**5. Hypoxia and HCC**

256 Hepatocellular Carcinoma – Basic Research

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

HCC progression requires the development and maintenance of adequate blood supply. This requires vascular endothelial cell proliferation and migration in order to establish a new vascular network. The transformation from a poorly vascular nodule to the hypervascular malignant phenotype of the disease has become a hallmark for diagnosis, treatment and possible future therapeutic approaches (Fernandez et al., 2009). Formation of new vessels with abnormal architecture is linked to fibrinogenesis and cirrhosis, processes that cause extensive cell damage and can lead to malignancy (Rosmorduc & Housset, 2010). The ability to form new vessels is critical not only for the emergence of HCC but also for the growth of metastatic nests and the transition from early to advanced stages of the disease. Indeed, impairing the blood supply that feeds the tumor with transarterial chemoembolization is an effective treatment for patients with advanced HCC and improves

Neoangiogenesis in HCC is a process stimulated by hypoxia, growth factors, oncogenes and nutrient concentration followed by the release of pro-angiogenic growth factors and activation of endothelial cells. The most prominent pro-angiogenic signal, as revealed in a number of studies, is vascular endothelial growth factor (VEGF) (Fernandez et al., 2009; Rosmorduc & Housset, 2010). Its expression can be induced as a result of hypoxia, oncogenic signaling and viral infection, involving MAPK pathway activation and transcriptional regulation by AP1 and HIF-1α (Hassan et al., 2009). Its significant role in HCC is shown by the fact that cells isolated from human tumors are able to produce VEGF by themselves. Hypoxia further enhances VEGF expression both by regulation at the transcriptional level and by stabilization of VEGF mRNA (von Marschall et al., 2001). Moreover, VEGF and its receptors (VEGFR-1 and VEGFR-2) are found over-expressed in patients' samples and their increased levels have been associated with the aggressive phenotype of the disease (Imura et al., 2004). In line with this observation, increased concentration of VEGF in serum samples is directly connected to poor patient outcome after

Other factors that promote tumor angiogenesis in HCC are FGFs (Fibroblast growth factors), angiopoietins (Ang-2) and platelet-derived growth factor (PDGF). There is evidence that bFGF over-expression is involved in vascular endothelial cell proliferation during tumor angiogenesis in human HCC (Imura et al., 2004). Furthermore, Ang-2 levels as well as its activity through the Tie-2 receptor are increased in human samples and have been correlated with neovascularization and microvessel density (MVD) (Mitsuhashi et al., 2003). Finally, animal studies have shown that the involvement of PDGF-C in HCC development is linked to its role in the initiation of fibrosis (Campbell et al., 2005). Interestingly, it appears that all these factors team up with the VEGF signaling cascade in order to provoke normal

resection or ablation (Poon, R.T. et al., 2004; Poon, R.T. et al., 2007).

et al., 2010).

**4. Angiogenesis and HCC** 

their survival (Llovet & Bruix, 2003).

or aberrant vessel formation in HCCs.

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

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

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 correlate to its progress.

The Involvement of the ERK-Hypoxia-Angiogenesis

Signaling Axis and HIF-1 in Hepatocellular Carcinoma 259

In terms of its oxygen-dependent or -independent regulation, HIF-1α is more extensively investigated. HIF-2α, on the other hand, is regulated in a similar fashion by oxygen but many elements of its oxygen-independent regulation still remain unclear. Under normal oxygen conditions, HIF-1α is constantly produced and destroyed in a process that involves von Hippel-Lindau (VHL)-mediated polyubiquitination and subsequent proteasomal degradation (Schofield & Ratcliffe, 2005). Interaction of HIF-1α with VHL, a tumor suppressor protein and a subunit of an E3 ubiquitin ligase complex, requires the hydroxylation of two proline residues within the ODD domain of HIF-1α. This hydroxylation is mediated by three conserved prolyl hydroxylases (termed PHDs or HPHs), the activity of which depends on the availability of oxygen, iron and 2-oxoglutarate (Semenza, 2001). When oxygen becomes sparse, hydroxylation is impaired, HIF-1α is stabilized and is imported into the nucleus, where it dimerizes with ARNT and activates the expression of its target genes. However, oxygen dependent-regulation does not only rely on HIF-1α destruction alone but also on the control of its activity. Another oxygen-sensitive hydroxylase called FIH (Factor Inhibiting HIF-1) modifies HIF-1α in one asparagine residue situated inside its C-terminal TAD region (Asn803) and interferes with the association of

HIF-1α with the transcriptional co-activator CBP/p300 (Lancaster et al., 2004a).

Apart from oxygen tension, HIF-1α expression and activity are additionally stimulated by oxygen-independent mechanisms that respond to oncogenic activation, growth factors, cytokines and variations of the cellular environment (e.g. pH). There are multiple levels at which cells can control HIF-1α activity, ranging from transcriptional and translational regulation to post-translational modifications. Its transcriptional activation responds to inflammatory stimuli as IKKβ activation causes increased HIF-1α gene expression in a NFκB-dependent fashion in the liver of hypoxic mice (Rius et al., 2008). The signal transducer and activator of transcription 3 (Stat3) is also involved in the regulation of HIF-1α mRNA synthesis (Niu et al., 2008) and mediates the transcriptional suppression of HIF-1α by the eIF2α kinase PKR (Papadakis et al., 2010). Activation of the phosphatidylinositol 3 kinase/AKT pathway leads to elevated translation of HIF-1α mRNA and increased HIF-1α protein levels (Bardos & Ashcroft, 2005). Post-translationally, HIF-1α is regulated either through its association with other proteins or its modification by number of different enzymes. Protein interactions like binding to the molecular chaperone HSP90 can increase HIF-1α stability by inhibiting VHL-independent degradation (Isaacs et al., 2002; Katschinski et al., 2004). On the other hand, the protein RACK1 competes with HSP90 for binding to the HIF-1α N-terminal PAS-A domain. The RACK1-HIF-1α interaction, which is stabilized by the protein SSAT1 and inhibited by Sept9-v1, promotes increased ubiquitination and degradation of HIF-1α irrespective of oxygen levels (Amir et al., 2009; Baek et al., 2007; Liu, Y.V. et al., 2007). MgcRacGAP (male germ cell RacGTPase Activating Protein), identified using the yeast two-hybrid system, is another protein that interacts with and inhibits HIF-1α. MgcRacGAP over-expression inhibits HIF-1α transcriptional activity, without lowering HIF-1α protein levels or altering its subcellular localization (Lyberopoulou et al., 2007).

Apart from hydroxylation, other posttranslational modifications of HIF-1α include SUMOylation, acetylation, S-nitrosylation and phosphorylation by a number of different kinases. HIF-1α can be SUMOylated but the role of this modification remains controversial; certain reports claim that SUMO conjugate stabilizes HIF-1α while others suggest that deSUMOylation of HIF-1α is necessary for its stability and activity (Bae et al., 2004; Berta et al., 2007; Carbia-Nagashima et al., 2007; Cheng et al., 2007). In contrast,

There is plenty of evidence linking HIF-1α to both early and late HCC stages. The genetic instability observed during the premalignant state of HCC can be caused by viral infection. HCV core protein induces HIF-1α and HIF-dependent transcriptional activation of the VEGF gene in HCC-derived cells (Hassan et al., 2009). Liver angiogenesis has been indeed observed in biopsy samples of HCV patients and is possibly an essential step for HCVrelated oncogenesis. HIF-1α mRNA and protein levels are also increased in premalignant dysplastic nodules, as observed in both human and animal samples, and cause upregulation of a number of genes that promote angiogenesis (VEGF), glucose transport (GLUT1) and PI3K/AKT pathway activation (growth factor IGF-II and hepatocyte growth factor receptor c-Met) (Nakamura et al., 2007). Interestingly, it has also been shown that HIF-1α expression at this stage is hypoxia-independent and its levels rise as the disease progresses (Tanaka et al., 2006). These findings indicate the important role of this transcription factor in abnormal gene expression that occurs during early HCC development.

As already mentioned, hypervascularity is a prominent feature of progression to HCC malignancy and HIF-1α over-expression is also directly associated with VEGF expression, microvessel density (MVD) and microvenous invasion in human HCC samples (Huang et al., 2005). Deregulation of the HIF pathway, which comes as a result of HIF-1α overexpression, is often associated with resistance to radiotherapy and chemotherapy, which renders hypoxic tumors highly aggressive and metastatic (Poon, E. et al., 2009). This is also true for primary HCC, in which HIF-1α expression is associated with poor response to radiotherapy, metastasis and low survival rates (Xiang et al., 2011). Therefore, understanding HIF-α regulation may provide valuable information in order to target the HIF pathway as a means for combinational therapeutic strategies against HCC.
