**Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in Hepatocellular Carcinoma**

Yasunobu Matsuda, Toshifumi Wakai, Masayuki Kubota, Mami Osawa and Shun Fujimaki

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55201

### **1. Introduction**

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide [Serag et al., 2007, Liovet et al., 2003, Yang et al., 2010]. More than 500,000 people are diagnosed with HCC every year, and it remains the leading cause of death among patients with hepatitis B virus (HBV), hepatitis C virus (HCV) and alcohol-induced liver cirrhosis. One of the main obstacles for treating HCC is late diagnosis of patients. Many unresectable HCC patients are treated with loco-regional therapies such as radiofrequency ablation and transarterial chemo‐ embolization (TACE), but the prognosis remains poor [Bruix et al., 2005]. A recent study in multiple clinical facilities in Japan reported that 5-year survival of patients treated with TACE was less than 30% [Takayasu et al., 2006]. Moreover, HCC is poorly responsive to chemother‐ apeutic drugs and radiotherapy [Arii et al., 2000, Kuwahara et al., 2009]; thus, effective therapeutic tools for HCC are long-awaited.

Sorafenib (Nexavar, BAY 43-9006, Bayer HealthCare Pharmaceuticals) is a new type of drug designed to target RAF signaling, and represents a new era of HCC treatment. However, accumulating evidence has revealed the limited effect of sorafenib, and many clinical trials of sorafenib-based combination therapy are now underway. It should be noted that, while sorafenib was originally designed to target RAF-mediated signaling, recent studies have strongly indicated that its effect is closely involved in various types of non-RAF signal‐ ing [Matsuda et al., 2011]. To explore safe and effective therapies combined with sorafe‐ nib, full understanding of the functional mechanism of sorafenib is necessary. Herein, we review recent findings from studies of sorafenib-mediated inhibition of RAF and non-

© 2013 Matsuda 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. RAF signaling pathways. We also discuss the possibility of administering sorafenib with other drugs in combination therapy, which might become a promising approach in the treatment of advanced HCC.

### **2. Clinical perspectives of sorafenib**

#### **2.1. The effects and limitations of sorafenib**

Sorafenib is an orally bioavailable inhibitor of multiple kinases including RAF, vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor (PDGF) receptor, and the v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene (*KIT*) and fms-like tyrosine kinase 3 (*FLT-3*) oncogene [Wilhelm et al., 2004, Hochhaus et al., 2011]. At present, sorafenib is the only oral drug shown to improve the survival of unresectable HCC. The SHARP trial (the Sorafenib HCC Assessment Randomized Protocol), a multicenter double-blind phase III trial in Europe, North America, South America, and Australasia conducted in 2008, reported overall survival in the sorafenib-treated group was significantly longer than in the placebo group (10.7 *vs.* 7.9 months) [Llovet et al., 2008]. An Asia-Pacific study, which was conducted in China, South Korea, and Taiwan in 2009, also reported that the median overall survival in the sorafenib-treated group was improved (6.5 *vs.* 4.2 months) [Cheng et al., 2009], suggesting that the effect of sorafenib is universal among different ethnic backgrounds. Unfortunately however, subsequent clinical studies have highlighted several issues. First, sorafenib treatment rarely results in tumor shrinkage [Jubb et al., 2010]. A partial tumor response was seen in only 2% and 3.3% of the SHARP and the Asia-Pacific studies, respectively, and there have been few reported cases that achieved complete remission after sorafenib treatment [SO et al., 2008, Yeganeh et al., 2009, Wang et al., 2010, Sacco et al., 2011]. Second, sorafenib is less effective when the patients are affected with medium to severe liver dysfunction (Child-Pugh class B and C) [Pinter et al., 2009, Schütte et al., 2011]. The reason for the influence of liver function on the efficacy of sorafenib should be determined in the near future. Because liver cirrhosis is a unique condition in which excessive inflammatory cytokines is produced, it is plausible that cancer microenvironment in liver disease might affect the sorafenib efficacy (Fig. 1). Third, sorafenib causes many side-effects, including diarrhea, skin eruption, and bone marrow dysfunction. All these lines of evidence strongly suggest that safer and more effective sorafenib therapy should be established for HCC patients.

2007]. Furthermore, combination of sorafenib and the chemotherapeutic drug doxorubicin was

**Figure 1.** Sorafenib and Raf signaling. Sorafenib inhibits C-Raf rather than B-Raf, and attenuates the activation of MAPK/ERK signaling. However, the effect of sorafenib might be influenced by the cancer microenvironment in liver cirrhosis. MEK, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular

Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in…

http://dx.doi.org/10.5772/55201

241

Basic studies have now evaluated the preclinical protocols of the combination of sorafenib with other therapeutic agents. The most prospective method for treating HCC is a combination of sorafenib with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) pathway. mTOR is known to be activated in many types of cancer cells, and around half of human HCC cases showed aberrant mTOR signaling [Villanueva et al., 2008]. Several studies using a human HCC xenograft mouse model have reported that the combination of rapamycin and sorafenib synergistically enhanced the anti-tumor effect, and resulted in tumor shrinkage [Wang et al., 2008, Huynh et al., 2009, Newell et al., 2009]. Thus far, a phase I trial of the combination therapy of sorafenib and temsirolimus (a rapamycin analog) is in progress for

**3. Sorafenib affects both RAF and non-RAF signaling pathways**

The *RAS* oncogene encodes a small guanosine triphosphate-binding protein (GTPase) that plays a central role in promoting the cell proliferation, survival and transformation [Karnoub

found to be effective in HCC [Abou-Alfa et al., 2010].

treating advanced HCC [Kelley et al., 2010].

**3.1. RAF signaling**

signal-regulated kinase.

*3.1.1. RAF signaling and HCC*

#### **2.2. Clinical trial of sorafenib-based combination therapy**

To improve the limited efficacy of sorafenib, many clinical trials of sorafenib-based combina‐ tion treatment have been undertaken. For example, a phase II multicenter study in Italy reported that the combination of sorafenib and long-acting octreotide (an analogue of soma‐ tostatin) resulted in a better survival rate as compared with sorafenib monotherapy [Prete et al., 2010]. This report suggests a possible synergic tumor killing effect by sorafenib, because octreotide monotherapy has been regarded as less effective in advanced HCC [Becker et al.,

Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in… http://dx.doi.org/10.5772/55201 241

**Figure 1.** Sorafenib and Raf signaling. Sorafenib inhibits C-Raf rather than B-Raf, and attenuates the activation of MAPK/ERK signaling. However, the effect of sorafenib might be influenced by the cancer microenvironment in liver cirrhosis. MEK, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase.

2007]. Furthermore, combination of sorafenib and the chemotherapeutic drug doxorubicin was found to be effective in HCC [Abou-Alfa et al., 2010].

Basic studies have now evaluated the preclinical protocols of the combination of sorafenib with other therapeutic agents. The most prospective method for treating HCC is a combination of sorafenib with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) pathway. mTOR is known to be activated in many types of cancer cells, and around half of human HCC cases showed aberrant mTOR signaling [Villanueva et al., 2008]. Several studies using a human HCC xenograft mouse model have reported that the combination of rapamycin and sorafenib synergistically enhanced the anti-tumor effect, and resulted in tumor shrinkage [Wang et al., 2008, Huynh et al., 2009, Newell et al., 2009]. Thus far, a phase I trial of the combination therapy of sorafenib and temsirolimus (a rapamycin analog) is in progress for treating advanced HCC [Kelley et al., 2010].

### **3. Sorafenib affects both RAF and non-RAF signaling pathways**

#### **3.1. RAF signaling**

RAF signaling pathways. We also discuss the possibility of administering sorafenib with other drugs in combination therapy, which might become a promising approach in the

Sorafenib is an orally bioavailable inhibitor of multiple kinases including RAF, vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor (PDGF) receptor, and the v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene (*KIT*) and fms-like tyrosine kinase 3 (*FLT-3*) oncogene [Wilhelm et al., 2004, Hochhaus et al., 2011]. At present, sorafenib is the only oral drug shown to improve the survival of unresectable HCC. The SHARP trial (the Sorafenib HCC Assessment Randomized Protocol), a multicenter double-blind phase III trial in Europe, North America, South America, and Australasia conducted in 2008, reported overall survival in the sorafenib-treated group was significantly longer than in the placebo group (10.7 *vs.* 7.9 months) [Llovet et al., 2008]. An Asia-Pacific study, which was conducted in China, South Korea, and Taiwan in 2009, also reported that the median overall survival in the sorafenib-treated group was improved (6.5 *vs.* 4.2 months) [Cheng et al., 2009], suggesting that the effect of sorafenib is universal among different ethnic backgrounds. Unfortunately however, subsequent clinical studies have highlighted several issues. First, sorafenib treatment rarely results in tumor shrinkage [Jubb et al., 2010]. A partial tumor response was seen in only 2% and 3.3% of the SHARP and the Asia-Pacific studies, respectively, and there have been few reported cases that achieved complete remission after sorafenib treatment [SO et al., 2008, Yeganeh et al., 2009, Wang et al., 2010, Sacco et al., 2011]. Second, sorafenib is less effective when the patients are affected with medium to severe liver dysfunction (Child-Pugh class B and C) [Pinter et al., 2009, Schütte et al., 2011]. The reason for the influence of liver function on the efficacy of sorafenib should be determined in the near future. Because liver cirrhosis is a unique condition in which excessive inflammatory cytokines is produced, it is plausible that cancer microenvironment in liver disease might affect the sorafenib efficacy (Fig. 1). Third, sorafenib causes many side-effects, including diarrhea, skin eruption, and bone marrow dysfunction. All these lines of evidence strongly suggest that safer and more effective sorafenib

To improve the limited efficacy of sorafenib, many clinical trials of sorafenib-based combina‐ tion treatment have been undertaken. For example, a phase II multicenter study in Italy reported that the combination of sorafenib and long-acting octreotide (an analogue of soma‐ tostatin) resulted in a better survival rate as compared with sorafenib monotherapy [Prete et al., 2010]. This report suggests a possible synergic tumor killing effect by sorafenib, because octreotide monotherapy has been regarded as less effective in advanced HCC [Becker et al.,

treatment of advanced HCC.

240 Hepatocellular Carcinoma - Future Outlook

**2. Clinical perspectives of sorafenib**

**2.1. The effects and limitations of sorafenib**

therapy should be established for HCC patients.

**2.2. Clinical trial of sorafenib-based combination therapy**

#### *3.1.1. RAF signaling and HCC*

The *RAS* oncogene encodes a small guanosine triphosphate-binding protein (GTPase) that plays a central role in promoting the cell proliferation, survival and transformation [Karnoub et al., 2008]. Four proteins, including H-RAS, N-RAS, K-RASA and K-RASB are encoded by the *RAS* gene, and all of these mutant forms have been known to lead to increased GTP-bound RAS (RAS-GTP). Of these, K-RAS is frequently activated by gene mutation in many types of cancer cells [Karnoub et al., 2008], indicating that *RAS* is a common oncogene in various cell types. Several growth factors such as epidermal growth factor (EGF), insulin-like growth factor-I (IGF-1) and PDGF induce cell proliferation through enhanced exchange of guanine nucleotides on RAS. RAS has a guanosine diphosphate (GDP) binding domain, and GDPbound RAS (RAS-GDP) is activated when converted into RAS-GTP. Downstream mediators of RAS (RAF, PI3K-bound RAL-GEF) bind RAS-GTP with higher affinity than RAS-GDP [Herrmann et al., 1995]. RAS recruits members of the RAF serine/threonine kinase family to the plasma membrane, whereupon they are activated by phosphorylation. The RAF kinase family is composed of three members: A-RAF, B-RAF, and RAF-1 (also termed C-RAF). Of these, many studies have suggested that RAF-1 plays a critical role in the early step of carcinogenesis. It has now been widely accepted that RAF signaling exerts a critical role on the progression in many of the incurable diseases. Good example might be an autosomal dominant polycystic kidney disease (ADPKD). Recent studies have unveiled that Ras/Raf signaling is hyper-activated in cyst epithelial cells in this disease, and both sorafenib and a novel Raf kinase inhibitor PLX5568 can attenuate the proliferation of ADPKD cyst epithelial cells [Yamaguchi T et al., 2010, Buchholz et al., 2011].

complex consisting of C-RAF and B-RAFV600E becomes decreased as compared with C-RAF

Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in…

http://dx.doi.org/10.5772/55201

243

Intriguingly, the difference in the kinase activities of each RAF complex affects the therapeutic effect of sorafenib. Sorafenib inhibits C-RAF at low doses, while it inhibits wild-type and mutated B-RAF at high doses [Wilhelm et al., 2004]. Therefore, high doses of sorafenib can inhibit the activities of both C-RAF and B-RAF (either wild-type or V600E), while at lower doses, sorafenib only inhibits C-RAF, resulting in disinhibition of B-RAF [Garnett et al., 2005]. More importantly, low doses of sorafenib has the unique ability in that it induces the formation of heterologous complexes between B-RAFV600E and wild-type B-RAF, leading to the enhancement of the kinase ability of B-RAFV600E [Garnett et al., 2005]. It has been recently reported that cells expressing oncogenic *RAS* are selectively inhibited, B-RAF, B-RAF-C-RAF heterodimers are induced, and RAF/MEK/ERK signaling is activated [Heidorn et al., 2010]. It has been also shown that B-RAF-ERK signaling and C-RAF signaling play dominant roles in the regulation of proliferation of lung cancer cells with wild-type or mutant *KRAS*, respec‐ tively. Intriguingly however, sorafenib can inhibit both cell types by targeting B-RAF-mediated ERK phosphorylation in cells with wild-type *KRAS*, and by targeting C-RAF in the cells with mutant *KRAS* [Takezawa et al., 2009]. These lines of evidence strongly suggest that clinicians should decide the dosage of sorafenib in reference to the level of each RAF kinase in the tumors.

Recently, several studies have reported that sorafenib has a significant effect on non-RAFsignaling pathways, as well as RAF-mediated signaling, particularly caspase-mediated apoptotic signaling. Apoptosis is mainly regulated by two major pathways; (1) tumor necrosis factor- alpha receptors (TNFRs) or the Fas-mediated caspase-8 signaling pathway, and (2) BCL-2 family members-regulated caspase-9 pathway [Ashkenazi, 2008, Leber et al., 2010]. It has been recently reported that sorafenib kills tumor cells by regulating MCL-1, which is a member of the BCL2 protein family [Akgul, 2009, Thomas et al., 2010]. MCL-1 is a repressor of apoptotic cell death via its interactions with the cell death inducer BAX, and overproduction of MCL-1 inhibits cell apoptosis induced by growth factor withdrawal, MYC overexpression, or cytotoxic agents. MCL-1 has been known to be overexpressed in many types of malignan‐ cies, and many studies have suggested that the levels of MCL-1 expression may determine the

Recent studies have led to the suggestion that MCL-1 might be one of the main targets for MEK/ERK-independent mechanisms of action of sorafenib. Sorafenib reduces MCL-1 in various types of cancer cells by proteasome-mediated degradation [Yo et al., 2005], and sorafenib-mediated MCL-1 downregulation is associated with MCL-1-translation and cyto‐ chrome-c release into the cytosol. [Rahmani et al., 2005] Intriguingly, MCL-1 might be a promising biomarker of therapeutic efficacy, because it was found to be upregulated in sorafenib-resistant cells [Ulivi et al., 2009]. It has also been reported that HCV can increase therapeutic response to sorafenib by miR-193b-dependent modulation of MCL-1 [Braconi et al., 2010], suggesting that MCL-1 might be involved in the virus-associated drug response in

and non-mutated B-RAF heterodimers [Garnett et al., 2005].

*3.1.3. Apoptotic pathways and sorafenib*

therapeutic efficacy of anti-tumor agents.

cancer cells.

In the case of HCC, point mutations of *RAS* have been reported to be infrequent in HCC [Challen et al., 1992]. However, it has been revealed that its downstream signaling is frequently activated during hepatocarcinogenesis. Hepatitis B virus X protein (HBx) and HCV core protein, both of which have been considered as strong promoters of hepatocarcinogenesis, increase the kinase activity of RAF-1 [Aoki et al., 2000, Chen et al., 2007]. It has been also reported that the C-terminal of HCV-encoded nonstructural protein 5A (NS5A) binds to and activates RAF-1 [Bürckstümmer et al., 2006]. More importantly, activated RAF-1 has been found in around 90% of liver cirrhosis cases and in 100% of HCC cases [Hwang et al., 2004].

#### *3.1.2. RAF signaling and sorafenib*

RAF-1 is a mitogen-activated protein kinase (MAPK) kinase, which phosphorylates and activates the serine/threonine-specific extracellular signal-regulated protein kinases ERK1 and ERK2 [Avruch et al., 1994]. In the nucleus, phosphorylated ERK activates transcription factors such as ELK-1 and c-JUN, leading to cell proliferation and survival. RAF-1 has been also reported to form a complex with Cdc25 and activates the cyclin E-Cdk2 complex, leading to progression through the G1-S phase transition [Kerkhoff et al., 1998, Hindley et al., 2002]. When activated, RAF members form homologous and heterologous complexes, leading to activation of the MEK/ERK pathway. It should be noted that the kinase activity of RAF complexes is defined by the type of RAF constituents. It has been reported that the kinase activity of B-RAF and c-RAF heterodimers is higher than that of B-RAF or C-RAF homodimers [Rushworth et al., 2006]. Moreover, when B-RAF is mutated at V600E (B-RAFV 600E), as observed in some types of cancer cells, it acquires strong kinase activity and can directly stimulate the MEK/ERK pathway [Wan et al., 2004, Garnett et al., 2005]. In turn, the kinase activity of a heterologous complex consisting of C-RAF and B-RAFV600E becomes decreased as compared with C-RAF and non-mutated B-RAF heterodimers [Garnett et al., 2005].

Intriguingly, the difference in the kinase activities of each RAF complex affects the therapeutic effect of sorafenib. Sorafenib inhibits C-RAF at low doses, while it inhibits wild-type and mutated B-RAF at high doses [Wilhelm et al., 2004]. Therefore, high doses of sorafenib can inhibit the activities of both C-RAF and B-RAF (either wild-type or V600E), while at lower doses, sorafenib only inhibits C-RAF, resulting in disinhibition of B-RAF [Garnett et al., 2005]. More importantly, low doses of sorafenib has the unique ability in that it induces the formation of heterologous complexes between B-RAFV600E and wild-type B-RAF, leading to the enhancement of the kinase ability of B-RAFV600E [Garnett et al., 2005]. It has been recently reported that cells expressing oncogenic *RAS* are selectively inhibited, B-RAF, B-RAF-C-RAF heterodimers are induced, and RAF/MEK/ERK signaling is activated [Heidorn et al., 2010]. It has been also shown that B-RAF-ERK signaling and C-RAF signaling play dominant roles in the regulation of proliferation of lung cancer cells with wild-type or mutant *KRAS*, respec‐ tively. Intriguingly however, sorafenib can inhibit both cell types by targeting B-RAF-mediated ERK phosphorylation in cells with wild-type *KRAS*, and by targeting C-RAF in the cells with mutant *KRAS* [Takezawa et al., 2009]. These lines of evidence strongly suggest that clinicians should decide the dosage of sorafenib in reference to the level of each RAF kinase in the tumors.

#### *3.1.3. Apoptotic pathways and sorafenib*

et al., 2008]. Four proteins, including H-RAS, N-RAS, K-RASA and K-RASB are encoded by the *RAS* gene, and all of these mutant forms have been known to lead to increased GTP-bound RAS (RAS-GTP). Of these, K-RAS is frequently activated by gene mutation in many types of cancer cells [Karnoub et al., 2008], indicating that *RAS* is a common oncogene in various cell types. Several growth factors such as epidermal growth factor (EGF), insulin-like growth factor-I (IGF-1) and PDGF induce cell proliferation through enhanced exchange of guanine nucleotides on RAS. RAS has a guanosine diphosphate (GDP) binding domain, and GDPbound RAS (RAS-GDP) is activated when converted into RAS-GTP. Downstream mediators of RAS (RAF, PI3K-bound RAL-GEF) bind RAS-GTP with higher affinity than RAS-GDP [Herrmann et al., 1995]. RAS recruits members of the RAF serine/threonine kinase family to the plasma membrane, whereupon they are activated by phosphorylation. The RAF kinase family is composed of three members: A-RAF, B-RAF, and RAF-1 (also termed C-RAF). Of these, many studies have suggested that RAF-1 plays a critical role in the early step of carcinogenesis. It has now been widely accepted that RAF signaling exerts a critical role on the progression in many of the incurable diseases. Good example might be an autosomal dominant polycystic kidney disease (ADPKD). Recent studies have unveiled that Ras/Raf signaling is hyper-activated in cyst epithelial cells in this disease, and both sorafenib and a novel Raf kinase inhibitor PLX5568 can attenuate the proliferation of ADPKD cyst epithelial cells [Yamaguchi

In the case of HCC, point mutations of *RAS* have been reported to be infrequent in HCC [Challen et al., 1992]. However, it has been revealed that its downstream signaling is frequently activated during hepatocarcinogenesis. Hepatitis B virus X protein (HBx) and HCV core protein, both of which have been considered as strong promoters of hepatocarcinogenesis, increase the kinase activity of RAF-1 [Aoki et al., 2000, Chen et al., 2007]. It has been also reported that the C-terminal of HCV-encoded nonstructural protein 5A (NS5A) binds to and activates RAF-1 [Bürckstümmer et al., 2006]. More importantly, activated RAF-1 has been found in around 90% of liver cirrhosis cases and in 100% of HCC cases [Hwang et al., 2004].

RAF-1 is a mitogen-activated protein kinase (MAPK) kinase, which phosphorylates and activates the serine/threonine-specific extracellular signal-regulated protein kinases ERK1 and ERK2 [Avruch et al., 1994]. In the nucleus, phosphorylated ERK activates transcription factors such as ELK-1 and c-JUN, leading to cell proliferation and survival. RAF-1 has been also reported to form a complex with Cdc25 and activates the cyclin E-Cdk2 complex, leading to progression through the G1-S phase transition [Kerkhoff et al., 1998, Hindley et al., 2002]. When activated, RAF members form homologous and heterologous complexes, leading to activation of the MEK/ERK pathway. It should be noted that the kinase activity of RAF complexes is defined by the type of RAF constituents. It has been reported that the kinase activity of B-RAF and c-RAF heterodimers is higher than that of B-RAF or C-RAF homodimers [Rushworth et al., 2006]. Moreover, when B-RAF is mutated at V600E (B-RAFV 600E), as observed in some types of cancer cells, it acquires strong kinase activity and can directly stimulate the MEK/ERK pathway [Wan et al., 2004, Garnett et al., 2005]. In turn, the kinase activity of a heterologous

T et al., 2010, Buchholz et al., 2011].

242 Hepatocellular Carcinoma - Future Outlook

*3.1.2. RAF signaling and sorafenib*

Recently, several studies have reported that sorafenib has a significant effect on non-RAFsignaling pathways, as well as RAF-mediated signaling, particularly caspase-mediated apoptotic signaling. Apoptosis is mainly regulated by two major pathways; (1) tumor necrosis factor- alpha receptors (TNFRs) or the Fas-mediated caspase-8 signaling pathway, and (2) BCL-2 family members-regulated caspase-9 pathway [Ashkenazi, 2008, Leber et al., 2010]. It has been recently reported that sorafenib kills tumor cells by regulating MCL-1, which is a member of the BCL2 protein family [Akgul, 2009, Thomas et al., 2010]. MCL-1 is a repressor of apoptotic cell death via its interactions with the cell death inducer BAX, and overproduction of MCL-1 inhibits cell apoptosis induced by growth factor withdrawal, MYC overexpression, or cytotoxic agents. MCL-1 has been known to be overexpressed in many types of malignan‐ cies, and many studies have suggested that the levels of MCL-1 expression may determine the therapeutic efficacy of anti-tumor agents.

Recent studies have led to the suggestion that MCL-1 might be one of the main targets for MEK/ERK-independent mechanisms of action of sorafenib. Sorafenib reduces MCL-1 in various types of cancer cells by proteasome-mediated degradation [Yo et al., 2005], and sorafenib-mediated MCL-1 downregulation is associated with MCL-1-translation and cyto‐ chrome-c release into the cytosol. [Rahmani et al., 2005] Intriguingly, MCL-1 might be a promising biomarker of therapeutic efficacy, because it was found to be upregulated in sorafenib-resistant cells [Ulivi et al., 2009]. It has also been reported that HCV can increase therapeutic response to sorafenib by miR-193b-dependent modulation of MCL-1 [Braconi et al., 2010], suggesting that MCL-1 might be involved in the virus-associated drug response in cancer cells.

#### *3.1.4. Endoplasmic reticulum stress and sorafenib*

Another RAF-independent mechanism involved in sorafenib-inhibited signaling is endoplas‐ mic reticulum (ER) stress [Rahmani et al., 2007]. The endoplasmic reticulum (ER) is a central organelle in each eukaryotic cell that serves many general functions, including lipid synthesis, protein folding and protein maturation, transportation of synthesized proteins, and activation of chaperone proteins. When cells are exposed to various types of stress such as hypoxia, oxidative stress, hypoglycemia and viral infection, unfolded protein aggregates (unfolded protein response, UPR) accumulate to interfere the function of ER, which is generally called ER stress [Tsukada et al., 1993, Kim et al., 2008]. ER stress causes decreased protein translation to prevent further accumulation of unfolded proteins. It should be noted, however, that the function of ER stress is complex because it can induce either cell survival or autophagy-related cell death [Schleicher et al., 2010]. Several UPR-involving signaling molecules have been identified; the PKR-like kinase (PERK) is an important inhibitor of protein translation through phosphorylation of eukaryotic initiation factor 2 (eIF2α). The kinase activity of PERK is induced by ER stress, and phosphorylation of PERK at Thr980 is regarded as a marker for ER stress. Endoplasmic oxidoreductin-1 (Ero1) is an ER membrane-associated N-glycoprotein that provides oxidizing potential and protein folding. Inositol requiring-1 (IRE1) and activating transcription factor-6 (ATF6) induce calcium-dependent protein chaperones such as GRP78/BiP to maintain correct protein folding [Kim et al., 2008, McConkey et al., 2008]. Calnexin is a calcium-binding protein that retains the synthesized glycoproteins inside the ER. CAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a dominant-negative inhibitor of C/EBP and LAP, which plays a role in cell cycle arrest during G1 to S phase. During ER stress, the level of CHOP is increased to induce the activation of GADD34, a downstream protein of P53 tumor suppressor that causes DNA excision repair and cell arrest, and ERO-1 expression. ERO-1 promotes oxidative stress inside the ER, leading to programmed cell death. suggesting that sorafenib-induced oxidative stress might not be the secondary phenomenon during cell death [Chiou et al., 2009]. Chiou et al. also reported that glutathione (GSH), an intracellular non-protein-thiol antioxidant, was decreased after treatment with sorafenib. Currently it is not known that why sorafenib results in the dysregulated balance of oxidants and anti-oxidants. Park et al. reported that low doses of sorafenib and vorinostat, a histone deacetylase inhibitor (HDACI) that has shown preclinical evidence of anti-tumor activity against hepatoma, rapidly increase ROS, Ca (2+), and ceramide levels in gastrointestinal tumor cells [Park et al., 2010]. In turn, Banerjee et al. reported that the anti-oxidant enzyme heme oxygenase-1 (HO-1) protected apoptosis of cells treated with sorafenib [Banerjee et al., 2012]. HO-1 was found to induce the expression of anti-apoptotic BCL-XL and decreased the expres‐ sion of autophagic proteins Beclin-1 and LC3B-II, indicating that ROS might determine the therapeutic efficacy of sorafenib. To improve the efficacy of sorafenib, further investigation of

Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in…

http://dx.doi.org/10.5772/55201

245

the relationship between ROS and sorafenib should be performed.

if caffeine could enhance the effect of sorafenib in HCC patients.

**5. Conclusions**

**4. Future perspectives of combination treatment with sorafenib**

Because accumulating evidence strongly indicated that the anti-tumor effect of sorafenib is mediated by both RAF and non-RAF signaling, recent studies have investigated the usefulness of sorafenib-based combination therapy via targeting of non-RAF signaling pathways [Peck-Radosavljevic et al., 2010, Shen et al., 2010, Kudo et al., 2010]. Some studies have reported that targeting non-RAF signaling such as TNF-related apoptosis-inducing ligand (TRAIL) [Meng et al., 2007, Rosato et al., 2007, Ricci et al., 2007, Kudo et al., 2010], histone deacetylase [Dasmahapatra et al., 2007] and BCL-2 [Lin et al., 2007] might be effective when combined with sorafenib. Moreover, several clinically available agents such as sulforaphane (SF) [Rausch et al., 2010], zoledronic acid [Zhang et al., 2010], and vitamin K1, K2, and K5 [Wei et al., 2010a, 2010b] have been also reported to be useful for combination treatment. Recently we found that caffeine, which is a well-known inhibitor of DNA damage-response kinase ataxia telangiec‐ tasia mutated (ATM), can effectively enhance the effect of sorafenib [Fujimaki et al., 2012]. ATM is a widely known DNA damage-stimulated serine/threonine kinase that phosphorylates several of the DNA damage checkpoint molecules [Lavin, 2008]. We found that ATM is activated by sorafenib-mediated non-genotoxic ROS production, resulting in the sorafenibinduced reciprocal activation of AKT signaling to help the cells to acquire drug resistance [Fujimaki et al., 2012]. Interestingly, it has been recently reported that intra-arterial local administration of caffeine could potentiate cisplatin-based chemotherapy, without severe sideeffects [Takeuchi et al., 2007]. Together with our finding, it would be intriguing to investigate

At present, sorafenib is the only molecular targeting agent proven to have a significant effect on the survival of patients with advanced HCC. Although its tumor-killing effect has been

Several studies have reported that sorafenib strongly induces ER stress. Sorafenib results in the phosphorylation of PERK and eIF2α, leading to decreases in protein synthesis [Tsukada, 1993]. ERK cannot rescue this cellular reaction, indicating that sorafenib-induced ER stress is RAF-independent [Rahmani et al., 2007]. It has been also reported that sorafenib-induced apoptosis is associated with the increase in the level of CHOP expression [Niessner et al., 2011]; therefore ER stress might be another important mechanism of sorafenib efficacy.

#### *3.1.5. Oxidative stress and sorafenib*

It is well known that reactive oxygen species (ROS) is an important player in the process of various types of cellular process. ROS is unique in its dual role; and it plays a critical role in maintaining cancer phenotype, cell proliferation and genetic instability [Radisky et al., 2005, Chen et al., 2005]. In turn, when produced at high concentrations, it activates the caspases to induce apoptosis. Although the functional mechanisms of these opposing effects of ROS is unknown, recent studies have revealed that the effect of cytotoxic anti-tumor agents is exclusively caused by elevated ROS production. Recently it was revealed that sorafenib induces mitochondria-dependent ROS production to induce hepatoma cell death. Chiou et al. reported that ROS could be generated just 30 minutes after cells were treated with sorafenib, suggesting that sorafenib-induced oxidative stress might not be the secondary phenomenon during cell death [Chiou et al., 2009]. Chiou et al. also reported that glutathione (GSH), an intracellular non-protein-thiol antioxidant, was decreased after treatment with sorafenib. Currently it is not known that why sorafenib results in the dysregulated balance of oxidants and anti-oxidants. Park et al. reported that low doses of sorafenib and vorinostat, a histone deacetylase inhibitor (HDACI) that has shown preclinical evidence of anti-tumor activity against hepatoma, rapidly increase ROS, Ca (2+), and ceramide levels in gastrointestinal tumor cells [Park et al., 2010]. In turn, Banerjee et al. reported that the anti-oxidant enzyme heme oxygenase-1 (HO-1) protected apoptosis of cells treated with sorafenib [Banerjee et al., 2012]. HO-1 was found to induce the expression of anti-apoptotic BCL-XL and decreased the expres‐ sion of autophagic proteins Beclin-1 and LC3B-II, indicating that ROS might determine the therapeutic efficacy of sorafenib. To improve the efficacy of sorafenib, further investigation of the relationship between ROS and sorafenib should be performed.

### **4. Future perspectives of combination treatment with sorafenib**

Because accumulating evidence strongly indicated that the anti-tumor effect of sorafenib is mediated by both RAF and non-RAF signaling, recent studies have investigated the usefulness of sorafenib-based combination therapy via targeting of non-RAF signaling pathways [Peck-Radosavljevic et al., 2010, Shen et al., 2010, Kudo et al., 2010]. Some studies have reported that targeting non-RAF signaling such as TNF-related apoptosis-inducing ligand (TRAIL) [Meng et al., 2007, Rosato et al., 2007, Ricci et al., 2007, Kudo et al., 2010], histone deacetylase [Dasmahapatra et al., 2007] and BCL-2 [Lin et al., 2007] might be effective when combined with sorafenib. Moreover, several clinically available agents such as sulforaphane (SF) [Rausch et al., 2010], zoledronic acid [Zhang et al., 2010], and vitamin K1, K2, and K5 [Wei et al., 2010a, 2010b] have been also reported to be useful for combination treatment. Recently we found that caffeine, which is a well-known inhibitor of DNA damage-response kinase ataxia telangiec‐ tasia mutated (ATM), can effectively enhance the effect of sorafenib [Fujimaki et al., 2012]. ATM is a widely known DNA damage-stimulated serine/threonine kinase that phosphorylates several of the DNA damage checkpoint molecules [Lavin, 2008]. We found that ATM is activated by sorafenib-mediated non-genotoxic ROS production, resulting in the sorafenibinduced reciprocal activation of AKT signaling to help the cells to acquire drug resistance [Fujimaki et al., 2012]. Interestingly, it has been recently reported that intra-arterial local administration of caffeine could potentiate cisplatin-based chemotherapy, without severe sideeffects [Takeuchi et al., 2007]. Together with our finding, it would be intriguing to investigate if caffeine could enhance the effect of sorafenib in HCC patients.

### **5. Conclusions**

*3.1.4. Endoplasmic reticulum stress and sorafenib*

244 Hepatocellular Carcinoma - Future Outlook

*3.1.5. Oxidative stress and sorafenib*

Another RAF-independent mechanism involved in sorafenib-inhibited signaling is endoplas‐ mic reticulum (ER) stress [Rahmani et al., 2007]. The endoplasmic reticulum (ER) is a central organelle in each eukaryotic cell that serves many general functions, including lipid synthesis, protein folding and protein maturation, transportation of synthesized proteins, and activation of chaperone proteins. When cells are exposed to various types of stress such as hypoxia, oxidative stress, hypoglycemia and viral infection, unfolded protein aggregates (unfolded protein response, UPR) accumulate to interfere the function of ER, which is generally called ER stress [Tsukada et al., 1993, Kim et al., 2008]. ER stress causes decreased protein translation to prevent further accumulation of unfolded proteins. It should be noted, however, that the function of ER stress is complex because it can induce either cell survival or autophagy-related cell death [Schleicher et al., 2010]. Several UPR-involving signaling molecules have been identified; the PKR-like kinase (PERK) is an important inhibitor of protein translation through phosphorylation of eukaryotic initiation factor 2 (eIF2α). The kinase activity of PERK is induced by ER stress, and phosphorylation of PERK at Thr980 is regarded as a marker for ER stress. Endoplasmic oxidoreductin-1 (Ero1) is an ER membrane-associated N-glycoprotein that provides oxidizing potential and protein folding. Inositol requiring-1 (IRE1) and activating transcription factor-6 (ATF6) induce calcium-dependent protein chaperones such as GRP78/BiP to maintain correct protein folding [Kim et al., 2008, McConkey et al., 2008]. Calnexin is a calcium-binding protein that retains the synthesized glycoproteins inside the ER. CAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a dominant-negative inhibitor of C/EBP and LAP, which plays a role in cell cycle arrest during G1 to S phase. During ER stress, the level of CHOP is increased to induce the activation of GADD34, a downstream protein of P53 tumor suppressor that causes DNA excision repair and cell arrest, and ERO-1 expression. ERO-1 promotes oxidative stress inside the ER, leading to programmed cell death.

Several studies have reported that sorafenib strongly induces ER stress. Sorafenib results in the phosphorylation of PERK and eIF2α, leading to decreases in protein synthesis [Tsukada, 1993]. ERK cannot rescue this cellular reaction, indicating that sorafenib-induced ER stress is RAF-independent [Rahmani et al., 2007]. It has been also reported that sorafenib-induced apoptosis is associated with the increase in the level of CHOP expression [Niessner et al., 2011]; therefore ER stress might be another important mechanism of sorafenib efficacy.

It is well known that reactive oxygen species (ROS) is an important player in the process of various types of cellular process. ROS is unique in its dual role; and it plays a critical role in maintaining cancer phenotype, cell proliferation and genetic instability [Radisky et al., 2005, Chen et al., 2005]. In turn, when produced at high concentrations, it activates the caspases to induce apoptosis. Although the functional mechanisms of these opposing effects of ROS is unknown, recent studies have revealed that the effect of cytotoxic anti-tumor agents is exclusively caused by elevated ROS production. Recently it was revealed that sorafenib induces mitochondria-dependent ROS production to induce hepatoma cell death. Chiou et al. reported that ROS could be generated just 30 minutes after cells were treated with sorafenib,

At present, sorafenib is the only molecular targeting agent proven to have a significant effect on the survival of patients with advanced HCC. Although its tumor-killing effect has been found to be limited, recent basic studies have revealed that this new agent acts upon multiple non-RAF signaling pathways as well as RAF-mediated signaling. More interestingly, recent studies have unveiled that sorafenib might also act as preventive agents against liver fibrosis. Wang et al. reported that sorafenib treatment attenuated liver fibrosis in rat liver fibrosis model, possibly due to its inhibitory role on the cell proliferation of hepatic satellite cells [Wang at al., 2010]. Thus, to further identify an efficient protocol for sorafenib treatment, clinicians should pay more attention to non-RAF signaling in cancer cells.

[5] Ashkenazi, A. (2008). Directing cancer cells to self-destruct with pro-apoptotic recep‐

http://dx.doi.org/10.5772/55201

247

Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in…

[6] Banerjee, P, Basu, A, Wegiel, B, Otterbein, L. E, Mizumura, K, Gasser, M, Waagagasser, A. M, Choi, A. M, & Pal, S. (2012). Heme oxygenase-1 promotes survival of renal cancer cells through modulation of apoptosis- & autophagy-regulating mole‐

[7] Becker, G, Allgaier, H. P, Olschewski, M, Zähringer, A, & Blum, H. E. HECTOR Study Group. ((2007). Long-acting octreotide versus placebo for treatment of ad‐ vanced HCC: a randomized controlled double-blind study. *Hepatology*, , 45, 9-15. [8] Braconi, C, Valeri, N, Gasparini, P, Huang, N, Taccioli, C, Nuovo, G, Suzuki, T, Croce, C. M, & Patel, T. (2010). Hepatitis C virus proteins modulate microRNA ex‐ pression and chemosensitivity in malignant hepatocytes. *Clin Cancer Res,* , 16,

[9] Bruix, J, & Sherman, M. (2005). Management of hepatocellular carcinoma. Hepatolo‐

[10] Buchholz, B, Klanke, B, Schley, G, Bollag, G, Tsai, J, Kroening, S, Yoshihara, D, Wal‐ lace, D. P, Kraenzlin, B, Gretz, N, Hirth, P, Eckardt, K. U, & Bernhardt, W. M. (2011). The Raf kinase inhibitor PLX5568 slows cyst proliferation in rat polycystic kidney disease but promotes renal and hepatic fibrosis. *Nephrol Dial Transplant, 26* ,

[11] Bürckstümmer, T, Kriegs, M, Lupberger, J, Pauli, E. K, Schmittel, S, & Hildt, E. (2006). Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication.

[12] Challen, C, Guo, K, Collier, J. D, Cavanagh, D, & Bassendine, M. F. (1992). Infrequent point mutations in codons 12 and 61 of ras oncogenes in human hepatocellular carci‐

[13] Chen, J, & Siddiqui, A. (2007). Hepatitis B virus X protein stimulates the mitochon‐

[14] Chen, Z, Trotman, L. C, Shaffer, D, Lin, H. K, Dotan, Z. A, Niki, M, Koutcher, J. A, Scher, H. I, Ludwig, T, Gerald, W, Cordon-cardo, C, & Pandolfi, P. P. (2005). Crucial role of cellular senescence in suppression of Pten-deficient tumorigenesis. *Nature*,

[15] Cheng, A. L, Kang, Y. K, Chen, Z, Tsao, C. J, Qin, S, Kim, J. S, Luo, R, Feng, J, Ye, S, Yang, T. S, Xu, J, Sun, Y, Liang, H, Liu, J, Wang, J, Tak, W. Y, Pan, H, Burock, K, Zou, J, Voliotis, D, & Guan, Z. (2009). Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised,

[16] Chiou, J. F, Tai, C. J, Wang, Y. H, Liu, T. Z, Jen, Y. M, & Shiau, C. Y. (2009). Sorafenib induces preferential apoptotic killing of a drug- and radio-resistant Hep G2 cells

drial translocation of Raf-1 via oxidative stress. *J Virol*, , 81, 6757-6760.

double-blind, placebo-controlled trial. *Lancet Oncol*, , 10, 25-34.

tor agonists. *Nat Rev Drug Discov*, , 7, 1001-1012.

cules. *J Biol Chem,* , 287, 32113-32123.

957-966.

3458-3465.

gy , 42, 1208-1236.

*FEBS Lett*, , 580, 575-580.

436: 725-730, 53.

nomas. *J Hepatol*, , 14, 342-346.

### **Author details**

Yasunobu Matsuda1\*, Toshifumi Wakai2 , Masayuki Kubota3 , Mami Osawa3 and Shun Fujimaki1

\*Address all correspondence to: yasunobu@med.niigata-u.ac.jp

1 Department of Medical Technology, Niigata University Graduate School of Health Scien‐ ces, Japan

2 Division of Digestive and General Surgery, Niigata University Graduate School of Medical and Dental Sciences, Japan

3 Division of Pediatric Surgery, Niigata University Graduate School of Medical and Dental Sciences, Japan

### **References**


[5] Ashkenazi, A. (2008). Directing cancer cells to self-destruct with pro-apoptotic recep‐ tor agonists. *Nat Rev Drug Discov*, , 7, 1001-1012.

found to be limited, recent basic studies have revealed that this new agent acts upon multiple non-RAF signaling pathways as well as RAF-mediated signaling. More interestingly, recent studies have unveiled that sorafenib might also act as preventive agents against liver fibrosis. Wang et al. reported that sorafenib treatment attenuated liver fibrosis in rat liver fibrosis model, possibly due to its inhibitory role on the cell proliferation of hepatic satellite cells [Wang at al., 2010]. Thus, to further identify an efficient protocol for sorafenib treatment, clinicians

, Masayuki Kubota3

1 Department of Medical Technology, Niigata University Graduate School of Health Scien‐

2 Division of Digestive and General Surgery, Niigata University Graduate School of Medical

3 Division of Pediatric Surgery, Niigata University Graduate School of Medical and Dental

[1] Abou-alfa, G. K, Johnson, P, Knox, J. J, Capanu, M, Davidenko, I, Lacava, J, Leung, T, Gansukh, B, & Saltz, L. B. (2010). Doxorubicin plus sorafenib vs doxorubicin alone in patients with advanced hepatocellular carcinoma: a randomized trial. *JAMA*, , 304,

[2] Akgul, C. (2009). Mcl-1 is a potential therapeutic target in multiple types of cancer.

[3] Aoki, H, Hayashi, J, Moriyama, M, Arakawa, Y, & Hino, O. (2000). Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1. *J Virol*, , 74,

[4] Arii, S, Yamaoka, Y, Futagawa, S, Inoue, K, Kobayashi, K, Kojiro, M, Makuuchi, M, Nakamura, Y, Okita, K, & Yamada, R. (2000). Results of surgical and nonsurgical treatment for small-sized hepatocellular carcinomas: a retrospective and nationwide survey in Japan. The Liver Cancer Study Group of Japan. *Hepatology,* , 32, 1224-1229.

, Mami Osawa3

and

should pay more attention to non-RAF signaling in cancer cells.

\*Address all correspondence to: yasunobu@med.niigata-u.ac.jp

**Author details**

246 Hepatocellular Carcinoma - Future Outlook

Shun Fujimaki1

ces, Japan

Sciences, Japan

**References**

and Dental Sciences, Japan

2154-2160.

1736-1741.

*Cell Mol Life Sci, 66* , 1326-1336.

Yasunobu Matsuda1\*, Toshifumi Wakai2


through a mitochondria-dependent oxidative stress mechanism. *Cancer Biol Ther*, , 8, 1904-1913.

[28] Karnoub, A. E, & Weinberg, R. A. (2008). Ras oncogenes: split personalities. *Nat Rev*

Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in…

http://dx.doi.org/10.5772/55201

249

[29] Kelley, R. K, Nimeiri, H. S, Vergo, M. T, Bergsland, E. K, Ko, A. H, Munster, P. N, Reinert, A, Mulcahy, M. F, Benson, A. B, & Venook, A. P. A Phase I trial of the combi‐ nation of temsirolimus (TEM) and sorafenib (SOR) in advanced hepatocellular carci‐

[30] Kerkhoff, E, & Rapp, U. R. (1998). Cell cycle targets of Ras/Raf signaling. *Oncogene*, ,

[31] Kim, I, Xu, W, & Reed, J. C. (2008). Cell death and endoplasmic reticulum stress: dis‐ ease relevance and therapeutic opportunities. *Nat Rev Drug Discov*, , 7, 1013-1030. [32] Kudo, M, & Ueshima, K. (2010). Positioning of a molecular-targeted agent, sorafenib, in the treatment algorithm for hepatocellular carcinoma and implication of many

[33] Kuwahara, Y, Li, L, Baba, T, Nakagawa, H, Shimura, T, Yamamoto, Y, Ohkubo, Y, & Fukumoto, M. (2009). Clinically relevant radioresistant cells efficiently repair DNA

[34] Lavin, M. F. (2008). Ataxia-telangiectasia: from a rare disorder to a paradigm for cell

[35] Leber, B, Geng, F, Kale, J, & Andrews, D. W. (2010). Drugs targeting Bcl-2 family

[36] Lin, X, Morgan-lappe, S, Huang, X, Li, L, Zakula, D. M, Vernetti, L. A, Fesik, S. W, & Shen, Y. (2007). Seed' analysis of off-target siRNAs reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-XL inhibitor ABT-737. *Oncogene*, , 26,

[37] Llovet, J. M, Burroughs, A, & Bruix, J. (2003). Hepatocellular carcinoma. Lancet, , 362,

[38] Llovet, J. M, Ricci, S, Mazzaferro, V, Hilgard, P, Gane, E, Blanc, J. F, De Oliveira, A. C, Santoro, A, Raoul, J. L, Forner, A, Schwartz, M, Porta, C, Zeuzem, S, Bolondi, L, Gret‐ en, T. F, Galle, P. R, Seitz, J. F, Borbath, I, Häussinger, D, Giannaris, T, Shan, M, Mos‐ covici, M, Voliotis, D, & Bruix, J. SHARP Investigators Study Group ((2008).

Sorafenib in advanced hepatocellular carcinoma. *N Engl J Med*, , 359, 378-390.

[39] Matsuda, Y, & Fukumoto, M. (2011). Sorafenib: complexities of Raf-dependent and Raf-independent signaling are now unveiled. *Med Mol Morphol*, , 44, 183-189.

[40] Mcconkey, D. J, & Zhu, K. (2008). Mechanisms of proteasome inhibitor action and re‐

[41] Meng, X. W, Lee, S. H, Dai, H, Loegering, D, Yu, C, Flatten, K, Schneider, P, Dai, N. T, Kumar, S. K, Smith, B. D, Karp, J. E, Adjei, A. A, & Kaufmann, S. H. (2007). Mcl-1

members as an emerging strategy in cancer. *Expert Rev Mol Med*, 12: e28

complete remission cases in Japan. *Oncology*, 78 Suppl , 1, 154-166.

double-strand breaks induced by X-rays. *Cancer Sci,* , 100, 747-752.

signalling and cancer. *Nat Rev Mol Cell Biol,* , 9, 759-669.

sistance in cancer. *Drug Resist Updat*, , 11, 164-179.

*Mol Cell Biol*, , 9, 517-531.

17, 1457-1462.

3972-3979.

1907-191.

noma (HCC). *J Clin Oncol,* 28 (suppl.):TPS213


[28] Karnoub, A. E, & Weinberg, R. A. (2008). Ras oncogenes: split personalities. *Nat Rev Mol Cell Biol*, , 9, 517-531.

through a mitochondria-dependent oxidative stress mechanism. *Cancer Biol Ther*, , 8,

[17] Dasmahapatra, G, Yerram, N, Dai, Y, Dent, P, & Grant, S. (2007). Synergistic interac‐ tions between vorinostat and sorafenib in chronic myelogenous leukemia cells in‐

[18] El Serag, H. B, & Rudolph, K. L. (2007). Hepatocellular carcinoma: epidemiology and

[19] Fujimaki, S, Matsuda, Y, Wakai, T, Sanpei, A, Kubota, M, Takamura, M, Yamagiwa, S, Yano, M, Ohkoshi, S, & Aoyagi, Y. (2012). Blockade of ataxia telangiectasia mutat‐ ed sensitizes hepatoma cell lines to sorafenib by interfering with Akt signaling. *Can‐*

[20] Garnett, M. J, Rana, S, Paterson, H, Barford, D, & Marais, R. (2005). Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimeri‐

[21] Heidorn, S. J, Milagre, C, Whittaker, S, Nourry, A, Niculescu-duvas, I, Dhomen, N, Hussain, J, Reis-filho, J. S, Springer, C. J, Pritchard, C, & Marais, R. (2010). Kinasedead BRAF and oncogenic RAS cooperate to drive tumor progression through

[22] Herrmann, C, Martin, G. A, & Wittinghofer, A. (1995). Quantitative analysis of the complex between and the Ras-binding domain of the human Raf-1 protein kinase. *J*

[23] Hindley, A, & Kolch, W. (2002). Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases. *J Cell Sci*, ,

[24] Hochhaus, A, Rosée, P. L, Müller, M. C, Ernst, T, & Cross, N. C. (2011). Impact of BCR-ABL mutations on patients with chronic myeloid leukemia. *Cell Cycle, 10* ,

[25] Huynh, H, Ngo, V. C, Koong, H. N, Poon, D, Choo, S. P, Thng, C. H, Chow, P, Ong, H. S, Chung, A, & Soo, K. C. (2009). Sorafenib and rapamycin induce growth sup‐ pression in mouse models of hepatocellular carcinoma. *J Cell Mol Med*, , 13,

[26] Hwang, Y. H, Choi, J. Y, Kim, S, Chung, E. S, Kim, T, Koh, S. S, Lee, B, Bae, S. H, Kim, J, & Park, Y. M. (2004). Over-expression of c-raf-1 proto-oncogene in liver cirrhosis

[27] Jubb, A. M, & Harris, A. L. (2010). Biomarkers to predict the clinical efficacy of beva‐

and hepatocellular carcinoma. *Hepatol Res,* , 29, 113-121.

cizumab in cancer. *Lancet Oncol, 11* , 1172-1183.

volve Mcl-1 and down-regulation. *Clin Cancer Res*, 13: 4280-4290, 21CIP1.

molecular carcinogenesis. *Gastroenterology*, , 132, 2557-2576.

1904-1913.

248 Hepatocellular Carcinoma - Future Outlook

*cer Lett*, , 319, 98-108.

zation. *Mol Cell*, , 20, 963-969.

CRAF. *Cell*, , 140, 209-221.

115, 1575-1581.

250-260.

2673-2683.

*Biol Chem*, 270: :2901-2905, 21ras.


as a buffer for proapoptotic Bcl-2 family members during TRAIL-induced apoptosis: a mechanistic basis for sorafenib (Bay 43-9006)-induced TRAIL sensitization. *J Biol Chem,* , 282, 29831-29846.

[50] Rahmani, M, Davis, E. M, Crabtree, T. R, Habibi, J. R, Nguyen, T. K, Dent, P, & Grant, S. (2007). The kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress. *Mol Cell Biol*, , 27, 5499-5513. [51] Rausch, V, Liu, L, Kallifatidis, G, Baumann, B, Mattern, J, Gladkich, J, Wirth, T, Schemmer, P, Büchler, M. W, Zöller, M, Salnikov, A. V, & Herr, I. (2010). Synergistic activity of sorafenib and sulforaphane abolishes pancreatic cancer stem cell charac‐

Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in…

http://dx.doi.org/10.5772/55201

251

[52] Ricci, M. S, Kim, S. H, Ogi, K, Plastaras, J. P, Ling, J, Wang, W, Jin, Z, Liu, Y. Y, Dick‐ er, D. T, Chiao, P. J, Flaherty, K. T, Smith, C. D, & Deiry, W. S. (2007). Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human

[53] Rosato, R. R, Almenara, J. A, Coe, S, & Grant, S. (2007). The multikinase inhibitor sor‐ afenib potentiates TRAIL lethality in human leukemia cells in association with Mcl-1

[54] Rushworth, L. K, Hindley, A. D, Neill, O, Kolch, E, & , W. (2006). Regulation and role

[55] Sacco, R, Bargellini, I, Giannelli, G, Bertini, M, Bozzi, E, Altomare, E, Battaglia, V, Ro‐ mano, A, Bertoni, M, Capria, A, Bresci, G, & Bartolozzi, C. (2011). Complete response for advanced liver cancer during sorafenib therapy: Case Report. *BMC Gastroenterol,*

[56] Schleicher, S. M, Moretti, L, Varki, V, & Lu, B. (2010). Progress in the unraveling of the endoplasmic reticulum stress/autophagy pathway and cancer: implications for

[57] Schütte, K, Zimmermann, L, Bornschein, J, Csepregi, A, Rühl, R, Ricke, J, & Malfer‐ theiner, P. (2011). Sorafenib Therapy in Patients with Advanced Hepatocellular Car‐

[58] Shen, Y. C, Hsu, C, & Cheng, A. L. (2010). Molecular targeted therapy for advanced hepatocellular carcinoma: current status and future perspectives. *J Gastroenterol,* , 45,

[59] So, B. J, Bekaii-saab, T, Bloomston, M. A, & Patel, T. (2008). Complete clinical re‐ sponse of metastatic hepatocellular carcinoma to sorafenib in a patient with hemo‐

[60] Takayasu, K, Arii, S, Ikai, I, Omata, M, Okita, K, Ichida, T, Matsuyama, Y, Nakanu‐ ma, Y, Kojiro, M, Makuuchi, M, & Yamaoka, Y. (2006). Liver Cancer Study Group of Japan. Prospective cohort study of transarterial chemoembolization for unresectable

[61] Takeuchi, A, Tsuchiya, H, Yamamoto, N, Hayashi, K, Yamauchi, K, Kawahara, M, Miyamoto, K, & Tomita, K. (2007). Caffeine-potentiated chemotherapy for patients

hepatocellular carcinoma in 8510 patients. *Gastroenterology*, , 131, 461-469.

cancer cells to TRAIL-induced death. *Cancer Cell*, , 12, 66-80.

and cFLIPL down-regulation. *Cancer Res*, , 67, 9490-9500.

of Raf-1/B-Raf heterodimerization. *Mol Cell Biol*, , 26, 2262-2272.

future therapeutic approaches. *Drug Resist Updat,* , 13, 79-86.

cinoma in Advanced Liver Cirrhosis. *Digestion,* , 83, 275-282.

chromatosis: a case report. *J Hematol Oncol*, 1:18

teristics. *Cancer Res*, , 70, 5004-5013.

11: 4

794-807.


[50] Rahmani, M, Davis, E. M, Crabtree, T. R, Habibi, J. R, Nguyen, T. K, Dent, P, & Grant, S. (2007). The kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress. *Mol Cell Biol*, , 27, 5499-5513.

as a buffer for proapoptotic Bcl-2 family members during TRAIL-induced apoptosis: a mechanistic basis for sorafenib (Bay 43-9006)-induced TRAIL sensitization. *J Biol*

[42] Newell, P, Toffanin, S, Villanueva, A, Chiang, D. Y, Minguez, B, Cabellos, L, Savic, R, Hoshida, Y, Lim, K. H, Melgar-lesmes, P, Yea, S, Peix, J, Deniz, K, Fiel, M. I, Thung, S, Alsinet, C, Tovar, V, Mazzaferro, V, Bruix, J, Roayaie, S, Schwartz, M, Friedman, S. L, & Llovet, J. M. (2009). Ras pathway activation in hepatocellular carcinoma and antitumoral effect of combined sorafenib and rapamycin in vivo. *J Hepatol,* , 51, 725-733.

[43] Niessner, H, Beck, D, Sinnberg, T, Lasithiotakis, K, Maczey, E, Gogel, J, Venturelli, S, Berger, A, Mauthe, M, Toulany, M, Flaherty, K, Schaller, M, Schadendorf, D, Proikascezanne, T, Schittek, B, Garbe, C, Kulms, D, & Meier, F. (2011). The farnesyl transfer‐ ase inhibitor lonafarnib inhibits mTOR signaling and enforces sorafenib-induced

[44] Park, M. A, Mitchell, C, Zhang, G, Yacoub, A, Allegood, J, Häussinger, D, Reinehr, R, Larner, A, Spiegel, S, Fisher, P. B, Voelkel-johnson, C, Ogretmen, B, Grant, S, & Dent, P. (2010). Vorinostat and sorafenib increase CD95 activation in gastrointestinal tumor cells through a Ca(2+)-de novo ceramide-oxygen species-dependent signaling path‐

[45] Peck-radosavljevic, M, Greten, T. F, Lammer, J, Rosmorduc, O, Sangro, B, Santoro, A, & Bolondi, L. (2010). Consensus on the current use of sorafenib for the treatment of

[46] Pinter, M, Sieghart, W, Graziadei, I, Vogel, W, Maieron, A, Königsberg, R, Weiss‐ mann, A, Kornek, G, Plank, C, & Peck-radosavljevic, M. (2009). Sorafenib in unresect‐ able hepatocellular carcinoma from mild to advanced stage liver cirrhosis.

[47] Prete, S. D, Montella, L, Caraglia, M, Maiorino, L, Cennamo, G, Montesarchio, V, Piai, G, Febbraro, A, Tarantino, L, Capasso, E, Palmieri, G, Guarrasi, R, Bianco, M, Mamone, R, Savastano, C, Pisano, A, Vincenzi, B, Sabia, A, Agostino, D, Faiola, A, & Addeo, V. R. ((2010). Sorafenib plus octreotide is an effective and safe treatment in advanced hepatocellular carcinoma: multicenter phase II So.LAR. study. *Cancer Che‐*

[48] Radisky, D. C, Levy, D. D, Littlepage, L. E, Liu, H, Nelson, C. M, Fata, J. E, Leake, D, Godden, E. L, Albertson, D. G, Nieto, M. A, Werb, Z, & Bissell, M. J. Rac1b and reac‐ tive oxygen species mediate MMP-3-induced EMT and genomic instability. *Nature*, ,

[49] Rahmani, M, Davis, E. M, Bauer, C, Dent, P, & Grant, S. (2005). Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation

of Mcl-1 through inhibition of translation. *J Biol Chem*, , 280, 35217-35227.

apoptosis in melanoma cells. *J Invest Dermatol*, , 131, 468-479.

hepatocellular carcinoma. *Eur J Gastroenterol Hepatol*, , 22, 391-398.

*Chem,* , 282, 29831-29846.

250 Hepatocellular Carcinoma - Future Outlook

way. *Cancer Res*, 70: 6313-6324, 2A.

*Oncologist,* , 14, 70-76.

436, 123-127.

*mother Pharmacol*, , 66, 837-844.


with high-grade soft tissue sarcoma: long-term clinical outcome. *Anticancer Res*, , 27, 3489-3495.

[73] Wilhelm, S. M, Carter, C, Tang, L, Wilkie, D, Mcnabola, A, Rong, H, Chen, C, Zhang, X, Vincent, P, Mchugh, M, Cao, Y, Shujath, J, Gawlak, S, Eveleigh, D, Rowley, B, Liu, L, Adnane, L, Lynch, M, Auclair, D, Taylor, I, Gedrich, R, Voznesensky, A, Riedl, B, Post, L. E, Bollag, G, & Trail, P. A. (2004). BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. *Cancer Res, 64* , 7099-7109.

Sorafenib-Inhibited Signaling: Emerging Evidence of RAF-Independent Pathways as Potential Therapeutic Targets in…

http://dx.doi.org/10.5772/55201

253

[74] Yamaguchi, T, Reif, G. A, Calvet, J. P, & Wallace, D. P. (2010). Sorafenib inhibits cAMP-dependent ERK activation, cell proliferation, and in vitro cyst growth of hu‐

[75] Yang, J. D, & Roberts, L. R. (2010). Hepatocellular carcinoma: A global view. *Nat Rev*

[76] Yeganeh, M, Finn, R. S, & Saab, S. (2009). Apparent remission of a solitary metastatic pulmonary lesion in a liver transplant recipient treated with sorafenib. *Am J Trans‐*

[77] Yu, C, Bruzek, L. M, Meng, X. W, Gores, G. J, Carter, C. A, Kaufmann, S. H, & Adjei, A. A. (2005). The role of Mcl-1 downregulation in the proapoptotic activity of the

[78] Zhang, W, Zhu, X. D, Sun, H. C, Xiong, Y. Q, Zhuang, P. Y, Xu, H. X, Kong, L. Q, Wang, L, Wu, W. Z, & Tang, Z. Y. (2010). Depletion of tumor-associated macrophag‐ es enhances the effect of sorafenib in metastatic liver cancer models by antimetastatic

multikinase inhibitor BAY 43-9006. *Oncogene*, , 24, 6861-6869.

and antiangiogenic effects. *Clin Cancer Res,* , 16, 3420-3430.

man ADPKD cyst epithelial cells. *Am J Physiol Renal Physiol,* 299: FF951, 944.

*Gastroenterol Hepatol*, , 7, 448-458.

*plant,* , 9, 2851-2854.


[73] Wilhelm, S. M, Carter, C, Tang, L, Wilkie, D, Mcnabola, A, Rong, H, Chen, C, Zhang, X, Vincent, P, Mchugh, M, Cao, Y, Shujath, J, Gawlak, S, Eveleigh, D, Rowley, B, Liu, L, Adnane, L, Lynch, M, Auclair, D, Taylor, I, Gedrich, R, Voznesensky, A, Riedl, B, Post, L. E, Bollag, G, & Trail, P. A. (2004). BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. *Cancer Res, 64* , 7099-7109.

with high-grade soft tissue sarcoma: long-term clinical outcome. *Anticancer Res*, , 27,

[62] Takezawa, K, Okamoto, I, Yonesaka, K, Hatashita, E, Yamada, Y, Fukuoka, M, & Na‐ kagawa, K. (2009). Sorafenib inhibits non-small cell lung cancer cell growth by target‐ ing B-RAF in KRAS wild-type cells and C-RAF in KRAS mutant cells. *Cancer Res*, , 69,

[63] Tang, Z. Y. (2008). Effect of rapamycin alone and in combination with sorafenib in an orthotopic model of human hepatocellular carcinoma. *Clin Cancer Res, 14* , 5124-5130.

[64] Thomas, L. W, Lam, C, & Edwards, S. W. (2010). Mcl-1; the molecular regulation of

[65] Tsukada, M, & Oshumi, Y. (1993). Isolation and characterization of autophagy-defec‐

[66] Ulivi, P, Arienti, C, Amadori, D, Fabbri, F, Carloni, S, Tesei, A, Vannini, I, Silvestrini, R, & Zoli, W. (2009). Role of RAF/MEK/ERK pathway, p-STAT-3 and Mcl-1 in sorafe‐

[67] Villanueva, A, Chiang, D. Y, Newell, P, Peix, J, Thung, S, Alsinet, C, Tovar, V, Roayaie, S, Minguez, B, Sole, M, Battiston, C, Van Laarhoven, S, & Fiel, M. I. Di Feo, A., Hoshida, Y., Yea, S., Toffanin, S., Ramos, A., Martignetti, JA., Mazzaferro, V., Bruix, J., Waxman, S., Schwartz, M., Meyerson, M., Friedman, SL., Llovet, JM. ((2008). Pivotal role of mTOR signaling in hepatocellular carcinoma. *Gastroenterology*, , 135,

[68] Wan, P. T, Garnett, M. J, Roe, S. M, Lee, S, Niculescu-duvaz, D, Good, V. M, Jones, C. M, Marshall, C. J, Springer, C. J, Barford, D, & Marais, R. (2004). Mechanism of acti‐ vation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. *Cell*, ,

[69] Wang, S. X, Byrnes, A, Verma, S, Pancoast, J. R, & Rixe, O. (2010). Complete remis‐ sion of unresectable hepatocellular carcinoma treated with reduced dose of sorafe‐

[70] Wang, Y, Gao, J, Zhang, D, Zhang, J, Ma, J, & Jiang, H. (2010). New insights into the antifibrotic effects of sorafenib on hepatic stellate cells and liver fibrosis. *J Hepatol,* ,

[71] Wei, G, Wang, M, & Carr, B. I. (2010a). Sorafenib combined vitamin K induces apop‐ tosis in human pancreatic cancer cell lines through RAF/MEK/ERK and c-Jun NH2-

[72] Wei, G, Wang, M, Hyslop, T, Wang, Z, & Carr, B. I. (2010b). Vitamin K enhancement of sorafenib-mediated HCC cell growth inhibition in vitro and in vivo. Int J Cancer, ,

nib activity in human pancreatic cancer cell lines. *J Cell Physiol,220* , 214-221.

tive mutants of Saccharomyces cerevisiae. *FEBS Lett,* , 333, 169-174.

protein function. *FEBS Lett*, , 584, 2981-2989.

nib: a case report. *Target Oncol,* , 5, 59-63.

terminal kinase pathways. *J Cell Physiol*, , 224, 112-119.

3489-3495.

252 Hepatocellular Carcinoma - Future Outlook

6515-6521.

1972-1983.

116, 855-867.

53, 132-144.

127, 2949-295.


**Chapter 13**

**Insulin-Like Growth Factor-II: Molecular-Targeted**

Hepatocellular carcinoma (HCC) is the third deadliest and 5th most common cancer world‐ wide. It ranks the second in China among all malignancies, and its mortality is almost equal to its morbidity [1-3]. Carcinogenesis of HCC is a multi-factor, multi-step and complex process, which is associated with a background of chronic and persistent infection of hepatitis B virus (HBV) or hepatitis C virus (HCV) [4-6]. Their infections along with alcohol and aflatoxin B1 intake are widely recognized etiological agents in HCC [7, 8]. Multiple genetic alterations, including the activation of oncogenes and inactivation of tumor suppressor genes, are required for malignancy in human cancers and are correlated with increased stages of carcinogenesis and further tumor progression with many characteristics, such as fast infiltrating growth, metastasis in early stage, high-grade malignancy, and poor therapeutic efficacy [9-11]. HCC prognoses are poor, and early detection is of the utmost importance [12, 13]. Most of HCC patients died quickly because of the rapid tumor progression, and hepatic resection or transplantation is the only potential curative treatment for HCC. Treatment options are severely limited by the frequent presence of metastases. Therefore, it is the 3rd leading cause of cancer-induced death worldwide with a very poor prognosis [14]. Growing understanding of the molecular mechanisms underlying the carcinogenesis of HCC is a multi-factor, multistep, and complex process, involving chromosomal aberrations, gene mutations, epigenetic

Recently, studies have discovered changes in the insulin-like growth factor (IGF) axis that affect the molecular pathogenesis of HCC, and IGF-II is a polypeptide hormone secreted by many organs of the fetus [17, 18]. IGF axis (Figure.1) has emerged as an important pathway in the development and progression of HCC and as a potential therapeutic target. Human IGF-

> © 2013 Yao 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.

**Therapy for Hepatocellular Carcinoma**

Min Yao, Li Wang, Meijuan Yan, Xiaodi Yan and

alterations, and activation of complex signaling pathways [15, 16].

Additional information is available at the end of the chapter

Dengfu Yao

**1. Introduction**

http://dx.doi.org/10.5772/55418
