**2.4 MAPK pathway**

Mitogen-activated protein kinase (MAPK) is serine-threonine kinase that is involved in a variety of cellular activities. There are three members in the mammalian MAPK family, extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 (Kim and Choi, 2010; Min et al., 2011). Among them, ERK signaling pathway is the most studied for its involvement in promoting cell proliferation, migration, survival, and its association with tumorigenesis and tumor progression (Gollob et al., 2006). Recent data also implicate JNK and p38 as playing important roles during HCC development (Min et al., 2011).

The ERK pathway is ubiquitous and can be activated by various receptors, particularly receptor tyrosine kinases (RTKs). Upon ligand binding, the RTKs dimerize which leads to activation of the intracellular tyrosine kinase domain. Activated kinase results in receptor phosphorylation on tyrosine residues, that then serves as docking sites for adaptor proteins such as GRB2 and SOS (Schulze et al., 2005; Zarich et al., 2006). Upon docking to the receptors, the GRB2 and SOS activate the small GTPase RAS (HRAS, NRAS and KRAS) which in turn will activate the serine/threonine kinase RAF. RAF is a MAPK kinase kinase (MAP3K), and it has three isoforms, ARAF, BRAF and CRAF. Activated RAF will phosphorylate and activate MEK, a MAPK kinase (MAP2K) and MEK is the kinase for ERK (ERK1 and ERK2) (Avruch et al., 2001; Malumbres and Barbacid, 2003).

There are more than 100 substrates downstream of ERK, and many of them are transcription factors. The altered levels and activities of the transcription factors by ERK activation can lead to altered expression levels of genes that are important for cell cycle progression (Davis, 1995). For example, ERK can phosphorylate and activate C-myc, a transcription factor that regulates the expression of many target genes involved in cell growth and proliferation. C-Myc is a strong proto-oncogene and can be found unregulated in many types of cancers (Penn et al., 1990). ERK also directly phosphorylates kinase substrates such as myosin light chain kinase, calpain, and focal adhesion kinase, which promotes cell migration (Huang et al., 2004). Furthermore, the ERK pathway can regulate proteins involved in apoptotic pathway such as BIM and MCL1, promoting survival of cancer cells (Balmanno and Cook, 2009).

Given the contribution of ERK signaling towards cell proliferation, migration and survival, it is not surprising to see constitutive activation of the ERK pathway in many tumors. In fact, genes along the ERK pathway, such as HRAS, KRAS, and CRAF, are often upregulated in HCC. One study has shown that CRAF is overexpresed among all 30 HCC tissue samples tested (Hwang et al., 2004). Immunostaining also showed around 7 fold increase of MEK phosphorylation in HCC tissues compared to surrounding benign liver tissues (Huynh et al., 2003). Other studies also found that phosphorylated ERK level is higher in HCC tissues and ERK activation is associated with aggressive tumor behavior (Schmitz et al., 2008). In addition, negative regulator proteins of MAPK/ERK pathway such as Sprouty and DUSP1 are down-regulated in HCC tumors (Calvisi et al., 2008) (Yoshida et al., 2006). Sustained

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).

Mitogen-activated protein kinase (MAPK) is serine-threonine kinase that is involved in a variety of cellular activities. There are three members in the mammalian MAPK family, extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 (Kim and Choi, 2010; Min et al., 2011). Among them, ERK signaling pathway is the most studied for its involvement in promoting cell proliferation, migration, survival, and its association with tumorigenesis and tumor progression (Gollob et al., 2006). Recent data also implicate

The ERK pathway is ubiquitous and can be activated by various receptors, particularly receptor tyrosine kinases (RTKs). Upon ligand binding, the RTKs dimerize which leads to activation of the intracellular tyrosine kinase domain. Activated kinase results in receptor phosphorylation on tyrosine residues, that then serves as docking sites for adaptor proteins such as GRB2 and SOS (Schulze et al., 2005; Zarich et al., 2006). Upon docking to the receptors, the GRB2 and SOS activate the small GTPase RAS (HRAS, NRAS and KRAS) which in turn will activate the serine/threonine kinase RAF. RAF is a MAPK kinase kinase (MAP3K), and it has three isoforms, ARAF, BRAF and CRAF. Activated RAF will phosphorylate and activate MEK, a MAPK kinase (MAP2K) and MEK is the kinase for ERK

There are more than 100 substrates downstream of ERK, and many of them are transcription factors. The altered levels and activities of the transcription factors by ERK activation can lead to altered expression levels of genes that are important for cell cycle progression (Davis, 1995). For example, ERK can phosphorylate and activate C-myc, a transcription factor that regulates the expression of many target genes involved in cell growth and proliferation. C-Myc is a strong proto-oncogene and can be found unregulated in many types of cancers (Penn et al., 1990). ERK also directly phosphorylates kinase substrates such as myosin light chain kinase, calpain, and focal adhesion kinase, which promotes cell migration (Huang et al., 2004). Furthermore, the ERK pathway can regulate proteins involved in apoptotic pathway such as BIM and MCL1, promoting survival of cancer cells (Balmanno and Cook,

Given the contribution of ERK signaling towards cell proliferation, migration and survival, it is not surprising to see constitutive activation of the ERK pathway in many tumors. In fact, genes along the ERK pathway, such as HRAS, KRAS, and CRAF, are often upregulated in HCC. One study has shown that CRAF is overexpresed among all 30 HCC tissue samples tested (Hwang et al., 2004). Immunostaining also showed around 7 fold increase of MEK phosphorylation in HCC tissues compared to surrounding benign liver tissues (Huynh et al., 2003). Other studies also found that phosphorylated ERK level is higher in HCC tissues and ERK activation is associated with aggressive tumor behavior (Schmitz et al., 2008). In addition, negative regulator proteins of MAPK/ERK pathway such as Sprouty and DUSP1 are down-regulated in HCC tumors (Calvisi et al., 2008) (Yoshida et al., 2006). Sustained

JNK and p38 as playing important roles during HCC development (Min et al., 2011).

(ERK1 and ERK2) (Avruch et al., 2001; Malumbres and Barbacid, 2003).

The contribution of FGFR4 in HCC progression requires further clarification.

**2.4 MAPK pathway** 

2009).

activation of ERK signaling can also occur due to point mutations in the RAS gene, which leads to constitutive CRAF activation (Downward, 2003; Whittaker et al., 2010). Mutation in the RAS gene has been reported in 10-30% of HCC tumors (Whittaker et al., 2010). The involvement of the ERK pathway in HCC is further confirmed by preclinical studies using the MEK inhibitor, AZD6244, which blocks proliferation and promotes apoptosis in primary HCC cells (Huynh et al., 2007a; Huynh et al., 2007b). AZD6244 also suppresses tumor growth in HCC xenograft model in a dose-dependent manner. And tumor growth inhibition after AZD6244 treatment correlates with inactivation of ERK, up-regulation of apoptotic genes such as caspase-3 and 7 and down-regulation of cell cycle regulators such as cyclin D1. It has also been shown that AZD6244 can induce a synergistic effect in tumor suppression when combined with chemotherapeutic agent doxorubicin (Huynh et al., 2007a; Huynh et al., 2007b).

JNK is another major MAPK signaling pathway. It can be activated by two MAPK kinases, MKK4 and MKK7, and its downstream substrates include c-Jun (Keshet and Seger, 2010). JNK signaling can be activated by various cytokines and environmental factors. It has been demonstrated that JNK1 and JNK2 regulate stress-induced apoptosis, and increased JNK activity has also been shown to enhance proliferation of mouse embryonic fibroblasts (MEFs) (Das et al., 2011). There is strong correlation between activated JNK signaling and HCC. For example, one study has shown that JNK1 is over-activated in 17 out of 31 samples (55%) from Chinese HCC patients (Chang et al., 2009). The activation in JNK1 is associated with increased tumor size and a lack of encapsulation of the tumors. In addition, JNK1 activation also associates with increased histone H3 lysines 4 and 9 tri-methylation, which leads to up-regulation of genes promoting cell growth (Chang et al., 2009).

Direct evidence demonstrating the role of JNK pathway in HCC development comes from studies in mouse models. JNK1 knockout mice had a significant reduction in liver tumorigenesis chemically induced by DEN and hepatocyte proliferation also decreased in those animals (Hui et al., 2008). It was proposed that mice lacking JNK1 have increased expression of p21, a cell cycle inhibitor. Blocking JNK activity using pharmacological inhibitor D-JNKI1 reduced growth of both xenografted human HCC cells and DEN-induced mouse liver cancers, further supporting the role played by JNK pathway in HCC development (Hui et al., 2008).

The activation of p38 is induced by MKK3, 4, and 6, as well as autophosphorylation. Its substrates include transcription factors such as p53 and protein kinases such as MK2 and MK5 (Min et al., 2011). Interestingly, unlike ERK and JNK pathways, p38 seems to play a suppressive role in HCC (Min et al., 2011). It was shown that human embryonic fibroblasts displayed enhanced proliferation upon treatment with a p38 inhibitor (SB203580) (Wang et al., 2002). p38 negatively regulates proliferation partly through suppression of the JNK pathway, which is known to promote cell proliferation. Depending upon the cellular context, p38 can enhance the protein level of JNK phosphatase and repress the activities of JNK kinases (Wagner and Nebreda, 2009). In p38 deficient fetal liver cells and liver tumor cells, the JNK pathway activity was found to be increased. Direct evidence supporting the suppressive role of p38 in HCC came from a study using mice with liver-specific deletion of p38, where enhanced hepatocyte proliferation and tumor development were observed during liver carcinogenesis (Hui et al., 2007). After DEN treatment, p38 deficient mice clearly developed more tumors in the liver and had larger average tumor size compared with control mice. Moreover, inactivation of JNK pathway with -JNKI1 suppressed the hyperproliferation in p38-deficient animals (Hui et al., 2007).

In summary, numerous data have clearly demonstrated the deep involvement of MAPK signaling pathways during liver carcinogenesis. Identifying pharmacological intervention points along these pathways could be considered as a very promising strategy for combating HCC.
