone. Furthermore, FGF19 requires Klotho as a co-receptor in activating FGF

benefit of the combined therapy using those compounds.

by DEN in a mouse model (Huang et al., 2006).

**2.3 FGF pathway** 

receptors. In the presence of Klotho, FGF19 is able to activate similar downstream signaling through FGF receptors (Kurosu et al., 2007; Wu et al., 2007).

First evidence connecting FGF19 with HCC came from a transgenic FGF19 mice model, where human FGF19 driven by myosin promoters was overexpressed from skeleton muscle, resulting in elevated serum FGF19 protein levels (Nicholes et al., 2002). HCC development was observed in these FGF19 transgenic mice at 8-10 months, while no tumors were observed in wild type control mice. Tumors occurred in different liver lobes and were either solitary or multifocal. Histological staining showed neoplastic cells invasion and replacement of normal hepatocytes (Nicholes et al., 2002). Hepatocytes are normally mitotically quiescent in the liver. However, in case of HCC, hepatocellular proliferation is a prerequisite for transformation. In vivo 5-bromo-2'-deoxyuridine (BrdU) labeling was performed to assess the proliferation in FGF19 transgenic mice. BrdU-labeling index of hepatocytes was eight fold higher in the transgenic mice than age-matched wild type mice at 2 to 4 months of age (Nicholes et al., 2002). Furthermore, recombinant FGF19 protein also induced a significant higher BrdU-labeling index after being injected into normal mice (Nicholes et al., 2002; Wu et al., 2010a; Wu et al., 2010b). These results strongly support the notion that FGF19 can induce hepatocellular proliferation which eventually leads to development of HCC.

FGF19 is able to activate the Wnt pathway in hepatocytes, and 44% of neoplastic hepatocytes in FGF19 transgenic mice have nuclear staining for -catenin (Nicholes et al., 2002; Pai et al., 2008). Since aberrant Wnt signaling correlates strongly with HCC tumorigenesis, this may be one mechanism whereby FGF19 induces liver tumor formation. To test the hypothesis of whether FGF19 could be a valid target in HCC treatment, a monoclonal anti-FGF19 antibody was generated and tested in tumor inhibition. Anti-FGF19 antibody or control antibody were used to treat DEN accelerated HCC formation in FGF19 transgenic mice (Desnoyers et al., 2008). After six months of antibody treatment, all the animals treated with control antibody developed multifocal, large HCCs throughout the liver lobes while almost none of the mice treated with FGF19 antibody had liver tumors. The same anti-FGF19 antibody was also tested in xenograft mice models (Desnoyers et al., 2008). Mice xenografted with colon cancer cell lines HCT116 and Colo201 were injected with anti-FGF19 antibody twice weekly. At day 35, FGF19 antibody suppressed tumor growth by 57% compared to the control in HCT116 group and 64% growth inhibition was achieved in Colo201 animals treated with the antibody. These data suggest that targeting FGF19 could be a valid strategy for HCC treatment.

The main candidate FGF receptor mediating FGF19 induced tumorigensis in liver is believed to be FGFR4, which is the predominant FGF receptor expressed in the liver. Strong FGFR4 mRNA was detected in hepatocytes adjacent to central vein by in situ hybridization, and the hepatic dysplasia foci and BrdU labeling from FGF19 transgenic livers are also located around central vein. In addition, activation of FGFR4 alone was sufficient to induce hepatocyte proliferation (Wu et al., 2010a; Wu et al., 2010b) and FGF19 induced hepatocytes proliferation was not observed in FGFR4 knockout mice, confirming the role played by FGFR4 in hepatocarcinogenesis (Wu et al., 2011).

FGFR4 has been implicated in HCC in many literature reports (Yang et al., 2011). FGFR4 is frequently overexpressed in patients with hepatocellular carcinoma (Yang et al., 2011). siRNA against FGFR4 in liver cancer lines HuH7 is able to suppress -fetoprotein production (Yang et al., 2011). However, there have also been reports showing genetic deletion of FGFR4 in mice results in faster progression of DEN-accelerated hepatocellular carcinoma, suggesting that FGFR4 suppresses hepatoma proliferation (Huang et al., 2009). The contribution of FGFR4 in HCC progression requires further clarification.
