**3.2.2 Up-regulated gene 11 (URG11) and hepatocarcinogenesis**

Another transcript identified by subtractive hybridization in HBx positive compared to negative HepG2 cells encoded a novel protein provisionally designated as URG11 (Lian et al., 2003). The protein product was about 70kDa (673 amino acids) in size and contained five von Willebrand factor type-C repeats and one C-type lectin domain. Functional characterization showed that over-expression of URG11 significantly stimulated cell growth in culture, anchorage-independent growth in soft agar, accelerated tumor formation, and yielded larger tumors in SCID mice injected subcutaneously with HepG2 cells. Further work showed that HBx *trans*-activated URG11, and that URG11 *trans*-activated the -catenin promoter. URG11 specific siRNA inhibited the growth of HBx expressing liver cells in serum free medium. The latter was associated with depressed levels of -catenin. As

Fig. 2. Model showing selected steps of how HBx inhibits apoptosis and promotes tumorigenesis. HBx alters pathways involving URG7 (in purple), URG11 (in green), and ErbB-2 (in red). See the text for additional details.

Additionally, the observation that HBx activates the expression of Fas ligand in HCC cell lines (Shin et al., 1999), may provide a way for virus infected cells to escape direct T cell killing by inducing apoptosis in such T cells. This would not only promote chronicity, but

Another transcript identified by subtractive hybridization in HBx positive compared to negative HepG2 cells encoded a novel protein provisionally designated as URG11 (Lian et al., 2003). The protein product was about 70kDa (673 amino acids) in size and contained five von Willebrand factor type-C repeats and one C-type lectin domain. Functional characterization showed that over-expression of URG11 significantly stimulated cell growth in culture, anchorage-independent growth in soft agar, accelerated tumor formation, and yielded larger tumors in SCID mice injected subcutaneously with HepG2 cells. Further work showed that HBx *trans*-activated URG11, and that URG11 *trans*-activated the -catenin promoter. URG11 specific siRNA inhibited the growth of HBx expressing liver cells in serum free medium. The latter was associated with depressed levels of -catenin. As

Fig. 2. Model showing selected steps of how HBx inhibits apoptosis and promotes tumorigenesis. HBx alters pathways involving URG7 (in purple), URG11 (in green), and

ErbB-2 (in red). See the text for additional details.

in tumor cells, an escape from immune elimination.

**3.2.2 Up-regulated gene 11 (URG11) and hepatocarcinogenesis** 

with URG7, there was extensive co-staining between HBx and URG11 in chronically infected liver (Lian et al., 2006) but not in tumor. This suggests that URG11 promotes hepatocellular growth prior to the appearance of HCC. The ability of URG11 specific siRNA to block the growth of liver tumor cells both *in vitro* and *in vivo*, not only underscores the importance of elevated URG11 to cell growth, but also suggests that it may be a novel target for the development of specific therapeutics against HCC (Fan et al., 2011). Independent work has recently shown that URG11 was induced under hypoxic conditions in human kidney tubule cells (Du et al., 2010). The latter was associated with increased levels of HIF-1, which is also known to be a target of HBx (Holotnakova et al., 2010). Importantly, HIF-1 is known to *trans*-activate VEGF *in vivo* (Yoo et al., 2003), suggesting that neovascularization may occur in cirrhotic nodules prior to the appearance of HCC. If this occurs during the pathogenesis of chronic hepatitis B, it would most likely be observed in cirrhotic nodules, since this represents a hypoxic environment characterized by high levels of HBx expression (Wang et al., 1991a, 1991b). Interestingly, elevated expression of URG11 in kidney tubule cells was also associated with suppression of E-cadherin, and upregulation of the mesenchymal markers vimentin and alpha-SMA, suggesting that URG11 is associated with EMT. In chronic HBV infection, the development of cirrhosis is accompanied by considerable alterations in the tissue architecture within the liver, implying that URG11 may also play a significant role in tissue remodeling during the pathogenesis of chronic infection.

#### **3.2.3 Elevated vascular endothelial growth factor receptor 3 (VEGFR-3)**

Vascular endothelial growth factor receptor 3 (VEGFR-3), which is associated with angiogenesis, is a receptor tyrosine kinase that is expressed in lymphatic endothelial cells (Iljin et al., 2001). Binding of VEGFR-3 to the ligands VEGF-C or VEGF-D stimulate lymphangiogenesis (Alitalo & Carmeliet, 2002), while in carcinogenesis, the production of VEGFs by tumors promote metastases and result in decreased survival (Su et al., 2006). Elevated VEGF has been found in patients with HCC (Dahr et al., 2002, Poon et al., 2003). VEGFR-3 is also expressed in tumor cells from several tumor types (Bando et al., 2004, Su et al., 2006), including HCC (Dahr et al., 2002), implying the existence of an autocrine/paracrine loop that promotes tumor development independent of lymphangiogenesis (Su et al., 2006). In HCC, elevated VEGFR-3 is associated with portal vein invasion of tumors, increased hepatic tumor recurrence, and shorter survival (Dhar et al., 2002), suggesting that VEGFR-3 is important in the pathogenesis of HCC. In this context, differential display of HBx positive compared to negative cells showed that HBx upregulated the expression of an mRNA which encoded a splice variant of VEGFR-3 (Lian et al., 1997). This was verified at the mRNA and protein levels in HBx positive compared to negative HepG2 cells. In infected liver, expression of VEGFR-3 was prominent in nodules of HCC and correlated with HBx expression. VEGFR-3 stimulated cell cycle in culture, anchorage independent growth in soft agar, and accelerated tumor formation and larger tumor size in SCID mice injected with HepG2 cells over-expressing VEGFR-3. Further work showed that over-expression of VEGFR-3 in the absence of HBx resulted in activation of PI3K/Akt, which then activated -catenin gene expression (Figure 2), and with inactivation of the tumor suppressor, PTEN. Interestingly, HBx also mediates these changes, suggesting that they may be actually carried out by up-regulation of VEGFR-3. These findings also suggest that in addition to lymphangiogenesis, VEGFR-3 may promote tumorigenesis in HBx associated HCC.

Mechanisms of HBx Mediated Liver Cancer: Multiple Pathways and Opportunities 297

tissues from other studies (Alitalo & Carmeliet, 2002; Hsu et al., 2002; Vlasoff et al., 2002). The finding that HBx up-regulates and stabilizes -catenin (Lian et al., 2006), which in some tumors is activated by elevated levels of ErbB-2, suggested that constitutive expression of catenin may be associated with elevated ErbB2. Accordingly, when HBx positive and negative cells were subjected to proteomics analysis, ErbB-2 was up-regulated in HBx expressing but not control cells. ErbB-2 was also strongly up-regulated in HBV infected liver, where it correlated with HBx expression, and weakly in some HCC nodules (Liu et al., 2009). Among tumor bearing patients, strong ErbB-2 staining in the liver was associated with dysplasia, and a shorter survival after tumor diagnosis. This implies that elevated ErbB-2 is an early marker of HCC. Treatment of HBx expressing cells with ErbB-2 specific siRNA not only reduced ErbB2 expression, but also reduced the expression of -catenin, suggesting that ErbB-2 contributed to the stabilization of -catenin. ErbB-2 specific siRNA also partially blocked the ability of HBx to promote DNA synthesis and growth of cells *in vitro* (Liu et al., 2009). These results suggested that ErbB-2/-catenin up-regulation contributed to HBx mediated hepatocellular growth. The additional finding that HBx stimulates expression of the epidermal growth factor receptor (EGFR or ErbB1) (Menzo et al.,1993), and that EGFR signaling stabilizes -catenin (Takahashi et al., 1997), suggested that EGF signaling may be strongly activated in patients at high risk for HCC or with already established tumors. This suggests that elevated ErbB-2 may be rate limiting in tumor formation, and if so, may be a therapeutic target (Altimari et al., 2003). Further, the accumulation of wild type -catenin in the presence of elevated ErbB-2 correlated with the activation of PI3K/Akt signaling, which is known to be activated by HBx and ErbB-2 (Lian et al., 2006; Shih et al., 2000; Yarden & Sliwkowski, 2001) (Figure 2). PI3K/Akt activity may also be stimulated by src, the latter of which is activated by HBx, early in tumor development (Lara-Pezzi et al., 2001b; Shih et al., 2003). Further, the peptidyl prolyl isomerase, Pin1, is up-regulated in HCC, and is known to stabilize both HBx (Pang et al., 2007) and ErbB-2 (Lam et al., 2008), suggesting a variety of possible mechanisms underlying

In addition to transcriptional regulation of gene expression, HBx up-regulates expression of the ribosomal protein, S15a (Lian et al., 2004) and down-regulates expression of the translation initiation factor, Sui1 (Lian et al., 1999). S15a is a highly conserved protein (Chan et al., 1994; Reed, 1980; Schaap et al., 1995) that promotes mRNA/ribosome interactions early in translation (Lavoie et al., 1994). S15a also stimulates growth in yeast (Pringle et al., 1981; Reed, 1980,), in plants (Bonhan-Smith & Moloney, 1994; Bonham-Smith et al., 1992) and in human lung carcinoma cells (Akiyama et al., 2000). The observation that S15a stimulates hepatocellular growth and survival *in vitro*, and tumor formation *in vivo*, suggests that it also plays a role in hepatocarcinogeneis, and that HBx contributes to transformation, in part, at the level of protein translation by up-regulated expression of S15a (Lian et al., 2004). As stated above,, HBx was also shown to depress the expression of the translation initiation factor, sui1. Sui1, whose function is to work with eIF-2 to enable the initiator tRNAMET to establish ribosomal recognition of an AUG codon (Yoon and Donahue, 1992), suggests that the expression of hu-sui1 contributes to the regulation of protein translation. *In* 

the close HBx/ErbB-2 relationship.

**3.2.6 Other natural target genes of HBx** 

#### **3.2.4 Elevation of -catenin and suppression of E-cadherin**

Constitutive activation of-catenin is characteristic of many tumor types (Fukuchi et al., 1998; Morin et al., 1997). This results in constitutive Wnt signaling, where -catenin translocates to the nucleus and stimulates the expression of genes that promote tumorigenesis (Clevers & van de Wetering, 1997; Peifer & Polakis, 2000; Terradillos et al., 1997). Importantly, -catenin mutations are found in small HCCs and in preneoplastic liver (Calvisi et al., 2001; Terris et al., 1999,), suggesting they occur early in tumor development. The finding of frequent -catenin mutations in a subset of human HCC (de La Costa et al., 1998, Miyoshi et al., 1998), especially in HBV-negative tumors (Hsu et al., 2000), implies that the majority of -catenin activation must occur by mechanisms other than mutation. In the chronically infected liver, HBx has been shown to be associated with the constitutive activation of wild type -catenin. The finding that the activation of wild type -catenin was associated with URG11 (Lian et al., 2006) and URG7 (Pan et al., 2007), underscores the importance of this activation in hepatocarcinogenesis. Moreover, -catenin appears to be stabilized by a number of mechanisms, including *trans*-activation of the -catenin promoter (Lian et al., 2006; Pan et al., 2007), inhibition of proteasomal degradation (Cui et al., 2006; Zhang et al., 2000), and suppression of E-cadherin expression (Arzumanyan et al., 2011; Lee et al., 2005; Liu et al., 2006) (Figure 2). The latter is of particular importance because suppression of the cell adhesion protein, E-cadherin, is a hallmark of EMT, which is important to the pathogenesis of CLD and HCC. The importance of suppressed E-cadherin expression is further underscored by the findings that this occurs by DNA methylation of the E-cadherin promoter (Lee et al., 2005; Liu et al., 2006), by the inhibition miR-373 expression by HBx, and by HBx mediated stimulation of histone deacetylase (HDAC) at the E-cadherin promoter (Arzumanyan et al., 2011). Independent of the mechanism involved, suppression of E-cadherin has important ramifications upon -catenin. Normally, -catenin participates in cell adhesion by serving as a link between E-cadherin and the cytoskeleton. When E-cadherin expression is suppressed, -catenin is released from this role and translocates to the nucleus where it activates genes that promote cell growth. Thus, in the presence of HBx, there is an inverse correlation with E-cadherin expression, and a direct correlation with the accumulation of cytoplasmic and nuclear -catenin at the expense of membranous -catenin, both in cultured cells and in clinical specimens (Arzumanyan et al., 2011; Lian et al., 2006; Liu et al., 2006). This suggests a tight coupling between EMT and the promotion of hepatocellular growth prior to the development of HCC (Du et al., 2010).

#### **3.2.5 Elevated expression of ErbB-2**

Another natural effector of HBx is ErbB-2 (Liu et al., 2009). ErbB-2 (HER2 or neu) is a member of the epidermal growth factor receptor tyrosine kinases that is involved in the transmission of differentiation and proliferation signals (Olayioye et al., 2000, Yarden & Sliwkowski, 2001). High levels of ErbB-2 have been shown in various types of cancers (Sauter et al., 1993; Slamon et al., 1987; Tanner et al., 1996), and in some tumors, overexpression is associated with poor prognosis. In breast cancer, up-regulated ErbB-2 appears to be an early event, since it appears in tumor and nontumor tissue (Menard et al., 2002). In HCC, elevated ErbB-2 has been reported in hyperplastic nodules (Niu & Wang, 2005) and in 30-40% of HCCs (Chen et al., 2002; Neo et al., 2004). However, ErbB-2 was not found in HCC

Constitutive activation of-catenin is characteristic of many tumor types (Fukuchi et al., 1998; Morin et al., 1997). This results in constitutive Wnt signaling, where -catenin translocates to the nucleus and stimulates the expression of genes that promote tumorigenesis (Clevers & van de Wetering, 1997; Peifer & Polakis, 2000; Terradillos et al., 1997). Importantly, -catenin mutations are found in small HCCs and in preneoplastic liver (Calvisi et al., 2001; Terris et al., 1999,), suggesting they occur early in tumor development. The finding of frequent -catenin mutations in a subset of human HCC (de La Costa et al., 1998, Miyoshi et al., 1998), especially in HBV-negative tumors (Hsu et al., 2000), implies that the majority of -catenin activation must occur by mechanisms other than mutation. In the chronically infected liver, HBx has been shown to be associated with the constitutive activation of wild type -catenin. The finding that the activation of wild type -catenin was associated with URG11 (Lian et al., 2006) and URG7 (Pan et al., 2007), underscores the importance of this activation in hepatocarcinogenesis. Moreover, -catenin appears to be stabilized by a number of mechanisms, including *trans*-activation of the -catenin promoter (Lian et al., 2006; Pan et al., 2007), inhibition of proteasomal degradation (Cui et al., 2006; Zhang et al., 2000), and suppression of E-cadherin expression (Arzumanyan et al., 2011; Lee et al., 2005; Liu et al., 2006) (Figure 2). The latter is of particular importance because suppression of the cell adhesion protein, E-cadherin, is a hallmark of EMT, which is important to the pathogenesis of CLD and HCC. The importance of suppressed E-cadherin expression is further underscored by the findings that this occurs by DNA methylation of the E-cadherin promoter (Lee et al., 2005; Liu et al., 2006), by the inhibition miR-373 expression by HBx, and by HBx mediated stimulation of histone deacetylase (HDAC) at the E-cadherin promoter (Arzumanyan et al., 2011). Independent of the mechanism involved, suppression of E-cadherin has important ramifications upon -catenin. Normally, -catenin participates in cell adhesion by serving as a link between E-cadherin and the cytoskeleton. When E-cadherin expression is suppressed, -catenin is released from this role and translocates to the nucleus where it activates genes that promote cell growth. Thus, in the presence of HBx, there is an inverse correlation with E-cadherin expression, and a direct correlation with the accumulation of cytoplasmic and nuclear -catenin at the expense of membranous -catenin, both in cultured cells and in clinical specimens (Arzumanyan et al., 2011; Lian et al., 2006; Liu et al., 2006). This suggests a tight coupling between EMT and the promotion of hepatocellular growth prior to the development of HCC (Du et al., 2010).

Another natural effector of HBx is ErbB-2 (Liu et al., 2009). ErbB-2 (HER2 or neu) is a member of the epidermal growth factor receptor tyrosine kinases that is involved in the transmission of differentiation and proliferation signals (Olayioye et al., 2000, Yarden & Sliwkowski, 2001). High levels of ErbB-2 have been shown in various types of cancers (Sauter et al., 1993; Slamon et al., 1987; Tanner et al., 1996), and in some tumors, overexpression is associated with poor prognosis. In breast cancer, up-regulated ErbB-2 appears to be an early event, since it appears in tumor and nontumor tissue (Menard et al., 2002). In HCC, elevated ErbB-2 has been reported in hyperplastic nodules (Niu & Wang, 2005) and in 30-40% of HCCs (Chen et al., 2002; Neo et al., 2004). However, ErbB-2 was not found in HCC

**3.2.4 Elevation of -catenin and suppression of E-cadherin** 

**3.2.5 Elevated expression of ErbB-2** 

tissues from other studies (Alitalo & Carmeliet, 2002; Hsu et al., 2002; Vlasoff et al., 2002). The finding that HBx up-regulates and stabilizes -catenin (Lian et al., 2006), which in some tumors is activated by elevated levels of ErbB-2, suggested that constitutive expression of catenin may be associated with elevated ErbB2. Accordingly, when HBx positive and negative cells were subjected to proteomics analysis, ErbB-2 was up-regulated in HBx expressing but not control cells. ErbB-2 was also strongly up-regulated in HBV infected liver, where it correlated with HBx expression, and weakly in some HCC nodules (Liu et al., 2009). Among tumor bearing patients, strong ErbB-2 staining in the liver was associated with dysplasia, and a shorter survival after tumor diagnosis. This implies that elevated ErbB-2 is an early marker of HCC. Treatment of HBx expressing cells with ErbB-2 specific siRNA not only reduced ErbB2 expression, but also reduced the expression of -catenin, suggesting that ErbB-2 contributed to the stabilization of -catenin. ErbB-2 specific siRNA also partially blocked the ability of HBx to promote DNA synthesis and growth of cells *in vitro* (Liu et al., 2009). These results suggested that ErbB-2/-catenin up-regulation contributed to HBx mediated hepatocellular growth. The additional finding that HBx stimulates expression of the epidermal growth factor receptor (EGFR or ErbB1) (Menzo et al.,1993), and that EGFR signaling stabilizes -catenin (Takahashi et al., 1997), suggested that EGF signaling may be strongly activated in patients at high risk for HCC or with already established tumors. This suggests that elevated ErbB-2 may be rate limiting in tumor formation, and if so, may be a therapeutic target (Altimari et al., 2003). Further, the accumulation of wild type -catenin in the presence of elevated ErbB-2 correlated with the activation of PI3K/Akt signaling, which is known to be activated by HBx and ErbB-2 (Lian et al., 2006; Shih et al., 2000; Yarden & Sliwkowski, 2001) (Figure 2). PI3K/Akt activity may also be stimulated by src, the latter of which is activated by HBx, early in tumor development (Lara-Pezzi et al., 2001b; Shih et al., 2003). Further, the peptidyl prolyl isomerase, Pin1, is up-regulated in HCC, and is known to stabilize both HBx (Pang et al., 2007) and ErbB-2 (Lam et al., 2008), suggesting a variety of possible mechanisms underlying the close HBx/ErbB-2 relationship.

#### **3.2.6 Other natural target genes of HBx**

In addition to transcriptional regulation of gene expression, HBx up-regulates expression of the ribosomal protein, S15a (Lian et al., 2004) and down-regulates expression of the translation initiation factor, Sui1 (Lian et al., 1999). S15a is a highly conserved protein (Chan et al., 1994; Reed, 1980; Schaap et al., 1995) that promotes mRNA/ribosome interactions early in translation (Lavoie et al., 1994). S15a also stimulates growth in yeast (Pringle et al., 1981; Reed, 1980,), in plants (Bonhan-Smith & Moloney, 1994; Bonham-Smith et al., 1992) and in human lung carcinoma cells (Akiyama et al., 2000). The observation that S15a stimulates hepatocellular growth and survival *in vitro*, and tumor formation *in vivo*, suggests that it also plays a role in hepatocarcinogeneis, and that HBx contributes to transformation, in part, at the level of protein translation by up-regulated expression of S15a (Lian et al., 2004). As stated above,, HBx was also shown to depress the expression of the translation initiation factor, sui1. Sui1, whose function is to work with eIF-2 to enable the initiator tRNAMET to establish ribosomal recognition of an AUG codon (Yoon and Donahue, 1992), suggests that the expression of hu-sui1 contributes to the regulation of protein translation. *In* 

Mechanisms of HBx Mediated Liver Cancer: Multiple Pathways and Opportunities 299

up-regulated expression of IGF-2, which appears to be a target of HBx *in vivo*, may promote

The finding that HBx interacts with and inhibits the function of the proteasome (Huang et al., 1996) suggests another mechanism whereby HBx could alter gene expression at a posttranslational level. This inhibition appears to be important in supporting HBx *trans*activation activity (Hu et al., 1999). Given that HBx *trans*-activates virus gene expression and replication, when mutants of the X protein that bound to and inhibit the proteasome were introduced into WHV, and the resulting virus used for experimental infection, no or transient viremia was observed. In contrast to wild type WHV, which resulted in a high carrier rate among experimentally infected woodchucks, none of the animals infected with the X mutant developed the carrier state (Zhang et al., 2001). Further work *in vitro* showed that in the presence of proteasome inhibitors, replication of the wild-type virus was not affected, while the replication of the X-negative HBV or WHV was enhanced and restored to the wild-type levels. Similar results were obtained in mouse models replicating wild type and X mutant HBV (Zhang et al., 2010). Thus, HBx appears to affect hepadnavirus replication through a proteasome-dependent pathway (Zhang et al., 2004). Moreover, in the livers of transgenic mice where the levels of HBx expression increased with age, there was a parallel age related decreases in the peptidase activities of the proteasome in the liver (Hu et al., 2006). Microarray analysis showed that many of the genes affected involved transcription and cell growth. For example, insulin-like growth factor-binding protein 1 was down-regulated in the HBx mouse liver (Hu et al., 2006), while *in vitro*, HBx stabilized c-myc (Kalra & Kumar, 2006) and the protooncoprotein, pituitary tumor-transforming gene 1 (PTTG1) (Molina-Jimenez et al., 2010), by blocking ubiquitination and proteasomal degradation. HBx also differentially regulated the level of -catenin through two ubiquitindependent proteasome pathways depending upon the status of p53 (Jung et al., 2007). Given that HBx expression is dominant in liver compared to HCC tissue (Wang et al., 1991a, 1991b), it was not surprising to find an elevated proteasomal activity in HCC compared to surrounding nontumor liver, both in HBx transgenic mice that developed tumors, and in clinical samples from patients with HCC (Cui et al., 2006). These observations suggest that changes in proteasome function accompany the pathogenesis of CLD and HCC, and that

Tumorigenesis is a multi-step process, and as outlined above, HBx impacts upon this process by targeting selected pathways and genes in natural infection. For most of the target genes presented here, up-regulated or down-regulated expression was established by comparison of gene expression profiles in HBx positive compared to negative cells, suggesting that they were due to the properties of HBx. Clinical validation was carried out on liver and tumor tissues obtained from HBV infected patients. For up-regulated genes, there was strong co-staining between HBx and the putative target, while for down-regulated genes, there was an inverse relationship by immunohistochemistry, and in many cases, northern blotting or RT/PCR analyses as well. Moreover, many of the natural targets of HBx discussed herein were characterized to gain at least a preliminary outline as to their contribution to the pathogenesis of HCC. The overall results show that HBx contributes to

hepatocarcinogenesis.

these changes appear to be related to the levels of HBx .

**4. Conclusions** 

*vivo* work showed that sui1 was expressed in nontumor liver but not in tumor cells from patients with HCC. Sui1 inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice, suggesting that suppression of sui1 may result in the abrogation of negative growth regulation that contributes to the development of HCC (Lian et al., 1999). Given that S15a and sui1 are both involved in regulating translation, it is likely that HBx also contributes to HCC by altering gene expression at multiple steps within translation, although the mRNAs that are differentially translated remain to be identified.

HBx also stimulates the expression of the novel protein, URG4 (Tufan et al., 2002). URG4, encodes a protein of about 104 kDa that was strongly expressed in HBV- infected liver and in HCC cells, where it co-stained with HBx, and was weakly expressed in uninfected liver, suggesting URG4 was an effector of HBx *in vivo*. Over-expression of URG4 without HBx in human hepatoblastoma cells promoted hepatocellular growth and survival in tissue culture and in soft agar, and accelerated tumor development in nude mice (Tufan et al., 2002). URG4 over-expression was associated with elevated cyclin D1 expression, and treatment of such cells with URG4 specific siRNA reduced both cyclin D1 expression and inhibited cell cycle progression (Tufan et al., 2010). These observations suggest that URG4 may be an oncogene that contributes to HBV associated HCC. Independent work showed that overexpression of URG4 in osteosarcoma tissues directly correlated with tumor recurrence and metastasis, as well as with the proliferative activity of osteosarcoma cells. Patients with high expression of URG4 had shorter survival time, suggesting that URG4 might be rate limiting in carcinogenesis and a valuable prognostic marker in osteosarcoma patients (Huang et al., 2009). Thus, URG4 may contribute to carcinogenesis outside of the liver.

HBx also appears to up-regulate the expression of insulin - like growth factor 2 (IGF-2) and the IGF-1 receptor in HCC (Kim et al., 1996; Su et al., 1994). The finding that insulin-like growth factor-2 expression, which is normally observed only in fetal liver (Soares et al., 1985), is elevated in HCCs (D'Arville et al., 1991, Cariani et al., 1991), and in premalignant proliferative nodules in the liver (Cariani et al., 1988; D'Arville et al., 1991), suggest that its reactivation may be an early step in the development of this tumor type. The elevation of IGF-2 expression in HCCs from HBV infected but not uninfected patients, combined with the finding of a strong correlation between IGF-2 and HBx in the liver by immunohistochemical staining (Su et al., 1994), suggest that IGF-2 may be a natural target of HBx during chronic infection. In human hepatoma cell lines, IGF-2 was expressed strongly in growing cells, but was undetectable in confluent cultures (Su et al., 1994), suggesting that it was associated with cell proliferation. At the molecular level, the tumor suppressor, PTEN normally suppresses IGF-2 expression (Kang-Park et al., 2003), but in the presence of HBx, PTEN expression is blocked, resulting in activation of IGF-2 (Chung et al., 2003). Normally, PTEN is up-regulated by another tumor suppressor, p53, but since HBx binds to and inactivates p53 (Feitelson et al., 1993b; Wang et al., 1994), PTEN expression also drops (Chung et al., 2003). In addition, HBx activation of Sp1 via protein kinase C (PKC) and p44/p42MAPK signaling pathways are also operative in promoting IGF-2 gene expression (Kang-Park et al., 2001). These multiple pathways underscore the importance of IGF-2 upregulation in hepatocarcinogenesis. Finally, the finding that HBx stimulates the expression of the IGF-1 receptor in human HCC cell lines (Kim et al., 1996), which binds both IGF-1 and IGF-2, suggests that HBx may set up an autocrine loop that enhances cell growth. Thus, the up-regulated expression of IGF-2, which appears to be a target of HBx *in vivo*, may promote hepatocarcinogenesis.

The finding that HBx interacts with and inhibits the function of the proteasome (Huang et al., 1996) suggests another mechanism whereby HBx could alter gene expression at a posttranslational level. This inhibition appears to be important in supporting HBx *trans*activation activity (Hu et al., 1999). Given that HBx *trans*-activates virus gene expression and replication, when mutants of the X protein that bound to and inhibit the proteasome were introduced into WHV, and the resulting virus used for experimental infection, no or transient viremia was observed. In contrast to wild type WHV, which resulted in a high carrier rate among experimentally infected woodchucks, none of the animals infected with the X mutant developed the carrier state (Zhang et al., 2001). Further work *in vitro* showed that in the presence of proteasome inhibitors, replication of the wild-type virus was not affected, while the replication of the X-negative HBV or WHV was enhanced and restored to the wild-type levels. Similar results were obtained in mouse models replicating wild type and X mutant HBV (Zhang et al., 2010). Thus, HBx appears to affect hepadnavirus replication through a proteasome-dependent pathway (Zhang et al., 2004). Moreover, in the livers of transgenic mice where the levels of HBx expression increased with age, there was a parallel age related decreases in the peptidase activities of the proteasome in the liver (Hu et al., 2006). Microarray analysis showed that many of the genes affected involved transcription and cell growth. For example, insulin-like growth factor-binding protein 1 was down-regulated in the HBx mouse liver (Hu et al., 2006), while *in vitro*, HBx stabilized c-myc (Kalra & Kumar, 2006) and the protooncoprotein, pituitary tumor-transforming gene 1 (PTTG1) (Molina-Jimenez et al., 2010), by blocking ubiquitination and proteasomal degradation. HBx also differentially regulated the level of -catenin through two ubiquitindependent proteasome pathways depending upon the status of p53 (Jung et al., 2007). Given that HBx expression is dominant in liver compared to HCC tissue (Wang et al., 1991a, 1991b), it was not surprising to find an elevated proteasomal activity in HCC compared to surrounding nontumor liver, both in HBx transgenic mice that developed tumors, and in clinical samples from patients with HCC (Cui et al., 2006). These observations suggest that changes in proteasome function accompany the pathogenesis of CLD and HCC, and that these changes appear to be related to the levels of HBx .

#### **4. Conclusions**

298 Hepatocellular Carcinoma – Basic Research

*vivo* work showed that sui1 was expressed in nontumor liver but not in tumor cells from patients with HCC. Sui1 inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice, suggesting that suppression of sui1 may result in the abrogation of negative growth regulation that contributes to the development of HCC (Lian et al., 1999). Given that S15a and sui1 are both involved in regulating translation, it is likely that HBx also contributes to HCC by altering gene expression at multiple steps within translation, although the mRNAs that are differentially translated remain to be identified.

HBx also stimulates the expression of the novel protein, URG4 (Tufan et al., 2002). URG4, encodes a protein of about 104 kDa that was strongly expressed in HBV- infected liver and in HCC cells, where it co-stained with HBx, and was weakly expressed in uninfected liver, suggesting URG4 was an effector of HBx *in vivo*. Over-expression of URG4 without HBx in human hepatoblastoma cells promoted hepatocellular growth and survival in tissue culture and in soft agar, and accelerated tumor development in nude mice (Tufan et al., 2002). URG4 over-expression was associated with elevated cyclin D1 expression, and treatment of such cells with URG4 specific siRNA reduced both cyclin D1 expression and inhibited cell cycle progression (Tufan et al., 2010). These observations suggest that URG4 may be an oncogene that contributes to HBV associated HCC. Independent work showed that overexpression of URG4 in osteosarcoma tissues directly correlated with tumor recurrence and metastasis, as well as with the proliferative activity of osteosarcoma cells. Patients with high expression of URG4 had shorter survival time, suggesting that URG4 might be rate limiting in carcinogenesis and a valuable prognostic marker in osteosarcoma patients (Huang et al.,

HBx also appears to up-regulate the expression of insulin - like growth factor 2 (IGF-2) and the IGF-1 receptor in HCC (Kim et al., 1996; Su et al., 1994). The finding that insulin-like growth factor-2 expression, which is normally observed only in fetal liver (Soares et al., 1985), is elevated in HCCs (D'Arville et al., 1991, Cariani et al., 1991), and in premalignant proliferative nodules in the liver (Cariani et al., 1988; D'Arville et al., 1991), suggest that its reactivation may be an early step in the development of this tumor type. The elevation of IGF-2 expression in HCCs from HBV infected but not uninfected patients, combined with the finding of a strong correlation between IGF-2 and HBx in the liver by immunohistochemical staining (Su et al., 1994), suggest that IGF-2 may be a natural target of HBx during chronic infection. In human hepatoma cell lines, IGF-2 was expressed strongly in growing cells, but was undetectable in confluent cultures (Su et al., 1994), suggesting that it was associated with cell proliferation. At the molecular level, the tumor suppressor, PTEN normally suppresses IGF-2 expression (Kang-Park et al., 2003), but in the presence of HBx, PTEN expression is blocked, resulting in activation of IGF-2 (Chung et al., 2003). Normally, PTEN is up-regulated by another tumor suppressor, p53, but since HBx binds to and inactivates p53 (Feitelson et al., 1993b; Wang et al., 1994), PTEN expression also drops (Chung et al., 2003). In addition, HBx activation of Sp1 via protein kinase C (PKC) and p44/p42MAPK signaling pathways are also operative in promoting IGF-2 gene expression (Kang-Park et al., 2001). These multiple pathways underscore the importance of IGF-2 upregulation in hepatocarcinogenesis. Finally, the finding that HBx stimulates the expression of the IGF-1 receptor in human HCC cell lines (Kim et al., 1996), which binds both IGF-1 and IGF-2, suggests that HBx may set up an autocrine loop that enhances cell growth. Thus, the

2009). Thus, URG4 may contribute to carcinogenesis outside of the liver.

Tumorigenesis is a multi-step process, and as outlined above, HBx impacts upon this process by targeting selected pathways and genes in natural infection. For most of the target genes presented here, up-regulated or down-regulated expression was established by comparison of gene expression profiles in HBx positive compared to negative cells, suggesting that they were due to the properties of HBx. Clinical validation was carried out on liver and tumor tissues obtained from HBV infected patients. For up-regulated genes, there was strong co-staining between HBx and the putative target, while for down-regulated genes, there was an inverse relationship by immunohistochemistry, and in many cases, northern blotting or RT/PCR analyses as well. Moreover, many of the natural targets of HBx discussed herein were characterized to gain at least a preliminary outline as to their contribution to the pathogenesis of HCC. The overall results show that HBx contributes to

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#### **5. Acknowledgements**

This work was supported by grants CA111427, CA104025, and AI076535 awarded to Dr. Mark Feitelson

#### **6. References**


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**15** 

*Japan* 

**Molecular Mechanism of DNA** 

**During Hepatic Carcinogenesis** 

**Damage Response Pathway** 

Yasunobu Matsuda1 and Toshifumi Wakai2

 *Niigata University Graduate School of Health Sciences* 

*Niigata University Graduate School of Medical and Dental Sciences* 

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The prevalence of HCC is still increasing in Asia and Africa, and represents a leading cause of death among patients with chronic liver diseases in industrial countries including Europe and North America (Llovet et al., 2003). The prognosis of HCC is considerably poor for following reasons: (1) aggressive treatment for HCC is not usually possible because most of the patients have impaired liver function, (2) hepatoma cells are refractory to standard chemotherapy drugs and radiation, and (3) HCC frequently recurs even after curative resection (Poon et al., 2009). Moreover, owing to the lack of reliable clinical HCC markers, fewer than 20% of patients are diagnosed at a stage where curative treatment can be

HCC is unique among the various types of malignancies, in that it frequently arises in individuals with hepatitis B virus (HBV)- and C virus (HCV)-related liver cirrhosis. Although the precise mechanism of the relationship between the hepatitis viruses and hepatocarcinogenesis is unknown, recent studies have suggested that an aberrant response against DNA damage might be involved in HBV- and HCV-induced carcinogenesis, as observed in many types of cancer cells. Therefore, for future development of treatments against HCC, understanding the functional role of the DNA damage response (DDR) in

Many previous studies have indicated a close relationship between metal overload and oxidative DNA damage (Imlay et al., 1988). For example, when DNA is exposed to hydrogen peroxide with iron, the Fenton reaction, in which hydrogen peroxide (H2O2) is catalysed to hydroxyl radicals (OH•) by iron (II) (Lloyd et al., 1998), causes the production of carcinogenic malondialdehyde, 4-hydroxynonenal (4-HNE) and other exocyclic DNA

**2. Impact of oxidative DNA damage during hepatocarcinogenesis** 

**1. Introduction** 

performed (Llovet et al., 2003).

HCC-prone individuals would be of value.

*1Department of Medical Technology,* 

*2Division of Digestive and General Surgery,* 

