TP53 Mutations and Hepatocellular Carcinoma: Insights Into the Etiology and Pathogenesis of Liver Cancer

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Oncogene (2007) 26, 2166–2176 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer

SP Hussain, J Schwank, F Staib1, XW Wang and CC Harris

Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA

Hepatocellular carcinoma (HCC) is one of the most Incidence and etiology of hepatocellular carcinoma common malignancies worldwide and the major risk factors include chronic infections with the hepatitis B Hepatocellular carcinoma (HCC) is one of the most (HBV) or C (HCV) virus, and exposure to dietary frequent and malignant diseases worldwide.With an aflatoxin B1 (AFB1) or alcohol consumption. Multiple estimated 560 000 newly diagnosed cases per year, it is genetic and epigenetic changes are involved in the the third most common cause of cancer deaths (Pisani molecular pathogenesis of HCC, for example, somatic et al., 1999; Parkin et al., 2005). Although this mutations in the p53 tumor suppressor gene (TP53) and malignancy is less common, but increasing in Western- the activation of the WNT signal transduction pathway. developed countries such as the USA, with an incidence AFB1 frequently induces G:C to T:A transversions at the of 2.8–6.1 (Caucasian vs African-American) per 100 000 third base in codon 249 of TP53 and cooperates with HBV (El-Serag and Mason, 1999), it is endemic in China, in causing p53 mutations in HCC. The detection of TP53 Taiwan, Korea and sub-Saharan Africa, where the mutant DNA in plasma is a biomarker of both AFB1 incidence is between 20 and 100 cases per 100 000 inhab exposure and HCC risk. Chronic infection with HBV and itants (Figure 1a and b) (Sherman, 1995; Dominguez- HCV viruses, and oxyradical disorders including hemo- Malagon and Gaytan-Graham, 2001).Furthermore, chromatosis, also generate reactive oxygen/nitrogen HCC is a leading cause of cancer-related death in these species that can both damage DNA and mutate cancer- countries, because less than 3% of these patients survive related genes such as TP53. Certain mutant p53 proteins more than 5 years (Di Bisceglie et al.,1988). may exhibit a ‘gain of oncogenic function’. The p53 The major HCC risk factors include various viruses biological networkis a keyresponder to this oxidative and and chemicals, and are summarized in Figure 2.Among nitrosative stress. Depending on the extent of the DNA those, chronic hepatitis B (HBV) and C (HCV) viruses damage, p53 regulates the transcription of protective infections attribute to HCC development in more than antioxidant genes and with extensive DNA damage, 80% of the HCC cases worldwide (Wang et al., 2002). transactivates pro-oxidant genes that contribute to Other known risk factors of HCC, including die- apoptosis. The X gene of HBV (HBx) is the most tary aflatoxin B1 (AFB1) intake, cigarette smoking or common open reading frame integrated into the host heavy alcohol consumption, can have synergistic effects genome in HCC and the integrated HBx is frequently (reviewed by Ross et al., 1992; Qian et al., 1994; Kew, mutated. Mutant HBx proteins still retain their ability to 2003; Yu and Yuan, 2004).Besides these risk factors, there bind to p53, and attenuate DNA repair and p53-mediated are several genetic disorders (Figure 2) such as hemochro- apoptosis. In summary, both viruses and chemicals are matosis (an iron overload disease) that are associated with implicated in the etiology of TP53 mutations during the an increased risk of HCC (Hofseth et al., 2002; Kowdley, molecular pathogenesis of HCC. 2004).Regardless of its etiology, cirrhosis alone is also an Oncogene (2007) 26, 2166–2176. doi:10.1038/sj.onc.1210279 independent risk factor for HCC. In recent years, we have gained a better understanding Keywords: hepatocellular carcinoma; p53; aflatoxin B1; of the genetic, environmental interactions and mechan- HBV; HCV; oxidative/nitrosative stress isms associated with the development of HCC.For example, genomic and gene expression analyses have identified key dysregulated signal transduction pathways involved in liver carcinogenesis (Thorgeirsson and Grisham, 2002; Ye et al., 2003; Budhu et al., 2006; Farazi and DePinho, 2006; Lee and Thorgeirsson, 2006).These studies and others have generated a new Correspondence: Dr CC Harris, Laboratory of Human Carcino- paradigm of metastasis (Figure 3) that is not mutually genesis, National Cancer Institute, NIH, Building 37, Room 3068, exclusive to the hypothesis of multistage carcinogenesis Bethesda, MD 20892-4258, USA. depicted in Figure 2. E-mail: [email protected] 1Current Address: I.Medizinische Klinik und Poliklinik, Johannes This review will focus on the various functions and Gutenberg-Universita¨ t Mainz, Langenbeckstr.1, 55131 Mainz, involvement of the p53 tumor suppressor gene (TP53) in Germany. liver carcinogenesis. TP53 mutations and HCC SP Hussain et al 2167 TP53 mutation archeology in HCC cells in nontumorous liver that has a dose-dependent relationship with the intake of AFB1 (Aguilar et al., The p53 response pathway is frequently defective in 1994).In geographic regions, such as Qidong and China, human cancers including HCC.The frequency of TP53 40% of HCC can be attributable to dietary AFB1 mutations and the mutation spectra vary in different exposure (Ming et al., 2002). geographic areas (Figure 1a–c), and presumably reflect Several studies support the findings of a positive differences in both etiological agents and host susceptibility association between the 249ser mutation of TP53 and factors (Hussain and Harris, 1998; Olivier et al., 2004). AFB1 exposure, whereas the analysis of HCCs in areas of low AFB1 intake always reported a different Causation evaluated by the Bradford–Hill criteria mutational spectrum (Ozturk, 1991; Kress et al., 1992; The presence of specific TP53 mutational hotspots in differ- Oda et al., 1992). In vitro studies exposing human liver ser ent types of human cancer implicates environmental cell lines to AFB1 presented the same 249 mutational carcinogens and endogenous processes in the etiology of pattern of TP53 (Aguilar et al., 1993; Mace et al., 1997), human cancer.The presence of a characteristic TP53 as it has been reported in the epidemiological studies mutation is a putative molecular link between the exposure (Figure 1c). to a particular carcinogen and a specific type of cancer, for There are at least two possible explanations for these ser example, AFB1 exposure and codon 249 mutations in findings.Aguilar et al.(1994) have shown a higher HCC (Figure 1b and c).In addition, the detection of such a relative abundance of the 249ser mutant liver cells, in particular mutation in normal-appearing tissue provides nontumorous liver, by using a highly sensitive genotypic further support for the involvement of a specific carcinogen mutation assay.They suggested that this early muta- in a specific human cancer.Once a careful assessment of tional event may be due to the high mutability of the causation has been carried out for a presumed carcinogen third base at codon 249 to AFB1, as suggested by association, its associated molecular damage, and the final previous in vitro studies in human liver cells (Aguilar disease, individuals with an increased cancer risk may et al., 1993; Cerutti et al., 1994). Another possible be identified.For this approach, appropriate criteria explanation might be that the 249ser mutant p53 protein as proposed by Bradford–Hill, might be used for the may provide a special growth and/or survival advantage assessment (Hill, 1965).Using the ‘weight of the evidence’ to these liver cells (Puisieux et al., 1995). The latter principle, these criteria include strength of association hypothesis is supported by the following findings: first, (consistency, specificity and temporality) and biological the transfection of a TP53 249ser mutant into p53- plausibility.One prime example for the use of the Bradford– negative human liver cancer cells resulted in enhanced Hill criteria is the association of AFB1 dietary intake cell growth that is consistent with a gain of function of followed by TP53 249ser mutations and liver carcinogenesis p53 249ser (Ponchel et al., 1994); second, the introduction (Table 1) (Hussain and Harris, 1998; Staib et al., 2003). of a murine TP53 mutation, corresponding to human However, geographic location, race and gender may also codon 249 into a murine hepatocyte cell line, resulted in influence an individual’s susceptibility for cancer risk. a selective growth advantage (Dumenco et al., 1995); third, the 249ser mutant inhibits wild-type p53-mediated apoptosis, resulting in increased cell survival (Wang ser Aflatoxin B1 et al., 1995) and fourth, the 249 mutant is more effective than other mutants in inhibiting wild-type p53 TP53 mutations indicate that the sites and features of transactivation activity in human liver cells (Forrester DNA base changes differ among the various human et al., 1995). tumor types.In human HCC, the mutational spectrum In addition to the direct mutagenicity of the has provided a strong molecular link between carcino- metabolically activated AFB1 to the 8, 9-epoxide that gen exposure and cancer.In geographical areas of AFB 1 binds to DNA and forms the promutagenic N7dG dietary exposure (a mycotoxin contaminating foods like adduct (Buss et al., 1990; Guengerich et al., 1996), AFB1 corn, rice and peanuts) and chronic viral hepatitis, like can cause oxidative and nitrosative stress, and may China and Africa, a point mutation at the third position indirectly induce TP53 249ser mutations by lipid perox- of codon 249ser resulting in a G:C to T:A transversion idation (Figure 4).Our recent studies indicate that was common in HCC (Figure 1a–c) (Bressac et al., 1991; oxidative-stress-generated 4-hydroxynonenal can induce Hsu et al., 1991). These results have been confirmed and TP53 249ser mutations in vitro (Hussain et al., 2004). extended by others (Scorsone et al., 1992; Li et al., 1993; However, although chronic inflammation associated Lunn et al., 1997). This codon 249 mutation can also be with HBV or HCV infection can also cause lipid detected in serum DNA from patients from the Gambia peroxidation, TP53 249ser mutations are uncommon with HCC, so that TP53 mutant DNA may be a without AFB1 exposure.These observations argue biomarker of exposure to AFB1 and possibly early HCC against this indirect mechanism as a major pathway to (Kirk et al., 2000). Their most recent results indicate TP53 249ser mutation in HCC.HCC occurring in a multiplicative effect on HCC risk between the AFB1- individuals with oxyradical overload diseases, such as induced TP53 249ser mutation, as monitored by the hemochromatosis, contain TP53 mutations occurring detection of plasma TP53 249ser, and chronic viral primarily at A:T base pairs and TP53 249ser mutations hepatitis (Kirk et al., 2005) (Table 2). These results are have not been observed (Marrogi et al., 2001). The ser consistent with the TP53 mutation load of 249 mutant combination of HBV and AFB1 exposure may be

Oncogene TP53 mutations and HCC SP Hussain et al 2168 critical to the high frequency of TP53 249ser mutations in sure in mice, but TP53 mutations are rare (Fujimoto HCC (Ming et al., 2002). We are not aware of HCC et al., 1992; Tong et al., 2006). Nevertheless, attenuated studies occurring in a population, if it exists, exposed p53 function cooperates with telomere-induced chromo- only to AFB1 without HBV infection.AFB 1 induces somal instability in a murine model of HCC carcino- HCC in nonhuman primates and with neonatal expo- genesis (Farazi et al., 2006).

Oncogene TP53 mutations and HCC SP Hussain et al 2169 HBV and HCV as oncoviruses in liver carcinogenesis Among these, the HBx protein has been the focus of attention, because it seems to play a strong causal role in The hepatitis viruses, HBV and HCV, induce liver HCC (Tang et al., 2006). The most commonly integrated injury, hepatocyte death and promote hepatocarcino- HBV gene is the HBx gene (Figure 5) (Unsal et al., 1994). genesis.It is still not clear whether the virus infection HBx decreases p53 binding to XPB (Wang et al.,1994), is causing the tumor initiation or the subsequent which is both involved in nucleotide excision repair and inﬂammation, leading to liver regeneration and cirrho- in transcription as a basic transcription factor (Schaeffer sis, which act as a tumor promoter in hepatocarcino- et al., 1993), and interacts with various other DNA genesis.Available data suggest that viral proteins repair-associated proteins or enzymes, including DNA undergo various interactions with host proteins helicases (Qadri et al., 1996; Jia et al., 1997). HBx itself leading to an alteration of the cellular gene expression, has been shown to attenuate nucleotide excision repair which may contribute to a virus-associated carcino- and alter the AFB1-induced mutation spectrum (Becker genesis. et al., 1998; Groisman et al., 1999; Jia et al.,1999; HBV is a hepadna virus with a partially double- Mathonnet et al., 2004). HBx transgenic mice have stranded DNA molecule, which is about 3.2 kb long and increased G:C to T:A transversions induced by AFB1 consists of four open reading frames encoding the enve- when compared with AFB1-exposed wild-type mice lope protein, the nucleocapsid (core) protein, the viral reverse (Madden et al., 2002). These interferences of HBx with transcriptase, and the X gene of HBV (HBx) protein. the cellular DNA repair system provide another mechan- HBx is required for the transcription of the viral genome. ism of how HBV contributes to liver carcinogenesis.

Figure 2 Multistage hepatocarcinogenesis.CMAR, cellular adhesion regulatory molecule; HBV, hepatitis B virus; HCV, hepatitis C virus; IGF-I, insulin-like growth factor-I; IGF-II, insulin-like growth factor-II; LOH, loss of heterozygosity; TGF-a, transforming growth factor a; TGF-b, transforming growth factor b.

Figure 1 (a) Worldwide incidences of HCC with pie diagrams of TP53 mutation patterns for various countries and continents according to the IARC TP53 mutation database (http://www.iarc.fe/p53/index.html). A high frequency of G:C to T:A mutations is found in high incidence areas of both endemic HBV infection and dietary AFB1 exposure.( b) TP53 mutational spectra in HCC from different geographic areas of Asia.TP53 249 ser mutations are found only in high incidence geographic areas with endemic HBV infections and dietary AFB1 exposure.( c) Schematic representation of the p53 molecule.The human p53 protein consists of 393 amino acids with functional domains, evolutionarily conserved domains and regions designated as mutational hotspots.Functional domains include the transactivation region (gold block), sequence-specific DNA-binding region (amino acids 100–293), nuclear localization sequence (amino acids 316–325, dark green block) and oligomerization region (amino acids 319–360, light green block).Evolutionarily conserved domains (amino acids 17–29, 97–292 and 324–352; rose areas) were determined using the Multiple Alignment Construction and Analysis Workbench (MACAW) program.Seven mutational hotspots and evolutionarily conserved regions within the large conserved domain are also identified (amino acids 130–142, 151–164, 171–181, 193–200, 213–233, 234–258 and 270–286, blue blocks). Vertical lines above the schematic represent missense mutations.The majority of these mutations are in the conserved hydrophobic midregion of the protein, which is required for the sequence-specific binding to DNA.The nonmissense mutations (nonsense, frameshift, splicing and silent) are distributed throughout the protein and determined primarily by sequence context, whereas deletions and insertions are more common in the N- and C-terminus of the TP53 gene.The pie charts on the left show the mutational spectra of codons 157 and 249, and the frequency evoked by AFB1 intake.In contrast, the pie chart on the right includes all TP53 mutations in HCC.

Oncogene TP53 mutations and HCC SP Hussain et al 2170 We and others have shown that HBx binds to p53 and proapoptotic ability, the mutant HBx enhanced the inactivates p53-dependent activities (Feitelson et al., transforming ability of ras and myc (Tu et al., 2001). 1993; Wang et al., 1994, 1995; Ueda et al., 1995; Elmore The abrogation of p53-mediated apoptosis by HBx et al., 1997), including p53 sequence-specific DNA- (also reviewed by Arbuthnot et al., 2000; Bergsland, binding activity in vitro (Wang et al., 1994), p53- 2001; Murakami, 2001) may provide a selective clonal mediated transcriptional activation in vivo (Wang et al., advantage for preneoplastic or neoplastic hepatocytes 1994) and represses the TP53 transcription (Lee and Rho, and contribute to hepatocellular carcinogenesis.Mole- 2000).Moreover, HBx deregulates cell cycle check cular epidemiological studies also support the hypoth- point controls and blocks p53-mediated apop- esis that mutant HBx integrated in HCC is positi- tosis (Lucito and Schneider, 1992; Feitelson et al., vely associated with TP53 249ser mutations.In both 1993; Bennett et al., 1995; Wang et al., 1995; Miura China and Thailand, analysis of plasma DNA revealed et al., 1997; Chan and Ng, 2006). Interestingly, tumor- a frequent concordance between HBV 1762T/1764A derived HBx mutants that lacked their transcriptional and TP53 249ser DNA in plasma at the time of HCC cotransactivation activity as well as proapoptotic diagnosis (Kuang et al., 2004, 2005). The double mutant activity (Sirma et al., 1999), still retained their p53- resides in the HBx open reading frame within HBV, binding functions and blocked p53-mediated apoptosis and results in lysine to methionine and valine to (Huo et al., 2001. (Figure 6) Furthermore, by losing the isoleucine at HBx codons 130 and 131, respectively. The functional significance of this HBx double mutant is not known. To gain a better insight into HCV-related hepatocarcinogenesis, microarray technology has been applied in several studies.Analysis of HCV-associated cirrhosis revealed an upregulation of proinflammatory, proapoptotic and pro-proliferative genes, which might reflect groups of genes being involved in HCV-related cirrhosis progressing to HCC (Honda et al., 2001; Shackel et al., 2002).Gene expression profiles of the HCV genotypes

Table 2 TP53 codon 249ser mutant in serum DNA and serum HBVSAg are biomarkers of liver cancer risk (Kirk et al., 2005) Figure 3 Clonal and dual proclivity models of tumor evolution to metastasis.In the clonal evolution model, a normal hepatocyte HBSAg/249TP53 mutant RR (95% CI) (green) progresses to a tumor through clonal expansion of an initiated cell with a mutation (yellow), followed by the acquisition Minus/minus 1 of sequential mutations (red) that support metastasis.In the dual Plus/minus 10 (5–20) proclivity model, multiple tumor-initiating events are concurrent in Minus/plus 13 (5–35) premalignant lesions before tumor formation and metastasis.These Plus/plus 399 (49–3272) events are inherent in the primary tumor and/or are modulated by the stromal environment.(Budhu et al., 2005). Abbreviations: CI, conﬁdence interval; RR, relative risk.

ser Table 1 Hypothesis: dietary AFB1 exposure can cause 249 (AGG-AGT) TP53 mutations during human liver carcinogenesis (Staib et al., 2003) Strength of association Biological plausibility

Consistency AFB1 is a potent mutagen and carcinogen in laboratory studies. J Positive dose–response correlation between estimated dietary AFB1 AFB1 is enzymatically activated by human hepatocytes and the ser exposure and frequency of 249 TP53 mutations in three 8,9-AFB1 oxide binds to the third base (G) in codon 249. ser different ethnic populations on three continents. AFB1 exposure to human liver cells in vitro produces codon 249 J 249ser TP53 mutant DNA is detected in sera from individuals exposed TP53 mutations. ser to AFB1 and infected with HBV. HBx gene expression increases the frequency of 249 TP53 ser J 249 TP53 mutations are found in HCC from individuals both exposed mutations in cells exposed to AFB1 in vitro. ser to dietary AFB1 and infection with HBV, but not with HBV alone. 249 TP53 expression inhibits apoptosis and p53-mediated transcription, and enhances liver cell growth in vitro. Speciﬁcity J 249ser TP53 mutations are uncommon in other cancer types. J 249ser TP53 mutation in serum and plasma is a biomarker of liver cancer risk.

Temporality J 249ser TP53 mutant cells are observed in nontumorous liver in high HCC incidence geographic areas.

Abbreviations: AFB1, aﬂatoxin B1; HBV, hepatitis B virus; HBx, X gene of HBV; HCC, hepatocellular carcinoma; TP53, p53 tumor suppressor gene.

Oncogene TP53 mutations and HCC SP Hussain et al 2171 1b, 2a and 4d core proteins in HepG2 and Huh-7 cells esis (Dou et al., 2005). In another study with transient indicated that each core protein has its own expression HCV core gene-transfected Huh-7 cells by Fukutomi profile and that each of them seems to be implicated in et al.(2005) most transcriptionally regulated genes were HCV replication as well as pathogenesis and oncogen- involved in cell growth or oncogenic signalling.Of particular interest were growth-related genes in the WNT-1 pathway.In addition, to further focus on the role of p53 in HCC, a number of TP53 mutant and TP53 wild-type HCC cases were analysed by microarrays identifying 83 p53-related genes in TP53 mutant HCCs when compared with wild-type TP53 HCCs (Okada et al., 2003). The authors conclude that the most striking finding of their study was that HCV-infected HCCs with wild-type TP53 and those with mutant TP53 differ significantly in their gene expression patterns.Cell cycle- related genes (CCNG2, BZAP45) and cell proliferation- related genes (SSR1, ANXA2, S100A10 and PTMA) were overexpressed in mutant TP53 tumors compared with wild-type TP53 tumors.Based on their results, they assume that mutant TP53 tumors have higher malignant potentials than those with wild-type TP53.Studies like the ones named above may help to identify major Figure 4 Overview of different reactive oxygen and nitrogen oxide pathways being involved in HCV-related hepatocarci- species (ROS/RNOS) and their reaction products leading to DNA nogenesis and may help to select potential targets for damage during inflammation. new potential therapies.

Chronic inﬂammation and cancer

Chronic inflammation and infection is frequently associated with increased cancer risk, although excep- tions can be cited, including rheumatoid arthritis and human papillomavirus infection (Table 3; Hussain et al., 2003). Infection with HBV and HCV viruses cause inflammation with the release of free radicals, chemokines and cytokines resulting in DNA damage, cell proliferation, fibrosis and angiogenesis.The p53 pathway is a key responder to inflammatory stress (Staib et al., 2005). Free radicals, for example, reactive nitrogen or oxygen species, can directly damage DNA and proteins, and indirectly damage these macromole- Figure 5 Integration of HBV genes in human HCC from different geographic regions.In all tested areas, the HBV-X gene (grey bars) cules via lipid peroxidation (Figure 4).The p53 pathway is the most commonly integrated HBV-gene compared with the responds to lower levels of DNA damage by cell cycle HBV-S gene (black bars) or the HBV-C gene (white bars). checkpoint arrest, facilitating DNA repair as an adapter

Figure 6 The HBV-X gene product and its mutations.Both missense mutations and deletions are frequent in tumor-derived HBV-X. The nuclear export signal (NES) and retention of p53 binding is found in the integrated HBV-X gene in human cancers.

Oncogene TP53 mutations and HCC SP Hussain et al 2172 Table 3 Chronic inﬂammation and infection can increase cancer risk (Stewart and Kleihues 2003) Inherited disease Tumor site OR Acquired disease Tumor site OR

Hemochromatosis Liver 219 Viral Crohn’s disease Colon 3 Hepatitis B Liver 88 Ulcerative Colitis Colon 6 Hepatitis C Liver 30

Bacterial Helicobacter pylori Gastric 11 ‘18% of human cancers, that is, 1.6 million per year, are related to PID Ovary 3 infection.’ Parasitic Schistosoma hematobium Urinary bladder 2–14 Caveats Schistosoma japonicum Colon 2–6 Rheumatoid arthritis is an example of a chronic inflammatory Liver fluke Liver 14 disease without an increased cancer risk, for example, joint sarcoma. Oncogenic human papilloma viruses are examples of cancer-prone Chemical/Physical/Metabolic chronic infections without inflammation. Acid reflux Esophagus 50–100 Asbestos Lung pleura >10 Obesity Multiple sites 1.3–6.5

Abbreviations: OR, odds ratio; PID, pelvic inﬂammatory disease.

Table 4 Examples of anti-oxidant and -nitrosative activities of the 1996; Ambs et al., 1997). NO is catalysed by a family of p53 tumor suppressor enzymes known as NOS (Marletta, 1993; Forstermann Transcriptional transrepression Transcriptional transactivation and Kleinert, 1995).The isoforms NOS1 and NOS3 are of pro-oxidant enzymes of anti-oxidant enzymes found to be constitutively expressed, for example, in neurons (NOS1) or endothelial cells (NOS3).They NOS2 GPx (Tan et al., 1999) Human and mouse cells ALDH4 (Yoon et al., 2004) produce NO levels ranging from pico- to nanomolar (Forrester et al., 1996) MnSOD (Hussain et al., 2004) concentrations.In contrast, NOS2 (also called iNOS) Mouse model (Ambs et al., SESN 1 and 2 (Sablina et al., requires induction generally, but is able to produce NO 1998b) 2005) concentrations in the micromolar range (reviewed by TIGAR (Bensaad et al., 2006) Ambs et al., 1997). NOS2 gene expression can be induced by bacterial endotoxins, proinflammatory cytokines or hypoxia (Wild et al., 1986; Nussler et al., 1992; Lombard and Guarente, 2000) in many cell types, in the formation of DNA repair protein complexes including macrophages (Xie et al., 1992) and hepato- and transcriptional transactivation of DNA repair genes cytes (Mowat et al., 1990; Lombard and Guarente, 2000; (Sengupta and Harris, 2005), and to extensive DNA Vodovotz et al., 2004) as well as in a variety of human damage by mediating cell death, p53 also contributes to tumors (reviewed by Ambs et al., 1997). During chronic these processes by switching from increased expression of viral hepatitis, the upregulation of certain proinflamma- anti- to pro-oxidant genes.p53 can both transcriptionally tory cytokines, like tumor necrosis factor-a (TNF-a) and transrepress pro-oxidant/nitrosative genes, for example, interferon-g (IFN-g), has been repeatedly demonstrated nitric oxide synthases (NOS)2, and transactivate (Gonzalez-Amaro et al., 1994; Mihm et al., 1996). These antioxidant genes expressing glutathione peroxidase, proinflammatory cytokines, TNF-a,IFN-g and IL-1, aldehyde dehydrogenase, and Mn-superoxide dismutase, induce NOS2 gene expression, which leads to increased sestrins, and TIGAR (TP53-induced glycolysis and NO concentrations in human hepatocytes (de Vera apoptosis regulator) (Table 4).An animal model et al., 1996; Laskin et al., 1998). In addition, NOS2 is of the Li–Fraumeni syndrome has provided new also induced directly by the HBV and HCV. In vitro insights into the protective antioxidative and ntirosative studies from Elmore et al.(1997); Amaro et al.(1999); function of p53.TP53 null mice are predisposed to Majano et al.(1998) demonstrate that the HBx protein lymphoma and leukemia (Donehower et al.,1992). is capable of transcriptional transactivation of NOS2. If these mice are fed anti-oxidative and -nitrosative An induction of the NOS2 expression has also been seen agent, that is, N-acetylcysteine, the number of tumors after hepatocytes are exposed to woodchuck hepatitis was markedly decreased in support of the protective virus surface antigen (Liu et al., 1994). In patients with function of p53 in tumor suppression (Sablina et al., HBV or HCV infection, a consistent upregulation of 2005). hepatic NOS2 has been shown (Kane et al., 1997; Growing evidence indicates that nitric oxide (NO), Vodovotz et al., 2004). an important bioregulatory and signalling molecule, We and others were able to show that this NO- may play a significant role in carcinogenesis (Moncada related DNA damage leads to p53 accumulation and et al., 1991; Bredt and Snyder, 1994; Nathan and Xie, p53-mediated apoptosis (Forrester et al., 1996; Messmer 1994; Hentze and Kuhn, 1996; Tamir and Tannenbaum, and Brune, 1996) (Figure 7).Moreover, we presented

Oncogene TP53 mutations and HCC SP Hussain et al 2173 transcriptionally activate both NOS2 and COX2 by T-cell factor (TCF)4-b-catenin binding to positive response elements in their promoters (Araki et al., 2003; Du et al., 2006). Therefore, the NOS2 and COX2 are concomitantly activated by pro-inflammatory cytokines, hypoxia and the WNT pathway.The WNT pathway is frequently activated in HCC associated with high AFB1 exposure (Devereux et al., 2001). COX2 activity is amplified by nitrosylation following its physical interaction with NOS2.NO can both damage DNA to induce the anticarcinogenic p53 stress response Figure 7 Model of the interactive pathways in nitrosative-stress- pathway and mutate cancer-related genes including induced TP53 mutation.NOS2 and COX2 are positively regulated TP53.PGE2 attenuates apoptosis (Gupta and DuBois, by hypoxia, proinflammatory cytokines, and the WNT signal 2001), which may allow survival of hepatocytes with transduction pathway.NOS2 produces a high concentration of NO that can both damage DNA to induce mutations in cancer-related DNA damage and increase the probability of somatic genes, for example, TP53, and induce p53-dependent cell cycle arrest mutations.As NO -induced apoptosis can be p53 and apoptosis, so that cells containing mutated TP53 attain selective dependent, cells with mutant TP53 have a clonal clonal growth and survival advantages.NOS2 can physically bind expansion advantage.Consistent with this model, we COX2 and activate it by nitrosylation.COX2 can increase have found a significant association as well as a dose– lipidperoxidation to produce unsaturated aldehydes that can induce TP53 mutations, and increase PGE2 concentrations.PGE2 can response relationship between TP53 mutations (G:C to attenuate p53-mediated apoptosis and activate the WNT pathway, A:T transition at cytosine guanine dinucleotide (CpG) resulting in increased b-catenin that with TCF-4, transcriptionally sites) and an increased NOS2 activity in patients with activates NOS2 and COX2.High amounts of p53 can participate in colon cancer (Ambs et al., 1999). Furthermore, we and negative feedback loops in the regulation of NOS2 and COX2. Lesser amounts of p53 can enhance COX2 accumulation. others have demonstrated a positive association of NOS2 expression and a comparable TP53 mutational spectrum in lung cancer (Fujimoto et al., 1998; Ambs results indicating that p53 is a transcriptional trans- et al., 1998a). This TP53 mutation spectrum is consistent repressor of NOS2 expression in vivo and attenuates an with increased rates of N2O3-induced deamination of excessive NO production in a regulatory negative 5-methylation at CpG sites.In summary, oxidative/ feedback loop (Forrester et al., 1996; Ambs et al., nitrosative stress, mediated by free radicals, which is 1998b; Hussain et al., 2001). NO can also activate the also induced by the cytokine profile of various arachidonic acid pathway by enhancing the enzymatic inflammatory hepatic disorders like HBV and HCV activity of cyclooxygenase 2 (COX2) (Figure 7).The infection, or hemochromatosis, can act as an endogen- mechanism of this activation is by NOS2 binding and ous carcinogen to increase TP53 mutations and provide nitrosylation of COX2 (Kim et al., 2005). Increased a microenvironment allowing a clonal growth advantage COX2 activity results in the release of prostaglandin 2E for cells with TP53 mutations. (PGE2), that diminishes the apoptotic pathway and activates the WNT pathway (Castellone et al., 2005; Buchanan and DuBois, 2006).The WNT signal Acknowledgements transduction and COX2 pathways are key contributors to liver carcinogenesis (Thorgeirsson and Grisham, We thank Dorothea Dudek-Creaven for editorial and graphic 2002), and also predict poor survival of patients with assistance, Karen MacPherson for bibliographical assistance, HCC (Kondo et al., 1999). The WNT pathway can also as well as Mohammed Khan for technical data.

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