**2.1 Wnt/-catenin pathway**

Wnt signaling pathway involves three main components which include the cell surface, the cytoplasm, and the nucleus (Giles et al., 2003). At the cell membrane, Wnt ligands form complexes with Frizzled receptors and LRP5 or LRP6 co-receptors (Dale, 1998; Wehrli et al., 2000). There are at least 19 known human Wnt family genes and 10 Frizzled family genes, and most Wnt proteins can bind to multiple Frizzled receptors and vice versa (Wang et al., 1996). This ligand receptor interaction can be additionally regulated by other secreted factors and cell surface proteins. For example, soluble forms of Frizzled proteins compete with cell membrane Frizzled receptor for Wnt ligands (Finch et al., 1997). And DKK proteins block Wnt signaling by binding to LRP6 co-receptor (Fedi et al., 1999).

In the cytoplasm, -catenin associates with a multiprotein complex which includes tumor suppressor proteins APC, axin, and Ser/Thr kinase GSK3. Axin and APC form a structural scaffold that allows GSK3 to phosphorylate -catenin. Phosphorylation of -catenin targets it for ubiquitination and protein degradation (Clevers, 2006; Gordon and Nusse, 2006). Therefore, under basal conditions, in the absence of Wnt ligands, -catenin is continuously

Fig. 1. Major cellular signaling pathways involved in hepatocellular carcinoma

block Wnt signaling by binding to LRP6 co-receptor (Fedi et al., 1999).

Wnt signaling pathway involves three main components which include the cell surface, the cytoplasm, and the nucleus (Giles et al., 2003). At the cell membrane, Wnt ligands form complexes with Frizzled receptors and LRP5 or LRP6 co-receptors (Dale, 1998; Wehrli et al., 2000). There are at least 19 known human Wnt family genes and 10 Frizzled family genes, and most Wnt proteins can bind to multiple Frizzled receptors and vice versa (Wang et al., 1996). This ligand receptor interaction can be additionally regulated by other secreted factors and cell surface proteins. For example, soluble forms of Frizzled proteins compete with cell membrane Frizzled receptor for Wnt ligands (Finch et al., 1997). And DKK proteins

In the cytoplasm, -catenin associates with a multiprotein complex which includes tumor suppressor proteins APC, axin, and Ser/Thr kinase GSK3. Axin and APC form a structural scaffold that allows GSK3 to phosphorylate -catenin. Phosphorylation of -catenin targets it for ubiquitination and protein degradation (Clevers, 2006; Gordon and Nusse, 2006). Therefore, under basal conditions, in the absence of Wnt ligands, -catenin is continuously

**2. Major signaling pathways** 

tumorigenesis

**2.1 Wnt/-catenin pathway** 

degraded in the cytosol. Upon Wnt binding, axin is recruited to the membrane to LPR5 and the -catenin destruction complex is then inactivated. This allows the unphosphorylated catenin to accumulate and to translocate into the nucleus (Clevers, 2006; Gordon and Nusse, 2006). The nuclear -catenin then forms a complex with TCF/LEF family of DNA binding transcription factors to activate TCF/LEF target genes. Many of the target genes are involved in cell proliferation, such as cyclin D1 (Shtutman et al., 1999).

Besides regulating gene transcription, -catenin also participates in cell–cell adhesion. Cellcell adhesion and separation are important physiological processes involved in development as well as tumor metastasis. It is known that cadherin mediated adhesion is regulated by -catenin. The formation of stable cell–cell adhesions depends on the integrity of a core complex including E-cadherin, -catenin and -catenin. -catenin binds directly to the cytoplasmic domain of E-cadherin and such association is regulated by the phosphorylation state of -catenin (Nelson and Nusse, 2004).

Wnt signaling clearly plays important roles in normal liver function as 8 out of the total 19 Wnt ligands are expressed in hepatocytes (Thompson and Monga, 2007). Both LRP5 and LRP6 and 9 of 10 Frizzled receptors are expressed in normal liver (Fujino et al., 2003; Zeng et al., 2007). Evidence has suggested that the Wnt signaling pathway is critically involved in liver development and postnatal liver homeostasis. In addition, this pathway is also associated with many other important liver functions, such as ammonia and nitrogen metabolism, bile acid homeostasis, drug detoxification and injury recovery (Behari, 2010; Takigawa and Brown, 2008).

Multiple lines of evidence link aberrant Wnt signaling to HCC. Wnt signaling pathway activation is frequently reported in liver carcinoma. It has been demonstrated that there are significant changes in subcellular localization of -catenin and -catenin-associated cell adhesion molecules in HCC (Ihara et al., 1996). Studies have shown that 50-70% of liver tumors have increased levels of -catenin in the cytoplasm and in the nucleus (Wong et al., 2001). Such accumulation can potentially provide tumor cells a growth advantage by promoting proliferation and inhibiting differentiation. A transgenic mouse model overexpressing -catenin developed severe hepatomegaly (Giles et al., 2003). There are also studies associating -catenin mutations or activation with worsened HCC outcome such as larger tumor size and increased vascular invasion (Behari, 2010).

Accumulation and stabilization of -catenin could be a direct result from point mutations or deletions in the -catenin gene, which is found in 12-26% of HCC (Giles et al., 2003). Such dominant gain-of-function mutations usually occur at the N-terminal phosphorylation sites on -catenin, including the sites phosphorylated by GSK3 that regulate -catenin degradation (Takigawa and Brown, 2008). Mutations at these positions disrupt recognition by GSK3resulting in more stable -catenin protein. Besides gain of function mutations in positive modulators of Wnt signaling such as -catenin, the Wnt pathway can also be activated from loss-of function mutations in negative modulators such as Axin and APC (Takigawa and Brown, 2008). However, frequencies of -catenin accumulation in HCC determined by immunostaining are much higher than the known incidence of Wnt pathway mutations. Thus, other causes of -catenin accumulation may also exist. Epigenetic changes could be a contributing factor, which leads to higher gene expression without any mutations in the gene (Takigawa and Brown, 2008).

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 intervention points for treating HCC.

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 inhibition of the pathway in HCC.
