**Targets and Approaches to Control Hepatocellular Carcinoma in Future**

Mukherjee Biswajit, Hossain Chowdhury Mobaswar , Bhattacharya Sanchari and Shampa Ghosh

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56867

### **1. Introduction**

Cancer is uncontrolled proliferation of cells, which results from the loss of proper balance between cell death and cell growth. The transformed phenotypes of cancer cells are caused by the accumulation of mutations in a variety of genes, products of which normally play a role in the biochemical pathways that regulate cell death and cell proliferation. Cancer is a broad term used to define a group of more than 250 different diseases (Roncalli et al. 2010). It is a slow multi-stage, multi-step process (Cammà et al. 2008; Calvisi et al. 2009; Sherman 2011). In the first instance, these cells, derived initially from a normal cell, form a primary tumor which comprises a growth-transformed population of cells. The cells acquire a set of mutations to a set of genes which allow them to divide repeatedly in a way that normal cells cannot (Besaratinia et al. 2009; Calvisi et al. 2009). Histologically, cancer is characterized by several morphological alterations, including changes in tissue architecture, cytological abnormalities of both the nucleus and cytoplasm and the presence of abnormal mitoses. A stepwise several biochemical, genetic and biological alterations eventually result in a cancer.

Primary liver cancer or hepatocellular carcinoma (HCC) is a very common malignant hepa‐ tobiliary disease and it represents the fifth most frequent neoplastic disease which causes approximately 1 million deaths per year (Yang and Roberts, 2010, Cha et al. 2010). HCC is the third leading cause of cancer related death worldwide (Raphael 2012). Viruses and chemicals have been identified as the most important etiological factor associated with the development of human liver cancer (Carr et al. 2010). The most common cause of HCC is hepatitis B and C (Woo et al. 2008; Masuzaki et al. 2008, Gouas et al. 2010; Iavarone and Colombo 2011) and a

© 2013 Biswajit et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. number of risk factors that have been identified (Shariff et al. 2009; Sherman 2010; Gomaa et al. 2008 and 2009). Most of HCC cases develop from a cirrhotic liver (Bartolomeo et al. 2011; Chagas et al. 2009; Orlando et al.2009; Cammà et al. 2008) with an annual incidence of 2-6% for hepatitis B virus carriers (Kew 2010; Lim et al. 2009; Hadziyannis 2011) and 3-5% for hepatitis C virus-infected patients (Masuzaki et al. 2008; Rosen 2011). Males are more suscep‐ tible to HCC.

receptors and the signal transduction pathways involved in the pathogenesis of cancer to provide potential target for therapeutic intervention. Many studies have focused to identify molecular pathways to elicit cancer cell proliferation, including HCC. Here many of them have been highlighted to identify fundamental targets of hepatocellular oncogenesis. Thus, the present chapter has been projected to the molecular targets and approaches to intervene the

Three generally considered fundamental but interrelated targets of controlling oncogenesis are regulation of deregulated energy metabolism and ion homeostasis; signal transduction, oncogenes and growth factors; and immunomodulation. One of the most characteristic phenotypes of rapidly growing cancer cells is their propensity to catabolise glucose at high rates. Rapidly growing activity to cancer cells has a reduced number of mitochondria and increased glycolytic activity with a shift from respiratory to fermentative ATP supply to cover

early response in most of the quiescent cells stimulated to proliferate by multiple combinations of growth promoting factors. Growth factors, cytokines exert their action on cell proliferation by modulation of cell signalling process. There is a strong relationship between the immune system and cell proliferation. Immune suppressive agents have a powerful effect on hepatocyte

Reviewing current literature, a selection of therapeutic targets of HCC has been described

Like most other cancers, growth factors, their receptors, and downstream signalling proteins play a pivotal role in the development and maintenance of HCC and are of significant interest for future therapeutic approaches. In foetal liver, a large number of growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), transforming growth factors-α and –β (TGF-α, TGF-β), and vascular endothelial growth factor (VEGF) (Höpfner et al. 2008; Hoshida et al. 2008 and 2009) are produced. Their secretion either declines or shuts down in adult liver. However, during hepatic regenera‐ tion due to the cause of hepatic injury or damage many such growth factors (Böhm et al. 2010), namely, EGF, TGF-α, IGFs, and VEGF are upregulated in normal hepatocytes. The transient upregulation of those factors is dysregulated in the chronic injured liver leading to sustained mitogenic/ oncogenic signalling, during the development of HCC. FGF and PDGF released from non-hepatocyte sources such as activated hepatic stellate cells, myofibroblasts, endothelial cells, Kupffer cells, and bile duct epithelia have been shown to play important roles in promoting hepatic fibrosis and HCC growth (Friedman 2008). The ubiquitin-proteasome pathway has emerged as a key player in the regulation of several diverse cellular processes. Inhibition of poly-ubiquitination using proteasome inhibitors has shown some light in HCC treatment. Besides, immunomodulation has been found to be

, H+

Targets and Approaches to Control Hepatocellular Carcinoma in Future

and Na+ fluxes is a general

http://dx.doi.org/10.5772/56867

99

targets for the management of HCC in humans in coming years.

most of their energy requirement. The stimulation of the K+

**2. Therapeutic targets for HCC**

growth regulation in HCC.

below.

Despite the advances in cancer treatments there is no effective chemotherapeutic protocol to treat HCC (Andreana et al. 2009; Arii et al. 2010). Advanced HCC has a poor prognosis ( Simile et al. 2011; Sonja et al. 2010). Historically, no effective systemic chemotherapy treatment options have been available for patients with advanced HCC (Bruix and Sherman 2011). Thus, proper understanding of the molecular basis of pathogenesis of HCC can lead us to plan for proper therapeutic strategies to combat the notorious disease.

Accumulating epidemiological evidence suggests that a pronounced predisposition to develop cancer as a consequence of a mutation in a single gene is rare (approximately 1-5%) (Frau et al. 2010). One possible explanation for this finding is that carcinogenesis is a multistage process involving a number of different genes and environmental factors (Chung et al. 2008; Forner et al. 2010; Frau et al. 2010). In connection with many distinct subtypes of cancer, some functional alterations are required for malignant transformation. They are, namely, sufficiency with respect to growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death (apoptosis), the potential for unlimited replication, sustained angiogenesis, tissue invasion and metastasis (Bergers and Hanahan 2008; Bartolomeo et al. 2011; Cao et al. 2010; Frau et al. 2010; Gouas et al. 2010). The exact number of distinct stages involved may vary from tumor to tumor, since some of these acquired characteristics probably interact with other processes (Roncalli 2010). Indeed, the heterogeneity of tumors, both with regards to morphology and pattern of gene expression, may even indicate the participation of many more sequential steps.

A highly regulatory network controls cellular proliferation in multicellular organisms. Normally cells in many tissues and organs remain in a non-proliferative state. In response to external stimuli such as growth factors, hormones or antigens, cells are stimulated to begin DNA synthesis and cellular proliferation according to the need of the living system. As soon as the need is fulfilled, the cell division stops. However, cancerous cells are characterized by the unrestrained cellular proliferation due to the alteration of normal cellular signalling process and they acquire complete or partial independence of mitogenic signals through production of growth factors (Garrett et al. 2008; Hironaka et al. 2009) and /or alteration in number or structure of cellular receptors (Lachenmayer et al. 2010) and/ or modulation in the activity of post receptor signalling pathway (Cavard et al. 2008; Chen et al. 2009). The com‐ munication of extracellular signals to the cells, then to the nucleus to modulate gene expression is governed by phosphorylation regulated signal transduction cascades which act to amplify the events generated at the cellular membrane by ligand-receptor interaction or cell stress. Therefore, identification of the extracellular factors that modulate cell proliferation and elucidation of the cellular molecular mechanism during the development of cancer can answer many fundamental questions in cancer cell biology. It is important to understand in details the receptors and the signal transduction pathways involved in the pathogenesis of cancer to provide potential target for therapeutic intervention. Many studies have focused to identify molecular pathways to elicit cancer cell proliferation, including HCC. Here many of them have been highlighted to identify fundamental targets of hepatocellular oncogenesis. Thus, the present chapter has been projected to the molecular targets and approaches to intervene the targets for the management of HCC in humans in coming years.

### **2. Therapeutic targets for HCC**

number of risk factors that have been identified (Shariff et al. 2009; Sherman 2010; Gomaa et al. 2008 and 2009). Most of HCC cases develop from a cirrhotic liver (Bartolomeo et al. 2011; Chagas et al. 2009; Orlando et al.2009; Cammà et al. 2008) with an annual incidence of 2-6% for hepatitis B virus carriers (Kew 2010; Lim et al. 2009; Hadziyannis 2011) and 3-5% for hepatitis C virus-infected patients (Masuzaki et al. 2008; Rosen 2011). Males are more suscep‐

Despite the advances in cancer treatments there is no effective chemotherapeutic protocol to treat HCC (Andreana et al. 2009; Arii et al. 2010). Advanced HCC has a poor prognosis ( Simile et al. 2011; Sonja et al. 2010). Historically, no effective systemic chemotherapy treatment options have been available for patients with advanced HCC (Bruix and Sherman 2011). Thus, proper understanding of the molecular basis of pathogenesis of HCC can lead us to plan for

Accumulating epidemiological evidence suggests that a pronounced predisposition to develop cancer as a consequence of a mutation in a single gene is rare (approximately 1-5%) (Frau et al. 2010). One possible explanation for this finding is that carcinogenesis is a multistage process involving a number of different genes and environmental factors (Chung et al. 2008; Forner et al. 2010; Frau et al. 2010). In connection with many distinct subtypes of cancer, some functional alterations are required for malignant transformation. They are, namely, sufficiency with respect to growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death (apoptosis), the potential for unlimited replication, sustained angiogenesis, tissue invasion and metastasis (Bergers and Hanahan 2008; Bartolomeo et al. 2011; Cao et al. 2010; Frau et al. 2010; Gouas et al. 2010). The exact number of distinct stages involved may vary from tumor to tumor, since some of these acquired characteristics probably interact with other processes (Roncalli 2010). Indeed, the heterogeneity of tumors, both with regards to morphology and pattern of gene expression, may even indicate the participation of

A highly regulatory network controls cellular proliferation in multicellular organisms. Normally cells in many tissues and organs remain in a non-proliferative state. In response to external stimuli such as growth factors, hormones or antigens, cells are stimulated to begin DNA synthesis and cellular proliferation according to the need of the living system. As soon as the need is fulfilled, the cell division stops. However, cancerous cells are characterized by the unrestrained cellular proliferation due to the alteration of normal cellular signalling process and they acquire complete or partial independence of mitogenic signals through production of growth factors (Garrett et al. 2008; Hironaka et al. 2009) and /or alteration in number or structure of cellular receptors (Lachenmayer et al. 2010) and/ or modulation in the activity of post receptor signalling pathway (Cavard et al. 2008; Chen et al. 2009). The com‐ munication of extracellular signals to the cells, then to the nucleus to modulate gene expression is governed by phosphorylation regulated signal transduction cascades which act to amplify the events generated at the cellular membrane by ligand-receptor interaction or cell stress. Therefore, identification of the extracellular factors that modulate cell proliferation and elucidation of the cellular molecular mechanism during the development of cancer can answer many fundamental questions in cancer cell biology. It is important to understand in details the

proper therapeutic strategies to combat the notorious disease.

tible to HCC.

98 Hepatocellular Carcinoma - Future Outlook

many more sequential steps.

Three generally considered fundamental but interrelated targets of controlling oncogenesis are regulation of deregulated energy metabolism and ion homeostasis; signal transduction, oncogenes and growth factors; and immunomodulation. One of the most characteristic phenotypes of rapidly growing cancer cells is their propensity to catabolise glucose at high rates. Rapidly growing activity to cancer cells has a reduced number of mitochondria and increased glycolytic activity with a shift from respiratory to fermentative ATP supply to cover most of their energy requirement. The stimulation of the K+ , H+ and Na+ fluxes is a general early response in most of the quiescent cells stimulated to proliferate by multiple combinations of growth promoting factors. Growth factors, cytokines exert their action on cell proliferation by modulation of cell signalling process. There is a strong relationship between the immune system and cell proliferation. Immune suppressive agents have a powerful effect on hepatocyte growth regulation in HCC.

Reviewing current literature, a selection of therapeutic targets of HCC has been described below.

Like most other cancers, growth factors, their receptors, and downstream signalling proteins play a pivotal role in the development and maintenance of HCC and are of significant interest for future therapeutic approaches. In foetal liver, a large number of growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), transforming growth factors-α and –β (TGF-α, TGF-β), and vascular endothelial growth factor (VEGF) (Höpfner et al. 2008; Hoshida et al. 2008 and 2009) are produced. Their secretion either declines or shuts down in adult liver. However, during hepatic regenera‐ tion due to the cause of hepatic injury or damage many such growth factors (Böhm et al. 2010), namely, EGF, TGF-α, IGFs, and VEGF are upregulated in normal hepatocytes. The transient upregulation of those factors is dysregulated in the chronic injured liver leading to sustained mitogenic/ oncogenic signalling, during the development of HCC. FGF and PDGF released from non-hepatocyte sources such as activated hepatic stellate cells, myofibroblasts, endothelial cells, Kupffer cells, and bile duct epithelia have been shown to play important roles in promoting hepatic fibrosis and HCC growth (Friedman 2008). The ubiquitin-proteasome pathway has emerged as a key player in the regulation of several diverse cellular processes. Inhibition of poly-ubiquitination using proteasome inhibitors has shown some light in HCC treatment. Besides, immunomodulation has been found to be effective in stabilizing HCC growth in patients. Number of immunomodulators has been investigated and few of them have been found to be effective. They have been discussed below under immunomodulation agents. Signal transduction, and growth factors; inhibi‐ tors of proteasome pathway, immunomodulation and antisense oligomer-mediated inhibition of targeted oncogenes have been projected as future targets and approaches of HCC (Table 1).

the inhibition of angiogenesis is a potential and promising therapeutic approach in HCC. Anti-VEGF therapy with sorafenib was the first systemic therapy against VEGF to demonstrate improved survival in patients with advanced-stage HCC (Cheng et al. 2009; Zhu et al. 2011, Miller et al. 2009; Zhu 2008, Llovet et al. 2008a). Sorafenib was also tested in advanced stage liver cirrhosis patients with unresectable HCC (Pinter et al. 2009). Bevacizumab alone or in combination with other agents showed promise in patients with advanced HCC (Siegel et al. 2008; Thomas et al. 2009; Thomas et al. 2008; Kaseb et al. 2012). However, the common bevacizumab-related side effects were hypertension, bleeding, and proteinuria (Thomas et al. 2009; Siegel et al. 2008a; Kopetz et al. 2009). Besides, inhibition of the tyrosine kinase activity of VEGFR has been tried as an effective measure to inhibit angeogenesis in HCC (Bhide et al. 2010). PTK787/ ZK222584 (vatalanib) is an oral angiogenesis inhibitor that targets tyrosine kinase activity of VEGFR (Gauler et al. 2012). Pan-VEGFR tyrosine kinase activity inhibitor

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101

Like all other cancers diverse signaling pathways in HCC are very complex. One of the key pathways regulating cellular proliferation is the mitogen activated protein Kinase (MAPK) pathway. Other pathways involved in the development of HCC include the PI3K/Akt/mTOR, hepatocyte growth factor (HGF)/c-MET, insulin-like growth factor (IGF) and its receptor (IGFR) pathways, and the Wnt-β catenin pathway (Cavard et al. 2008;Chen et al. 2009; Desbois-Mouthon et al. 2009; Takigawa and Nouso 2008; Zhang et al. 2008). The Raf family of kinases are central to this pathway where the transduction of extracellular growth signals from the cell surface to the nucleus occurs via the ras-raf-MEK-ERK signaling cascade. The several experi‐ ments have shown that Raf, MEK, MAP Kinase are downstream effector molecules of Ras and their sequential order in the pathway. The Raf serine/threonine kinases are the principal effectors of Ras in this mitogen activated protein Kinase (MAPK) signaling pathway. As serine/ threonine kinases, Raf proteins phosphorylate and activate serine and threonine residues on subsequent downstream effector proteins of Ras. Therefore, molecularly targeted agents that interact with multiple signaling pathways/effectors appear to be very promising in the treatment of patients with HCC (Cervello et al. 2012; Cheng et al. 2009). The novel bi-aryl urea sorafenib, an orally available multi-kinase inhibitor, targets kinases of wild-type B-Raf, mutant V559EB-Raf and cRaf, thereby blocking tumor growth (Spangenberg et al. 2008). There are three ras protooncogenes that encode 21 Kd proteins – H-Ras (Harvey murine sarcoma virus), N-Ras (neuroblastoma cell line) and two alternatively spliced K-Ras, K-Ras 4A, and K-Ras 4B; These isoforms are capable of differentially activating various critical effectors, thereby exerting distinct biologic effects. Sorafenib, an inhibitor of receptor tyrosine kinases was found to stabilize the advanced unresectable HCC patients by regulating angiogenesis, and was approved by regulatory agencies in 2007. It has a role on human VEGF receptors-2 and -3 (VEGFR-2/-3) and PDGF-βR. However, sorafenib has been also suggested to provide antitu‐ mor action in HCC by inhibition of the Raf/MEK/ERK pathway (Llovet and Bruix, 2008 and 2009). Multikinase inhibitor sunitinib is a small molecule that inhibits members of the splitkinase domain family of receptor tyrosine kinase including VEGFR types 1 and 2 (Llovet et al. 2008a). Antiangiogenic effects of sunitinib have been suggested through VEGFR and PDGFR.

with activity against PDGFRs also carries a new hope.

**3.3. Multi-kinase inhibitor**


**Table 1.** Growth factors, proteasome-inhibitors, immunomodulators and antisense oligomers in HCC

### **3. Approaches**

A selection of agents currently in the development and/or testing stages for the clinical application in targeted HCC treatment is summarized in the following section.

#### **3.1. Therapies against EGFR**

There are two classes of anti-EGFR agents found to have antitumor activity against HCC. One of them belongs to monoclonal antibodies (as an example cetuximab) which competitively inhibit extracellular endogenous ligand binding. The other class belongs to chemicals such as gefitinib, erlotinib which inhibit the intracellular tyrosine kinase domain. EGF, TGF-α, heparin binding-EGF and EGFR have been shown to involve in the pathogenesis of HCC. Thus, EGFR signalling pathways have become a potential investigating area of research to identify the target (s) to inhibit proliferation of HCC and metastasis. Gefitinib, erlotinib, cetuximab were tested in patients with advanced HCC (Thomas et al. 2007; Philip et al. 2005; Asnacios et al. 2008; Wu et al. 2011, Levêque 2011) and were reported to possess signals of activity in controlling the progress of HCC in a variable extent.

#### **3.2. Targeting approaches towards VEGF and VEGFR**

HCCs rely on the formation of new blood vessels for growth, and VEGF is critical in this process (Zhu et al. 2011). HCCs are with high vascular architecture and VEGF is a key factor in tumor angiogenesis (Bergers and Hanahan, 2008; Garrett et al. 2008; Hironaka et al. 2009). Therefore, the inhibition of angiogenesis is a potential and promising therapeutic approach in HCC. Anti-VEGF therapy with sorafenib was the first systemic therapy against VEGF to demonstrate improved survival in patients with advanced-stage HCC (Cheng et al. 2009; Zhu et al. 2011, Miller et al. 2009; Zhu 2008, Llovet et al. 2008a). Sorafenib was also tested in advanced stage liver cirrhosis patients with unresectable HCC (Pinter et al. 2009). Bevacizumab alone or in combination with other agents showed promise in patients with advanced HCC (Siegel et al. 2008; Thomas et al. 2009; Thomas et al. 2008; Kaseb et al. 2012). However, the common bevacizumab-related side effects were hypertension, bleeding, and proteinuria (Thomas et al. 2009; Siegel et al. 2008a; Kopetz et al. 2009). Besides, inhibition of the tyrosine kinase activity of VEGFR has been tried as an effective measure to inhibit angeogenesis in HCC (Bhide et al. 2010). PTK787/ ZK222584 (vatalanib) is an oral angiogenesis inhibitor that targets tyrosine kinase activity of VEGFR (Gauler et al. 2012). Pan-VEGFR tyrosine kinase activity inhibitor with activity against PDGFRs also carries a new hope.

#### **3.3. Multi-kinase inhibitor**

effective in stabilizing HCC growth in patients. Number of immunomodulators has been investigated and few of them have been found to be effective. They have been discussed below under immunomodulation agents. Signal transduction, and growth factors; inhibi‐ tors of proteasome pathway, immunomodulation and antisense oligomer-mediated inhibition of targeted oncogenes have been projected as future targets and approaches of

Growth factors, e.g., EGFR, EGF, TGF-α, TGF-β, VEGF Cetuximab, Gefitinib, Erlotinib, Vandetanib. mTOR Rapamycin, Temsirolimus, Salirasib, RAD001 Multikinase Sorafenib, Everolimus, AP23573, RAD001.

**Table 1.** Growth factors, proteasome-inhibitors, immunomodulators and antisense oligomers in HCC

application in targeted HCC treatment is summarized in the following section.

Immunomodulators used in HCC Thymostimulin, Retinoids, Everolimus, Azathioprine, 6-

A selection of agents currently in the development and/or testing stages for the clinical

There are two classes of anti-EGFR agents found to have antitumor activity against HCC. One of them belongs to monoclonal antibodies (as an example cetuximab) which competitively inhibit extracellular endogenous ligand binding. The other class belongs to chemicals such as gefitinib, erlotinib which inhibit the intracellular tyrosine kinase domain. EGF, TGF-α, heparin binding-EGF and EGFR have been shown to involve in the pathogenesis of HCC. Thus, EGFR signalling pathways have become a potential investigating area of research to identify the target (s) to inhibit proliferation of HCC and metastasis. Gefitinib, erlotinib, cetuximab were tested in patients with advanced HCC (Thomas et al. 2007; Philip et al. 2005; Asnacios et al. 2008; Wu et al. 2011, Levêque 2011) and were reported to possess signals of activity in

HCCs rely on the formation of new blood vessels for growth, and VEGF is critical in this process (Zhu et al. 2011). HCCs are with high vascular architecture and VEGF is a key factor in tumor angiogenesis (Bergers and Hanahan, 2008; Garrett et al. 2008; Hironaka et al. 2009). Therefore,

Proteasome Bortezomeb

controlling the progress of HCC in a variable extent.

**3.2. Targeting approaches towards VEGF and VEGFR**

Antisense oligonucleotides used in HCC ISIS5132. ISIS2513

**Molecular targets in HCC Inhibitors / Modulators / Antisense oligonucleotides**

mercaptopurine.

HCC (Table 1).

100 Hepatocellular Carcinoma - Future Outlook

**3. Approaches**

**3.1. Therapies against EGFR**

Like all other cancers diverse signaling pathways in HCC are very complex. One of the key pathways regulating cellular proliferation is the mitogen activated protein Kinase (MAPK) pathway. Other pathways involved in the development of HCC include the PI3K/Akt/mTOR, hepatocyte growth factor (HGF)/c-MET, insulin-like growth factor (IGF) and its receptor (IGFR) pathways, and the Wnt-β catenin pathway (Cavard et al. 2008;Chen et al. 2009; Desbois-Mouthon et al. 2009; Takigawa and Nouso 2008; Zhang et al. 2008). The Raf family of kinases are central to this pathway where the transduction of extracellular growth signals from the cell surface to the nucleus occurs via the ras-raf-MEK-ERK signaling cascade. The several experi‐ ments have shown that Raf, MEK, MAP Kinase are downstream effector molecules of Ras and their sequential order in the pathway. The Raf serine/threonine kinases are the principal effectors of Ras in this mitogen activated protein Kinase (MAPK) signaling pathway. As serine/ threonine kinases, Raf proteins phosphorylate and activate serine and threonine residues on subsequent downstream effector proteins of Ras. Therefore, molecularly targeted agents that interact with multiple signaling pathways/effectors appear to be very promising in the treatment of patients with HCC (Cervello et al. 2012; Cheng et al. 2009). The novel bi-aryl urea sorafenib, an orally available multi-kinase inhibitor, targets kinases of wild-type B-Raf, mutant V559EB-Raf and cRaf, thereby blocking tumor growth (Spangenberg et al. 2008). There are three ras protooncogenes that encode 21 Kd proteins – H-Ras (Harvey murine sarcoma virus), N-Ras (neuroblastoma cell line) and two alternatively spliced K-Ras, K-Ras 4A, and K-Ras 4B; These isoforms are capable of differentially activating various critical effectors, thereby exerting distinct biologic effects. Sorafenib, an inhibitor of receptor tyrosine kinases was found to stabilize the advanced unresectable HCC patients by regulating angiogenesis, and was approved by regulatory agencies in 2007. It has a role on human VEGF receptors-2 and -3 (VEGFR-2/-3) and PDGF-βR. However, sorafenib has been also suggested to provide antitu‐ mor action in HCC by inhibition of the Raf/MEK/ERK pathway (Llovet and Bruix, 2008 and 2009). Multikinase inhibitor sunitinib is a small molecule that inhibits members of the splitkinase domain family of receptor tyrosine kinase including VEGFR types 1 and 2 (Llovet et al. 2008a). Antiangiogenic effects of sunitinib have been suggested through VEGFR and PDGFR. However, a randomized phase 3 study in HCC failed to show a significant survival benefit as compared to sorafenib and study stopped in 2011.

ways, including modulation of splicing and inhibition of protein translation by disruption of ribosome assembly. Single stranded synthetic nucleic acid (oligonucleotide) when hybridize with DNA or RNA alters transcription or prevents translation thus, preventing or modifying protein production. Because of the volume of information nowadays available on gene sequencing, there has been burst of exploration of capacity for oligomers to inhibit gene/ protein expression. Thus, antisense therapies focus on controlling the production of the proteins on a genetic level. A strand of mRNA is transcribed from DNA, and is a copy of the "coding" or "sense" strand of the gene. The main form of therapy uses the complementary or antisense strand to hybridize the sense strand or mRNA and thus it prevents production of the protein by blocking or altering transcription or translation (Figure 1). With the backbone chemical modifications (in phosphate linkage), antisense oligonucleotides increase resistance to nuclease digestion, prolong their biological half-lives and significantly suppress target-gene expression. Antisense oligonucleotides have been studied for several years as treatments for many diseases and genetic disorders. The therapy is based on the principles of genetic expression. The most widely used modified oligomers in antisense therapies is phosphoro‐ thioate oligonucleotides, which have much greater resistance to digestion by nucleases. Phosphorothioate oligonucleotides are rapidly and extensively absorbed and distributed from

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**Figure 1.** Antisense oligomer-mRNA duplex inhibiting to synthesize peptide chain

The first antisense treatment to get FDA approval to date has been Formivirsen (Vitravene), which is a treatment for cytomegalovirus (CMV) retinitis in people with acquired immuno‐ deficiency disease (AIDS) (Rahman et al. 2008). Several antisense oligonucleotides were shown to target various oncogenes, to overcome tumour escape and to improve therapeutic activity.

blood.

#### **3.4. mTOR inhibitors**

mTOR inhibitors are potential anti-HCC agents for future (Zhou et al. 2009). Promising mTOR inhibitors are rapamycin and its analogues such as sirolimus, temsirolimus (CCI-779), everolimus (RAD001) and AP23573 (Nocera et al 2008; Rizell et al. 2008). Rapamycin and its analogues such as temsirolimus ( the cell cycle inhibitor) and everolimus and AP23573 (an orally bioavailable derivative of rapamycin) modulate angiogennesis to improve survival of patients in advanced HCC (Heuer 2009, Huynh et al. 2008 ). RAD001, an orally-administered, novel mTOR inhibitor was evaluated in a phase I study (Huynh et al. 2008 and 2008a; Chen et al 2009). Treatment of patients with the combination of rapamycin/ rapamycin-analogue(s) with conventional anticancer drug(s) such as doxorubicin, vinblastine has been found to improve survival in advanced HCC patients (Spangenberg et al. 2008).

#### **3.5. Proteasome inhibition**

HCC is highly ubiquitinated. The ubiquitination is important to the development and progression of HCC. Proteasome inhibitor such as bortezomib blocks multi-ubiquitinated protein degradation by reversible and competitive inhibition of the active site threonine residue of the 26S proteasome (Cao and Mao, 2011; Boozari et al. 2009). Antineoplastic activity of bortezomib approved for the treatment of mantle cell lymphoma has already been shown to stabilize advanced HCC in patients (Höpfner et al 2008).

#### **3.6. Immunomodulatory agents**

An immunomodulator is a substance which has an effect on the immune system. An immu‐ nomodulator may be at the same time an immunosuppressant or an immunostimulant and can act on different targets within the immune system. Cell signalling process regulates immune system consisting of immunomodulatory endogenic chemicals and cells. Immuno‐ modulators interfere with the signalling process by shifting the homeostasis of the immune system to reduce or eliminate disease symptoms. Thus these compounds are the obvious choice for therapeutic intervention of HCC. Thymostimulin (a standardized low molecular protein fraction containing thymosin alpha 1 and thymic humoral factor) has been shown to produce cytotoxic immune reaction against HCC. Phase II trials using thymostimulin in patients with advanced and metastasised HCC have shown to control metastatic HCC without predominant side-effects (Dollinger et al. 2010). However, thymostimulin administration in some patients was found to accumulate ascites and cause renal failure (Dollinger et al. 2010).

#### **3.7. Antisense therapy**

Antisense oligonucleotides offer one approach to target genes involved in cancer progression. They are typically less than 50 nucleotides long and are specifically designed to hybridize to corresponding gene/ mRNA by Watson-Crick binding. They inhibit mRNA function in several ways, including modulation of splicing and inhibition of protein translation by disruption of ribosome assembly. Single stranded synthetic nucleic acid (oligonucleotide) when hybridize with DNA or RNA alters transcription or prevents translation thus, preventing or modifying protein production. Because of the volume of information nowadays available on gene sequencing, there has been burst of exploration of capacity for oligomers to inhibit gene/ protein expression. Thus, antisense therapies focus on controlling the production of the proteins on a genetic level. A strand of mRNA is transcribed from DNA, and is a copy of the "coding" or "sense" strand of the gene. The main form of therapy uses the complementary or antisense strand to hybridize the sense strand or mRNA and thus it prevents production of the protein by blocking or altering transcription or translation (Figure 1). With the backbone chemical modifications (in phosphate linkage), antisense oligonucleotides increase resistance to nuclease digestion, prolong their biological half-lives and significantly suppress target-gene expression. Antisense oligonucleotides have been studied for several years as treatments for many diseases and genetic disorders. The therapy is based on the principles of genetic expression. The most widely used modified oligomers in antisense therapies is phosphoro‐ thioate oligonucleotides, which have much greater resistance to digestion by nucleases. Phosphorothioate oligonucleotides are rapidly and extensively absorbed and distributed from blood.

However, a randomized phase 3 study in HCC failed to show a significant survival benefit as

mTOR inhibitors are potential anti-HCC agents for future (Zhou et al. 2009). Promising mTOR inhibitors are rapamycin and its analogues such as sirolimus, temsirolimus (CCI-779), everolimus (RAD001) and AP23573 (Nocera et al 2008; Rizell et al. 2008). Rapamycin and its analogues such as temsirolimus ( the cell cycle inhibitor) and everolimus and AP23573 (an orally bioavailable derivative of rapamycin) modulate angiogennesis to improve survival of patients in advanced HCC (Heuer 2009, Huynh et al. 2008 ). RAD001, an orally-administered, novel mTOR inhibitor was evaluated in a phase I study (Huynh et al. 2008 and 2008a; Chen et al 2009). Treatment of patients with the combination of rapamycin/ rapamycin-analogue(s) with conventional anticancer drug(s) such as doxorubicin, vinblastine has been found to

HCC is highly ubiquitinated. The ubiquitination is important to the development and progression of HCC. Proteasome inhibitor such as bortezomib blocks multi-ubiquitinated protein degradation by reversible and competitive inhibition of the active site threonine residue of the 26S proteasome (Cao and Mao, 2011; Boozari et al. 2009). Antineoplastic activity of bortezomib approved for the treatment of mantle cell lymphoma has already been shown

An immunomodulator is a substance which has an effect on the immune system. An immu‐ nomodulator may be at the same time an immunosuppressant or an immunostimulant and can act on different targets within the immune system. Cell signalling process regulates immune system consisting of immunomodulatory endogenic chemicals and cells. Immuno‐ modulators interfere with the signalling process by shifting the homeostasis of the immune system to reduce or eliminate disease symptoms. Thus these compounds are the obvious choice for therapeutic intervention of HCC. Thymostimulin (a standardized low molecular protein fraction containing thymosin alpha 1 and thymic humoral factor) has been shown to produce cytotoxic immune reaction against HCC. Phase II trials using thymostimulin in patients with advanced and metastasised HCC have shown to control metastatic HCC without predominant side-effects (Dollinger et al. 2010). However, thymostimulin administration in some patients

Antisense oligonucleotides offer one approach to target genes involved in cancer progression. They are typically less than 50 nucleotides long and are specifically designed to hybridize to corresponding gene/ mRNA by Watson-Crick binding. They inhibit mRNA function in several

was found to accumulate ascites and cause renal failure (Dollinger et al. 2010).

improve survival in advanced HCC patients (Spangenberg et al. 2008).

to stabilize advanced HCC in patients (Höpfner et al 2008).

compared to sorafenib and study stopped in 2011.

**3.4. mTOR inhibitors**

102 Hepatocellular Carcinoma - Future Outlook

**3.5. Proteasome inhibition**

**3.6. Immunomodulatory agents**

**3.7. Antisense therapy**

**Figure 1.** Antisense oligomer-mRNA duplex inhibiting to synthesize peptide chain

The first antisense treatment to get FDA approval to date has been Formivirsen (Vitravene), which is a treatment for cytomegalovirus (CMV) retinitis in people with acquired immuno‐ deficiency disease (AIDS) (Rahman et al. 2008). Several antisense oligonucleotides were shown to target various oncogenes, to overcome tumour escape and to improve therapeutic activity. Several studies have shown the anticancer potential of antisense oligonucleotides (Das et al. 2010; Rayburn and Zhang, 2008) and many of them are in clinical trial. They have less cytotoxic side-effects than conventional chemotherapy agents. Systemic treatment with fomivirsen is a milestone in the field of antisense treatment with antisense oligonucleotides. This has led the way for development of antisense oligonucleotides for various new potential targets for the treatment of cancer, including HCC.

[2] Arii S, Sata M, Sakamoto M, Shimada M, Kumada T, Shiina S. Management of hepa‐ tocellular carcinoma Report of Consensus Meeting in the 45th Annual Meeting of the

Targets and Approaches to Control Hepatocellular Carcinoma in Future

http://dx.doi.org/10.5772/56867

105

[3] Asnacios A, Fartoux L, Romano O, Tesmoingt C, Louafi SS, Mansoubakht T. Gemci‐ tabine plus oxaliplatin Gemox combined with cetuximab in patients with progressive advanced stage hepatocellular carcinoma: results of a multicenter phase 2 studies.

[4] Bartolomeo N, Trerotoli P, Serio G. Progression of liver cirrhosis to HCC: an applica‐

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[13] Cao B, Mao X. The ubiquitin-proteasomal system is critical for multiple myeloma:

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implications in drug discovery. Am J Blood Res 2011; 1 (1) 46–56.

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growth factors and cytokines. EMBO Mol Med 2010; 2 (8) 294–305.

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#### **4. Conclusion**

Several experimental evidences have established that targeted inhibition of genes/ proteins involved in controlling HCC growth combined with cytostatic anticancer treatments is a promisingapproachforHCCtherapy.Blockingof singlegene/proteinhasbeenfoundtocontrol neoplastic cellular proliferation *in vitro* effectively. However, considering the multitude of molecular entities and signalling pathways that regulate the proliferation and the life/death decisionincancer cells,inhibitionof a single target genemaynot be sufficientto suppress tumor growth. The preclinical/ clinical trials of several potential compounds targeting liver cancerrelevant genes/ proteins may address more specific and adequate future therapies for HCC.

### **Acknowledgements**

Authors are indebted to the grants from Department of Science Technology (Govt. of India) (DST/Inspire Fellowship/ 2010/ 87) and Indian Council of Medical Research (58/7/2009-BMS) for funding the related projects and findings of which have helped us partially to write this work.

### **Author details**

Mukherjee Biswajit\* , Hossain Chowdhury Mobaswar , Bhattacharya Sanchari and Shampa Ghosh

\*Address all correspondence to: biswajit55@yahoo.com

Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India

#### **References**

[1] Andreana L, Isgr Ó G, Pleguezuelo M. Surveillance and diagnosis of hepatocellular carcinoma in patients with cirrhosis. World J Hepatol 2009; 1 (1) 48–61.

[2] Arii S, Sata M, Sakamoto M, Shimada M, Kumada T, Shiina S. Management of hepa‐ tocellular carcinoma Report of Consensus Meeting in the 45th Annual Meeting of the Japan Society of Hepatology. Hepatol Res 2010; 40 (7) 667–685.

Several studies have shown the anticancer potential of antisense oligonucleotides (Das et al. 2010; Rayburn and Zhang, 2008) and many of them are in clinical trial. They have less cytotoxic side-effects than conventional chemotherapy agents. Systemic treatment with fomivirsen is a milestone in the field of antisense treatment with antisense oligonucleotides. This has led the way for development of antisense oligonucleotides for various new potential targets for the

Several experimental evidences have established that targeted inhibition of genes/ proteins involved in controlling HCC growth combined with cytostatic anticancer treatments is a promisingapproachforHCCtherapy.Blockingof singlegene/proteinhasbeenfoundtocontrol neoplastic cellular proliferation *in vitro* effectively. However, considering the multitude of molecular entities and signalling pathways that regulate the proliferation and the life/death decisionincancer cells,inhibitionof a single target genemaynot be sufficientto suppress tumor growth. The preclinical/ clinical trials of several potential compounds targeting liver cancerrelevant genes/ proteins may address more specific and adequate future therapies for HCC.

Authors are indebted to the grants from Department of Science Technology (Govt. of India) (DST/Inspire Fellowship/ 2010/ 87) and Indian Council of Medical Research (58/7/2009-BMS) for funding the related projects and findings of which have helped us partially to write this

, Hossain Chowdhury Mobaswar , Bhattacharya Sanchari and

[1] Andreana L, Isgr Ó G, Pleguezuelo M. Surveillance and diagnosis of hepatocellular

carcinoma in patients with cirrhosis. World J Hepatol 2009; 1 (1) 48–61.

Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India

treatment of cancer, including HCC.

104 Hepatocellular Carcinoma - Future Outlook

**4. Conclusion**

**Acknowledgements**

work.

**Author details**

Mukherjee Biswajit\*

\*Address all correspondence to: biswajit55@yahoo.com

Shampa Ghosh

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**Chapter 7**

**Early Chronic Inflammation and Subsequent Somatic**

**Mutations Shift Phospho-Smad3 Signaling from Tumor-**

**Suppression to Fibro-Carcinogenesis in Human Chronic**

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide [1]. HCC is strongly associated with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, which are implicated in about 80% of HCCs in certain geographic area [2]. Risk of HCC is increased 5- to 15-fold in chronic HBV carriers [1] and 11.5 to 17-fold in HCV-infected patients [3]. In addition, epidemiological studies have shown that chronic inflammation of the liver predisposes individuals to HCC. Most HCCs are associated with severe fibrosis or cirrhosis caused by unresolved inflammation. Both HBV and HCV show a wide spectrum of clinical manifestations, ranging from a healthy carrier state to chronic hepatitis, cirrhosis and HCC. Notably, HCC occurs less often in chronic viral hepatitis with‐ out cirrhosis. As liver fibrosis progresses from chronic hepatitis to cirrhosis, HCC occurrence increases [4]. Thus, unresolved inflammation with long-term viral infection leads to HCC associated with cirrhosis. Approaches to understanding how human HCC develops in chronic inflammatory liver diseases should therefor focus on molecular mechanisms shared between

Transforming growth factor (TGF)-β is a key regulator of many important biologic processes. TGF-β can inhibit epithelial cell growth, physiologically acting as a tumor suppressor, but it also can promote neoplasia. TGF-β has been shown to play both tumor-suppressive and tumor promoting roles [5-7]. As disease progresses toward malignancy, cancer cells gain advantage

> © 2013 Murata et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Liver Diseases**

Koichi Matsuzaki

**1. Introduction**

http://dx.doi.org/10.5772/56739

Miki Murata, Katsunori Yoshida and

Additional information is available at the end of the chapter

liver fibrosis and carcinogenesis (fibro-carcinogenesis).

**Early Chronic Inflammation and Subsequent Somatic Mutations Shift Phospho-Smad3 Signaling from Tumor-Suppression to Fibro-Carcinogenesis in Human Chronic Liver Diseases**

Miki Murata, Katsunori Yoshida and Koichi Matsuzaki

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56739

**1. Introduction**

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide [1]. HCC is strongly associated with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, which are implicated in about 80% of HCCs in certain geographic area [2]. Risk of HCC is increased 5- to 15-fold in chronic HBV carriers [1] and 11.5 to 17-fold in HCV-infected patients [3]. In addition, epidemiological studies have shown that chronic inflammation of the liver predisposes individuals to HCC. Most HCCs are associated with severe fibrosis or cirrhosis caused by unresolved inflammation. Both HBV and HCV show a wide spectrum of clinical manifestations, ranging from a healthy carrier state to chronic hepatitis, cirrhosis and HCC. Notably, HCC occurs less often in chronic viral hepatitis with‐ out cirrhosis. As liver fibrosis progresses from chronic hepatitis to cirrhosis, HCC occurrence increases [4]. Thus, unresolved inflammation with long-term viral infection leads to HCC associated with cirrhosis. Approaches to understanding how human HCC develops in chronic inflammatory liver diseases should therefor focus on molecular mechanisms shared between liver fibrosis and carcinogenesis (fibro-carcinogenesis).

Transforming growth factor (TGF)-β is a key regulator of many important biologic processes. TGF-β can inhibit epithelial cell growth, physiologically acting as a tumor suppressor, but it also can promote neoplasia. TGF-β has been shown to play both tumor-suppressive and tumor promoting roles [5-7]. As disease progresses toward malignancy, cancer cells gain advantage

© 2013 Murata et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. by selective reduction of the tumor-suppressive activity of TGF-β together with augmentation of TGF-β oncogenic activity [6]. In concert with mitogens, TGF-β induces accumulation of extracellular matrix (ECM), while mitogenic signaling antagonizes cytostatic TGF-β function [8,9]. These results indicate that perturbation of TGF-β signaling by mitogens can promote hepatic fibro-carcinogenesis.

The TGF-β superfamily includes many multifunctional cytokines including TGF-β, activin, and others [6,10]. Progress over the past 10 years has disclosed important details of how the TGF-β family elicits its responses [11-14]. Smads, central mediators conveying signals from receptors for TGF-β superfamily members to the nucleus, are modular proteins with conserved Mad-homology (MH)1, intermediate linker, and MH2 domains [13]. In cell-signaling path‐ ways, various transcription factors are phosphorylated at multiple sites by upstream kinases. Catalytically active TGF-β type I receptor (TβRI) phosphorylates COOH-tail serine residues of receptor-activated Smads (R-Smads), which include Smad2 and the highly similar protein Smad3 [12]. Mitogenic signals alternatively cause phosphorylation of R-Smad at specific sites in their middle linker regions [15-20]. After a phosphorylated R-Smad rapidly oligomerizes with Smad4, this complex translocates to the nucleus, where it regulates transcription of target genes.

Monitoring phosphorylation status of signaling molecules is a key step in dissecting their pathways. In Smad signaling, phosphorylation of not only the COOH-tail but also the linker regions of R-Smads are likely to be important in regulating Smad activity under physiologic and pathologic conditions [21]. Understanding of molecular mechanisms underlying hepatitis virus-induced fibro-carcinogenesis can help to guide early management and improve therapy for patients with chronic liver diseases. This review describes current knowledge of the molecular pathogenesis of human fibro-carcinogenesis, especially concerning Smad3 phos‐ phorylation profiles. We further consider how enhanced understanding of phospho-Smad3 signaling could lead to more effective prevention of human fibro-carcinogenesis.

### **2. Smad3 phosphoisoforms**

The canonical TGF-β pathway involves Smad2 and Smad3 signaling through direct serine phosphorylation of COOH termini by TβRI upon TGF-β binding (Figure 1A), [10,13]. TβRImediated phosphorylation of Smad2 and Smad3 induces their association with the shared partner Smad4, followed by translocation into the nucleus where these complexes activate transcription of specific genes [10-14]. Smad2 and Smad3 proteins contain a conserved Madhomology (MH)1 domain that binds DNA, and a conserved MH2 domain that binds to receptors, Smad4, and transcription co-activators.

1B), [15-26]. TGF-β alternatively elicits signaling responses through non-Smad pathways representing important effectors for TGF-β activated kinase (TAK) 1 in response to proinflammatory cytokines. TAK1 activates JNK and p38 MAPK signaling through mitogenactivated kinase kinase (MKK) 4/7 and MKK3/6 [27,28]. JNK and p38 MAPK have been linked to modification of TGF-β signaling by pro-inflammatory cytokines through their regulation of distinct processes such as cytoskeleton organization, cell growth, survival, migration and invasion [29]. Imbalances between signaling through non-Smad and Smad pathways may occur during fibro-carcinogenesis, with interaction between these pathways mediating pro-

**Figure 1. Reversible phospho-Smad3 signaling between tumor-suppression and fibro-carcinogenesis** A) TGF-β treatment activates TβRI, further leading to direct phosphorylation of Smad3C, which inhibits normally hepatocytic growth by up-regulating p21WAF1 transcription. B) Mitogens drastically alter phospho-Smad3 signaling via the JNK pathway, increasing basal nuclear fibro-carcinogenic pSmad3L activity while shutting down TGF-β-dependent cyto‐ static pSmad3C. Although TGF-β signal weakly phosphorylates Smad3L in normal hepatocytes (dotted line), hepatitis viral components including HBx, pro-inflammatory cytokines including TNF-α, and somatic mutations such as Ras addi‐ tively transmit fibro-carcinogenic signal through the JNK-dependent pSmad3L pathway to participate in hepatocytic growth and ECM deposition, possibly by stimulating transcription of *c-Myc* and *PAI-1* genes. Linker phosphorylation of Smad3 indirectly prevents COOH-tail phosphorylation, pSmad3C-mediated p21WAF1 transcriptions and cytostatic func‐ tion. C) Either various JNK inhibitors or a Smad3 mutation causing lack of JNK phosphorylation sites in the linker re‐ gion can eliminate fibro-carcinogenic pSmad3L signaling, restoring or maintaining the tumor-suppressive pSmad3C

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Findings in mice with targeted deletion of Smad3 and JNK1 indicate that both Smad3 and JNK1 pathways promote hepatic fibro-carcinogesis. When acute liver injury was induced by administration of CCl4, *Smad3*-/- mice showed approximately half as much of the induction of collagen type I mRNA as seen in wild-type mice [31]. *JNK1*-/- mice resisted not only liver fibrosis

fibrogenic and pro-tumorigenic effects of TGF-β [30]

signaling characteristic of mature hepatocytes.

More divergent linker regions separate the two domains [13]. The linker domain undergoes regulatory phosphorylation by Ras/mitogen-activated protein kinase (MAPK) pathways including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 MAPK, and cyclin-dependent kinase (CDK)-2/4, as well as glycogen synthase kinase 3-β, Ca (2+)-calmodulin-dependent protein kinase II, and G protein-coupled receptor kinase-2 (Figure Early Chronic Inflammation and Subsequent Somatic Mutations Shift Phospho-Smad3 Signaling from… http://dx.doi.org/10.5772/56739 115

by selective reduction of the tumor-suppressive activity of TGF-β together with augmentation of TGF-β oncogenic activity [6]. In concert with mitogens, TGF-β induces accumulation of extracellular matrix (ECM), while mitogenic signaling antagonizes cytostatic TGF-β function [8,9]. These results indicate that perturbation of TGF-β signaling by mitogens can promote

The TGF-β superfamily includes many multifunctional cytokines including TGF-β, activin, and others [6,10]. Progress over the past 10 years has disclosed important details of how the TGF-β family elicits its responses [11-14]. Smads, central mediators conveying signals from receptors for TGF-β superfamily members to the nucleus, are modular proteins with conserved Mad-homology (MH)1, intermediate linker, and MH2 domains [13]. In cell-signaling path‐ ways, various transcription factors are phosphorylated at multiple sites by upstream kinases. Catalytically active TGF-β type I receptor (TβRI) phosphorylates COOH-tail serine residues of receptor-activated Smads (R-Smads), which include Smad2 and the highly similar protein Smad3 [12]. Mitogenic signals alternatively cause phosphorylation of R-Smad at specific sites in their middle linker regions [15-20]. After a phosphorylated R-Smad rapidly oligomerizes with Smad4, this complex translocates to the nucleus, where it regulates transcription of target

Monitoring phosphorylation status of signaling molecules is a key step in dissecting their pathways. In Smad signaling, phosphorylation of not only the COOH-tail but also the linker regions of R-Smads are likely to be important in regulating Smad activity under physiologic and pathologic conditions [21]. Understanding of molecular mechanisms underlying hepatitis virus-induced fibro-carcinogenesis can help to guide early management and improve therapy for patients with chronic liver diseases. This review describes current knowledge of the molecular pathogenesis of human fibro-carcinogenesis, especially concerning Smad3 phos‐ phorylation profiles. We further consider how enhanced understanding of phospho-Smad3

The canonical TGF-β pathway involves Smad2 and Smad3 signaling through direct serine phosphorylation of COOH termini by TβRI upon TGF-β binding (Figure 1A), [10,13]. TβRImediated phosphorylation of Smad2 and Smad3 induces their association with the shared partner Smad4, followed by translocation into the nucleus where these complexes activate transcription of specific genes [10-14]. Smad2 and Smad3 proteins contain a conserved Madhomology (MH)1 domain that binds DNA, and a conserved MH2 domain that binds to

More divergent linker regions separate the two domains [13]. The linker domain undergoes regulatory phosphorylation by Ras/mitogen-activated protein kinase (MAPK) pathways including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 MAPK, and cyclin-dependent kinase (CDK)-2/4, as well as glycogen synthase kinase 3-β, Ca (2+)-calmodulin-dependent protein kinase II, and G protein-coupled receptor kinase-2 (Figure

signaling could lead to more effective prevention of human fibro-carcinogenesis.

hepatic fibro-carcinogenesis.

114 Hepatocellular Carcinoma - Future Outlook

**2. Smad3 phosphoisoforms**

receptors, Smad4, and transcription co-activators.

genes.

**Figure 1. Reversible phospho-Smad3 signaling between tumor-suppression and fibro-carcinogenesis** A) TGF-β treatment activates TβRI, further leading to direct phosphorylation of Smad3C, which inhibits normally hepatocytic growth by up-regulating p21WAF1 transcription. B) Mitogens drastically alter phospho-Smad3 signaling via the JNK pathway, increasing basal nuclear fibro-carcinogenic pSmad3L activity while shutting down TGF-β-dependent cyto‐ static pSmad3C. Although TGF-β signal weakly phosphorylates Smad3L in normal hepatocytes (dotted line), hepatitis viral components including HBx, pro-inflammatory cytokines including TNF-α, and somatic mutations such as Ras addi‐ tively transmit fibro-carcinogenic signal through the JNK-dependent pSmad3L pathway to participate in hepatocytic growth and ECM deposition, possibly by stimulating transcription of *c-Myc* and *PAI-1* genes. Linker phosphorylation of Smad3 indirectly prevents COOH-tail phosphorylation, pSmad3C-mediated p21WAF1 transcriptions and cytostatic func‐ tion. C) Either various JNK inhibitors or a Smad3 mutation causing lack of JNK phosphorylation sites in the linker re‐ gion can eliminate fibro-carcinogenic pSmad3L signaling, restoring or maintaining the tumor-suppressive pSmad3C signaling characteristic of mature hepatocytes.

1B), [15-26]. TGF-β alternatively elicits signaling responses through non-Smad pathways representing important effectors for TGF-β activated kinase (TAK) 1 in response to proinflammatory cytokines. TAK1 activates JNK and p38 MAPK signaling through mitogenactivated kinase kinase (MKK) 4/7 and MKK3/6 [27,28]. JNK and p38 MAPK have been linked to modification of TGF-β signaling by pro-inflammatory cytokines through their regulation of distinct processes such as cytoskeleton organization, cell growth, survival, migration and invasion [29]. Imbalances between signaling through non-Smad and Smad pathways may occur during fibro-carcinogenesis, with interaction between these pathways mediating profibrogenic and pro-tumorigenic effects of TGF-β [30]

Findings in mice with targeted deletion of Smad3 and JNK1 indicate that both Smad3 and JNK1 pathways promote hepatic fibro-carcinogesis. When acute liver injury was induced by administration of CCl4, *Smad3*-/- mice showed approximately half as much of the induction of collagen type I mRNA as seen in wild-type mice [31]. *JNK1*-/- mice resisted not only liver fibrosis but also HCC development. Remarkable collagen deposition in wild-type and *JNK2*-/- was less evident in *JNK1*-/- mice, suggesting importance of JNK1 in development of liver fibrosis [32]. *JNK1*-/- mice exhibited impaired liver carcinogenesis with reduced tumor mass, size, and number [33]. Importantly, *JNK1*-/- mice displayed decreased HCC proliferation in a carcino‐ genic model and decreased hepatocytic growth in a model of liver regeneration. In both cases, the impaired proliferation was caused by increased expression of p21WAF1, a cell-cycle inhibitor, and reduced expression of c-Myc, a negative regulator of p21WAF1.

linker regions [15]. Smad2 phosphorylation at the linker region inhibits nuclear accumulation of Smad2 without interfering with TGF-β-induced phosphorylation of its COOH-tail [19,41-50]. In contrast, linker phosphorylation does not retain Smad3 in the cytoplasm, permitting further consequences of Ras/JNK signaling. Mechanisms underlying this difference between the two R-Smads are not known, but phosphorylation sites of Smad3 at clusters of 3 serine residues in its linker region (Ser204, Ser208, and Ser213) somewhat differ in sequence location from the corresponding linker phosphorylation sites of Smad2 (Ser245, Ser250, and

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Several lines of evidence indicate that JNK transmits carcinogenic (mitogenic) signal via the pSmad3L pathway. First, JNK can directly phosphorylate Smad3 linker sites *in vitro*, while JNK inhibitors block Smad3 linker phosphorylation *in vivo* [16,19]. Second, mitogens translo‐ cate pSmad3L into the nucleus [16,19,20]. Third, nuclear pSmad3L forms a hetero-complex with Smad4 [16,23]. Fourth, nuclear pSmad3L binds to the Smad-binding element in the promoter with high affinity and specificity [23,51-53]. Finally, mitogens induce growth of normal epithelial cells by up-regulating c-Myc, and such mitogenic effects are blocked in Smad3 mutants lacking linker phosphorylation sites and by JNK inhibitors [19,54]. These

results strongly support the notion that JNK specifically signals via Smad3 [55].

**5. Reversible shifts in phospho-Smad3 signaling between tumor-**

JNK/pSmad3L and TβRI/pSmad3C signals oppose each other; most importantly, the balance between carcinogenesis and tumor-suppression can shift (Figure 1C). Linker phosphorylation of Smad3 blocks COOH-tail phosphorylation induced by TβRI [16,19,24,54,56]. Mitogenic signaling accelerates nuclear transport of pSmad3L from the cytoplasm, while preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and anti-proliferative effects of TGF-β [16,19]. Smad3 mutants lacking linker phosphorylation sites, as well as JNK inhibitors, can restore growth inhibitory and transcriptional responses to TGF-β in Ras-transformed cells and pre-neoplastic hepatocytes, both *in vitro* and *in vivo* [19,54,56]. Our model implies that the JNK pathway directly or indirectly modulates pSmad3C- and pSmad3L-mediated signaling to regulate target genes, resulting in an antagonistic relationship between carcinogenesis and tumor-suppression. Thus, effectiveness of tumor-suppressive TGF-β signaling can depend on

**6. Homeostatic termination of mitogenic JNK/pSmad3L/c-Myc signaling after liver regeneration by hepatocytic TβRI/pSmad3C/p21WAF1 signaling**

Carcinogenesis is currently thought to occur as a sequence of steps termed initiation, promo‐ tion, and progression. Each step is characterized by disruption of normal cellular control mechanisms. Thus, development of HCC involves sequential alterations of physiological

**suppression and carcinogenesis**

extent of Smad3 phosphorylation at the linker region.

Ser255).

Mitogens simultaneously activate linker-phosphorylated R-Smad and non-Smad signaling, with both usually operating in parallel. Biologic significance of linker-phosphorylated R-Smad pathways is therefore difficult to assess in isolation. Here we will review recent work in this area with a particular focus on how mitogens modulate TGF-β signaling through Smad3 linker phosphorylation, using hepatic fibro-carcinogenesis as an example. Antibod‐ ies (Abs) reactive with structurally related phosphorylated peptides are emerging as valuable tools for determining phosphorylation sites *in vivo* and for investigating distinct signals via phosphorylated domains. Domain-specific phospho-Smad3 Abs have allowed us to reveal that TβRI and JNK differentially phosphorylate Smad3 to create 2 phosphorylated forms (phosphoisoforms): COOH-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L) [34-37]. Linker phosphorylation can modify COOHterminally phosphorylated R-Smad signaling [15-17,19-24]. Differential localization of kinases and phosphatases in the cytoplasm or nucleus raises the intriguing possibility of differen‐ ces in temporal dynamics between cytoplasmic and nuclear R-Smad phosphoisoforms, adding to the repertoire of signaling responses that determine cell-fate decisions [8,9]. Immunohistochemical and immunofluorescence analyses using specific Abs in human tissues can examine the clinical significance of context-dependent and cell type-specific signaling mediated by R-Smad phosphoisoforms by comparison of their tissue and cellular localiza‐ tion in pathologic specimens.

### **3. Tumor-suppressive (cytostatic) TGF-β signaling: the pSmad3C pathway**

TGF-β inhibits proliferation of normal hepatocytes, a crucial function in hepatic homeostasis [38]. In the context of cell cycle control, the most important targets of action by TGF-β are the genes encoding two CDK inhibitors (*p21WAF1* and *p15INK4B*) and *c-Myc* [39]. The pSmad3C signal induces expression of these CDK inhibitors and represses expression of c-Myc, shutting down cell cycle progression in the early to mid G1 phase of the cell cycle (Figure 1A). Development of HCC is ordinarily blocked through actions of the pSmad3C pathway, which causes normal hepatocytes to cease growth and enter apoptosis after hepatocytic proliferation.

### **4. Carcinogenic (mitogenic) JNK signaling: the pSmad3L pathway**

Mitogens strongly activate the JNK pathway, as TGF-β does more weakly (Figure 1B) [40]. Ras/ MAPK signaling has been shown to induce phosphorylation of Smad2 and Smad3 at their linker regions [15]. Smad2 phosphorylation at the linker region inhibits nuclear accumulation of Smad2 without interfering with TGF-β-induced phosphorylation of its COOH-tail [19,41-50]. In contrast, linker phosphorylation does not retain Smad3 in the cytoplasm, permitting further consequences of Ras/JNK signaling. Mechanisms underlying this difference between the two R-Smads are not known, but phosphorylation sites of Smad3 at clusters of 3 serine residues in its linker region (Ser204, Ser208, and Ser213) somewhat differ in sequence location from the corresponding linker phosphorylation sites of Smad2 (Ser245, Ser250, and Ser255).

but also HCC development. Remarkable collagen deposition in wild-type and *JNK2*-/- was less evident in *JNK1*-/- mice, suggesting importance of JNK1 in development of liver fibrosis [32]. *JNK1*-/- mice exhibited impaired liver carcinogenesis with reduced tumor mass, size, and number [33]. Importantly, *JNK1*-/- mice displayed decreased HCC proliferation in a carcino‐ genic model and decreased hepatocytic growth in a model of liver regeneration. In both cases, the impaired proliferation was caused by increased expression of p21WAF1, a cell-cycle inhibitor,

Mitogens simultaneously activate linker-phosphorylated R-Smad and non-Smad signaling, with both usually operating in parallel. Biologic significance of linker-phosphorylated R-Smad pathways is therefore difficult to assess in isolation. Here we will review recent work in this area with a particular focus on how mitogens modulate TGF-β signaling through Smad3 linker phosphorylation, using hepatic fibro-carcinogenesis as an example. Antibod‐ ies (Abs) reactive with structurally related phosphorylated peptides are emerging as valuable tools for determining phosphorylation sites *in vivo* and for investigating distinct signals via phosphorylated domains. Domain-specific phospho-Smad3 Abs have allowed us to reveal that TβRI and JNK differentially phosphorylate Smad3 to create 2 phosphorylated forms (phosphoisoforms): COOH-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L) [34-37]. Linker phosphorylation can modify COOHterminally phosphorylated R-Smad signaling [15-17,19-24]. Differential localization of kinases and phosphatases in the cytoplasm or nucleus raises the intriguing possibility of differen‐ ces in temporal dynamics between cytoplasmic and nuclear R-Smad phosphoisoforms, adding to the repertoire of signaling responses that determine cell-fate decisions [8,9]. Immunohistochemical and immunofluorescence analyses using specific Abs in human tissues can examine the clinical significance of context-dependent and cell type-specific signaling mediated by R-Smad phosphoisoforms by comparison of their tissue and cellular localiza‐

**3. Tumor-suppressive (cytostatic) TGF-β signaling: the pSmad3C pathway**

TGF-β inhibits proliferation of normal hepatocytes, a crucial function in hepatic homeostasis [38]. In the context of cell cycle control, the most important targets of action by TGF-β are the genes encoding two CDK inhibitors (*p21WAF1* and *p15INK4B*) and *c-Myc* [39]. The pSmad3C signal induces expression of these CDK inhibitors and represses expression of c-Myc, shutting down cell cycle progression in the early to mid G1 phase of the cell cycle (Figure 1A). Development of HCC is ordinarily blocked through actions of the pSmad3C pathway, which causes normal

hepatocytes to cease growth and enter apoptosis after hepatocytic proliferation.

**4. Carcinogenic (mitogenic) JNK signaling: the pSmad3L pathway**

Mitogens strongly activate the JNK pathway, as TGF-β does more weakly (Figure 1B) [40]. Ras/ MAPK signaling has been shown to induce phosphorylation of Smad2 and Smad3 at their

and reduced expression of c-Myc, a negative regulator of p21WAF1.

tion in pathologic specimens.

116 Hepatocellular Carcinoma - Future Outlook

Several lines of evidence indicate that JNK transmits carcinogenic (mitogenic) signal via the pSmad3L pathway. First, JNK can directly phosphorylate Smad3 linker sites *in vitro*, while JNK inhibitors block Smad3 linker phosphorylation *in vivo* [16,19]. Second, mitogens translo‐ cate pSmad3L into the nucleus [16,19,20]. Third, nuclear pSmad3L forms a hetero-complex with Smad4 [16,23]. Fourth, nuclear pSmad3L binds to the Smad-binding element in the promoter with high affinity and specificity [23,51-53]. Finally, mitogens induce growth of normal epithelial cells by up-regulating c-Myc, and such mitogenic effects are blocked in Smad3 mutants lacking linker phosphorylation sites and by JNK inhibitors [19,54]. These results strongly support the notion that JNK specifically signals via Smad3 [55].

### **5. Reversible shifts in phospho-Smad3 signaling between tumorsuppression and carcinogenesis**

JNK/pSmad3L and TβRI/pSmad3C signals oppose each other; most importantly, the balance between carcinogenesis and tumor-suppression can shift (Figure 1C). Linker phosphorylation of Smad3 blocks COOH-tail phosphorylation induced by TβRI [16,19,24,54,56]. Mitogenic signaling accelerates nuclear transport of pSmad3L from the cytoplasm, while preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and anti-proliferative effects of TGF-β [16,19]. Smad3 mutants lacking linker phosphorylation sites, as well as JNK inhibitors, can restore growth inhibitory and transcriptional responses to TGF-β in Ras-transformed cells and pre-neoplastic hepatocytes, both *in vitro* and *in vivo* [19,54,56]. Our model implies that the JNK pathway directly or indirectly modulates pSmad3C- and pSmad3L-mediated signaling to regulate target genes, resulting in an antagonistic relationship between carcinogenesis and tumor-suppression. Thus, effectiveness of tumor-suppressive TGF-β signaling can depend on extent of Smad3 phosphorylation at the linker region.

### **6. Homeostatic termination of mitogenic JNK/pSmad3L/c-Myc signaling after liver regeneration by hepatocytic TβRI/pSmad3C/p21WAF1 signaling**

Carcinogenesis is currently thought to occur as a sequence of steps termed initiation, promo‐ tion, and progression. Each step is characterized by disruption of normal cellular control mechanisms. Thus, development of HCC involves sequential alterations of physiological mechanisms regulating hepatocytic growth. Before consideration of molecular mechanisms of hepatocarcinogenesis, examination of the physiologic role of phospho-Smad3 signaling in liver regeneration is instructive. A unique feature of adult mammalian liver is its ability to accurately regenerate lost mass, which occurs following surgical resection or diffuse liver injury [57]. Although precise identities of cytokines and molecular mechanisms involved in liver regen‐ eration are largely unknown, TGF-β and tumor necrosis factor (TNF)-α apparently act as positive and negative regulators of hepatocytic growth, respectively (Figure 1 A and 1B).

**7. Liver fibrosis as the largest single risk factor for HCC occurrence**

in NASH cirrhosis [72].

present in the space of Disse [79].

Liver fibrosis usually precedes the multistage process of HCC development. Liver fibrosis is strongly associated with HCC, with 80 to 90% of HCCs arising in cirrhotic livers [67]. In hepatitis B infection is a risk factor for HCC, along with age, gender, viral DNA load, and viral core promoter mutation [68]. Fibrosis has also been identified as risk factor in hepatitis C infection, where cancer risk is directly related to fibrosis severity [69]. Similarly, HCC devel‐ opment is linked to alcoholic cirrhosis [70], nonalcoholic steatohepatitis (NASH) [70], and hemochromatosis [71], with a yearly HCC incidence of 1.7% in alcoholic cirrhosis [70] and 2.6%

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**8. Involvement of both myofibroblasts and hepatocytes in liver fibrosis**

Hepatic fibrosis is characterized by accumulation of excess ECM proteins, regardless of underlying etiology. Amount of matrix deposition reflects a balance between matrix synthesis and degradation [73,74]. When synthesis of ECM exceeds degradation, pathologic accumula‐ tion of ECM leads to liver fibrosis. Reversibility of experimental hepatic fibrosis and a striking decrease in collagenolytic activity observed in liver fibrosis models suggest crucial importance of impaired matrix degradation in hepatic fibrogenesis [75]. The plasminogen activator/ plasmin system, which is situated upstream of the fibrolysis system, can directly degrade matrix components, and indirectly inhibit ECM deposition [76]. Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of plasminogen activator, is a potent promoter of fibrosis. Introduction of a PAI-1 small interfering RNA attenuates deposition of

Liver fibrosis is one of the most common pathologic processes occurring in response to increased inflammatry factors. A complex interplay among different hepatic cell types takes place in injured livers. Hepatocytes are the targets for most hepatotoxic agents, including hepatitis viruses, alcohol metabolites, and chemical toxins [78]. Damaged hepatocytes induce recruitment of white blood cells by local inflammatory cells. Apoptosis of damaged hepato‐ cytes stimulates fibrogenesis by Kupffer cells. Activated Kupffer cells secrete pro-inflamma‐ tory cytokines including TNF-α and IL, as well as TGF-β. Intensive studies have shown that hepatic stellate cells (HSC) are the major cell type responsible for matrix production in damaged liver tissues [75]. HSC, characterized by retinoid droplets in the cytoplasm, are

Standardized methods of obtaining HSC from livers have been developed [80]. Long-term culture of HSC on plastic substrates is widely accepted as a model of liver fibrosis [79]. HSC spontaneously transdifferentiate to a myofibroblast (MFB) phenotype on plastic dishes, and this response reproduces the features of activation *in vivo*. MFB usually retain fibrogenic TGFβ signaling component, but have lost the capacity to respond to TGF-β with growth arrest [81]. Such a state of altered TGF-β responsiveness is also observed in pre-neoplastic hepatocytes,

ECM and hydroxyproline content in experimental hepatic fibrosis [77].

Hepatocytes undergo transition from a resting to a proliferative state after acute liver injury or partial hepatectomy [57]. Loss of parenchyma rapidly induces a wave of hepatocytic proliferation capable of restoring the total mass of the liver to normal. Several converging lines of evidence have established that pro-inflammatory cytokines such as TNF-α and interleukin (IL)-6 are important components of the mitogenic pathways leading to regeneration after acute liver injury [58]. Treatment of hepatocytes with antibodies against TNF-α resulted in decreased DNA synthesis and JNK activity [38]. DNA synthesis during liver regeneration was severely impaired in mice with a TNF-α type I receptor deficiency [59]. After acute liver injury, TGFβ increases in damaged livers within a time frame similar to that of increases in pro-inflam‐ matory cytokines [60-62]. This raises the problem of how hepatocytes manage to proliferate in response to a mitogenic pro-inflammatory cytokine signal despite elevated TGF-β concentra‐ tion. During liver regeneration, hepatocytes acquire temporary resistance to cytostatic effect of TGF-β, allowing them to proliferate [61-63]. The phosphorylation pattern of Smad3 in regenerative hepatocytes after acute liver injury suggested important participation of phos‐ pho-Smad3 in hepatocytic growth regulation. In actively growing hepatocytes, intracellular phosphorylation at Smad3L was found to be high [54,56,64]. Translocated to the nucleus, inflammatory cytokine-induced pSmad3L stimulated c-Myc transcription [54,64,65], which increased proliferation of hepatocytes and opposed the cytostatic action of the pSmad3C/ p21WAF1 pathway (Figure 1B). Accordingly, pSmad3C/p21WAF1 was undetectable in regenera‐ tive hepatocytic nuclei; escape from TGF-β-induced cytostasis was crucial in a subset of progenitor cells devoted to ensuring epithelial renewal. Thus, pSmad3L signaling can permit liver regeneration in response to mitogenic pro-inflammatory cytokines even though TGF-β concentration is elevated after acute liver injury.

Liver regeneration is tightly controlled by a delicate balance between hepatocytic growth and inhibition. Anti-mitotic effects of TGF-β contribute to the termination of hepatocyte prolifer‐ ation observed following the wave of DNA synthesis in the regenerating liver. Post regener‐ ation, return of TGF-β sensitivity thus limits hepatocyte proliferation and terminates liver regeneration [61,63]. After TNF-α and pSmad3L decreased, hepatocytic proliferation ceased, as decreases in pSmad3L allowed increased sensitivity to phosphorylation at Smad3C by TβRI (Figure 1C). TGF-β-dependent pSmad3C appears to limit the proliferative response of regenerating hepatocytes through inhibition of the G1 to S phase transition in the cell-cycle. Such signaling represents a highly effective defense mechanism against development of HCC, since nonproliferating hepatocytes containing pSmad3C that might have sustained any mutations are destined to die [66].

### **7. Liver fibrosis as the largest single risk factor for HCC occurrence**

mechanisms regulating hepatocytic growth. Before consideration of molecular mechanisms of hepatocarcinogenesis, examination of the physiologic role of phospho-Smad3 signaling in liver regeneration is instructive. A unique feature of adult mammalian liver is its ability to accurately regenerate lost mass, which occurs following surgical resection or diffuse liver injury [57]. Although precise identities of cytokines and molecular mechanisms involved in liver regen‐ eration are largely unknown, TGF-β and tumor necrosis factor (TNF)-α apparently act as positive and negative regulators of hepatocytic growth, respectively (Figure 1 A and 1B).

Hepatocytes undergo transition from a resting to a proliferative state after acute liver injury or partial hepatectomy [57]. Loss of parenchyma rapidly induces a wave of hepatocytic proliferation capable of restoring the total mass of the liver to normal. Several converging lines of evidence have established that pro-inflammatory cytokines such as TNF-α and interleukin (IL)-6 are important components of the mitogenic pathways leading to regeneration after acute liver injury [58]. Treatment of hepatocytes with antibodies against TNF-α resulted in decreased DNA synthesis and JNK activity [38]. DNA synthesis during liver regeneration was severely impaired in mice with a TNF-α type I receptor deficiency [59]. After acute liver injury, TGFβ increases in damaged livers within a time frame similar to that of increases in pro-inflam‐ matory cytokines [60-62]. This raises the problem of how hepatocytes manage to proliferate in response to a mitogenic pro-inflammatory cytokine signal despite elevated TGF-β concentra‐ tion. During liver regeneration, hepatocytes acquire temporary resistance to cytostatic effect of TGF-β, allowing them to proliferate [61-63]. The phosphorylation pattern of Smad3 in regenerative hepatocytes after acute liver injury suggested important participation of phos‐ pho-Smad3 in hepatocytic growth regulation. In actively growing hepatocytes, intracellular phosphorylation at Smad3L was found to be high [54,56,64]. Translocated to the nucleus, inflammatory cytokine-induced pSmad3L stimulated c-Myc transcription [54,64,65], which increased proliferation of hepatocytes and opposed the cytostatic action of the pSmad3C/ p21WAF1 pathway (Figure 1B). Accordingly, pSmad3C/p21WAF1 was undetectable in regenera‐ tive hepatocytic nuclei; escape from TGF-β-induced cytostasis was crucial in a subset of progenitor cells devoted to ensuring epithelial renewal. Thus, pSmad3L signaling can permit liver regeneration in response to mitogenic pro-inflammatory cytokines even though TGF-β

Liver regeneration is tightly controlled by a delicate balance between hepatocytic growth and inhibition. Anti-mitotic effects of TGF-β contribute to the termination of hepatocyte prolifer‐ ation observed following the wave of DNA synthesis in the regenerating liver. Post regener‐ ation, return of TGF-β sensitivity thus limits hepatocyte proliferation and terminates liver regeneration [61,63]. After TNF-α and pSmad3L decreased, hepatocytic proliferation ceased, as decreases in pSmad3L allowed increased sensitivity to phosphorylation at Smad3C by TβRI (Figure 1C). TGF-β-dependent pSmad3C appears to limit the proliferative response of regenerating hepatocytes through inhibition of the G1 to S phase transition in the cell-cycle. Such signaling represents a highly effective defense mechanism against development of HCC, since nonproliferating hepatocytes containing pSmad3C that might have sustained any

concentration is elevated after acute liver injury.

118 Hepatocellular Carcinoma - Future Outlook

mutations are destined to die [66].

Liver fibrosis usually precedes the multistage process of HCC development. Liver fibrosis is strongly associated with HCC, with 80 to 90% of HCCs arising in cirrhotic livers [67]. In hepatitis B infection is a risk factor for HCC, along with age, gender, viral DNA load, and viral core promoter mutation [68]. Fibrosis has also been identified as risk factor in hepatitis C infection, where cancer risk is directly related to fibrosis severity [69]. Similarly, HCC devel‐ opment is linked to alcoholic cirrhosis [70], nonalcoholic steatohepatitis (NASH) [70], and hemochromatosis [71], with a yearly HCC incidence of 1.7% in alcoholic cirrhosis [70] and 2.6% in NASH cirrhosis [72].

### **8. Involvement of both myofibroblasts and hepatocytes in liver fibrosis**

Hepatic fibrosis is characterized by accumulation of excess ECM proteins, regardless of underlying etiology. Amount of matrix deposition reflects a balance between matrix synthesis and degradation [73,74]. When synthesis of ECM exceeds degradation, pathologic accumula‐ tion of ECM leads to liver fibrosis. Reversibility of experimental hepatic fibrosis and a striking decrease in collagenolytic activity observed in liver fibrosis models suggest crucial importance of impaired matrix degradation in hepatic fibrogenesis [75]. The plasminogen activator/ plasmin system, which is situated upstream of the fibrolysis system, can directly degrade matrix components, and indirectly inhibit ECM deposition [76]. Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of plasminogen activator, is a potent promoter of fibrosis. Introduction of a PAI-1 small interfering RNA attenuates deposition of ECM and hydroxyproline content in experimental hepatic fibrosis [77].

Liver fibrosis is one of the most common pathologic processes occurring in response to increased inflammatry factors. A complex interplay among different hepatic cell types takes place in injured livers. Hepatocytes are the targets for most hepatotoxic agents, including hepatitis viruses, alcohol metabolites, and chemical toxins [78]. Damaged hepatocytes induce recruitment of white blood cells by local inflammatory cells. Apoptosis of damaged hepato‐ cytes stimulates fibrogenesis by Kupffer cells. Activated Kupffer cells secrete pro-inflamma‐ tory cytokines including TNF-α and IL, as well as TGF-β. Intensive studies have shown that hepatic stellate cells (HSC) are the major cell type responsible for matrix production in damaged liver tissues [75]. HSC, characterized by retinoid droplets in the cytoplasm, are present in the space of Disse [79].

Standardized methods of obtaining HSC from livers have been developed [80]. Long-term culture of HSC on plastic substrates is widely accepted as a model of liver fibrosis [79]. HSC spontaneously transdifferentiate to a myofibroblast (MFB) phenotype on plastic dishes, and this response reproduces the features of activation *in vivo*. MFB usually retain fibrogenic TGFβ signaling component, but have lost the capacity to respond to TGF-β with growth arrest [81]. Such a state of altered TGF-β responsiveness is also observed in pre-neoplastic hepatocytes, which typically exhibit a limited growth inhibitory response to TGF-β, instead responding to TGF-β with pro-fibrogenic behavior [9].

promotes ECM deposition in both hepatocytes and MFB. Thus, hepatocytes affected by chronic inflammation undergo transition from the tumor-suppressive pSmad3C pathway, character‐ istic of mature hepatocytes, to the JNK/pSmad3L/PAI-1 pathway, which favors a state of flux

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Our findings support many important papers reporting that hepatocytes can promote fibrogenesis via TGF-β/Smad signaling. Dooley et al. reported that overexpression of inhibi‐ tory Smad7 in hepatocytes attenuated TGF-β-mediated fibrogenesis by blocking Smad signaling [93]. Since the large latent TGF-β complex consisting of TGF-β, the N-terminal part of its precursor, and the latent TGF-β binding protein exists in not only HSC but also hepato‐ cytes, the complex can transmit a pro-fibrogenic signal [94], although intracellular functions of the TGF-β complex are poorly understood. TGF-β down-stream mediator connective tissue growth factor (CTGF) also involves hepatic fibro-carcinogenesis [95]. CTGF expression increases in fibrotic livers and various tumor tissues [96]. More importantly, *in vivo* knockdown of CTGF by small interfering RNA leads to substantial attenuation of experimental liver fibrosis. Differential regulation of CTGF expression in hepatocytes and HSC by Smad2 signaling may contribute to hepatic fibro-carcinogenesis [97]. Interestingly, a methylxanthine, caffeine, inhibits synthesis of CTGF in hepatocytes and HSC, primarily by inducing degrada‐

**10. Additive promotion of human carcinogenesis by persistent hepatitis**

Various experiments support the notion that a single promoting agent is insufficient for development of cancer. Hepatocarcinogenesis is multi-factorial, involving collaboration between 2 or more promoting agents in HCC occurrence [98]. Among tumor-promoting agents, hepatitis viruses and chronic inflammation directly participate in HCC pathogenesis,

Many clinical observations suggest that persistent hepatitis viral infection and chronic inflammation additively influence development of human HCC. For example, alcohol consumption is a recognized major cause of liver disease, and plays an important role in progression to HCC. However, alcoholic hepatitis progresses less frequently to HCC than HBV- or HCV- related hepatitis. In addition, patients with both viral infection and alcohol consumption have a higher risk of developing HCC than those with alcohol consumption alone [3,99,100]. Autoimmune hepatitis (AIH) and primary billiary cirrhosis (PBC) are chronic inflammatory disorders that proceed to cirrhosis. However, HCC only rarely arises from AIH or PBC, particularly in the absence of HBV or HCV infection [101,102]. Conversely, asympto‐ matic HBV or HCV carriers maintaining normal alanine aminotransferase (ALT) levels despite intensive viral replication less frequently develop HCC than patients with chronic hepatitis B. The annual risk of HCC occurrence in HBV healthy carriers is 0.26% to 0.6%, while risk increases to 1% in patients with chronic active hepatitis B [103]. Moreover, HBV can act synergistically with HCV. Patients co-infected with HBV and HCV have a 2- to 6-fold higher

characterized by MFB.

tion of Smad2 [96].

**viral infection and chronic inflammation**

which frequently occurs during long-standing hepatitis viral infection.

Hepatic fibrosis results from a wound-healing response to repeated injury in chronic liver diseases [82], in which HSC undergo dramatic phenotypic activation, with acquisition of fibrogenic properties. Patients develop liver fibrosis as a result of chronic liver damage, characterized by ECM accumulation that distorts hepatic architecture by forming a fibrous scar [79]. Ultimately, nodules of regenerating hepatocytes become enclosed by scar tissue, an event defining cirrhosis. Excess deposition of ECM of which type I collagen predominates disrupts the normal architecture of the liver, resulting in pathologic damage with pathophy‐ siologic consequences.

A new concept has been proposed that epithelial cells undergo a phenotypical change termed epithelial-mesenchymal transition (EMT), acquiring a fibroblastic phenotype. EMT facilitates metastasis and cancer development [83]. Pioneering studies on EMT in organ fibrosis were carried out in kidney, ocular lens, and lung [84,85]. Involvement of EMT also has been proposed in liver fibrosis. Zeisberg et al. demonstrated that hepatocytes acquire expression of fibroblast-specific protein 1 in response to CCl4 injury *in vivo* or TGF-β *in vitro* [86].

### **9. Fibrogenic pSmad3L signaling shared between MFB and pre-neoplastic hepatocytes**

As a result of chronic liver damage, HSC undergo progressive activation to become MFB-like cells. During transdifferentiation in culture, pSmad3C-mediated signal decreases while the pSmad3L pathway predominates [23]. These observations complement the finding of pSmad3L rather than pSmad3C in nuclei of α-smooth muscle actin (SMA)-immunoreactive MFB in portal tracts of chronically HCV-infected liver specimens [64]. The presence of α-SMA is associated with transdifferentiation of HSC into scar-forming MFB, an event considered pivotal in the fibrogenic response [75].

Plasma TGF-β, TNF-α, and PAI-1 concentrations are usually elevated in patients with chronic liver diseases [87-89]. Since pSmad3L can transmit a fibrogenic signal by stimulating PAI-1 transcription (Figure 1B) [23], we investigated the pSmad3L pathway in human chronic liver disease. The results indicated nuclear localization of pSmad3L in PAI-1-immunoreactive MFBs and hepatocytes in chronic hepatitis specimens [64]. Thus, hepatocytes are regulated by the same pSmad3L pathway as are MFBs. Hepatocytes in HCV-infected livers, particularly those adjacent to inflamed portal tracts, exhibited phosphorylation at Smad3L [64]. Extent of phosphorylation at Smad3L was less in hepatocytes distant from portal tracts, in sharp contrast to pSmad3C, which was predominantly located in hepatocytic nuclei distant from portal tracts [64]. Extent of hepatocytic pSmad3L/PAI-1 increased in proportion to fibrotic stage in chronic liver diseases [56,74]. TGF-β and pro-inflammatory cytokines are released from infiltrating Kupffer cells in portal tracts to activate JNK [90,91]. Considering these findings together with a previous observation showing transcriptional activation of the *PAI-1* gene by JNK [92], TGFβ and TNF-α can mediate JNK/pSmad3L signaling that in turn induces PAI-1 expression and promotes ECM deposition in both hepatocytes and MFB. Thus, hepatocytes affected by chronic inflammation undergo transition from the tumor-suppressive pSmad3C pathway, character‐ istic of mature hepatocytes, to the JNK/pSmad3L/PAI-1 pathway, which favors a state of flux characterized by MFB.

which typically exhibit a limited growth inhibitory response to TGF-β, instead responding to

Hepatic fibrosis results from a wound-healing response to repeated injury in chronic liver diseases [82], in which HSC undergo dramatic phenotypic activation, with acquisition of fibrogenic properties. Patients develop liver fibrosis as a result of chronic liver damage, characterized by ECM accumulation that distorts hepatic architecture by forming a fibrous scar [79]. Ultimately, nodules of regenerating hepatocytes become enclosed by scar tissue, an event defining cirrhosis. Excess deposition of ECM of which type I collagen predominates disrupts the normal architecture of the liver, resulting in pathologic damage with pathophy‐

A new concept has been proposed that epithelial cells undergo a phenotypical change termed epithelial-mesenchymal transition (EMT), acquiring a fibroblastic phenotype. EMT facilitates metastasis and cancer development [83]. Pioneering studies on EMT in organ fibrosis were carried out in kidney, ocular lens, and lung [84,85]. Involvement of EMT also has been proposed in liver fibrosis. Zeisberg et al. demonstrated that hepatocytes acquire expression of

**9. Fibrogenic pSmad3L signaling shared between MFB and pre-neoplastic**

As a result of chronic liver damage, HSC undergo progressive activation to become MFB-like cells. During transdifferentiation in culture, pSmad3C-mediated signal decreases while the pSmad3L pathway predominates [23]. These observations complement the finding of pSmad3L rather than pSmad3C in nuclei of α-smooth muscle actin (SMA)-immunoreactive MFB in portal tracts of chronically HCV-infected liver specimens [64]. The presence of α-SMA is associated with transdifferentiation of HSC into scar-forming MFB, an event considered

Plasma TGF-β, TNF-α, and PAI-1 concentrations are usually elevated in patients with chronic liver diseases [87-89]. Since pSmad3L can transmit a fibrogenic signal by stimulating PAI-1 transcription (Figure 1B) [23], we investigated the pSmad3L pathway in human chronic liver disease. The results indicated nuclear localization of pSmad3L in PAI-1-immunoreactive MFBs and hepatocytes in chronic hepatitis specimens [64]. Thus, hepatocytes are regulated by the same pSmad3L pathway as are MFBs. Hepatocytes in HCV-infected livers, particularly those adjacent to inflamed portal tracts, exhibited phosphorylation at Smad3L [64]. Extent of phosphorylation at Smad3L was less in hepatocytes distant from portal tracts, in sharp contrast to pSmad3C, which was predominantly located in hepatocytic nuclei distant from portal tracts [64]. Extent of hepatocytic pSmad3L/PAI-1 increased in proportion to fibrotic stage in chronic liver diseases [56,74]. TGF-β and pro-inflammatory cytokines are released from infiltrating Kupffer cells in portal tracts to activate JNK [90,91]. Considering these findings together with a previous observation showing transcriptional activation of the *PAI-1* gene by JNK [92], TGFβ and TNF-α can mediate JNK/pSmad3L signaling that in turn induces PAI-1 expression and

fibroblast-specific protein 1 in response to CCl4 injury *in vivo* or TGF-β *in vitro* [86].

TGF-β with pro-fibrogenic behavior [9].

120 Hepatocellular Carcinoma - Future Outlook

siologic consequences.

**hepatocytes**

pivotal in the fibrogenic response [75].

Our findings support many important papers reporting that hepatocytes can promote fibrogenesis via TGF-β/Smad signaling. Dooley et al. reported that overexpression of inhibi‐ tory Smad7 in hepatocytes attenuated TGF-β-mediated fibrogenesis by blocking Smad signaling [93]. Since the large latent TGF-β complex consisting of TGF-β, the N-terminal part of its precursor, and the latent TGF-β binding protein exists in not only HSC but also hepato‐ cytes, the complex can transmit a pro-fibrogenic signal [94], although intracellular functions of the TGF-β complex are poorly understood. TGF-β down-stream mediator connective tissue growth factor (CTGF) also involves hepatic fibro-carcinogenesis [95]. CTGF expression increases in fibrotic livers and various tumor tissues [96]. More importantly, *in vivo* knockdown of CTGF by small interfering RNA leads to substantial attenuation of experimental liver fibrosis. Differential regulation of CTGF expression in hepatocytes and HSC by Smad2 signaling may contribute to hepatic fibro-carcinogenesis [97]. Interestingly, a methylxanthine, caffeine, inhibits synthesis of CTGF in hepatocytes and HSC, primarily by inducing degrada‐ tion of Smad2 [96].

### **10. Additive promotion of human carcinogenesis by persistent hepatitis viral infection and chronic inflammation**

Various experiments support the notion that a single promoting agent is insufficient for development of cancer. Hepatocarcinogenesis is multi-factorial, involving collaboration between 2 or more promoting agents in HCC occurrence [98]. Among tumor-promoting agents, hepatitis viruses and chronic inflammation directly participate in HCC pathogenesis, which frequently occurs during long-standing hepatitis viral infection.

Many clinical observations suggest that persistent hepatitis viral infection and chronic inflammation additively influence development of human HCC. For example, alcohol consumption is a recognized major cause of liver disease, and plays an important role in progression to HCC. However, alcoholic hepatitis progresses less frequently to HCC than HBV- or HCV- related hepatitis. In addition, patients with both viral infection and alcohol consumption have a higher risk of developing HCC than those with alcohol consumption alone [3,99,100]. Autoimmune hepatitis (AIH) and primary billiary cirrhosis (PBC) are chronic inflammatory disorders that proceed to cirrhosis. However, HCC only rarely arises from AIH or PBC, particularly in the absence of HBV or HCV infection [101,102]. Conversely, asympto‐ matic HBV or HCV carriers maintaining normal alanine aminotransferase (ALT) levels despite intensive viral replication less frequently develop HCC than patients with chronic hepatitis B. The annual risk of HCC occurrence in HBV healthy carriers is 0.26% to 0.6%, while risk increases to 1% in patients with chronic active hepatitis B [103]. Moreover, HBV can act synergistically with HCV. Patients co-infected with HBV and HCV have a 2- to 6-fold higher risk of HCC occurrence than those with either infection alone [104,105]. Accordingly, we will consider how the oncogenic JNK/pSmad3L pathway induces development of HCC, with particular attention to potential synergy between hepatitis viruses and inflammation in formation of pre-neoplastic hepatocytes.

mic pathways [113,114,123]. For example, HBx protein was found to activate the JNK-

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To investigate whether HBx alters phospho-Smad3 signaling in hepatocytes, we stably transfected immortalized rat hepatocytes using a construct of HBx with a mammalian expression vector, resulting in high HBx-expressing cells [56]. High expression of HBx protein in hepatocytes tended to shut down pSmad3C-mediated signaling and favored acquisition of constitutively active JNK-mediated pSmad3L signaling, which fostered hepatocytic growth

In transgenic models, HBx played an important role in hepatocarcinogenesis via the pSmad3L/ c-Myc pathway [56]. HBx transgenic mouse livers progressed through hyperplasia to HCC. HBx, pSmad3L, and c-Myc were not detected in normal mouse livers. Beginning at the age of 2 months, HBx transgenic mouse liver showed centrilobular foci of cellular alteration with cytoplasmic vacuolation surrounding central veins where Bromodeoxyuridine (BrdU) was uptaken into the hepatocytes [121]. Smad3L was phosphorylated in hepatocytic nuclei of the centrilobular region, where HBx and c-Myc were expressed. Hepatocytic HBx, pSmad3L, and

Positivity of hepatocytic nuclei for pSmad3L in early chronic hepatitis B specimens increases with amount of HBV-DNA [56]. Taken together with results of *in vitro* experiments using HBxexpressing hepatocytes and HBx transgenic livers, these human findings indicate that HBx oncoprotein participates directly in hepatocarcinogenesis by shifting hepatocytic phospho-Smad3 signaling from the tumor-suppressive pSmad3C/p21WAF1 pathway to the oncogenic

Unlike HBV, HCV is a positive-single-strand RNA virus, apparently incapable of integration into the host's genome. The HCV genome has a long open reading frame, which encodes a polyprotein precursor [125,126]. This polyprotein is cleaved by both host and viral proteases to generate 4 structural proteins (C, E1, E2, and P7) and 6 nonstructural proteins (xlink, NS3, NS4A, NS4B, NS5A, and NS5B) [127,128]. The HCV components modulate a number of cellular regulatory functions by targeting a wide spectrum of cellular signaling pathways [129-136]. HCV core expression has been shown to induce activation of the JNK pathway in regulation of vascular endothelial growth factor [136]. NS5A acts as a positive regulator of the JNK signaling pathway by interacting with tumor necrosis factor receptor-associated factor 2, which may play a key role in HCV pathogenesis [137]. In an HCV infection model, Lin *et al.* demonstrated that HCV directly induced TGF-β release from hepatocytes in reactive oxygen species (ROS)-dependent and JNK-dependent manner [138]. Moreover, recent studies using transgenic mouse models indicate that HCV directly involves hepatocarcinogenesis. Three different HCV core transgenic lines develop liver steatosis and HCC [139-141]. Accordingly, future studies are expected to prove that the HCV components can activate the oncogenic JNK/

dependent pathway and up-regulate oncogenic c-Myc gene expression [124].

c-Myc increased as mouse liver progressed through hyperplasia to HCC.

by up-regulating c-Myc (Figure 1B).

JNK/pSmad3L/c-Myc pathway (Figure 1B), [56].

pSmad3L pathway.

### **11. Hepatitis virus components can activate oncogenic JNK/pSmad3L pathway**

One of the earliest evidence linking HBV to development of HCC was obtained in the wood‐ chuck hepatitis virus model, in which 100% of rodents infected with woodchuck hepatitis virus developed HCC [106]. Because HBV contains partially double stranded-DNA, it can directly cause HCC by integrating its DNA into the host genome. HBV genomic integration is present in over 85% to 90% of HBV-related HCC, usually even before development of HCC [107]. Integration of HBV DNA is not restricted to HCC but also is found in non-tumor tissue in patients with chronically HBV infection [108,109]. HBV integration induces a wide range of genetic alterations within the host genome, including chromosomal deletions, translocations, production of fusion transcripts, amplification of cellular DNA, and generalized genomic instability [110,111]. Many integration events occur near or within fragile sites or other cancerassociated regions of the human genome that are prone to instability in tumor development and progression. Genetic instability associated with integration may alter expression of oncogenes, tumor suppressor genes, and microRNAs [111]. A recent large-scale analysis of HBV DNA integration sites in cellular DNA found a preference for sites regulating cell signaling, proliferation, and viability [112]. A large proportion of HCC have integrated HBV sequences encoding HBV X (HBx) and/or truncated envelope pre-S2/S proteins.

The HBx protein encoded by the X gene has been long suspected as a viral oncoprotein participating in hepatocarcinogenesis. This protein is involved in liver cell transformation because of its pleiotropic activities on cell cycle regulation, cell signaling pathways and DNA repair [113-115]. Numerous attempts have been made to examine the oncogenic potential of HBx in cell culture. However, its transforming ability was barely measurable evident only when cells were immortalized by other oncogenes, such as SV40 T-antigen [116,117] or TGFα [118]. Furthermore, most transgenic mice harboring the HBx gene did not develop serious liver diseases or tumors [119]. Only in a certain transgenic lineage of CD-1 strain, HBx weakly promoted carcinogenesis, where HBx was highly expressed [120]. A second mouse lineage with lower HBx expression developed liver tumors at the same rate as normal CD-1 mice [121]. HBx was shown to potentiate c-Myc-induced liver carcinogenesis in transgenic mice [122]. Thus, HBx does not have strong transforming activity, but HBx overexpression in a certain genetic background might induce tumor formation in a multistage transformation, most likely in collaboration with other cellular oncogenic pathways.

HBx is mainly located in the cytoplasm and exhibits pleiotropic effects that modulate cell responses to oncogenic signaling pathways [114]. HBx protein do not bind directly to DNA, but rather acts on cellular promoters. Such protein-protein interaction can modulate cytoplas‐ mic pathways [113,114,123]. For example, HBx protein was found to activate the JNKdependent pathway and up-regulate oncogenic c-Myc gene expression [124].

risk of HCC occurrence than those with either infection alone [104,105]. Accordingly, we will consider how the oncogenic JNK/pSmad3L pathway induces development of HCC, with particular attention to potential synergy between hepatitis viruses and inflammation in

**11. Hepatitis virus components can activate oncogenic JNK/pSmad3L**

sequences encoding HBV X (HBx) and/or truncated envelope pre-S2/S proteins.

in collaboration with other cellular oncogenic pathways.

The HBx protein encoded by the X gene has been long suspected as a viral oncoprotein participating in hepatocarcinogenesis. This protein is involved in liver cell transformation because of its pleiotropic activities on cell cycle regulation, cell signaling pathways and DNA repair [113-115]. Numerous attempts have been made to examine the oncogenic potential of HBx in cell culture. However, its transforming ability was barely measurable evident only when cells were immortalized by other oncogenes, such as SV40 T-antigen [116,117] or TGFα [118]. Furthermore, most transgenic mice harboring the HBx gene did not develop serious liver diseases or tumors [119]. Only in a certain transgenic lineage of CD-1 strain, HBx weakly promoted carcinogenesis, where HBx was highly expressed [120]. A second mouse lineage with lower HBx expression developed liver tumors at the same rate as normal CD-1 mice [121]. HBx was shown to potentiate c-Myc-induced liver carcinogenesis in transgenic mice [122]. Thus, HBx does not have strong transforming activity, but HBx overexpression in a certain genetic background might induce tumor formation in a multistage transformation, most likely

HBx is mainly located in the cytoplasm and exhibits pleiotropic effects that modulate cell responses to oncogenic signaling pathways [114]. HBx protein do not bind directly to DNA, but rather acts on cellular promoters. Such protein-protein interaction can modulate cytoplas‐

One of the earliest evidence linking HBV to development of HCC was obtained in the wood‐ chuck hepatitis virus model, in which 100% of rodents infected with woodchuck hepatitis virus developed HCC [106]. Because HBV contains partially double stranded-DNA, it can directly cause HCC by integrating its DNA into the host genome. HBV genomic integration is present in over 85% to 90% of HBV-related HCC, usually even before development of HCC [107]. Integration of HBV DNA is not restricted to HCC but also is found in non-tumor tissue in patients with chronically HBV infection [108,109]. HBV integration induces a wide range of genetic alterations within the host genome, including chromosomal deletions, translocations, production of fusion transcripts, amplification of cellular DNA, and generalized genomic instability [110,111]. Many integration events occur near or within fragile sites or other cancerassociated regions of the human genome that are prone to instability in tumor development and progression. Genetic instability associated with integration may alter expression of oncogenes, tumor suppressor genes, and microRNAs [111]. A recent large-scale analysis of HBV DNA integration sites in cellular DNA found a preference for sites regulating cell signaling, proliferation, and viability [112]. A large proportion of HCC have integrated HBV

formation of pre-neoplastic hepatocytes.

122 Hepatocellular Carcinoma - Future Outlook

**pathway**

To investigate whether HBx alters phospho-Smad3 signaling in hepatocytes, we stably transfected immortalized rat hepatocytes using a construct of HBx with a mammalian expression vector, resulting in high HBx-expressing cells [56]. High expression of HBx protein in hepatocytes tended to shut down pSmad3C-mediated signaling and favored acquisition of constitutively active JNK-mediated pSmad3L signaling, which fostered hepatocytic growth by up-regulating c-Myc (Figure 1B).

In transgenic models, HBx played an important role in hepatocarcinogenesis via the pSmad3L/ c-Myc pathway [56]. HBx transgenic mouse livers progressed through hyperplasia to HCC. HBx, pSmad3L, and c-Myc were not detected in normal mouse livers. Beginning at the age of 2 months, HBx transgenic mouse liver showed centrilobular foci of cellular alteration with cytoplasmic vacuolation surrounding central veins where Bromodeoxyuridine (BrdU) was uptaken into the hepatocytes [121]. Smad3L was phosphorylated in hepatocytic nuclei of the centrilobular region, where HBx and c-Myc were expressed. Hepatocytic HBx, pSmad3L, and c-Myc increased as mouse liver progressed through hyperplasia to HCC.

Positivity of hepatocytic nuclei for pSmad3L in early chronic hepatitis B specimens increases with amount of HBV-DNA [56]. Taken together with results of *in vitro* experiments using HBxexpressing hepatocytes and HBx transgenic livers, these human findings indicate that HBx oncoprotein participates directly in hepatocarcinogenesis by shifting hepatocytic phospho-Smad3 signaling from the tumor-suppressive pSmad3C/p21WAF1 pathway to the oncogenic JNK/pSmad3L/c-Myc pathway (Figure 1B), [56].

Unlike HBV, HCV is a positive-single-strand RNA virus, apparently incapable of integration into the host's genome. The HCV genome has a long open reading frame, which encodes a polyprotein precursor [125,126]. This polyprotein is cleaved by both host and viral proteases to generate 4 structural proteins (C, E1, E2, and P7) and 6 nonstructural proteins (xlink, NS3, NS4A, NS4B, NS5A, and NS5B) [127,128]. The HCV components modulate a number of cellular regulatory functions by targeting a wide spectrum of cellular signaling pathways [129-136]. HCV core expression has been shown to induce activation of the JNK pathway in regulation of vascular endothelial growth factor [136]. NS5A acts as a positive regulator of the JNK signaling pathway by interacting with tumor necrosis factor receptor-associated factor 2, which may play a key role in HCV pathogenesis [137]. In an HCV infection model, Lin *et al.* demonstrated that HCV directly induced TGF-β release from hepatocytes in reactive oxygen species (ROS)-dependent and JNK-dependent manner [138]. Moreover, recent studies using transgenic mouse models indicate that HCV directly involves hepatocarcinogenesis. Three different HCV core transgenic lines develop liver steatosis and HCC [139-141]. Accordingly, future studies are expected to prove that the HCV components can activate the oncogenic JNK/ pSmad3L pathway.

### **12. Activation of the oncogenic JNK/pSmad3L pathway by chronic inflammation**

Inflammatory microenvironments are present in human hepatocarcinogenesis before malig‐ nant change occurs. A hepatitis virus infection triggers chronic inflammation, increasing the risk of HCC development. Several studies have discussed how chronic inflammation affects the proliferation and survival of hepatocytes [142,143]. TNF-α, IL-1β and IL-6 are multifunc‐ tional pro-inflammatory cytokines largely responsible for the hepatic response to chronic inflammation [144-146]. Serum concentrations of these cytokines are increased in chronic liver inflammation including hepatitis viral infection and steatohepatitis [147]. JNK is a key signal transducer for inflammatory cytokines and has emerged as an important endogenous tumor promoter [148,149].

glycoprotein Mdr2 [153]. HCC follows cholestatic inflammation in these mice. Incidence of HCC can be enhanced by another member of the TNF family, lymphotoxin β [154]. Tumorpromoting cytokines produced by Kupffer cells activate several transcription factors, includ‐ ing NF-kB, STAT3, and AP-1, in pre-malignant hepatocytes [155]. The activated transcription factors stimulate transcription of their target genes involved in hepatocytic proliferation and survival, representing a major tumor-promoting mechanism. Similarly to these transcription factors, tumor-promoting actions of hepatocytic Smad3 in human chronic liver disease rarely result from direct mutations [156]. Instead, pSmad3L depends on mitogenic pro-inflammatory

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**13. Constitutive phosphorylation at Smad3L in pre-neoplastic hepatocytes**

The mechanism regulating regeneration, which avoids accumulation of deleterious mutations in genes that promote cell growth and division, must be disrupted before hepatocytes can throw off normal restraints and behave as an asocial HCC. Constitutive phosphorylation at Smad3L is observed in pre-malignant hepatocytes in cirrhosis [56,64]. Constitutively active pSmad3L stimulates hepatocytes to proliferate continuously in human livers that normally experience little proliferation because hepatocytic regeneration is tightly regulated by cyto‐ static pSmad3C signaling. Since JNK is constitutively activated in pre-neoplastic hepatocytes in cirrhotic human liver [157], constitutive Smad3L phosphorylation in pre-malignant lesions can be a direct consequence of proto-oncogene-mediated JNK signaling. Somatic mutations in pre-neoplastic hepatocytes include changes in the *Ras* pathway that favor progression from cirrhosis toward HCC [158]. In pre-neoplastic hepatocyte nuclei, pSmad3L/c-Myc can accu‐ mulate when somatic mutations constitutively activate the JNK pathway to phosphorylate Smad3 at the linker region (Figure 1B). Then, the proliferative effect mediated via the pSmad3L/ c-Myc pathway constitutively keeps on suppresses the growth-inhibitory pSmad3C/p21WAF1

Pre-neoplastic hepatocytes and HCC show reduction of anti-mitogenic responses to TGF-β [20,37]. Escaping the cytostatic action of pSmad3C is a critical step for progression to full malignancy in cancers, which must overcome multiple fail-safe genetic controls [39,159,160]. The TGF-β/pSmad3C pathway is required for maintenance of genomic stability, induction of replicative senescence, and suppression of telomerase [161-163]. Selection for genetic instabil‐ ity occurs in clones of aberrant cells able to produce tumors, since genetic instability greatly accelerates accumulation of further genetic and epigenetic changes required for tumor progression. In this regard, the TGF-β/pSmad3C pathway contributes to tumor suppression

cytokine signals produced by neighboring Kupffer cells.

pathway in the nuclei of pre-neoplastic hepatocytes.

along with its cytostatic effect.

**in cirrhotic human liver**

TGF-β is also released by infiltrating Kupffer cells, the liver's resident macrophages, in portal tracts during chronic inflammation [150]. These findings suggest that elevated pro-inflamma‐ tory cytokines might alter hepatocytic TGF-β signaling in inflammatory microenvironments. We investigated this hypothesis using rat cultured hepatocytes [64]. Pretreatment of hepato‐ cytes with SP600125, a JNK inhibitor, reduced the subsequent increase in pSmad3L, c-Myc transcription, and hepatocytic growth triggered by pro-inflammatory cytokine stimulation (Figure 1C), suggesting a direct role of the JNK/pSmad3L/c-Myc pathway in facilitating hepatocytic growth in response to cytokine stimulation (Figure 1B).

Experimental models of HCC including inflammation can elucidate how chronic inflammation contributes to hepatocarcinogenesis. In a rat model involving diethylnitrosamine (DEN) induced carcinogenesis, chronic inflammation liver accompanies abnormalities that progress to HCC [151]. This DEN-induced rat HCC is histologically and genetically similar to human HCC, and also is associated with chronic inflammation [152]. In this chemical model, JNK act participates importantly in hepatocarcinogenesis via pSmad3L/c-Myc signaling. In DENtreated livers, the JNK/pSmad3L/c-Myc pathway was activated in early pre-neoplastic hepatocytes (Figure 1B), [54]. Moreover, a JNK inhibitor SP600125 suppressed HCC develop‐ ment in DEN-treated rat livers by restoring carcinogenic pSmad3L/c-Myc to the basal pSmad3C/p21WAF1 pathway in the pre-neoplastic hepatocytes (Figure 1C), [54].

In human chronic hepatitis C specimens, mainly in groups of hepatocytes adjoining inflam‐ matory cells in portal tracts, Smad3 was found to be phosphorylated at the linker region [64]. Furthermore, positivity of hepatocytic nuclei for pSmad3L/c-Myc in chronic hepatitis C specimens showed a significant relationship with necrosis and inflammatory activity [64]. Taken together with the results of *in vitro* experiments and DEN-treated rat livers, the human findings indicate that chronic inflammation directly participates in hepatocarcinogenesis by shifting hepatocytic phospho-Smad3 signaling from the tumor-suppressive pSmad3C/ p21WAF1 pathway to the oncogenic JNK/pSmad3L/c-Myc pathway [54,64].

Many tumor-enhancing effects of pro-inflammatory cytokines on hepatocytes are exerted at the level of tumor promotion [58]. TNF-α promotes HCC occurrence in mice lacking the P- glycoprotein Mdr2 [153]. HCC follows cholestatic inflammation in these mice. Incidence of HCC can be enhanced by another member of the TNF family, lymphotoxin β [154]. Tumorpromoting cytokines produced by Kupffer cells activate several transcription factors, includ‐ ing NF-kB, STAT3, and AP-1, in pre-malignant hepatocytes [155]. The activated transcription factors stimulate transcription of their target genes involved in hepatocytic proliferation and survival, representing a major tumor-promoting mechanism. Similarly to these transcription factors, tumor-promoting actions of hepatocytic Smad3 in human chronic liver disease rarely result from direct mutations [156]. Instead, pSmad3L depends on mitogenic pro-inflammatory cytokine signals produced by neighboring Kupffer cells.

**12. Activation of the oncogenic JNK/pSmad3L pathway by chronic**

Inflammatory microenvironments are present in human hepatocarcinogenesis before malig‐ nant change occurs. A hepatitis virus infection triggers chronic inflammation, increasing the risk of HCC development. Several studies have discussed how chronic inflammation affects the proliferation and survival of hepatocytes [142,143]. TNF-α, IL-1β and IL-6 are multifunc‐ tional pro-inflammatory cytokines largely responsible for the hepatic response to chronic inflammation [144-146]. Serum concentrations of these cytokines are increased in chronic liver inflammation including hepatitis viral infection and steatohepatitis [147]. JNK is a key signal transducer for inflammatory cytokines and has emerged as an important endogenous tumor

TGF-β is also released by infiltrating Kupffer cells, the liver's resident macrophages, in portal tracts during chronic inflammation [150]. These findings suggest that elevated pro-inflamma‐ tory cytokines might alter hepatocytic TGF-β signaling in inflammatory microenvironments. We investigated this hypothesis using rat cultured hepatocytes [64]. Pretreatment of hepato‐ cytes with SP600125, a JNK inhibitor, reduced the subsequent increase in pSmad3L, c-Myc transcription, and hepatocytic growth triggered by pro-inflammatory cytokine stimulation (Figure 1C), suggesting a direct role of the JNK/pSmad3L/c-Myc pathway in facilitating

Experimental models of HCC including inflammation can elucidate how chronic inflammation contributes to hepatocarcinogenesis. In a rat model involving diethylnitrosamine (DEN) induced carcinogenesis, chronic inflammation liver accompanies abnormalities that progress to HCC [151]. This DEN-induced rat HCC is histologically and genetically similar to human HCC, and also is associated with chronic inflammation [152]. In this chemical model, JNK act participates importantly in hepatocarcinogenesis via pSmad3L/c-Myc signaling. In DENtreated livers, the JNK/pSmad3L/c-Myc pathway was activated in early pre-neoplastic hepatocytes (Figure 1B), [54]. Moreover, a JNK inhibitor SP600125 suppressed HCC develop‐ ment in DEN-treated rat livers by restoring carcinogenic pSmad3L/c-Myc to the basal

In human chronic hepatitis C specimens, mainly in groups of hepatocytes adjoining inflam‐ matory cells in portal tracts, Smad3 was found to be phosphorylated at the linker region [64]. Furthermore, positivity of hepatocytic nuclei for pSmad3L/c-Myc in chronic hepatitis C specimens showed a significant relationship with necrosis and inflammatory activity [64]. Taken together with the results of *in vitro* experiments and DEN-treated rat livers, the human findings indicate that chronic inflammation directly participates in hepatocarcinogenesis by shifting hepatocytic phospho-Smad3 signaling from the tumor-suppressive pSmad3C/

Many tumor-enhancing effects of pro-inflammatory cytokines on hepatocytes are exerted at the level of tumor promotion [58]. TNF-α promotes HCC occurrence in mice lacking the P-

hepatocytic growth in response to cytokine stimulation (Figure 1B).

pSmad3C/p21WAF1 pathway in the pre-neoplastic hepatocytes (Figure 1C), [54].

p21WAF1 pathway to the oncogenic JNK/pSmad3L/c-Myc pathway [54,64].

**inflammation**

124 Hepatocellular Carcinoma - Future Outlook

promoter [148,149].

### **13. Constitutive phosphorylation at Smad3L in pre-neoplastic hepatocytes in cirrhotic human liver**

The mechanism regulating regeneration, which avoids accumulation of deleterious mutations in genes that promote cell growth and division, must be disrupted before hepatocytes can throw off normal restraints and behave as an asocial HCC. Constitutive phosphorylation at Smad3L is observed in pre-malignant hepatocytes in cirrhosis [56,64]. Constitutively active pSmad3L stimulates hepatocytes to proliferate continuously in human livers that normally experience little proliferation because hepatocytic regeneration is tightly regulated by cyto‐ static pSmad3C signaling. Since JNK is constitutively activated in pre-neoplastic hepatocytes in cirrhotic human liver [157], constitutive Smad3L phosphorylation in pre-malignant lesions can be a direct consequence of proto-oncogene-mediated JNK signaling. Somatic mutations in pre-neoplastic hepatocytes include changes in the *Ras* pathway that favor progression from cirrhosis toward HCC [158]. In pre-neoplastic hepatocyte nuclei, pSmad3L/c-Myc can accu‐ mulate when somatic mutations constitutively activate the JNK pathway to phosphorylate Smad3 at the linker region (Figure 1B). Then, the proliferative effect mediated via the pSmad3L/ c-Myc pathway constitutively keeps on suppresses the growth-inhibitory pSmad3C/p21WAF1 pathway in the nuclei of pre-neoplastic hepatocytes.

Pre-neoplastic hepatocytes and HCC show reduction of anti-mitogenic responses to TGF-β [20,37]. Escaping the cytostatic action of pSmad3C is a critical step for progression to full malignancy in cancers, which must overcome multiple fail-safe genetic controls [39,159,160]. The TGF-β/pSmad3C pathway is required for maintenance of genomic stability, induction of replicative senescence, and suppression of telomerase [161-163]. Selection for genetic instabil‐ ity occurs in clones of aberrant cells able to produce tumors, since genetic instability greatly accelerates accumulation of further genetic and epigenetic changes required for tumor progression. In this regard, the TGF-β/pSmad3C pathway contributes to tumor suppression along with its cytostatic effect.

### **14. Chronic inflammation together with hepatitis virus effects in shifting phospho-Smad3 signaling into oncogene-dependent fibro-carcinogenic signaling**

while inactivating the TGF-β-dependent cytostatic actions of pSmad3C (Figure 1B), pharma‐ cologic interference with JNK/pSmad3L signaling could interrupt carcinogenesis. A key therapeutic aim in chronic liver disorders is restoration of lost tumor-suppressive function observed in normal hepatocytes, at the expense of effects promoting hepatic carcinogenesis [169]. To accomplish this difficult aim, Nagata *et al*. (2009) administered a JNK inhibitor SP600125 to rats and were able to suppress chemical carcinogenesis by shifting hepatocytic Smad3 signaling from the carcinogenic pSmad3L pathway to the tumor-suppressive pSmad3C pathway (Figure 1C), [54]. These studies provide evidence that JNK/pSmad3L is an important target for development of chemopreventive and therapeutic measures to reduce emergence of HCC in the context of chronic liver injury and to slow progression of existing tumors. We must also consider whether long-term use of any drug inhibiting C-terminal phosphorylation of R-

Early Chronic Inflammation and Subsequent Somatic Mutations Shift Phospho-Smad3 Signaling from…

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127

**Figure 2. After hepatitis virus infection, early chronic inflammation and subsequent somatic mutations shift hepatocytic phospho-Smad3 signaling from the tumor-suppressive TβRI/pSmad3C made to the fibro-carcino‐ genic JNK/pSmad3L mode characteristic of MFB, accelerating liver fibrosis while increasing risk of HCC**. Both hepatitis virus infection and chronic inflammation represent early fibro-carcinogenic steps representing non-mutagenic tumor-promoting stimuli. In advanced liver fibrosis, mitogenic genetic or epigenetic alterations can drive multistep fibrocarcinogenesis via the pSmad3L pathway. Escaping the cytostatic action of pSmad3C is a critical step for progression to

Human fibro-carcinogenesis is a complex multistep process, which involves dysregulation of physiological signal transduction pathways. To maintain hepatic homeostasis, hepatocyt‐ ic TβRI/pSmad3C/p21WAF1 terminates mitogenic JNK/pSmad3L/c-Myc signaling after liver regeneration. During progression of chronic liver diseases, however, early pro-inflammato‐ ry cytokines together with hepatitis viruses and subsequent somatic mutations switch hepatocytic phospho-Smad3 signaling from the tumor-suppressive TβRI/pSmad3C to the

full malignancy in cancers, which must overcome multiple fail-safe genetic controls.

**16. Conclusion and perspectives**

Smads might cause cancer development [7].

In the pathogenesis of HCC, continuous viral infection and chronic inflammation have a prominent role. On the other hand, detailed analysis of HCC development in experimental animals and correlation of these results with HCC in humans has identified a variety of genomic and molecular alterations in fully developed HCC [164]and to a lesser extent in morphologically defined pre-neoplastic precursor lesions [165]. Thus, a series of mutations may accumulate in individual hepatocytes over time. Finally, hepatocytes come to carry somatic mutations that lead to focal uncontrolled hepatocytic growth and eventual malignant cell transformation, in some cases, HCC [166].

Chronic inflammation associated with hepatitis virus infection may be the primary initial requirement in multistep hepatocarcinogenesis. If pSmad3L-positive and pSmad3C-negative hepatocytes survive in the course of chronic hepatitis, such hepatocytes and their descendants can accumulate, and acquire various mutated alleles. Mutations may involve genes in *Ras* pathway [158]that impel pre-neoplastic hepatocytes with constitutive phosphorylation at Smad3L toward a neoplastic growth [8]. Tumor promotion results in further selective clonal expansion of initiated cells, thereby enhancing the likelihood of additional genetic damage as a consequence of endogenous mutations. During tumor progression, premalignant cells continue to develop progressive phenotypic changes and genomic instability, developing into overt HCC.

### **15. The JNK/pSmad3L pathway as a therapeutic target to avert HCC development**

Clinical analyses of pSmad3L and pSmad3C in human tumor formation have provided substantial mechanistic insights. For example, specimens from patients with chronic hepatitis B who develop HCC show abundant Smad3L but limited Smad3C phosphorylation in hepatocytic nuclei, while other patients with abundant hepatocytic pSmad3C but limited pSmad3L do not develop HCC [56]. The same relationships are observed in human HCVrelated hepatocarcinogenesis [64]. These clinical observations support roles for pSmad3C as a tumor-suppressor and pSmad3L as a promoter during human carcinogenesis.

HCC is a highly chemoresistant cancer with no effective systemic cytotoxic chemotherapy [167]. Despite surgical or locoregional therapies, the prognosis remains poor because of high likelihood of tumor recurrence or progression and there are no well-established effective adjuvant therapies [168]. Molecular events that affect carcinogenesis need to be identified and targeted to validate new treatment approaches and expand available therapeutics to include chemoprevention to other therapeutics. Since JNK acts as an important regulator of Smad3 signaling that increases the basal amount of hepatocytic pSmad3L available for cell growth while inactivating the TGF-β-dependent cytostatic actions of pSmad3C (Figure 1B), pharma‐ cologic interference with JNK/pSmad3L signaling could interrupt carcinogenesis. A key therapeutic aim in chronic liver disorders is restoration of lost tumor-suppressive function observed in normal hepatocytes, at the expense of effects promoting hepatic carcinogenesis [169]. To accomplish this difficult aim, Nagata *et al*. (2009) administered a JNK inhibitor SP600125 to rats and were able to suppress chemical carcinogenesis by shifting hepatocytic Smad3 signaling from the carcinogenic pSmad3L pathway to the tumor-suppressive pSmad3C pathway (Figure 1C), [54]. These studies provide evidence that JNK/pSmad3L is an important target for development of chemopreventive and therapeutic measures to reduce emergence of HCC in the context of chronic liver injury and to slow progression of existing tumors. We must also consider whether long-term use of any drug inhibiting C-terminal phosphorylation of R-Smads might cause cancer development [7].

**Figure 2. After hepatitis virus infection, early chronic inflammation and subsequent somatic mutations shift hepatocytic phospho-Smad3 signaling from the tumor-suppressive TβRI/pSmad3C made to the fibro-carcino‐ genic JNK/pSmad3L mode characteristic of MFB, accelerating liver fibrosis while increasing risk of HCC**. Both hepatitis virus infection and chronic inflammation represent early fibro-carcinogenic steps representing non-mutagenic tumor-promoting stimuli. In advanced liver fibrosis, mitogenic genetic or epigenetic alterations can drive multistep fibrocarcinogenesis via the pSmad3L pathway. Escaping the cytostatic action of pSmad3C is a critical step for progression to full malignancy in cancers, which must overcome multiple fail-safe genetic controls.

### **16. Conclusion and perspectives**

**14. Chronic inflammation together with hepatitis virus effects in shifting phospho-Smad3 signaling into oncogene-dependent fibro-carcinogenic**

In the pathogenesis of HCC, continuous viral infection and chronic inflammation have a prominent role. On the other hand, detailed analysis of HCC development in experimental animals and correlation of these results with HCC in humans has identified a variety of genomic and molecular alterations in fully developed HCC [164]and to a lesser extent in morphologically defined pre-neoplastic precursor lesions [165]. Thus, a series of mutations may accumulate in individual hepatocytes over time. Finally, hepatocytes come to carry somatic mutations that lead to focal uncontrolled hepatocytic growth and eventual malignant

Chronic inflammation associated with hepatitis virus infection may be the primary initial requirement in multistep hepatocarcinogenesis. If pSmad3L-positive and pSmad3C-negative hepatocytes survive in the course of chronic hepatitis, such hepatocytes and their descendants can accumulate, and acquire various mutated alleles. Mutations may involve genes in *Ras* pathway [158]that impel pre-neoplastic hepatocytes with constitutive phosphorylation at Smad3L toward a neoplastic growth [8]. Tumor promotion results in further selective clonal expansion of initiated cells, thereby enhancing the likelihood of additional genetic damage as a consequence of endogenous mutations. During tumor progression, premalignant cells continue to develop progressive phenotypic changes and genomic instability, developing into

**15. The JNK/pSmad3L pathway as a therapeutic target to avert HCC**

tumor-suppressor and pSmad3L as a promoter during human carcinogenesis.

Clinical analyses of pSmad3L and pSmad3C in human tumor formation have provided substantial mechanistic insights. For example, specimens from patients with chronic hepatitis B who develop HCC show abundant Smad3L but limited Smad3C phosphorylation in hepatocytic nuclei, while other patients with abundant hepatocytic pSmad3C but limited pSmad3L do not develop HCC [56]. The same relationships are observed in human HCVrelated hepatocarcinogenesis [64]. These clinical observations support roles for pSmad3C as a

HCC is a highly chemoresistant cancer with no effective systemic cytotoxic chemotherapy [167]. Despite surgical or locoregional therapies, the prognosis remains poor because of high likelihood of tumor recurrence or progression and there are no well-established effective adjuvant therapies [168]. Molecular events that affect carcinogenesis need to be identified and targeted to validate new treatment approaches and expand available therapeutics to include chemoprevention to other therapeutics. Since JNK acts as an important regulator of Smad3 signaling that increases the basal amount of hepatocytic pSmad3L available for cell growth

**signaling**

126 Hepatocellular Carcinoma - Future Outlook

overt HCC.

**development**

cell transformation, in some cases, HCC [166].

Human fibro-carcinogenesis is a complex multistep process, which involves dysregulation of physiological signal transduction pathways. To maintain hepatic homeostasis, hepatocyt‐ ic TβRI/pSmad3C/p21WAF1 terminates mitogenic JNK/pSmad3L/c-Myc signaling after liver regeneration. During progression of chronic liver diseases, however, early pro-inflammato‐ ry cytokines together with hepatitis viruses and subsequent somatic mutations switch hepatocytic phospho-Smad3 signaling from the tumor-suppressive TβRI/pSmad3C to the

fibro-carcinogenic JNK/pSmad3L mode characteristic of MFB, which accelerates liver fibrosis while increasing risk of HCC (Figure 2). Our model is likely to represent a crucial molecu‐ lar mechanism by which most HCCs arise in from fibrosis or cirrhosis caused by chronic inflammation associated with persistent hepatitis virus infection [164]. Thus, Smad phosphoi‐ soforms function as an important orchestrator of a human chronic inflammation-fibrosis-HCC axis [9,170].

[2] McGlynn KA, London WT. Epidemiology and natural history of hepatocellular carci‐

Early Chronic Inflammation and Subsequent Somatic Mutations Shift Phospho-Smad3 Signaling from…

http://dx.doi.org/10.5772/56739

129

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Recent studies in animal models using conditional transgenic expression have suggested an intriguing reversibility of malignant transformation at specific time points if the primary inciting cause of the neoplasia is eliminated [171,172]. However, the fibro-carcinogenic stage in human chronic liver at which the process becomes irreversible. Chronic hepatitis B and C can be cured if patients are treated with antiviral therapy that arrests chronic inflammation by eradicating hepatic HBV and HCV populations. Continued histologic improvement and reversal of fibrosis by antiviral therapy can lead to reduction of HCC development [173,174], but prevention appears most effective when therapy is given before development of cirrhosis. Chronic hepatitis is clearly dependent on continued promoter stimulation - involving in this case the presence of hepatitis viruses and chronic inflammation. However, many patients with cirrhosis have evolved beyond dependence on inflammation because hepatocytes have acquired genetic and epigenetic carcinogenic properties. We are carrying out several trials to determine whether or not antiviral therapy can decrease liver fibrosis and lower HCC incidence. The trials will bear upon important questions regarding relative participation in fibro-carcinogenesis of inflammation-dependent and oncogene-dependent Smad3 phosphoi‐ soform signaling in HBV- and HCV-related chronic liver disorders. In the trials, pathologic analyses using domain-specific phospho-Smad3 Abs, together with clinical data, will be used to evaluate the benefit from antiviral therapy, which decreases stimulation of the inflamma‐ tion-dependent Smad phosphorisoform pathway. After antiviral therapy, hepatocytic pSmad3L and pSmad3C assessment in liver specimens should prove clinically useful for predicting progression of fibrosis and risk of HCC.

### **Author details**

Miki Murata\* , Katsunori Yoshida and Koichi Matsuzaki

\*Address all correspondence to: muratami@takii.kmu.ac.jp

Departments of Gastroenterology and Hepatology, Kansai Medical University, Osaka, Japan

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Recent studies in animal models using conditional transgenic expression have suggested an intriguing reversibility of malignant transformation at specific time points if the primary inciting cause of the neoplasia is eliminated [171,172]. However, the fibro-carcinogenic stage in human chronic liver at which the process becomes irreversible. Chronic hepatitis B and C can be cured if patients are treated with antiviral therapy that arrests chronic inflammation by eradicating hepatic HBV and HCV populations. Continued histologic improvement and reversal of fibrosis by antiviral therapy can lead to reduction of HCC development [173,174], but prevention appears most effective when therapy is given before development of cirrhosis. Chronic hepatitis is clearly dependent on continued promoter stimulation - involving in this case the presence of hepatitis viruses and chronic inflammation. However, many patients with cirrhosis have evolved beyond dependence on inflammation because hepatocytes have acquired genetic and epigenetic carcinogenic properties. We are carrying out several trials to determine whether or not antiviral therapy can decrease liver fibrosis and lower HCC incidence. The trials will bear upon important questions regarding relative participation in fibro-carcinogenesis of inflammation-dependent and oncogene-dependent Smad3 phosphoi‐ soform signaling in HBV- and HCV-related chronic liver disorders. In the trials, pathologic analyses using domain-specific phospho-Smad3 Abs, together with clinical data, will be used to evaluate the benefit from antiviral therapy, which decreases stimulation of the inflamma‐ tion-dependent Smad phosphorisoform pathway. After antiviral therapy, hepatocytic pSmad3L and pSmad3C assessment in liver specimens should prove clinically useful for

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128 Hepatocellular Carcinoma - Future Outlook

**Author details**

Miki Murata\*

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, Katsunori Yoshida and Koichi Matsuzaki

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**Chapter 8**

**Surgical Treatment Strategies and**

Alessandro Uzzau, Maria Laura Pertoldeo,

Additional information is available at the end of the chapter

Vittorio Cherchi, Serena Bertozzi, Claudio Avellini and Giorgio Soardo

http://dx.doi.org/10.5772/55890

**1. Introduction**

**Prognosis of Hepatocellular Carcinoma**

Hepatocellular carcinoma (HCC is the fifth most common cause of mortality worldwide and the third cancer related cause and is responsible for about 1 million deaths yearly [1]. The ageadjusted worldwide incidence is 5.5-14.9 per 100.000 population. In some areas of the world, such as sub-Saharan Africa and Southeast Asia, HCC represents the first cause of cancer death with an incidence of 52 per 100.000. Furthermore, in Europe and USA, HCC incidence has

HCC is one of the few cancers for which a number of risk factors are known in great detail [2, 3]. HCC is almost always (80%) associated with cirrhosis, at least in developed countries, and chronic hepatitis C and B infection, alcoholic cirrhosis and haemocromatosis are some of the established risk factors [4]. The metabolic syndrome related to hypertension, central obesity, diabetes and obesity has been identified as a new risk factor. As a result, screening programs have developed, with the use of ultrasound and α-fetoprotein (AFP), with a hope to increase

Definitive diagnosis relies on the demonstration of a typical vascular pattern per liver imaging techniques (triple-phase CT-scan or MRI) of tumors larger than 2 cm with arterial hypervascu‐ larity and venous wash- out. Nodules, smaller than 2 cm, should be rechecked every six months or, if highly suspect, subjected to needle biopsy. It's likely that the study of tumor-specific tissue

Over the past 20 years, surgical treatment of hepatocellular carcinoma has seen an immense boost and improvement, with good survival outcomes and reduced morbidity and mortality.

> © 2013 Uzzau et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

the chances of diagnosing small HCC and unltimately increase the rate of curability.

markers with prognostic value could introduce a systematic use of needle biopsy.

progressively raised in the past decade representing a burden problem.


**Chapter 8**
