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Biology, Epidemiology, Clinical Aspects of Hepatocellular Carcinoma and the Role of Sorafenib Gianluigi Mazzoccoli1,*, Luca Miele2,§, Jude Oben3,§, Antonio Grieco2,§ and Manlio Vinciguerra1,3,4,5 1

Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo (FG) Italy; 2Division of Internal Medicine, Catholic University of the Sacred Heart, Rome, Italy; 3University College London (UCL) – Institute for Liver and Digestive Health, Division of Medicine, Royal Free Hospital, London, UK; 4 Istituto EuroMEditerraneo di Scienza e Tecnologia (IEMEST), Palermo, Italy; 5School of Science and Technology, Nottingham Trent University, Nottingham, UK

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Abstract: Sorafenib is a small molecular inhibitor of intracellular tyrosine and serine/threonine protein kinases (VEGFR, PDGFR, CRAF and BRAF), and is thought also to induce autophagy, a chief mechanism influencing tumor growth. Sorafenib shows efficacy in the management of non-resectable hepatocellular carcinoma (HCC), which is refractory to other chemotherapeutic drugs. HCC represents a major end point of chronic liver diseases and the third leading cause of cancer-related death. In HCC patients Sorafenib increases overall survival compared to placebo. The most common chronic liver disease affecting up to 30% of the population in Western countries is non-alcoholic fatty liver disease (NAFLD), an intra-hepatic amassing of triglycerides deemed as the hepatic manifestation of insulin resistance and obesity. NAFLD encompasses a range of disorders with grades of liver damage varying from steatosis to non-alcoholic steatohepatitis (NASH), hallmarked by hepatocellular injury/inflammation in the presence or not of fibrosis. NAFLD patients progress to NASH in 10% of cases, which may progress to cirrhosis and HCC. Recent exciting studies uncovered a potential therapeutic role for Sorafenib that goes beyond HCC, and extends to cirrhotic portal hypertensive syndrome during cirrhosis, and to selective anti-fibrotic effects mediated through direct inhibition of activated hepatic stellate cells (HSC), the cellular mediators of intra-hepatic matrix deposition. The aim of this review is to concisely summarize our current knowledge of the biology, epidemiology and clinical aspects of HCC, as well as the previously under-appreciated therapeutic efficacy of Sorafenib beyond HCC. The review therefore utilizes data along the spectrum of liver diseases, including from experimental via pre-clinical to clinical.

Keywords: Sorafenib, hepatocellular cancer, NASH, HBV, HCV, cirrhosis. INTRODUCTION Hepatocellular cancer (HCC) is one of the most important and common neoplastic diseases in the world [1, 2], being the most common histological type of primary liver tumor [3]. Annually, there are approximately 700000 new cases of liver cancers worldwide with considerable geographical variation. Areas of high incidence include SouthEast Asia (Korea, China and Vietnam) and some areas of Africa (Mozambique), while it is rare in Europe and America [4]. However, in the last 30 years HCC incidence has gradually increased, even in industrialized countries, currently representing up to 3-6% of all solid tumors. Males are most commonly affected (4:1 ratio), with HCC being a rare malignancy for those under the age of 40, mainly occurring in low risk Western populations in people aged 55 years and *Address correspondence to this author at the Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS “Casa Sollievo della Sofferenza”, Opera di Padre Pio da Pietrelcina, Cappuccini Av., San Giovanni Rotondo (FG), - 71013 – Italy; Tel: 0039 (0) 882 410255; Fax: 0039 (0) 882 410255; E-mail: [email protected] §

These authors contributed equally to this work. 1389-4501/16 $58.00+.00

above [5]. Specifically, HCC is the fifth most frequently diagnosed cancer and the second most frequent cause of cancer death in men, whilst it is the seventh most commonly diagnosed cancer and the sixth leading cause of cancer death in women [5]. Experimental evidence suggests that the genesis of HCC has a multiphasic dynamic, in that it proceeds through chronologically distinct phases characterized by specific morphological and molecular alterations [6]. In most cases, HCC develops in the presence of pathological conditions of the liver, usually hepatitis or cirrhosis, although, in some rare cases, it can also arise in healthy liver tissue. Recent data highlight non-alcoholic steatohepatitis (NASH) as an important risk factor, whilst risk is augmented by smoking and reduced by coffee [1]. In particular, a recent meta-analysis of 14 epidemiological studies calculated the summary relative risk (RR) for any, low, and high coffee consumption versus no consumption and supported the protective role played by coffee drinking, considering that it was associated with a HCC risk reduction of approximately 40%. The cut-off point for low versus high consumption was set to 3 cups per day in 9 studies and 1 cup per day in 5 studies. Precisely, the sum© 2016 Bentham Science Publishers

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mary RR was 0.72 (95% CI, 0.61-0.84) for low consumption and 0.44 (95% CI, 0.39-0.50) for high consumption. The summary RR was 0.80 (95% CI, 0.77-0.84) for an increment of 1 cup of coffee per day. The protective effect of coffee was reliable across different populations and subgroups at increased HCC risk, anyway causal relationships and effects of specific coffee components are still to be defined [7]. However, cirrhosis is the most important risk factor in the onset and susceptibility to HCC, which is associated with liver cirrhosis in up to 90% of HCC patients [1]. Tissue alteration resulting in HCC may be triggered by a number of factors, including infection with the hepatitis B virus (HBV) and hepatitis C virus (HCV), alcohol abuse and cirrhosis secondary to genetic and metabolic diseases, such as hereditary hemochromatosis [8]. The results of a meta-analysis of 32 case-control studies reported in the literature showed a HCC relative risk of 20 for carriers of HBV, 24 for HCV carriers and 135 for the simultaneous presence of both markers [9]. Chronic hepatitis and cirrhosis constitute a fertile ground for the emergence of dysplastic and frankly malignant hepatocellular lesions through a characterized multistep process [6]. Telomere shortening plays a crucial role in hepatocarcinogenesis, with an accelerated shortening inducing accumulation in the cirrhotic stage of senescent hepatocytes that are particularly sensitive to undergoing the molecular alterations predisposing to the development of neoplasia, such as chromosomal instability (CIN) [10, 11]. In particular, telomere shortening and inactivation of cell cycle checkpoints concur at specific stages of human hepatocarcinogenesis leading to an accumulation of DNA damage and promoting the development of CIN, which leads to an increase in aneuploidy and chromosomal aberrations [12]. Besides, single-nucleotide polymorphisms (SNPs) of telomere maintenance genes were shown to impact the development of HCC and the clinical outcomes of patients with HBVassociated HCC influencing hepatocarcinogenesis and survival of HCC patients with chronic HBV infection [13]. Importantly, somatic mutations activating telomerase reversetranscriptase promoter were recently identified in 59% of human HCC (significantly associated with activating mutations of CTNNB1, the gene encoding -catenin), 25% of cirrhotic preneoplastic macronodules and 44% of hepatocellular adenomas with malignant transformation in HCC. Up to now, telomerase reverse-transcriptase promoter mutations represent the most initial genetic change frequently found in cirrhotic pre-neoplastic lesions and correspond to the most frequent genetic modification in HCC either associated or not with cirrhosis [14]. The natural history of HCC is complex with its course seemingly relentless, as evidenced by the overlap of incidence and mortality rates of this cancer worldwide [15]. The incidence and mortality overlap reflects the prevalence of cases diagnosed late, as a consequence of frequent occurrence in geographical areas of high incidence where there are no screening programs and in which more than half of patients have metastases at diagnosis compared with 30% in industrialized countries [16]. The natural history of HCC is intricate and inexorable due also to the presence of cirrhosis, which aggravates the clinical course and, in general, restricts therapeutic interventions. For this reason, the most widely used staging systems of HCC considers not only tumor pro-

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gression, but also of the severity of the underlying cirrhosis. The prognosis of cirrhosis is different according to whether it is in a decompensated stage (i.e. in the presence of ascites, portosystemic encephalopathy, gastrointestinal bleeding due to portal hypertension, spontaneous bacterial peritonitis, hepatorenal syndrome, HCC), or in a compensated stage (in the absence of the above complications). The Child-Pugh score includes three different classes: A (5, 6 points), B (7-9 points) and C (10-15 points), each of which is associated with prognosis variability. The sum of the points associated with the values and the degree of each of the individual parameters determine the prognostic Child-Pugh class. In the United States, the Barcelona Clinic Liver Cancer (BCLC) system is the principal system, on account of its simultaneous consideration of tumor stage, liver function, and physical status, in addition to its ability to classify patients with earlystage disease who may benefit from available therapies [17]. MOLECULAR MECHANISMS OF HEPATOCELLULAR CARCINOGENESIS The traditional classification of tumors based on clinical and pathological criteria has been enriched by molecular parameters thanks to an explosion of knowledge in the field of tumor biology over the past 20 years. Unlike clinicopathological data, molecular data take into account the biological substrate of the tumor, providing additional information on the diagnosis, prognosis and therapy. In fact, the type and amount of molecular aberrations of a given tumor are the result of the pathogenic mechanisms and determine the biological behaviour, reflected accordingly in clinical disease presentations [18]. Notwithstanding the complexity of HCC genetics, biomolecular markers have been proposed to be incorporated into clinical management algorithms as indicators for disease diagnosis or prognostic stratification [19, 20]. In addition, thanks to the identification of specific molecular aberrations, the approach to antineoplastic pharmacology has changed dramatically, from a pharmacological treatment based on the disease to therapy addressing the molecular defect [21]. These pharmacological concepts are the basis of targeted therapy, whereby drugs are directed against molecular targets, thereby impinging on signalling pathways active in the tumor [22]. Investigations of the molecular and genetic bases underlying the malignant transformation indicate that many of the abnormalities associated with HCC are due to the abnormal function of receptors with tyrosine kinase activity and their downstream signalling that regulate cell proliferation [23, 24]. The heterogeneity of the etiologic factors highlights the complexity of the molecular mechanisms underpinning HCC (Fig. 1). Tumor development and promotion is accompanied by multifaceted changes in the expression profiles of genes within tumor tissue versus healthy tissue. Even diverse parts of the same tumor differ genetically, molecularly and/or morphologically [6, 24]. Several genes, whose molecular structures are modified over the course of tumor progression, are implicated in cell cycle regulation, which is of importance given that neoplastic tissues rely on hyperproliferation. In particular, c-Myc encodes a nuclear phosphoprotein acting as a multifunctional transcription factor playing a role

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Fig. (1). Scheme rendering the molecular mechanisms and oncogenic pathways involved in hepatocellular carcinogenesis and Sorafenib action in hepatocellular cancer and non-tumor liver diseases; A) WNT / -catenin signalling pathway; B) Ras-MAP kinase signalling pathway; C) PI3K/AKT/mTOR signalling pathway; D) Hedgehog signalling pathway; E) p53 signaling pathway; F) Bcl2 pathway; G) FAS pathway; H) HIF pathway (refer to article text for details and acronyms explanation; source: modified by http://www.genome.jp/kegg/).

in cell cycle progression, apoptosis and cellular transformation. When constitutively expressed c-Myc aberrantly drives the expression of many genes involved in cell proliferation leading to carcinogenesis. In addition, cyclins act as regulators of cyclin dependent kinases (CDK) and contribute to the temporal coordination of mitotic events. In particular, cyclin D1 is encoded by CCND1 and works as a regulatory subunit in complex with CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. Cyclin D1 interacts with tumor suppressor retinoblastoma protein (pRb); in turn the expression of CCND1 is regulated positively by pRb. Cell cycle progression is altered by CCND1 mutations, amplification and overexpression, which play a role in hepatocarcinogenesis. The over-expression of the genes encoding c-Myc, cyclin D1, cyclin A, and E2F1, which leads to the formation of the complexes cyclin D1-CDK4, E2F1-DP1, and pRb hyperphosphorilation, was detected in pre-neoplastic liver lesions in c-Myc/transforming growth factor (TGF)- transgenic (Tg) mice [25] and Fischer 344 rats with induced carcinogenesis through the resistant hepatocyte model [26]. The overall effect of such dysregulation, which is also evident in human HCC, lies in alterations in the transition from the G1 to S phase of the cell cycle, when DNA replication occurs [27].

The absence of a clear hereditary predisposition for the development of HCC, unlike the paradigmatic model of adenoma turning into colorectal carcinoma, has prevented the identification of keys genes and the definition of a hierarchy of genetic events involved in the various stages of liver carcinogenesis. The significant advancement of knowledge and technological tools in the field of molecular genetics and cytogenetics led in the second half of the 90s to a significant increase in the information available about the genetic alterations underlying the genesis of HCC. Molecular biology studies, analysis by MSA (PCR-based Microsatellite Marker Analysis) of "microsatellites" (repetitions of small polymorphic sequences of 2-6 nucleotides scattered throughout the genome), comparative genomic hybridization (CGH), highdensity arrays of single nucleotide polymorphisms (SNPs) and haplotype typization studies have revealed a wide variety of chromosomal alterations, ranging from genomic rearrangements associated with HBV-DNA integration, to the loss of heterozygosity (LOH, loss of one allele) in several loci on a large number of chromosomes, as well as gene amplification (increase in the number of alleles) [28, 29]. The chromosomal alterations in HCC include LOH of the chromosomes 1p, 4q, 8p, 13q, 16q, 17p and amplification or increase of DNA content in the regions of 1q, 8q and 17q [30, 31]. A LOH at 1p is more common in small and well differentiated HCC whereas LOH at 16p and 17q are more frequent in advanced and metastatic tumors. The amplification

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of the distal region at 8q24 is in relation to the overexpression of the proto-oncogene c-myc [29]. Overall, many tumor suppressor genes are inactivated by mutations or deletions (TP53, RB1, CDKN2A, IGF2R, AXIN1, CDH1, BRCA2, PTEN). As regards the tumor suppressor gene encoding p53 (TP53), LOH at 17p13 was observed in a percentage varying from 2% to 60% of cases, with evidence of marked geographical differences [30, 31]. The frequency of TP53 mutation is typically linked to the mycotoxin aflatoxin B1, which cooperates with HBV in causing TP53 mutations in HCC, and should be considered in all respects as a molecular exposure and HCC risk marker. Somatic mutations in TP53 are point mutations at the third base of codon 249 of exon 7, with GT transversion or, more rarely, GC transition [32]. Anyway, p53 can be altered in HCC from different etiologies, including chronic HBV and HCV infection and oxyradical disorders such as hemochromatosis, which produce reactive oxygen/nitrogen species that can induce gainof-function mutations of TP53. The p53 signaling pathway thwarts oxidative and nitrosative stress and drives the transcription of protective antioxidant genes, and in case of extensive DNA damage transactivates pro-oxidant genes that contribute to apoptosis. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC and is involved in the etiology of TP53 mutations during the molecular pathogenesis of HCC. The integrated HBx is frequently mutated, but maintains the ability to bind to p53 and decrease DNA repair and p53-mediated apoptosis [33]. Mutations in p53, a late event in the multiple stages of HCC development, are more frequent in larger high "grade" or poorly differentiated tumors, with worse prognosis and with a short tumor-free interval. The p53 related protein, namely p73, has also been investigated. This structural and functional homologue of p53 is present as two major forms that must be properly coordinated to guarantee the appropriate cellular fate: the transactivation-proficient, proapoptotic full-length TAp73 or the transactivation-deficient, antiapoptotic, NH(2)-terminally truncated DeltaNp73. Genotoxic insults leading to cell death induce the stabilization of TAp73, principally via posttranslational modifications, and the concomitant degradation of DeltaNp73, by still unclear mechanisms. Several observations indicate that during hepatocarcinogenesis there is a prevalence of dominant negative p73 isoforms (DeltaNp73 and DeltaTAp73) (anti-apoptotic and pro-proliferative) that are capable of blocking the functions of wild-type p53 [34]. In particular, DeltaNp73 holds up the transcriptional activation function of the transactivating isoforms of the p53 family, negatively regulates apoptosis-related genes of the extrinsic and intrinsic apoptosis signaling pathways, including the genes encoding the death receptors CD95, TNF-R1, TRAIL-R2 and TNFRSF18, as well as caspase-2,-3,-6,-8 and -9, concurrently hindering apoptosis emanating from mitochondria [35]. Inactivation of the product of TP73 does not usually occur by mutation but rather through expression of truncated isoforms that have dominant-negative effects on p73 and p53 [35]. The truncated oncogenic isoform DeltaEx2p73 is expressed in HCC and is produced through the alternative splicing of p73 premessenger RNA. Autocrine activation of EGFR signaling by amphiregulin triggers c-Jun N-terminal kinase-1 activity,

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leading to expression inhibition of the splicing regulator Slu7, which causes accumulation of DeltaEx2p73 transcripts in HCC cells [36]. Another mechanism may be represented by promoter methylation. A study that evaluated promoter methylation of 11 genes in peripheral blood lymphocytes and hepatic tissues of Egyptian patients with HCV associated HCC and chronic hepatitis showed that the methylation status was not dependent on the assayed specimen (lymphocyte or liver) and the methylation frequency and index increased with disease progression. In particular, methylation frequency of TP73 was significantly higher in HCC respect to adjacent normal tissue and a panel of 4 genes including TP73 (APC, TP73, p14, O6-MGMT) classified the cases independently into HCC and chronic hepatitis with high accuracy, sensitivity and specificity [37]. LOH at chromosome 1p includes many putative tumor suppressor genes, including TP73, which maps to 1p36 and is an imprinted gene expressed only from a maternal allele, suggesting that loss of the single functional copy of TP73 may contribute to increased tumor susceptibility. However, TP73 mutations in HCC are infrequent and p73 expression is up-regulated in HCC, hinting that TP73 is not the target of LOH at 1p [29]. This evidence seems corroborated by a study that investigated by PCR-RFLP analysis the allelic status of TP733 in 63 patients with HCC. The results were compared with LOH on chromosome 1p surrounding TP73 locus, mutations of TP53 and TP73, and clinicopathological characteristics. LOH on TP73 was observed in 33% of informative tumors, but not always between the regions with LOH on chromosome 1p examined despite the significant association of LOH in TP73 with LOH on chromosome 1p. No mutations were detected in TP73. Tumors with LOH in TP73 were more frequently detected in liver without cirrhosis than that with cirrhosis, disease-free survival rates were significantly poorer in patients with LOH in TP73 and multivariate analysis indicated that presence of LOH in TP73 was an independent prognostic factor in HCC patients. The results implied that TP73 should not be considered a candidate gene on chromosome 1p of HCC and does not function as a tumor suppressor gene. Anyway, the association of elevated expression in HCC with a significantly poorer survival suggests that p73 might play some role in HCC progression influencing the response to physiological stresses that accompany tumor growth, such as nutrient deprivation and hypoxia, and could assist prediction of early recurrence and stratification of patients requiring adjuvant therapy after surgery [29, 38]. The protein encoded by the RB1 gene (retinoblastoma protein, pRb), and the inhibitors of cyclin-dependent kinases are altered in HCC [29]. Cell cycle regulators are strongly implicated in cancer progression: p16 and p27 interact in a complex way and are key CDK inhibitors entailed in G1 phase progression. Abnormal DNA methylation represents the most important mechanism for p16 inactivation, whilst proteasomal degradation inactivates p27. In approximately 50% of HCC cases p27 is decreased, and when p27 is inactivated by inappropriate interaction with cyclin D1/CDK4 complexes p16 is simultaneously inactivated via DNA methylation [39].

Biology, Epidemiology, Clinical Aspects of Hepatocellular Carcinoma

The functions of pRb are inactivated in hepatocarcinogenesis due to multiple mechanisms, related to genetic alterations (LOH of RB1 locus in 25-48%, loss of the antiproliferative response to TGF- in 10%, loss of CDKN2A in 50% for mutations or more often through promoter hypermethylation) and epigenetic alterations of the expression of its different modulators (over-expression of cyclin D1 and cyclin E, decreased levels of p27). The described changes are present in up to 50-60% of cases, demonstrating the genetic heterogeneity of HCC, with most caused by chromosomal instability that especially occurs in more advanced stages of hepatocarcinogenesis and which is, in turn, mainly a consequence of telomere shortening and dysregulation of cell cycle control mechanisms [4, 6]. The study of global profiles of gene expression using microarrays has allowed the identification of numerous new genes modulated in hepatocarcinogenesis. In particular genes encoding proteins whose expression correlates with tumor progression (CDKN2A, SOCS1, PEG10), tumor dissemination and metastasis formation (NME1, SPP1, RHOC, KAI1, MMP14), relapse after radical treatment (REL, TNFAIP3, VIM, PDGFRA) have been identified. However, such data still does not allow the identification of unique "markers" that can be used in current clinical practice at present [40]. ONCOGENIC PATHWAYS INVOLVED IN HEPATOCARCINOGENESIS WNT / -catenin Signalling Pathway The WNT gene family encodes growth factors playing a critical role in cell fate determination during embryogenesis. The cell surface receptors for WNT proteins belong to the seven-pass transmembrane Frizzled (Fz) protein. When the WNT ligand binds its receptor by a mechanism that is still to be fully clarified, an intracellular protein called Dishvelled (DVL) is activated and recruited to the membrane where it destabilizes the Adenomatous Polyposis Coli (APC), glycogen synthase kinase-3 (GSK-3), Axin complex, which is no longer able to phosphorylate -catenin. Consequently, cytosolic -catenin increases, moving to the nucleus and binding to the transcription factors lymphocyte enhancer factor (Lef)-1 and T-cell factor (TCF) [41-43] (Fig. 1), in turn increasing the expression of genes engaged in cell migration, such as the gene encoding matrix metalloproteinase (MMP)-7, as well as the expression of active Rho family members, such as CDC42 and RAC, or inducing the redistribution of E-cadherin and the rearrangement of actin filaments by targeting CCND1 [44, 45]. The activation of the WNT/-catenin signalling pathway in colon cancer depends on APC gene mutations. When APC is mutated, the accumulation of -catenin in the cytosol and nucleus can directly activate the expression of the gene encoding cyclin D1, leading to uncontrolled cell proliferation that contributes to tumor genesis. CTNNB1 and AXIN1 mutations are far more common in HCC. Mutations of CTNNB1 have been detected in exon 3 of the gene, in codons 32-37 and 41-45. These mutations hamper protein degradation, thereby increasing its stabilization. CTNNB1 mutations and subsequent -catenin nuclear accumulation are reported in 13-34% and in 11-43% of hepatocarcinomas, respectively [46-48]. HCC occurring in HCV-positive patients more fre-

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quently show CTNNB1 mutations, compared with those occurring in HBV-positive patients. Furthermore, CTNNB1 mutations are typical in HCC when genomic instability is not present. AXIN1 mutations are reported in approximately 10% of HCC. They are mainly point mutations and/or small deletions that stabilize -catenin by preventing the formation of the APC/GSK-3/-catenin complex. Other gene dysegulations thwarting the WNT/-catenin pathway in HCC include the epigenetic silencing of soluble Fz Related Protein (sFRP1)-1, overexpression of DVL1 and inhibition of two DVL1 inhibitors such as human homologue of Dapper (HDPR)-1 and Prickle-1 [49, 50]. Hedgehog Signalling Pathway Hedgehog (Hh) signalling is a critical mediator of normal developmental processes, including embryo and cellular differentiation. The Hh protein was discovered in Drosophila, where a mutation produces larva with spinous processes that resemble a hedgehog. The key molecule of the Hh signalling system is the receptor Smoothened (SMO), which is the signal transducer. In the absence of the Hh ligand, its receptor function is inhibited by Patched, another transmembrane protein encoded by PTCH1. In mammals three Hh molecules have been described: Indian, Desert, and Sonic (Fig. 1). The Indian gene is predominantly expressed in the liver. The binding of the Hh ligand to the receptor Patched initiates signal transmission: the SMO activated protein directs a cascade of intracellular signals that lead to the activation of transcription factors, as well as blocking the production of their inhibitors. Consequently Hh regulates the function of Gli proteins and their activation. Data suggest that the Hh signalling pathway has an important role in hepatocarcinogenesis [51]. Hh over-expression occurs in approximately 60% of HCC patients. Moreover, SMO activation seems able to induce the over-expression of c-Myc, which also plays a relevant role ins hepatocarcinogenesis [51]. Ras-MAP Kinase Signalling Pathway KRAS was the first oncogene discovered and is mutated in approximately 30% of human tumors [52]. KRAS was initially described as a retroviral oncogene and encodes Ras GTPase-activating proteins maintained on the inner face of the plasma membrane by a lipid group. The Ras proteins are present in two different forms, one active bound to GTP and one inactive bound to GDP. KRAS mutations prevent GTP hydrolysis, thereby continuously subjecting the cell to a proliferative stimulus. There are several pathways activated by Ras, but a well characterized effector that promotes the progression of the cell cycle is the mitogen-activated protein kinase (MAPK) cascade [53]. This pathway transmits extracellular signals from the cell membrane through the cytoplasm to the nucleus, being activated when a growth factor such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF), binds to the extracellular domain of its receptor activating tyrosine kinase (Fig. 1). Many of these activated receptors contain phosphorylated tyrosine residues that serve to recruit the protein GRB2, which in turn binds to the guanine nucleotide exchange factor SOS and promotes the displacement of the GRB2-SOS complex from the cytoplasm to the cytoplasmic surface of the plasma membrane, near Ras. The interaction of SOS with Ras opens the nucleo-

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tides binding site of Ras. GDP is released and replaced by GTP. Ras induction sequentially determines the activation of the protein kinase activity of RAF, MEK and ERK (extracellular signal-regulated kinase) [54]. The protein kinase ERK1/2, which is activated by MEK phosphorylation, can be transported through the nuclear membrane. Once in the nucleus, pERK phosphorylates and activates the nuclear transcription factor ELK and other transcription factors. These factors determine the transcription of several genes whose products increase proliferation, survival and angiogenesis, via the activation of the transcription factors c-Fos, c-Jun, cMyc, driving cell proliferation and survival, and vascular endothelial growth factor (VEGF-), hypoxia-inducible factor (HIF)-1 and hexokinase II for angiogenesis and exploitation of energy intake in the absence of blood vessels (i.e. under anaerobic conditions). An increasing number of experimental observations confers an important role to the kinase cascade of the Ras-MAPK pathway in the genesis and progression of human HCC [55]. High levels of pERK1/2 expression have been observed in human HCC with poor versus good prognosis, although in both higher pERK1/2 levels are found compared with normal liver tissue and with surrounding non-tumoral hepatic tissue. HBV, HCV and HEV proteins may also modulate MAPK signalling, with the E2 protein of HCV activating the MAPK in human hepatoma Huh-7 cell line, thereby promoting cell proliferation. The RAF/MEK/ERK pathway is also crucial for angiogenesis in HCC, being a significant target of some anti-angiogenic therapies. Preclinical studies have demonstrated that Sorafenib, a molecule capable of inhibiting RAF at nanomolar concentrations, reduces cell proliferation with a concomitant reduction in angiogenesis, as well as promoting cell apoptosis in HCC [56]. PI3K/AKT/mTOR Signalling Pathway AKT is a serine/threonine kinase, also known as RAC (Related to A and C kinases) or PKB for its homology with the catalytic domain of PKA and PKC. AKT was initially identified as the product of the oncogene v-Akt, isolated from the AKT-8 retrovirus. The structure of the protein is characterized by an N-terminal pleckstrin homology (PH) domain, a central kinase domain and a C-terminal regulatory domain [57]. The PH domain consists of about 100 aminoacids, originally identified in the protein Pleckstrin, the substrate phosphorylated by PKC in platelets. The PH domain is capable of interacting with phosphoinositides produced by the lipid kinase phosphoinositide 3-kinase (PI3K), phosphatidylinositol (3,4,5) trisphosphate (PIP3) and phosphatidylinositol (3,4) bisphosphate (PIP2) [57]. The kinase domain is localized in the central portion of the molecule and shows a high similarity with that found in other protein kinases of the AGC [cAMP-dependent protein kinase (PKA)}protein kinaseG}protein kinase C (PKC)] protein kinase extended family. AKT has a C-terminal extension of approximately 40 amino acids containing the hydrophobic motif, characteristic of the AGC kinase family, whose phosphorylation is necessary for complete enzymatic activation. AKT activation and decreased expression of phosphatase and tensin homolog (PTEN) have been reported in 40-60% of HCC cases [58]. AKT activation suppresses apoptosis induced by TGF- and the growth inhibitory activity of

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CCAAT/enhancer binding protein . AKT activation may also turn on the -catenin pathway, indicating a connection in hepatocarcinogenesis between these two frequent oncogenic pathways. An important mediator of the PI3K-AKT pathway is mammalian target of rapamycin (mTOR). This protein kinase phosphorylates proteins that control mRNA translation, playing a central role in the regulation of protein synthesis. The activity of mTOR kinase depends on the phosphorylation of PRS40 (Proline rich Akt substrate 40kDa), which is part of the mTORC1 complex and is a direct substrate of Akt [59] (Fig. 1). The AKT-mTOR pathway promotes the transcription of cyclin D by increasing its mRNA translation via CREB. The mTOR pathway is activated in a subset of HCC cases and its block via rapamycin and/or the analogous everolimus inhibited the growth of HCC xenografts in preclinical models [60], but did not ameliorate overall survival in patients with advanced HCC patients [61]. SYSTEMIC THERAPY CARCINOMA

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The fact that HCC is substantially a neoplasm that grows for years confined to the liver constitutes the rationale for the success of loco-regional treatments (surgical or ablative, such as resection, transplantation, radiofrequency ablation, chemoembolization), whilst the systemic treatment has always represented the only therapeutic option for patients suffering from HCC at an advanced stage [62-64]. However, HCC is a chemoresistant tumor. In recent decades, a large number of cytotoxic drugs have been tested, either as monotherapy or in combination, although few agents have shown response rates over 20% and are therefore rarely used in clinical practice [64]. Patients with untreated advanced HCC have a median survival of 6-7 months [65]. Compelling evidence showing that systemic chemotherapy increases survival in these patients is lacking [66]. Advanced stage HCC is a disease difficult to treat for a variety of reasons, including the altered expression of proteins that carry chemotherapeutic drugs across the plasma membrane and the increased presence of active drug metabolizing systems that help to make this cancer resistant to conventional cytotoxic chemotherapy and impinge on the response to new therapeutic agents [67, 68]. In particular, down-regulation of solute carrier (SLC) family member 22A1 (SLC22A1) gene encoding the organic cation transporter (OCT)-1 influences the response of HCC to Sorafenib, a cationic drug, and aberrant OCT-1 variants in conjunction with decreased OCT-1 expression may severely impact the ability of Sorafenib to reach active intracellular concentrations. Two OCT-1 variants, R61S fs*10 and C88A fs*16, shut down Sorafenib transport, and screening of these SNPs in 23 HCC biopsies revealed that R61S fs*10 and C88A fs*16 were present in 17% of HCC specimens. In considering all SLC22A1 variants, at least one inactivating SNP was found in 48% of HCC cases [69]. Furthermore, HCC develops from chronically damaged tissue that contains large amounts of inflammation and fibrosis. Such factors contribute to an interaction of the stromal component with cancer cells, in turn supporting tumor progression and therapy resistance. Treatment is further complicated by the hepatotoxicity of many drugs, which adversely

Biology, Epidemiology, Clinical Aspects of Hepatocellular Carcinoma

affects the underlying liver disease. Finally, leukopenia and thrombocytopenia caused by splenic sequestration due to portal hypertension compromise the use of agents that induce bone marrow suppression. When used as a single agent, doxorubicin as has a response rate of 10-15% without any survival benefit, with significant grade 3 and 4 toxicity, in particular neutropenia [70]. Other chemotherapeutic agents, such as epirubicin, cisplatin, 5-fluorouracil and etoposide, have been studied with low response rates and no survival benefit [71]. Even the latest generation of chemotherapeutic agents such as gemcitabine, the pegylated liposomal doxorubicin and irinotecan, have produced disappointing results [72, 73]. The combination chemotherapy regimens have shown promise in phase II studies, but have not shown survival benefits in randomized phase III trials. The combination of cisplatin, interferon-2b, doxorubicin, and fluorouracil (PIAF) generated a lot of enthusiasm, showing, in the phase II study, 26% partial response and with 4 patients showing a complete response. However, in the phase III study this combination did not demonstrate any benefit in terms of survival in the face of significant toxicity [74]. In the past, hormonal therapies, including tamoxifen and antiandrogens, as well as octreotide, interferon, immunotherapy with interleukins and lymphokine activated killer cells have also been tested, although it is of note that before 2008 there were no treatments of proven efficacy for advanced HCC [20, 75]. MECHANISMS OF ACTION OF SORAFENIB AND CLINICAL TRIALS In recent years, the increasing knowledge of the pathways that regulate HCC onset and progression and the acquisition of information on the molecular alterations of proliferative signals in cancer cells provided a rationale for the development of anticancer molecules directed against new and more specific biological targets. For years, the medical treatment of HCC has remained one of the biggest black holes of oncology. This dreary picture has suddenly changed as a result of positive results obtained with Sorafenib, which has proven to prolong overall survival, so that it has become the standard first-line treatment of advanced HCC. International guidelines of the National Comprehensive Cancer Network (NCCN) highly recommend the use of Sorafenib in advanced disease. Several studies have assessed different innovative targeted therapies for HCC, but none have been found superior to Sorafenib [76], with no combinations of novel agents, such as everolimus or erlotinib, having exceeded the clinical benefits of Sorafenib monotherapy [20]. Sorafenib, a bisaryl urea, is an oral multi-kinase inhibitor that interferes with the intracellular cascade Ras/RAF/ MEK/ERK pathway involved in cancer cell proliferation, potently inhibiting the serine–threonine kinase RAF and both wild-type and V600E mutant variants of BRAF [77-79]. Sorafenib also acts on cell surface tyrosine kinase receptors involved in tumor growth and angiogenesis, including VEGFR-1,-2,-3, PDGFR-, c-Kit, FMS-like tyrosine kinase 3 (FLT-3) and RET [77-79]. In a phase I study in patients with advanced refractory solid tumors, treatment with Sorafenib resulted in a partial response [80]. Such data suggested the potential use of this drug in a broad spectrum of tumors,

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even with different molecular aetiologies, and in fact the results of phase III randomized trials have shown that Sorafenib is able to increase survival in patients with advanced HCC (Table 1). Sorafenib, therefore, has become the standard treatment in these patients [81]. The first Phase II clinical trial with Sorafenib monotherapy [82] involved 137 patients with advanced HCC, histologically confirmed, previously untreated with systemic therapy, with Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1, Child-Pugh A (72%) or B (28%) and life expectancy of more than 12 months. The dose of drug used was 400 mg of Sorafenib bis in die. Patients received treatment until disease progression or toxicity; partial response to treatment was observed in 2% of patients, a minor response in 6% of patients and disease stability exceeding 16 weeks in 34% of cases. Treatment with Sorafenib was also associated with a median time to progression of 4.2 months with a median overall survival of 9.2 months [82]. The efficacy of Sorafenib and its reduced toxicity have stimulated the further study of Sorafenib monotherapy in the treatment of advanced HCC. Two multicenter, randomized phase III double-blind trials have recently confirmed the benefit of Sorafenib in these patients: the study of Sorafenib Hepatocellular Carcinoma Assessment Randomized Protocol (SHARP) [83], conducted in 121 centers in 21 countries in Europe, America, Australia and New Zealand, where the onset of cancer is mainly due to HCV infection and alcohol, and the AsiaPacific study [84], conducted in 23 centres in China, Taiwan and South Korea, where chronic HBV infection is responsible for the onset of the majority of HCC cases. Both studies included patients with advanced HCC, histologically confirmed, with measurable lesions, without prior systemic therapy, Child-Pugh A, ECOG PS 0-2, with a life expectancy of at least 12 weeks, and adequate renal, hepatic, haematological and bone marrow function. In the SHARP trial, of 902 screened patients, 602 were randomized 1:1 to receive Sorafenib 400 mg bis in die (299 patients) or placebo (303 patients) per os. Patients continued the treatment until radiological progression, evaluated in accordance with the Response Evaluation Criteria in Solid Tumors (RECIST), and symptomatic progression, assessed through the questionnaire Functional Assessment of Cancer Therapy-Hepatobiliary Symptom Index 8 (FHSI-8). Prior to randomization, patients were stratified according to the presence of vascular invasion and/or extrahepatic disease, ECOG PS and geographic region. In the Asia-Pacific study 226 patients were enrolled, randomized 2:1 to receive Sorafenib (150 patients) or placebo (76 patients). The Asia-Pacific study was designed in parallel with the SHARP study to assess the efficacy and safety of Sorafenib in this population with clinical and epidemiological features of HCC different from the Western countries, for the purposes of drug registration in Asia. In this study primary goals were not defined. The primary objectives of the SHARP study were survival and time to symptomatic progression, calculated as an increase by 4 points in the score on the FHSI-8 questionnaire or worsening ECOG PS up to 4 or death. Time to tumor progression as the primary end point is the more valuable indicator of response to treatment, since this circumvents the confounding influence of underlying liver cirrhosis, which is important for progression-free survival [85]. Secondary endpoints were time to radiological progression and the rate of disease

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Table 1.

Mazzoccoli et al.

Clinical trials with Sorafenib as monotherapy and in combination with other drugs or treatments.

Type of Study

Doses of the Drug and Duration of Treatment

Clinical Findings

Adverse Events

Phase I study

50 to 800 mg per os administered once or twice daily on a varying weekly schedule

Sorafenib was well tolerated and afforded some clinical benefits. Forty-five patients were assessable for efficacy; one patient had a partial response (hepatocellular carcinoma at 400 mg bid continuous), 25 patients had stable disease, with eight lasting > 6 months and five for >12 months. Eighteen patients had progressive disease, and tumor response could not be evaluated in one patient. Based on the results of this study, sorafenib at 400 mg twice daily continuous was recommended for ongoing and future studies

Mild to moderate diarrhea (55%). The maximumtolerated dose was 400 mg twice daily continuous. Dose-limiting toxicities were grade 3 diarrhea and fatigue at 800 mg bid, and grade 3 skin toxicity at 600 mg twice daily.

[80]

Phase II study

400 mg per os twice daily in 4-week cycles.

On the basis of independent assessment, three (2.2%) patients achieved a partial response, eight (5.8%) had a minor response, and 46 (33.6%) had stable disease for at least 16 weeks. Investigator-assessed median time to progression was 4.2 months, and median overall survival was 9.2 months.

Grade 3/4 drug-related toxicities included fatigue (9.5%), diarrhea (8.0%), and hand-foot skin reaction (5.1%).

[82]

Phase III study

400 mg per os twice daily

Median overall survival 10.7 months in the sorafenib group and 7.9 months in the placebo group (hazard ratio in the sorafenib group, 0.69; 95% confidence interval, 0.55 to 0.87; P70% of the time with a half dose versus 9.6 months in the 219 patients treated for >70% of the time with a full dose. At month 2 of treatment, the overall radiologic response was 8%. Eastern Cooperative Oncology Group performance status, macrovascular invasion, extrahepatic spread of the tumor, radiologic response at month 2, and sorafenib dosing were independent predictors of shortened survival.

Two hundred sixty-nine (91%) patients experienced at least one adverse event, whereas 161 (54%) had to reduce dosing.

[94,95]

Phase IV study

82.4% of 1113 evaluable patients treated with sorafenib started on 400 mg twice daily, 15.4% started on 200 mg twice daily. Treatment duration (18.0 vs 13.0 weeks) and median overall survival (12.1 vs 9.4 months) were longer in patients receiving 400 mg twice daily

patients starting on 200 mg twice daily were slightly older and had baseline characteristics indicative of greater disease progression

patients starting on 200 mg twice daily had higher adverse events incidences (96 vs 88%).

[91-93]

control (defined as the percentage of complete responses, partial responses and stable disease obtained for at least 28 days). Enrolment was stopped early in the second interim analysis, when a benefit in terms of survival was shown in patients receiving Sorafenib. In both phase III studies, the median overall survival was significantly greater in patients who received Sorafenib: after 2 years, the group treated with Sorafenib had a median overall survival of 10.7 months versus 7.9 months for patients who received placebo in the SHARP trial and 6.5 months versus 4.2 months in the AsiaPacific study. The disease progression-free survival was 5.5 months in patients receiving Sorafenib and 2.8 months in patients who received placebo in the SHARP trial and 2.8 months in patients receiving Sorafenib versus 1.4 months in the patients who received placebo in the Asia-Pacific, with a statistically significant difference in both studies. The rates of disease control were 43% and 32% in favour of Sorafenib in the SHARP trial and 35% and 16%, respectively, in the Asia-Pacific study. An analysis of subgroups showed a better performance in patients with Child-Pugh class A compared

with Child-Pugh B in terms of efficacy, with similar tolerability profiles of the two groups. In the SHARP trial, the incidence of adverse events of any grade correlated to the drug was higher in the Sorafenib group than in the placebo group (80% versus 52%). The most common adverse events during treatment with Sorafenib were diarrhea (43% of patients), hand-foot syndrome (31%), fatigue (30%), skin desquamation (17%), nausea (16%), anorexia (14%), stomatitis (11%), vomiting (10%) and alopecia (10%). In terms of laboratory abnormalities, a grade 3 hypophosphatemia and grade 3-4 thrombocytopenia were more frequently observed. In the Asia-Pacific study, adverse events of grade 3 that were most frequently reported included diarrhea and hand-foot skin reaction. In the management of diarrhea in the patient receiving Sorafenib is necessary to investigate bowel habits, identify and possibly eliminate the foods that can foster it, prescribe drugs (loperamide) and carefully avoid dehydration [86]. However, the appearance of grade 2 or 3 diarrhea associates with a better

Biology, Epidemiology, Clinical Aspects of Hepatocellular Carcinoma

overall survival of HCC patients undergoing Sorafenib treatment [87]. The hand-foot syndrome refers to a group of signs and symptoms that involve inflammation of the hands and/or feet, usually bilaterally. The severity of hand-foot syndrome can be classified: Grade 1, characterized by numbness, dysesthesia, paresthesia, tingling, painless swelling and erythema, which do not compromise the normal daily activities of the patient; Grade 2, characterized by painful erythema, swelling, hyperkeratosis of hands and feet, which interfere with normal daily activities of the patient; Grade 3, characterized by exfoliation or desquamation, ulceration, blistering, hyperkeratosis, pain, preventing the patient to perform normal daily activities. Hand-foot syndrome usually occurs during the first 6 weeks of therapy, with a generally decreasing intensity spontaneously occurring over the course of treatment. Acneiform rash, a typical adverse event of anti-EGFR therapy, can also occur during treatment with Sorafenib. It is a macular, papular or erythematous rash which appears on the face and back, often without associated symptoms (grade 1); can evolve into scaling in less than 50% of body surface (grade 2) or more than half of the body (grade 3) [88]. Remarkably, development of dermatologic adverse events within sixty days of Sorafenib initiation associated with better survival and should not be considered harmful events or a motivation to suspend treatment [89, 90]. With the aim to evaluate the safety and efficacy of Sorafenib in different subgroups, especially Child-Pugh B patients who were not well represented in clinical trials, an international, prospective, open-label, multicentre, noninterventional post-marketing prospective study of patients with unresectable HCC treated with Sorafenib under real-life practice conditions was planned. From January 2009 to April 2011, the Global Investigation of Therapeutic DEcisions in Hepatocellular Carcinoma and Of its Treatment with Sorafenib (GIDEON) study enrolled more than 3000 patients from 39 countries. The prespecified first interim analysis performed on 479 patients treated with Sorafenib and followed up for 4 months or more pointed out global and regional differences in patient characteristics, disease aetiology and practice patterns. The starting dose of Sorafenib was not influenced by Child-Pugh status and the type and incidence of adverse events corroborated findings from previous clinical studies and were not different between Child-Pugh subgroups (Lencioni et al., 2012). The prespecified second interim analysis was performed on 1571 patients (61% ChildPugh A and 23% Child-Pugh B disease status). Not considering Child-Pugh disease status, 74% received the approved 800 mg initial Sorafenib dose. The median duration of therapy was shorter in patients Child-Pugh B disease status, but drug-related adverse events were similar across Child-Pugh, Barcelona Clinic Liver Cancer (BCLC) and initial dosing subgroups, reliable with the overall population, grade 1 or 2 for the most part and in line with earlier reports [91]. Data suggest that the safety profile of the molecule is preserved and duration of therapy, overall survival (OS) and time to progression (TTP) are improved when Sorafenib treatment is started at the full recommended dose of 800 mg/day versus a 400 mg/day starting dose [92]. The suggestion seems corroborated by the results obtained from the analysis performed on the European subpopulation (1113 evaluable patients) enrolled in the GIDEON study, of which 82% started

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on 800 mg/day Sorafenib. Treatment duration (18 versus 13 weeks) and median overall survival (12.1 versus 9.4 months) were longer in patients receiving 800 mg/day, but patients treated with an initial dosage of 400 mg/day presented higher median age, baseline characteristics (worse ECOG performance status and Child-Pugh disease status) predicting greater disease progression and greater adverse events incidences (96 versus 88%) [93]. A smaller multicenter, investigator driven, observational, non-interventional study conducted in six referral Italian centres, the Sorafenib Italian Assessment (SOFIA) study, was planned with the aim to assess Sorafenib safety in clinical practice and to evaluate treatment efficacy in terms of OS, early radiologic response and TTP. The study enrolled 296 consecutive patients (88% Child-Pugh A) with advanced stage HCC (75%) or intermediate-stage HCC and/or not eligible to or who failed ablative therapies (25%). Sorafenib was administered at a starting dose of 800 mg/day in all patients and treatment was down-dosed or interrupted according to the drug label. The median duration of treatment was 3.8 months, 90 patients (30%) were treated for more than 6 months and 41 patients (14%) were treated for more than 12 months. OS was 21.6 months in the 77 patients treated for more than 70% of the time with a half dose versus 9.6 months in the 219 patients treated for more than 70% of the time with a full dose. These results confirm the safety and effectiveness of Sorafenib in a real-life scenario. Notwithstanding the post hoc nature of the analysis of the effectiveness of full- versus half-dose Sorafenib and the lack of stratification before treatment of survival predictors, the effectiveness of half dose Sorafenib could impact clinical practice, in particular in patients who do not tolerate full-dose treatment [94]. The advantages of tailored therapy seem corroborated by the evaluation of the cost-effectiveness of Sorafenib in the treatment of HCC patients incorporating up to date prices and SOFIA study results: in daily practice dose-adjusted, but not full-dose, Sorafenib is a cost-effective treatment compared to best supportive care in intermediate and advanced HCC [95]. The activity of Sorafenib in blocking tumor growth and angiogenesis may explain the delay in the progression of the disease and the benefit in terms of survival despite the low rate of objective responses. Several studies have investigated the efficacy of Sorafenib in combination with chemotherapeutic agents in patients with advanced HCC. A randomized phase III double-blind study compared the efficacy of Sorafenib plus doxorubicin with doxorubicin plus placebo [96]. In 2 of the 47 patients (4%) treated with Sorafenib and doxorubicin a clinical response (complete or partial) was observed, while only 1 of 49 patients (2%) treated with doxorubicin had a clinical response. Treatment with Sorafenib also increased overall survival and progression-free survival. This study showed encouraging results, but did not show any synergism between Sorafenib and the chemotherapy agent. This study should be seen as preliminary, given the lack of a control group treated with Sorafenib monotherapy. Combinatorial drug delivery through a transferrin-targeted core-shell nanomedicine formed by encapsulating doxorubicin in poly(vinyl alcohol) nano-core and Sorafenib in albumin nano-shell, both formed by a sequential freezethaw/coacervation method, showed enhanced cellular uptake

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and synergistic cytotoxicity in ~92% of cells, particularly in an iron-deficient microenvironment, with studies using 3D spheroids of liver tumor indicating efficient penetration of targeted core-shell nanoparticles throughout the tissue that uniformly killed cells [97]. The results of this study seem to corroborate the report that the depletion of the intracellular iron stores achieved by using the iron chelating agent deferoxamine strikingly protects HCC cells from the cytotoxic effects of Sorafenib, suggesting that this multikinase inhibitor causes a cytotoxic effect that looks like ferroptosis, a type of cell death in which iron-dependent oxidative mechanisms are crucial [98]. Besides, the combination of Sorafenib and gemcitabine in advanced HCC was generally well tolerated and afforded modest clinical efficacy and median overall survival equal to one year [99]. Identification of predictors of response is necessary to optimize trial design and use of Sorafenib in HCC patients [81]. Monitoring the response to Sorafenib has utilized measures of -fetoprotein levels and the use of PET to assess early response to therapy. In a study conducted in 6 patients [100], in which response evaluation by means of PET was performed before initiation of therapy and after 3 weeks of treatment, 3 patients showed a reduction in PET uptake; 2 of them developed a partial response on a CT scan at 12 weeks. A CT scan at 12 weeks also showed stable disease in a patient who had not showed any change in PET uptake between baseline examination and at 3 weeks. A patient with clinical progression, showed multiple lesions upon PET examination at the third week and died before the 12th week. It is necessary, however, to replicate such studies with larger patient samples. At the moment there are no fully valuable and reliable clinical parameters or properly validated biomarkers to help identify which patients will benefit from Sorafenib treatment [20, 101]. Given that 7%-10% of HCC is hallmarked by focal amplifications in 6p21 containing the VEGF-A locus, it is of note that data suggest that this amplification seems to work via heterotypic paracrine interactions. VEGF-A binding to VEGF receptors stimulates paracrine secretion of hepatocyte growth factor (HGF) by stromal cells, which induces tumor progression. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs, so that response to Sorafenib is predicted in HCC patients with VEGFA amplification [102-104]. Over and above paracrine signaling, also autocrine stimulation may play a role in tumor phenotype and response to therapy with Sorafenib. In a recent study, significant nuclear and cytoplasmic staining for active, phosphorylated VEGF receptor 1 (pVEGFR1) and pVEGFR2 was found in HCC tissues by immunohistochemistry, and western blotting showed increase membrane VEGFR1 and VEGFR2. Autocrine VEGF promoted phosphorylation of VEGFR1 and VEGFR2 and internalization of pVEGFR2 in HCC cells, with pro-proliferative effects via a protein lipase Cextracellular kinase pathway and self-sustainment through increasing VEGF, VEGFR1, and VEGFR2 mRNA expressions. Sorafenib treatment inhibited cell proliferation, reduced VEGFR2 mRNA expression in vitro, and delayed xenograft tumor growth in vivo in high VEGFR1/2expressing HepG2 cells, but not in low VEGFR1/2expressing Hep3B cells. Accordingly, the absence of

Mazzoccoli et al.

VEGFR1 or VEGFR2 expression in resected tumor tissues before Sorafenib treatment predicted poorer overall survival in an advanced HCC population on Sorafenib treatment for postoperative recurrence [105]. The role of VEGF and VEGFR polymorphisms in influencing the clinical outcome of HCC patients who receive Sorafenib was evaluated by the multicentre ALICE-1 study that tested 148 samples (tumor or blood samples) of HCC patients receiving Sorafenib for VEGF-A, VEGF-C and VEGFR-1,-2,-3 SNPs and analyzed associations with patients' progression-free survival and overall survival. At univariate analysis VEGF-A alleles C of rs25648, T of rs833061, C of rs699947, C of rs2010963, VEGF-C alleles T of rs4604006, G of rs664393, VEGFR-2 alleles C of rs2071559, C of rs2305948 were important predictors of progression-free survival and overall survival. At multivariate analysis rs2010963, rs4604006 and BCLC stage were shown to be independent factors influencing progression-free survival and overall survival [106]. Furthermore, serial measurement of plasma VEGF concentrations showed that patients who had a VEGF reduction at week 8 had a longer median survival than those who did not have a VEGF reduction. In univariate analysis, decreased VEGF, radiological findings classified as progressive disease, and major vascular invasion were significantly associated with 1-year survival. In multivariate analysis, a VEGF decrease was identified as an independent factor significantly associated with survival [106]. A recent study reported that inhibition of Notch3 signaling sensitizes HCC cells to Sorafenib, in effect overcoming drug resistance. Notch3 inhibition considerably augmented the apoptosis inducing effect of Sorafenib in HCC cells via p21 down-regulation and pGSK3Ser9 up-regulation. Notch3 depletion combined with 21 days of Sorafenib treatment in a mouse xenograft model induced a significant antitumor effect. Upon exposure to Sorafenib treatment, Notch3 depleted xenografts maintained lower levels of p21 and higher levels of pGSK3Ser9 compared to control xenografts. Interestingly, in primary human HCC specimens Notch3 protein expression positively correlated with p21 protein expression and negatively correlated with pGSK3Ser9 expression, supporting the utility of Notch3 inhibition in combination with Sorafenib HCC treatment [107]. In addition, the unresponsiveness to Sorafenibinduced cell death in some in vitro studies, has also been shown in hepatic tumor cells with a mesenchymal-like phenotype, resistance to the suppressor effects of TGF- and with high expression of the stem cell marker CD44 [108]. Furthermore, NORE1A, a Ras effector with tumor suppressor function, was found down-regulated in human HCC cell lines and tissues, with statistically significant association with poor differentiation, advanced stage, high level of serum -fetoprotein, vascular invasion and incomplete involucrum, as well as independently predicting poor overall survival and recurrence-free survival. NORE1A overexpression reduced cell viability, inhibited colony formation, attenuated cell invasion in vitro and sensitized cancer cells to Sorafenib-induced apoptosis via the activation of the MST1/AKT pathway [109].

Biology, Epidemiology, Clinical Aspects of Hepatocellular Carcinoma

Clinical response including OS under Sorafenib treatment did not correlate with global microRNA (miR) expression patterns in liver biopsy specimens of Sorafenib treated patients. However, several miRs were identified that correlated with and could be considered candidate predictors of disease control rate or overall survival [110]. In HCC cell lines treated in vitro with Sorafenib an association was found between increased miR-425.3p, cell death and reduced cell motility, with higher levels of miR-425.3p in liver biopsy specimens being associated with longer time to progression and progression free survival following Sorafenib treatment [111]. Clinical features associated with a good radiological response and potential independent predictors of good effect following Sorafenib treatment in unresectable HCC include female gender and decreased serum -fetoprotein level [112]. As well as ECOG performance status, extrahepatic spread, macrovascular invasion, -fetoprotein or alkaline phosphatase levels at admission have been shown to be independent predictors of overall survival in patients with cirrhosis and HCC treated with Sorafenib [113]. A phase II study evaluating the efficacy of radiation therapy with concurrent and sequential Sorafenib therapy in patients with unresectable HCC showed an initial complete or partial response rate in 55% of cases with a 2-year infield progression-free survival of 39% with severe hepatic toxicity primary determinant of the safety of this combination [114]. In patients affected by advanced unresectable HCC with portal vein tumor thrombosis radiotherapy came out as a better first-line therapy with respect to Sorafenib [115], although a case of complete response after Sorafenib treatment was reported [116]. In particular, stereotactic body radiation therapy has been shown to afford good local tumor control and higher overall survival rates than other historical controls (best supportive care or Sorafenib), although high -fetoprotein levels were associated with poorer local control. However, a higher treatment dose improved local control [117]. A comparison of the efficacies of transarterial chemoembolization (TACE) combined with Sorafenib versus TACE monotherapy for treating advanced HCC patients showed that Sorafenib plus TACE was more effective than TACE monotherapy for treating patients with advanced HCC without main portal vein invasion, ameliorating time to progression more than overall survival [118, 119]. Likewise, a phase II study was conducted on patients with HCC not amenable to curative therapies in order to evaluate the safety and efficacy of sequential treatment using Sorafenib (400 mg twicedaily) initiated 14 days post-radioembolization with yttrium90 resin microspheres given as a single procedure. Results showed sequential radioembolization-Sorafenib therapy to have potential efficacy and manageable toxicity [120]. The safety of radioembolization with yttrium-90 resin microspheres followed by Sorafenib has been confirmed by the SORAMIC study, in which radioembolization was administered using a sequential lobar approach; on day 3 after the last radioembolization procedure, Sorafenib 200 mg twice daily was initiated escalating to 400 mg twice daily 1 week later. The incidence of total and grade 3 adverse events was similar in combination-treatment arm and control arm respectively [121].

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Sorafenib is thought also to bring on autophagy, which over and above representing a pathway for lysosomal degradation, is able to prompt programmed cell death called autophagic cell death (programmed cell death type II). Sorafenib was shown able to bring on autophagy in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B by inducing degradation of myeloid cell leukemia-1 (Mcl-1) and thwarting its association with Beclin 1; in turn, Mcl-1–Beclin 1 complex disassociation upholds considerable autophagic cell death via a mechanism involving increased activity of Small Heterodimer Partner (SHP)-1 and down-regulation of signal transducers and activator of transcription 3 (STAT3) phosphorylation [122]. Regarding use of Sorafenib in the transplant setting, we suggest to refer to a comprehensive recent review [123]. BEYOND HCC: SORAFENIB IN THE TREATMENT OF LIVER DISEASES Chronic hepatic cellular injury, arising from a variety of aetiologies including viral hepatitis, alcoholic liver disease, and NASH, as well as inherited liver disorders, such as hemochromatosis, induce a ‘wound healing’ response resulting in liver fibrosis. Unrelenting damage enhances production and deposition of extracellular matrix proteins by hepatic stellate cells (HSC), a liver non-parenchymal cell type, producing scarring. Unnoticed or untreated damage leads to the progression of fibrosis to cirrhosis, with an accompanying increased risk of hepatocarcinogenesis. The development of effective anti-fibrotic therapies to prevent the development of the end stage disease would be a significant advance. Experiments in rats with liver fibrosis showed Sorafenib treatment to hamper the phosphorylation of ERK, AKT and 70-kDa ribosomal S6 kinase (p70S6K), whils also reducing intrahepatic fibrogenesis, hydroxyproline accumulation, collagen deposition, and HSC proliferation, as well as iducing significantly higher levels of apoptosis. Furthermore, Cyclin D1 and CDK4 were down-regulated, whereas Fas, Fas-L, and Caspase-3 were increased, with concurrent decrease in the Bcl-2/Bax ratio. Sorafenib treatment also increased MMPs/tissue inhibitor of MMPs (TIMPs) ratio and reduced collagen synthesis in HSCs [124]. Fibrosis and intrahepatic hyperresponsiveness to vasoconstrictors caused by the activation and proliferation of HSCs lead to increased intrahepatic vascular resistance and portal hypertension in cirrhosis. Experiments performed in an animal model of secondary biliary cirrhosis induced by bile duct ligation showed that Sorafenib (60mg/kg/day for 1 week) can decrease portal hypertension, diminishing the number of activated HSCs and reducing hepatic -smooth muscle actin (SMA) and procollagen-1 mRNA expression, as well as perisinusoidal deposition of extracellular matrix, although at the cost of liver damage at the dosage used [125]. Likewise, in a murine model of liver fibrosis induced by carbon tetrachloride (CCl4), Sorafenib significantly reduced chronic liver injury and fibrosis, with reduction in liver inflammation and histopathology as well as decreased expression of liver fibrosis-related genes, including collagen, SMA, MMP and TIMP-1. The molecular mechanism involved the upregulation of STAT3 phosphorylation that was

14 Current Drug Targets, 2016, Vol. 17, No. 5

dependent on Kupffer cell-derived IL-6 and the translocation of cytoplasmic STAT3 to the nucleus in its active form [126]. Fibrogenesis, angiogenesis, and portal hypertension are influenced by paracrine signaling between HSCs and liver endothelial cells. In bile duct ligation-induced rat chronic liver injury Sorafenib influences such paracrine signalling. Sorafenib reduced the changes in both matrix and vascular compartments decreasing cell-cell apposition and junctional complexes and therefore the closeness between these sinusoidal barrier cells. The molecular mechanism was represented by down-regulation of Kruppel-like factor 6-induced discharge of angiopoietin-1 and fibronectin by HSCs [127]. A mouse model of liver fibrosis induced by hepatic PDGF-C over-expression, the PDGF-C Tg mice, is similar to human fibrosis produced by chronic alcoholism and NASH [128, 129]. Sorafenib was shown to hinder normal and abnormal hepatic cellular proliferation after 2/3 partial hepatectomy, and in PDGF-C Tg mice, respectively. In both models, Sorafenib inhibited non-parenchymal cell and especially Kupffer cell replication, but not hepatocyte proliferation, after partial hepatectomy. This evidence suggests that Sorafenib inhibits different cellular targets depending on the nature of the proliferative stimuli, and corroborates the notion that blocking the activation of hepatic non-parenchymal cells or stromal cells may be an effective therapeutic strategy to prevent or delay the development of HCC [130]. NASH also aggravates ischemia/reperfusion (IR) injury during liver transplantation by activating various kinases and subsequently releasing cytokines and chemokines. Sorafenib shows hepatoprotective effects in NASH rats with IR injury through the inhibition of the Rho-kinase-dependent RAF/MEK/ERK pathway, which is up-regulated during IR injury in the livers of NASH rats, and interferes with the inflammation, necrotic, and apoptotic responses causing leukocyte-dependent hepatic microcirculatory dysfunction [131]. Furthermore, in NASH-cirrhotic rats Sorafenib improved hepatic blood flow and hepatic venous deregulation, inhibited leucocytes recruitment/activation, splanchnic blood pooling and ascites formation, suggesting that it might be an useful therapeutic tool in cirrhotic patients suffering from severe haemodynamic derangement and ascites [132]. CONCLUSIONS AND PERSPECTIVES HCC is the fifth cause of cancer death in the world and shows a multiphasic dynamic, usually developing in the presence of liver diseases, such as hepatitis or cirrhosis. The genomic landscape of HCC is not completely revealed, but genetic drivers found more frequently deregulated are involved in biological processes including telomere maintenance, cell-cycle regulation, chromatin remodeling, as well as Wnt/-catenin, Hedgehog, RAS/RAF/MAPK kinase and PI3K/AKT/mTOR signaling pathways. The multikinase inhibitor Sorafenib has been shown to significantly increase survival in advanced HCC patients. Sorafenib interferes with tumor cell proliferation and angiogenesis by acting on intracellular Ras/RAF/MEK/ERK cascade pathway and on the receptor tyrosine kinases VEGFR1,-2,-3 and PDGFR-. To date there are no clinical or biological parameters that can be consistently considered pre-

Mazzoccoli et al.

dictive of response to treatment. Sorafenib therapy in advanced HCC patients is thwarted by premature discontinuation due to tumor progression, liver decompensation, or adverse effects. The pattern of tumor progression settles on post-progression survival, whilst post-Sorafenib survival in compensated patients is predicted by discontinuation due to adverse effects in the absence of macrovascular invasion, extrahepatic metastases, and deteriorated performance status; at the same time these predictors entail further patient counseling and selection for second-line therapy [133]. There is need of studies that analyse the role of tissue markers in predicting the clinical efficacy of Sorafenib in patients with advanced HCC to ameliorate patient selection and enhance response to treatment of this common and dreadful neoplastic disease. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS We apologize for not being able to cite all the relevant original research and review articles due to space limitations. We thank George Anderson for manuscript proofreading. This work was funded by the “5x1000” voluntary contribution, by a grant (to GM) from the Italian Ministry of Health (RC1201ME04, RC1203ME46, RC1302ME31, RC1403 ME50 and RC1504ME53) through Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS Scientific Institute and Regional General Hospital “Casa Sollievo della Sofferenza”, Opera di Padre Pio da Pietrelcina, San Giovanni Rotondo (FG), Italy, by Wellcome Trust (to JAO), and by the Associazione Italiana per la Ricerca sul Cancro (AIRC) (to MV) program MFAG-13419. REFERENCES [1] [2] [3]

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Accepted: November 12, 2015