**3. ERK pathway and HCC**

Extracellular-signal regulated kinases 1 and 2 (ERK 1/2 or else p44/42 MAPK) are serine/threonine protein kinases both homologous and highly similar in their regulatory

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

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

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

Extracellular-signal regulated kinases 1 and 2 (ERK 1/2 or else p44/42 MAPK) are serine/threonine protein kinases both homologous and highly similar in their regulatory

type of cancer (Villanueva et al., 2010).

another contributing to the progression of the disease.

aggressiveness of cancer cells.

**3. ERK pathway and HCC** 

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 (Calvisi et al., 2008).

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 survival time (Calvisi et al., 2008).

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

The Involvement of the ERK-Hypoxia-Angiogenesis

**5. Hypoxia and HCC** 

correlate to its progress.

advanced hepatocellular carcinoma in 2007 (Fernandez et al., 2009).

hypoxia as opposed to other cancer cell lines (Gwak et al., 2005).

within the cell are the hypoxia inducible factors (HIFs).

Signaling Axis and HIF-1 in Hepatocellular Carcinoma 257

Since vascular formation is very important for tumor progression and transition to malignancy, it represents an intriguing therapeutic target for the treatment of HCC. At present, there are several drugs undergone investigation in experimental models or humans and some of them are already in use in various types of cancer. Most of the tested inhibitors target selectively the VEGF pathway and induce arrest of endothelial cell proliferation, regression of the existing vessels or suppress the mobilization of endothelial progenitor cells from bone marrow. These agents range from monoclonal antibodies (mAb) targeting VEGF-A, like bevacizumab (Avastin), to small molecules that inhibit autophosphorylation of VEGF receptors like sorafenib (Nexavar), the single agent that was approved for the treatment of

As many other solid tumors, HCC is characterized by the development of hypoxia. Even in normal liver, emergence of hypoxic conditions can result from its division into areas with different capabilities of oxygen delivery (i.e., 60 to 65 mmHg in the portal area to 30 to 35 mmHg in the perivenous area) and increased cellular metabolic activity, which leads to high oxygen consumption (Rosmorduc & Housset, 2010). Apart from that, the response to trauma or inflammation can cause aberrant vessel formation, irregular blood flow and, finally, poor oxygenation of the cells. Cells respond to oxygen deprivation by activating a number of genes that allow them to adapt and survive. More importantly, hepatocellular carcinoma cells not only survive but also show significant stimulation of their proliferation under

Hypoxia promotes tumor progression through several mechanisms. Under hypoxic conditions, cells shift their metabolism from oxidative phosphorylation to anaerobic glycolysis by the induction of genes that encode glucose transporters, glycolytic enzymes and proteins that remove glycolysis by-products (e.g. lactic acid) from cancer cells. As mentioned above, HCC tumors exhibit epigenetic alterations and signaling pathways activation that stimulate proliferation. These may be supported by tumor hypoxia, which affects the methylation status of HCC cells and, more importantly, activates the ERK pathway (Liu, Q. et al., 2011; Minet et al., 2000). Another very important contribution of hypoxia to HCC development is the stimulation of angiogenesis. Two of the central proangiogenic factors in HCC, namely VEGF and angiopoietin 2, are induced in hypoxic conditions and promote the abnormal vessel formation and branching observed in these tumors. Key mediators of these responses and central components of hypoxia signaling

HIFs are heterodimeric transcriptional complexes that respond to changes of cellular oxygen concentration and activate the expression of hypoxia target genes. These genes encode for proteins involved in processes critical for oncogenesis such as survival, proliferation, invasion and metastasis. Active HIF heterodimers are composed of the constitutively expressed HIF-β subunit (or ARNT; Aryl hydrocarbon Receptor Nuclear Translocator) and the regulated HIF-α subunit, which is over-expressed in many tumors and causes HIF activation and increased transcription of its targets (Semenza, 2010). HCC is not an exception and expression levels of HIF-1α are increased in all stages of the disease and

shown that treatment with MEK inhibitors (AZD6244, PD0325901) inhibited cancer cell proliferation and tumor growth in human HCC cell lines and HCC xenografts in animal models (Hennig et al., 2010; Huynh et al., 2007). Moreover, when AZD6244 was tested in combination with sorafenib, it enhanced the anti-tumor activity of sorafenib, suggesting the potential value of ERK pathway inhibition in combinational therapeutic approaches (Huynh et al., 2010).
