Molecular targets and oxidative stress biomarkers in hepatocellular carcinoma: an overview

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer representing the 85% of liver cancers. Other types of liver cancer include cholangiocarcinoma, which starts in the cells that line the bile duct, angiosarcoma (or haemangiosarcoma), which starts in the blood vessels of the liver, and hepatoblastoma which is very rare and usually affects young children.

HCC accounts for up to 75% to 85% of primary liver cancer in the United States (U.S.) [1] and for over 90% in high-risk areas. It predominantly affect people in developing countries, such as sub-Saharan Africa, China, Taiwan, Korea, or Vietnam [2, 3].

The incidence has been increasing in recent years in the Mediterranean countries, including Italy, where the incidence and mortality rates are at a median frequency compared to other populations, and it represents the seventh cause of death for tumor, with about 5,000 deaths per year [4‐6].

Liver cirrhosis is present in about 90% of HCC [7] mainly caused by chronic infection by hepatitis B (HBV) and C (HCV) viruses [2, 8‐12] and/or alcohol assumption.

Race, heavy alcohol use, cigarette smoking, obesity, and mellitus diabetes have also been associated with an increased risk of developing HCC. HCC is now more often associated with HCV, particularly in developed countries. On the other hand, HCC is now decreasing in HBV endemic countries due to the implementation of vaccination programs while it is increasing in cohorts who have been infected with chronic HCV [13‐22].

The molecular mechanism of hepatocarcinogenesis is very intricated. Cancer cells have defects in regulatory genes that govern normal cell proliferation and homeostasis due to a progressive accumulation of mutations. The alterations in cell physiology that collectively dictate malignant growth are: i) self-sufficiency in growth signals (activation of oncogenes); ii) insensitivity to growth-inhibitory signals (inactivation of anti-oncogenes or tumor suppressor genes); iii) escape from apoptosis; iv) limitless replicative potential; v) neo-angiogenesis and tissue invasion and metastases [23].

In fact, hepatocarcinogenesis is considered a multistep process involving subsequent mutations of genes that control proliferation and/or apoptosis in the hepatocytes subjected to continuous inflammatory and regenerative stimuli, starting from the initial phases of chronic hepatitis and then of liver cirrhosis.

HCC is associated with, and preceded by, a number of morphologically distinct lesions. The latter are collectively described as 'preneoplastic lesions', and include dysplastic foci and dysplastic nodules. Hepatic nodules in patients with chronic liver diseases are subdivided into regenerative nodules (mono acinus and multi acinus), low-grade dysplastic nodules, high-grade dysplastic nodules, well-differentiated HCC, moderately-differentiated HCC, and poorly-differentiated HCC, in an ascending order of histologic grades, representing a sequence of multistep hepatocarcinogenesis. Accumulation of genetic alterations in the preneoplastic lesions is believed to lead to the development of HCC. Genomic alterations occur randomly, and they accumulate in dysplastic hepatocytes and HCC. Although genetic changes may occur independently of etiologic conditions, some molecular mechanisms have been more frequently related to a specific etiology [24‐26].

Under normal physiological conditions, hepatocyte turnover is very low with a half-life estimated at 6 months. However, adult liver cells retain the remarkable capacity to proliferate in response to injury or to the loss of liver mass. Progenitor cells (also referred to as oval cells) do not play a major role in this growth response but, the same 'resting' differentiated hepatocytes re-enter the cell cycle and replicate once or twice during the period of mass restoration before returning to a state of quiescence. In about 40% of HCC, progenitor cells express peculiar bio-markers (CK-7, CK-19, CD34) associated with a poor prognosis and with disease recurrence [27].

The carcinogenesis and progression of HCC is a complex multistep process that involves multiple genetic aberrations. The molecular mechanisms involved in development and progression of HCC are still largely unknown. On the other hand, different molecular markers have been considered as prognostic factors for HCC. To deepen the molecular mechanisms underlying HCC carcinogenesis and progression is important for improving prognosis and treatment strategies.

Several molecular pathways involved in the regulation of proliferation and cell death are implicated in the hepatocarcinogenesis (Figure 2). In fact, experimental studies have shown structural genomic changes in very early stages of hepatocarcinogenesis. Genomic instability, rearrangements and transactivation of Ras and β-catenin signaling are induced by the integration of HBV into hepatocyte genome [26, 56]. HCV core protein also upregulates TGF-α and IGF-2 [57‐60].

The most common genetic alterations in HCC can be grouped into 3 main routes: i) p53- ii) Wnt- and iii) RB1-dependent pathways [61]

The binding of Wnt proteins to specific Frizzled receptors on the surface of target cells activates distinct intracellular pathways. This results in the accumulation and nuclear localization of the β-catenin protein characteristic of canonical Wnt pathway activation that targets specific genes including cyclin D1, c-Myc, and survivin, which are critical for cancer development [62, 63]. In fact, a transgenic mice model suggested that high expression of Wnt-1 could be the major cause for nuclear accumulation of β-catenin, which subsequently contributes to c-myc/E2F1-driven hepatocarcinogenesis [64]. Clinical studies have reported that abnormal activation of Wnt/β-catenin pathway is frequently involved in hepatocarcinogenesis. About 33-67% of HCC tissues show accumulation of β-catenin in the cytoplasm and nucleus, whereas no accumulation was observed in the corresponding normal tissues [65, 66]. In addition, upregulation of upstream elements such as Frizzled receptors was reported to be involved in HCC development and progression [67, 68]. The activation of Wnt/β-catenin signaling was abolished by a knockdown of Frizzled-7 receptor expression by siRNA. More important, a specific Wnt3-Frizzled-7 receptor interaction was observed by co-immunoprecipitation experiments, which suggest that the action of Wnt3 was mediated via Frizzled-7 receptor [69].

In HCC, proteomics results suggested that enhanced Wnt-1 expression associated with NF-kB might be an important mechanism underlying hepatocarcinogenesis [70].

MAPK cascade transduces signals from tyrosine kinase receptors, such as EGFR, IGFR, Platelet-derived growth factor receptor (PDGFR), Hepatocyte growth factor receptor (HGF/MET), and Vascular endothelial growth factor receptor (VEGFR). In this cascade, active Ras (Ras-GTP) triggers the sequential activation of RAF-1, MEK-1/2, and ERK-1/2. The activation/phosphorylation of ERK1/2 allow to enter into the nucleus where transactivates numerous growth-related genes, including c-JUN, c-FOS, c-MYC (involved in the proliferation and survival mechanisms), vascular endothelial growth factor (VEGF) and hypoxia-induced factor (HIF-1α) that regulates angiogenesis, and HKII (Hexokinase II) [71‐73]. The constitutive activation of ERK1/2 can determine an increase of cell proliferation also in absence of growth factor. This condition can lead to tumour progression.

Genes that are components of MAPK cascade, such as Ras-GTP, c-RAF, c-FOS, and c-JUN, may be upregulated in HCC induced in rodents [58, 74]. 3-Hydroxy-3-methylglutaryl-CoA reductase gene, encoding a key enzyme for de novo synthesis of mevalonate, a precursor of isoprenoid residues necessary for activation of Ras, is upregulated in rat and human liver lesions [75].

Recent studies have shown high levels of active Ras, accompanied by modest/no increase in active RAF-1 and pMEK-1/2, in HCC. This is compatible with the strong induction of the inhibitors of phosphorylation/activation of RAF-1 and MEK-1/2: disabled homolog 2 (Dab2), and RAF kinase inhibitory protein (RKIP), respectively [73].

Up-regulation of principal mediators of the pathway, H-ras and B-RAF, was detected in HCC confirming their role in cancer. Different mechanisms account for Ras signaling in HCC, including:

i) H-ras overexpression; ii) DNA copy number gains in B-RAF genomic locus (chromosome 7q34); iii) epigenetic mechanisms involving the methylation of tumor suppressor genes RASSF1A and NORE1A [76].

The Ras-RAF-ERK-dependent pathway is implicated in the molecular pathogenesis of HCC for three reasons: i) Ras protein is activated in the 30% of cases of HCC [77]; ii) the over-expression of Raf kinase is in the majority of HCC [78]; iii) several upstream growth factors, such as EGF, VEGF, PDGF, TGFα, generally over-expressed in HCC, can activate this pathway binding proper tyrosin kinase receptors [79].

Recently developed technology, such as DNA microarrays and other molecular profiling techniques, has provided new insights into the molecular genetics of HCC [80, 12].

HCC are classified in metabolic pathways, and the most represented are the Aryl Hydrocarbon receptor signalling (AHR), involved in the activation of the cytosolic aryl hydrocarbon receptor by structurally diverse xenobiotic ligands (including dioxin, and polycyclic or halogenated aromatic hydrocarbons) and mediating their toxic and carcinogenic effects [81] and, protein Ubiquitination pathways, involved in cell-cycle regulation as well as cell death/apoptosis [82] through modification of target proteins.

Moreover, molecular profiling has been successfully used to identify candidate genes for HCC such as genes correlated with tumour progression (p16, SOCS1, PEG10), metastatization (NM23-H1, osteopontin, RhoC, KAI1, MMP14) or recurrence (REL, A20, vimentin, PDGFRA) [83].

Studies of mechanisms of oxidative stress have shown that it activates signaling cascades (including MAPK pathway), which can seriously influence regulation of cell growth and transformation processes [84]. Particularly, MAP kinases may be involved in pathogenesis of some diseases associated with oxidative stress.

It is known that the oxidative stress status has a key role in HCC development and progression.

The most important reactive oxygen species (ROS) derived by molecular oxygen include free oxygen radicals [e.g., superoxide (O₂•-), hydroxyl radical (OH^.), nitric oxide (NO^.) radicals] as well as nonradical ROS [e.g., hydrogen peroxide (H₂O₂), organic hydroperoxides, and hypochloride].

A low level of ROS is indispensable in several physiologic processes of the cell including proliferation, apoptosis, cell cycle arrest, cell senescence, etc. [85]. However, an increased level of ROS causes oxidative stress and creates a potentially toxic environment to the cells. In normal physiologic condition, a balance between ROS generation and oxidative defences exists in a cell. A significant role is played by endogenous antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) that act on O₂•- and H₂O₂, respectively, and glutathione peroxidase (Gpx1) that uses glutathione as co-substrate. Despite the basal production of radicals is hampered by the anti-oxidant defences, the generation of ROS is amplified in response to various environmental perturbations.

This stressful condition is known to play a major role in cancer development mainly by enhancing DNA damage and by modifying some key cellular processes, such as DNA damage caused primarily by hydroxyl radicals [64], cell proliferation, apoptosis, and cell motility cascades by superoxide radicals and hydrogen peroxides playing an important role in cancer development.

Although extensive or limited damage may trigger cell death, many cells can tolerate and repair the occasional hit from ROS. In the Fruehauf model [86], when the balance tips further in favour of ROS, programmed cell death becomes a near certainty.

The increase of ROS is associated with the increase of the inducible mitochondrial manganese SOD (MnSOD) expression. Elevated serum MnSOD levels have been found in patients with HCC [87] and relatively high values of the enzyme have also been observed in patients with chronic hepatitis and liver cirrhosis. Therefore, it could be hypothesized that during induction of the malignant process in cirrhotic liver, the increase in MnSOD activity can already occur in the precancerous phase.

In cancer biology, NO can be involved either in promotion or in prevention of tumour occurrence dependently from tumour microenvironment, NO concentration and time of exposure [88]. NO is a product of endothelial cells that binds and activates the guanylate cyclase, which catalyzes the conversion of GTP to the second messenger molecule cyclic GMP (cGMP). Concentrations of NO ranging between 1 and 30 nM produce high levels of cGMP promoting angiogenesis and proliferation of endothelial cells. In these conditions, ERK phosphorylation stimulates the proliferation of endothelial cells. Concentrations of NO ranging between 30 and 100 nM correspond to an increase of proliferative and anti-apoptotic AKT and ERK-dependent pathways in tumour cells [89‐91]. This range of concentrations seems to protect tumour cells from apoptosis and enhance angiogenic effects. In these conditions, the molecules activated by NO can be considered as factors correlated to poor prognosis events. On the other hand, higher NO levels (> 300 nM) promote apoptosis and are responsible for anti-tumour activity. NO levels are influenced also by ROS and, specifically, by superoxide anions that can attenuate the NO-mediated pathway. In fact, superoxide anions and ROS, through the scavenging of NO, can lower NO levels favouring its tumour-promoting activity [92]. Accordingly, tumours have high levels of ROS and low levels of SOD.

Similarly to oxidative stress, the expression of nitrosative stress supports the de-regulated synthesis or overproduction of NO and NO-derived products and its toxic physiological consequences [93]. The main source of NO in the mammals is the enzymatic oxidation of L-arginine by NO synthases [94]. As ROS, NO may limit oxidative damage by acting as a chain-breaking radical scavenger or may cause damage and kill cells by mechanisms that include inhibition of protein [95] and DNA [96] synthesis, downregulation of antioxidative enzymes [97] and depletion of intracellular GSH [98]. Nitrosative insult may occur in vivo also in pathologies associated with inflammatory processes, neurotoxicity and ischaemia [99].

NO is able to reduce oxidative injury via several mechanisms. NO reacts with peroxy and oxy radicals generated during the process of lipid peroxidation. The reactions between NO and these ROS can terminate lipid peroxidation and protect tissues from ROS-induced injuries [100]. Through the Fenton reaction, hydrogen peroxide oxidizes iron (II) and the process generates an extremely reactive intermediate (the hydroxyl radical) which then carries out oxidations of different substrates [H₂O₂ + Fe²⁺ → Fe³⁺ + OH- + hydroxyl radical (·OH)]. NO prevents hydroxyl radical formation by blocking the predominant iron catalyst in the Fenton reaction. In fact, NO reacts with iron and forms an iron-nitrosyl complex, inhibiting iron's catalytic functions in the Fenton reaction [101].

Treatment of rat hepatocytes with NO induces resistance to H₂O₂-induced cell death by induction of the rate-limiting antioxidant enzyme, heme oxygenase (HO-1) [102]. In addition, NO prevents the induction of some ROS-induced genes during tissue injury such as early growth response-1 (EGR-1), which activates a number of adhesion molecules and accelerates oxidative tissue injuries [103].

Regulatory events and their alterations depend on the magnitude and duration of the change in ROS or RNS concentration. ROS and RNS normally occur in living tissues at relatively low steady-state levels. The increase in superoxide or NO production leads to a temporary imbalance that forms the basis of redox regulation. The persistent production of abnormally large amounts of ROS or RNS, however, may lead to persistent changes in signal transduction and gene expression, which, in turn, may give rise to pathological conditions [104].

Ablative therapies, surgical resection or liver transplantation are the first-line treatment for patients affected by HCC [112, 113]. Nonetheless, advanced tumour stage and poor liver function preclude the majority of patients from these surgical interventions [114]. Moreover, transplantation is indicated only for early small HCC, and its application is limited by the availability of liver grafts [115]. Therefore, it is mandatory to develop an effective systemic therapy for patients with advanced HCC.

HCC is a chemo-resistant tumour and conventional cytotoxic chemotherapy has not provided clinical benefit or prolonged survival for patients with advanced HCC [116].

In recent years, emerging insights into the biology and molecular signalling pathways in cancer cells have led to the identification of potential targets for intervention and the advent of promising targeted therapy for the treatment of HCC (Table 1).

One strong mechanistic link between chronic inflammation and cancer is through the increased production of free radicals at the site of inflammation and the resulting molecular changes, which include lipid peroxidation and oxidative DNA damage [151]. Indeed, markers of DNA damage, such as 8-hydroxydeoxyguanosine (8-OHdG), and lipid peroxidation, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), are commonly elevated in liver of patients with chronic HCV infection and correlate well with the degree of viral infection and inflammation, known risk factors for HCC [152].

In addition to the classical genetic mechanisms of deletion or inactivating point mutations, epigenetic alterations, such as hyperacetylation of the chromatin-associated histones are believed to be involved in the development and progression of HCC. Histone deacetylases (HDACs) are important regulators of many oxidative stress pathways including those involved with both sensing and coordinating the cellular response to oxidative stress. In particular aberrant regulation of these pathways by HDACs may play critical roles in cancer progression. Infact, HA-But, an HDAC inhibitor in which butyric acid residues are esterified to a hyaluronic acid backbone and characterized by a high affinity for the membrane receptor CD44, valproic acid and ITF2357, exhibiting inherent therapeutic activity against HCC may represent a promising approach for HCC treatment [153, 154].

It is well known that inflammation is one of the biological responses driven by oxidative stress. Modulation of oxidative damage as well as inflammation protect against hepatocarcinogenesis. It has been shown that resveratrol, a compound present in grapes and red wine, has potent antioxidant [155] and anti-inflammatory [156] properties, which might play an important role in protecting the liver against carcinogen-induced neoplasia. Recently, it was reported that resveratrol significantly prevents diethylnitrosamine (DENA)-induced liver tumorigenesis in rats [157].

HCC is a disease that presents two relevant concerns: i) the presence of a cirrhotic background that severely affects both the quality of life and the survival of the patients and ii) the pleiotropic pathogenesis that has as common background: the chronic inflammation and the oxidative stress. The pharmacological weapons against HCC are still limited and efficacy has been established only for the multiple kinase inhibitor sorafenib. We have recently demonstrated that sorafenib plus octreotide is a safe and effective option in advanced HCC patients with compromised metabolic scores and/or low performance status. A still limited covered area of research in HCC is represented by the oxidative stress that underlies primary liver tumour development and that occurs through the generation of ROS and/or RNS and that is regulated by several scavenger mechanisms. On this view, we have found that the determination of oxidative stress status has high value in the prediction of response to sorafenib plus octreotide therapy in HCC patients. These data could have a profound impact in the determination of the sensitivity of the patients to this pharmacological strategy and could have a role in the selection of the patients to be subjected to this treatment. This could reduce both the relevant side effects correlated to the therapy and the relevant costs derived from the use of expensive drugs such as the new target based agents such as sorafenib. The factors involved in the oxidative stress could have a role not only in the prediction of response to pharmacological treatments but could be themselves targets of drugs as in the case of the stress-dependent kinases p38 kinase and Jun kinase or in the case of the use of anti-oxidant agents such as resveratrol or silibin. The investigations on oxidative stress and on its connection with signal transduction pathways correlated to survival and/or proliferation could disclose new scenarios of interventions based on the rational use of anti-oxidant agents in combination with target based drugs.