**The Involvement of the ERK-Hypoxia-Angiogenesis Signaling Axis and HIF-1 in Hepatocellular Carcinoma**

Ilias Mylonis and George Simos

*Laboratory of Biochemistry, School of Medicine, University of Thessaly & Institute of Biomedical Research and Technology (BIOMED), Centre for Research and Technology – Thessaly (CE.RE.TE.TH) Greece* 

#### **1. Introduction**

252 Hepatocellular Carcinoma – Basic Research

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Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is associated with high resistance to drugs and high mortality. There are multiple factors that influence its molecular pathogenesis but two well established characteristics of malignant transformation in HCC are its hypervascular nature and the upregulation of the Raf/MEK/ERK signaling cascade. MAPK pathway activation can be triggered by important risk factors of hepatocarcinogenesis such as HBV or HCV infection. Furtermore, its deregulation is well documented in human HCC patients and is associated with poor prognosis (Bruix & Llovet, 2009). Constitutive stimulation of both ERK isoforms has been frequently observed in both HCC samples and hepatocarcinoma-derived cell lines and plays a prominent role in the proliferation, invasion and metastasis of HCC cells (Min et al., 2010). These processes are also associated with neoangiogenesis and aberrant vessel formation, which in turn depend on the development of hypoxic regions and VEGF overexpession frequently observed in tumor samples (Rosmorduc & Housset, 2010). The master regulators of the cellular response to oxygen deprivation are the hypoxia inducible transcription factors (HIFs). Their activation results in expression of many genes that contribute to survival and proliferation of malignant cells and, more importantly, resistance to conventional treatments and poor patient outcome (Poon, E. et al., 2009). Interestingly, hypoxia has been observed to lead to ERK activation, which can further stimulate HIF transcriptional activity. This can result in increased expression of HIF target genes that include pro-angiogenic factors and other proteins that facilitate adaptation of tumor cells to their environment (Dimova et al., 2009). In this chapter, we will discuss the cross-talk between these pathways, their contribution to HCC development and progression and their potential as targets of combined therapeutic approaches.

#### **2. Hepatocellular carcinoma**

Hepatocellular Carcinoma (HCC) is the fifth most common and third in lethality cancer. It is characterized by intrinsic drug-metabolizing activity that confers resistance to

The Involvement of the ERK-Hypoxia-Angiogenesis

(Calvisi et al., 2008).

survival time (Calvisi et al., 2008).

Signaling Axis and HIF-1 in Hepatocellular Carcinoma 255

mechanisms and functions. They are expressed in all cell types and they integrate extracellular signals such as growth factors and cytokines into cellular responses that promote gene expression, proliferation, survival and migration. ERK activation relies on the binding of ligands to cell membrane receptors and the subsequent activation of the RAS/RAF/MEK signaling cascade resulting in the phosphorylation and activation of ERK1/2 (Pearson et al., 2001). It is well documented that sustained ERK activity is important for the development of many types of tumors including hepatocellular carcinoma. As it has been already mentioned, there is activation of Ras pathway and significant increase of phosphorylated ERK levels in all HCC-derived cell lines. However, Ras mutations that could account for sustained pathway activation are rare in human HCCs (Min et al., 2010). One explanation for constistutive ERK activation in HCCs can be that HBV or HCV viral infection interferes and activates the ERK signaling cascade facilitating thus hepatocarcinogenesis (Chin et al., 2007; Zhao et al., 2005). Another reason may lie with the fact that there is global suppression of negative regulators that control the ERK signaling cascade in HCC cell lines. It has been shown that Ras inhibitors are inactivated in HCCderived cell lines probably as a result of deregulated methylation and genomic instability (Calvisi et al., 2006). Furthermore, the Raf kinase inhibitor protein (RKIP) has been observed to be downregulated in HCC cells (Lee, H.C. et al., 2006). Significant decrease has also been detected in the phosphatase DUSP1, which restrains ERK activity by dephosphorylation, as a result of ubiquitin-mediated proteolysis triggered by sustained ERK activation in HCC

The constitutive activation of ERK signaling is essential for cell survival, proliferation and invasion of human HCC cells since Ras pathway inhibition results in growth suppression and cell death. Moreover, siRNA-mediated silencing of ERK2 or elimination of ERK1/2 phosphorylation by silencing of MEK1 resulted in inhibition of cell proliferation and tumor growth suppression in xenograft animal models (Bessard et al., 2008; Gailhouste et al., 2010). Activation of the Raf/MEK/ERK pathway in human HCC biopsy samples can be used as a biomarker of the disease because nuclear phosphorylated ERK levels are higher in tumor samples (Abou-Alfa et al., 2006). Finally, ERK pathway activation is associated with poor patient outcome whereas, inversely, elevated expression levels of pathway inhibitors such as DUSP1 are observed in tumor samples of patients with longer

The importance of the ERK pathway for HCC occurrence and progression made it an important candidate for targeted therapeutic approaches as shown in case of sorafenib. Sorafenib, a multikinase inhibitor, impedes cell proliferation by targeting the Raf/MEK/ERK signaling cascade at the level of Raf kinase (for which it was originally developed) and also exhibits antiangiogenic properties by targeting the tyrosine kinase activities of the vascular endothelial growth factor receptor-2/-3 (VEGFR-2/-3) and the platelet derived growth factor receptor beta (PDGFR-β) (Liu, L. et al., 2006; Wilhelm et al., 2004). Sorafenib has been recently approved as the first effective systemic drug for treating advanced HCC showing a significant (almost 3 months) increase in patient survival. Also, due to its tolerability in single agent trials, it has been evaluated in combination with other anticancer therapies, including cytotoxic chemotherapy and anti-angiogenic therapy (Llovet et al., 2008). The effectiveness of sorafenib in clinical evaluation highlights the potential of targeting the ERK pathway in the treatment of HCC. Recent experimental studies have

chemotherapeutic treatment. Mortality is associated with metastasis, recurrence and new tumor development and diagnosis is usually made at intermediate or late stage so that only ~ 20% of cases can undergo surgery that includes resection, ablation or transplantation. Unfortunately, due to the genomic heterogeneity of HCC tumors, the exact molecular pathogenetic and oncogenic alterations that lead to HCC initiation and progression are not completely understood and require further investigation. However, there are several risk factors and pathways known to be associated with the occurrence and development of this type of cancer (Villanueva et al., 2010).

Probably the most important predisposing factor for the development of HCC is liver cirrhosis that usually results from viral infection (HBV or HCV), alcohol abuse or contamination with Aflatoxin B1. Other contributing factors include inflammation and non-alcoholic fatty liver disease (NAFLD). All of these factors can lead to HCC development by triggering cellular events such as proto-oncogene activation, ROS generation and genetic alterations or instability (Frau et al., 2010). Genetic studies of human HCCs resulted in the identification of gene mutations and expression profile alterations. The identified deregulated genes can be associated with important signaling pathways and shed more light in the molecular events that contribute to HCC pathogenesis. According to these analyses, there are three subgroups of human HCC cancers, genetic profile of which corresponds to the deregulation of specific signaling events (Hoshida et al., 2010). One of them is characterized by over-expression of growth factors (EGF, IGF II and HGF being the most prominent) and induction of major signaling pathways such as PI3K/AKT/mTOR and RAS/RAF/MAPK, which affect cell proliferation and survival and, moreover, contribute to the aggressive phenotype of the disease. In another subgroup, the affected genes are related to cell differentiation and liver development. These genes are found downstream of the WNT or the highly similar Hedgehog pathway and their involvement in human hepatocarcinogenesis is still under investigation. Whereas, in the third less-defined group, the early stages of the disease are linked to inflammation-related pathways, with interleukin-6 being a major signaling molecule (Villanueva et al., 2010; Zender et al., 2010). However, all these signaling pathways do not function independently in the context of HCC tumors but they cooperate and influence one another contributing to the progression of the disease.

From these and other studies it has been made clear that ERK pathway activation and neoangiogenesis are two characteristics of HCCs that greatly facilitate malignant transformation, as they are involved in tumor development, growth and metastasis. Another aspect of HCC, common to many solid tumors, is the creation of hypoxic areas as a result of increased metabolic rate, irregular angiogenesis and tissue inflammation (Rosmorduc & Housset, 2010). The central regulatory elements of cell response to oxygen deprivation are the hypoxia inducible transcription factors (HIFs). After their activation, HIFs induce the expression of their targets, which in their turn facilitate adaption of the cells to the hypoxic environment of the tumor and contribute to survival, proliferation and aggressiveness of cancer cells.
