- Email: [email protected]
Hepatitis viruses and hepatocarcinogenesis
Journal of Physiology - Paris 95 (2001) 417–422 www.elsevier.com/locate/jphysparis
Hepatitis viruses and hepatocarcinogenesis Ga´bor Lotz, Andra´s Kiss, Pa´l Kaposi Nova´k, Ga´bor Sobel, Zsuzsa Schaff* Second Department of Pathology, Semmelweis University of Budapest, U¨llo¨i street 93, H-1091 Budapest, Hungary
Abstract Hepatocellular carcinoma (HCC) is among the most frequent malignancies worldwide. Hepatitis viruses, such as the hepatitis B virus (HBV) and hepatitis C virus (HCV) are undoubtedly listed in the etiology of HCC. Studies show that, in the near future, viral hepatitis will carry increasing weight in the etiology of HCC. This review briefly discusses the known carcinogenic effects of HBV and HCV in the light of experimental and human studies. The data show that viral proteins may directly interfere with gene products responsible for cell proliferation and cell growth. Many other signal transduction cascades may be affected as well. Direct integration of HBV viral sequences into the host genome increases the genomic instability. The genomic inbalance allows the development and survival of malignant clones bearing defected genomic information. HBV and HCV infection induces indirect and direct mechanisms through cellular damage, increased regeneration and cell proliferation, therefore enhancing the development of HCC. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Hepatitis B virus; Hepatitis C virus; Hepatocellular carcinoma
Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide, representing 4% of all malignancies and being the seventh most frequent carcinoma in males and the ninth most frequent in females [6,12,19,23,26]. The death rate due to HCC has been increasing over the past 20–30 years. HCC shows a wide degree of variability in occurrence rates in different parts of the world [9]. These patterns of HCC occurrence are in close correlation with the prevalence of infection with both hepatitis B virus (HBV) and hepatitis C virus (HCV) [34]. HCCs associated with HBV infection are very frequent in Southeast Asia and sub-Saharan Africa, whereas HCCs associated with HCV are most prevalent in southern Europe and Japan [1,4,20,21,22]. In Italy, Spain and Japan, 50–70% of HCC cases are associated with HCV infection [21]. Long-term natural history studies have clearly demonstrated links between chronic HBV or HCV infection and HCC. Chronic infections by these viruses lead to slowly progressive liver disease that over a period of up to 30 years, may result in cirrhosis and perhaps HCC (Table 1). Patients with more active and severe liver disease seem to be at higher risk of developing cancer [1]. * Corresponding author. Tel./fax: +36-1-215-6921. E-mail address: schaff@korb2.sote.hu (Z. Schaff).
The mechanism of HBV and HCV infection is still not exactly clear [30]. Expression of viral proteins stimulates the host immune response and triggers liver inflammation [14,32]. The importance of cell death, in the form of apoptosis, is well known and recognized in association with HBV and HCV infection [32]. It is hypothesized that cycles of necroinflammatory lesions and hepatocellular regeneration might be prerequisites for genetic alterations resulting in malignant changes leading to HCC. Certain viral components, and insertion of the viral DNA into the liver cell genome, also directly interfere with cell proliferation and viability and induce genetic alterations [2,3,5,7,8,10,13, 14,16,18,20–22,25, 28,35,37,38,40,41]. Liver cell progression into the cell cycle can overcome DNA repair mechanisms in the presence of mutational events. This may induce fixed DNA mutations and chromosomal rearrangements which are major determinants of cell transformation. Thus, any pathological conditions associated with chronic stimulation of the entry of quiescent liver cells into the cell cycle should be viewed as a risk factor for liver cancer.
1. The role of HBV infection in hepatocarcinogenesis HBV is a wide spread human pathogen that causes acute and persistent liver disease. In a recent estimate,
0928-4257/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(01)00057-2
418
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
about 350 million people in the world suffer from chronic HBV infection [6] (Table 2). Prospective epidemiological studies have shown that the risk of developing HCC is increased by 100-fold in chronic HbsAg carriers [6]. HBV, the founding member of a small family of viruses called hepadna-viruses, is a small, enveloped, partially double stranded DNA virus [7–10,13]. With a size of 3.2 kb, the HBV genome is one of the smallest of
all animal virus genomes and shows a very compact organization. HBV replicates its genome by reverse transcription. The entire coding minus strand carries four overlapping open reading frames that encode seven different peptides such as: core, HBeAg, HBsAg (preS1, preS1, S), Polygene (DNA polymerase with RT activity). Finally the X gene encodes the viral transcriptional transactivator HbxAg which is required for infection of liver cells in vivo [2,8,10,13,18,37].
Table 1 Pathomechanism of virus induced HCCa
a
Based on Bre´chot et al. [8], Buendia [11], Feitelson [18], Feo et al. [19], Idilman et al. [22], Ozturk [26].
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
Mechanisms of HBV replication have led to the discovery of common features with the replication of retroviruses, especially a step of reverse transcription. The question whether HBV is directly involved in the multistep hepatocarcinogenesis remains to be answered. It is known that viral DNA integration into the cellular DNA is not necessary for viral replication but allows persistence of the viral genome in the cell. Viral DNA insertion as well as cellular DNA replication occur during liver-cell proliferation, secondary to the necrosis/apoptosis of adjacent hepatocytes. The HBVDNA sequences are integrated into cellular DNA in most (90%) liver tumor samples deriving from HBV positive patients. The data available to date have failed to demonstrate any preferential integration site for HBV-DNA (however, there is a trend towards a high level of integrants in chromosome 11) [8–11] (Table 3). It has been shown that integration of viral DNA into the cellular genome preceded development of HCC. At
the time of HCC development, tumor cells no longer allow viral DNA replication and do not express HBcAg, although HBsAg can be detected in approximately 20% of the cases. The consequences of HBV-DNA integration are chromosomal DNA instability, synthesis of X and truncated preS2/S proteins and insertional mutagenesis (cis-activation) [2,8–10,18]. The HBx gene is highly conserved among all mammalian hepadnaviruses and encodes a small polypeptide which is expressed at low levels during acute and chronic hepatitis. The observation that HBx transactivates cellular genes controlling cell growth suggested that HBx might participate in the transformation of hepatocytes by HBV [2,8]. The biological actions of HBx are complex; it activates several cytoplasmic and nuclear transduction cascades (majority being cytosolic), binds to cellular proteins (including p53, RB suppressor genes) and has
Table 2 The features of HBV and HCV, and the infection caused by these virusesa HBV
HCV
Viral family
Hepadnaviridae
Flaviviridae
Nucleic acid Viral DNA integration Reverse transcriptional activity Size Number of infected individuals
ds DNA, circular 3.2 kb Yes Yes 42 nm total of 2 billion people 350 million chronically (chronicity: 5%) 1 million/year Parental, sexual, perinatal, etc.
ss RNA 9.4 kb No No 28 nm total of 170 million people 140 million chronically (chronicity: 80%) ? Parental, ?
Number of death cases Transmission a
Based on Buendia [11], Chisari [14], Cohen [16].
Table 3 The features and consequences of HBV-DNA integrationa HBV DNA integration Consequences
Features
Chromosomal DNA instability Insertional mutagenesis (cis-activation) Production of incomplete viral proteins Increased production of HBx protein Alteration of cellular genes and proto-oncogenes
Present in 90% of HBV-associated HCCs Random Present previous to HCC development
a
Based on Feitelson [18], Idilman et al. [22], Sirma et al. [37].
Table 4 The role of HBxa Transactivation of cellular genes controlling cell proliferation (proto-oncogenes; c-jun, c-fos, c-myc) Deregulation of the cell cycle check points Tumor promotion Activation of cytoplasmic and nuclear signal transduction cascades Induces HCC in HBx-transgenic mice Binding and inactivation of proteins produced by tumor suppressor genes (p53, RB, etc.) a
419
Based on Andrisani and Barnabas [2], Bre´chot et al. [8], Feitelson [18], ldilman et al. [22].
420
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
an effect on both apoptosis and cell proliferation [2,8] (Table 4). P53 mutation and binding of p53 to HBx result intracellular accumulation of the protein, which is detectable by immunohistochemistry [38]. Studies (including ours) have shown that p53 mutation is associated with progression, rather than early development [31,38]. It has been suggested that the association of HBx and p53 may cause the functional inactivation of p53 [8,26]. Persistence of viral genomes in hepatocytes is a requirement for the development of HBV-related HCC, and is due to a combination of several factors. The study of certain growth factors as transforming growth factor alpha (TGFa) [24] and transforming growth factor beta (TGFb) [33] probably add to our understanding of the pathomechanism of tumor development. HBV may act at two complementary stages of liver cell transformation: it may exert a direct role through a combination of cis- and transactivating mechanisms and in addition, HBV may induce promotion and clonal expansion in initiated cells by inducing liver cell death and proliferation [8,17,22,26].
2. The role of HCV in hepatocarcinogenesis More recently chronic infection with HCV has been recognized as an important cause of HCC [1,4,12, 20,22,34,39]. HCV is an ss RNA virus within the Flaviviridae family, without any RT activity [15]. There is no evidence that HCV-RNA is integrated into the host genome. The structural proteins are encoded at the 5 end, followed by the non-structural proteins that have various functions, including a helicase, or protease (NS3) and an RNA polymerase (NS5). Different isolates of HCV show substantial nucleotide sequence variability distributed throughout the viral genome [15]. The low
fidelity of the RNA-dependent RNA polymerase is responsible for genetic heterogeneity, leading to the evolution of quasispecies, being a complex population of closely related, but distinct virions in a given host [29]. It is known that HCV has many genomic mutation. In fact, the mutation rate of the HCV base is about 106 times higher than that of human DNA [20]. The development of accurate molecular assays for assessing circulating HCV viraemia or ‘viral load’ has become increasingly important for confirming the diagnosis of HCV infection. HCV-RNA testing has been particularly useful in seronegative patients with chronic hepatitis, especially in immunocompromized individuals who might lack serological evidence of HCV infection despite clinical and molecular evidence of HCV. Our observation concerning the association of HCV to the erythrocytes might be important from this point of view [36]. Detection of PCR-amplified hepatitis C virus RNA on human erythrocytes suggests that the association might serve as an internal pool for the virus [36]. The lack of a practical system for the propagation of HCV in cultured cells has hampered our understanding of the life cycle of HCV. Recently, a novel hepatitis C virus-positive cell line has been developed from a chimpanzee with chronic HCV infection, which might help to solve the problem [27]. HCV is considered as a non-cytopathic virus. However, HCV proteins might modulate cell proliferation [34, 42] (Table 5). There are factors interacting with HCV proteins which might modify cell proliferation [34] (Table 5). The HCV core protein seems to be important in several viral functions (Table 5). The core protein acts as a transcriptional regulator of various viral and cellular genes including c-myc, c-fos, RB, p53, etc. All these functions suggest that the HCV core protein may directly modulate cellular signaling [34]. Full-length core protein (of 191 aa) is located in the cytoplasm, in the perinuclear region [5]; core 151 mainly
Table 5 Modulatory effects of HCV proteins on cell proliferationa HCV protein
Factors interacting with HCV proteins
Functions of HCV proteins
Core
Apolipoprotein All Interaction with p53 (?) TNF receptor 1 DEAD domain of RNA helicase
Modulates lipid metabolism Changes in p21/waf1 gene expression Modulates apoptosis, steatosis Inhibits host-cell mRNA translation Induces HCC in transgenic mice Steatosis in transgenic mice Represses p53 transactivation function Inhibits PKR functions Transforms NIH 3T3 Transforms NIH 3T3 Modulates interferon sensitivity Anti-apoptotic and oncogenic potential Represses PKR activity Transactivator
Lymphotoxin receptor E2 NS3 NS4B NS5A
a
Based on Shimotohno [34].
PKR p53 ? ? PKR
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
in the nucleus, core 173 in the nucleus and the perinuclear cytoplasm. It has been shown by deletion analysis that the subcellular localization of the core protein could be shifted from the cytoplasm to the nucleus. It is possible that the HCV core protein, in regenerating hepatocytes, enhances growth stimuli and repeated hepatocyte proliferation may cause disorders of the genes in the hepatocytes, thus causing HCC. The core protein can alter lipid metabolism. In vivo and in vitro studies have revealed lipid droplets to accumulate in core transfected cell lines and in hepatocytes infected with HCV [5]. It has been shown that the HCV core is a predominantly cytoplasmic protein, localized in part around lipid droplets in vitro in HCV core expressing cells, and in vivo in chimpanzees [5]. Studies have been performed on the mechanism underlying the development of liver steatosis in an in vivo transgenic murine model of HCV core protein expression [25]. It has been shown that transgenic mice expressing HCV core protein develop microvesicular steatosis and HCC, in the absence of an immune response [25]. This suggests that the HCV protein probably has direct cytopathic effects in the pathogenesis of HCC.
3. The ‘‘common’’ pathomechanism of HBV and HCV associated HCC The above discussed data suggest that both hepatotropic viruses are able to drive the hepatocytes in an accelerated cycle through increased virus-induced apoptosis and proliferation. Depending on the host immune response and the viral factors, chronic inflammation may develop varying in intensity and propensity. Viral proteins such as HBx and HCV core protein may directly interfere with gene products responsible for cell proliferation and growth. The genomic integration of HBV-DNA may increase the genomic instability which favors the development of clones bearing genetic defects. The increased growth stimulus may help the clonal expansion of the damaged cell, finally leading to hepatocellular carcinoma. HBV and HCV might therefore lead to the development of hepatocellular carcinoma, directly and in part indirectly through increased cell proliferation—by strengthening the impact of each mechanism.
References [1] Y. Aizawa, Y. Shibamoto, I. Takagi, M. Zeniya, G. Toda, Analysis of factors affecting the appearance of hepatocellular carcinoma in patients with chronic hepatitis C, Cancer 89 (2000) 53–59. [2] O.M. Andrisani, S. Barnabas, The transcriptional function of the hepatitis B virus X protein and its role in hepatocarcinogenesis, Int. J. Oncol. 15 (1999) 373–379.
421
[3] H. Aoki, J. Hayashi, M. Moriyama, Y. Arakawa, O. Hino, Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1, J. Virol. 74 (2000) 1736–1741. [4] A.M. Bisceglie, Hepatitis C and hepatocellular carcinoma, Hepatology 26 (1997) 345–385. [5] G. Barba, F. Harper, T. Harada, M. Kohara, S. Goulinet, Y. Matsuura, G. Eder, Zs. Schaff, M.J. Chapman, T. Miyamura, C. Bre´chot, Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets, Proc. Natl. Acad. Sci. USA 94 (1997) 1200–1205. [6] F.X. Bosch, Global epidemiology of hepatocellular carcinoma, in: K. Okuda, E. Tabor (Eds.), Liver Cancer, Churchill Livingstone, New York, 1997, pp. 13–28. [7] C. Bre´chot, R.H.P. Mario Rizzetto, J.L. Gerin, G. Verme, Molecular mechanisms of hepatitis B and C viruses related liver carcinogenesis, Viral Hepatitis and Liver Diseases (1998) 490–508. [8] C. Bre´chot, D. Gozuacik, Y. Murakami, P. Paterlini-Bre´chot, Molecular bases for the development of hepatitis B virus (HBV)related hepatocellular carcinoma (HCC), Semin. Cancer Biol. 10 (2000) 211–231. [9] M.A. Buendia, Hepatitis B viruses and hepatocellular carcinoma, Adv. Cancer Res. 59 (1992) 167–226. [10] M.A. Buendia, Hepatitis B viruses and cancerogenesis, Biomed. & Pharmacother. 52 (1998) 34–43. [11] M.A. Buendia, Genetics of hepatocellular carcinoma, Semin. Cancer Biol. 10 (2000) 185–200. [12] W.H. Caselmann, M. Alt, Hepatitis C virus infection as a major risk factor for hepatocellular carcinoma, J. Hepatol. 24 (1996) 61–66. [13] P.-J. Chen, D.-S. Chen, Hepatitis B virus infection and hepatocellular carcinoma: molecular genetics and clinical perspectives, Semin. Liver Dis. 19 (1999) 253–262. [14] F.V. Chisari, Viruses, immunity, and cancer: Lessons from hepatitis B, Am. J. Path. 156 (2000) 1118–1132. [15] B. Clarke, Molecular virology of hepatitis C virus, J. Gen. Virol. 78 (1997) 2397–2410. [16] J. Cohen, The scientific challenge of hepatitis C, Science 285 (1999) 26–30. [17] N. Fausto, Mouse liver tumorigenesis: models, mechanisms, and relevance to human disease, Semin. Liver Dis. 19 (1999) 243–252. [18] M.A. Feitelson, Hepatitis B virus in hepatocarcinogenesis, J. Cell. Physiol. 181 (1999) 188–202. [19] F. Feo, R.M. Pascale, M.M. Simile, M.R. DeMiglio, M.R. Muroni, D. Calvisi, Genetic alterations in liver carcinogenesis: implications for new preventive and therapeutic strategies, Crit. Rev. Oncogen 11 (2000) 19–62. [20] J. Hayashi, H. Aoki, Y. Arakawa, O. Hino, Hepatitis C virus and hepatocarcinogenesis, Intervirology 42 (1999) 205–210. [21] H. Huang, H. Fujii, A. Sankila, B.M. Mahler-Araujo, M. Matsuda, G. Cathomas, H. Ohgaki, b-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection, Am. J. Pathol. 155 (1999) 1795–1801. [22] R. Idilman, N. DeMaria, A. Colantoni, D.H. Van Thiel, Pathogenesis of hepatitis B and C-induced hepatocellular carcinoma, J. Viral Hepat. 5 (1998) 285–299. [23] M.C. Kew, M.C. Yu, M.A. Kedda, A. Coppin, A. Sarkin, J. Hodkinson, The relative roles of hepatitis B anc C viruses in the etiology of hepatocellular carcinoma in Southern African Blacks, Gastroenterology 112 (1997) 184–187. [24] A. Kiss, A´. Szepesi, G. Lotz, P. Nagy, Zs. Schaff, Expression of transforming growth factor-a in hepatoblastoma, Cancer 83 (1998) 690–697. [25] H. Lerat, K. Loesch, M. Honda, R. Gosert, G.L.H. Pin, A.B. West, S.A. Wienman, S.M. Semon, Transgenic mice expressing HCV proteins develop microvesicular steatosis and hepatocellular carcinoma (HCC), 6th Int. Symp. on Hepatitis C and Related Viruses. NIH, June 6–9, 1999, Bethesda, MD, p. 204.
422
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
[26] M. Ozturk, Genetic aspects of hepatocellular carcinogenesis, Semin. Liver Dis. 19 (1999) 235–242. [27] B. Pavlova, Zs. Schaff, G. Eder, Novel hepatitis C virus-positive cell line derived from a chimpanzee with chronic HCV infection, in: R. Schinazi, C. Rice, J.-P. Sommadossi (Eds.), Frontiers in Viral Hepatitis, Int. Med. Press Ltd., London, 2000 (in press). [28] K. Poussin, H. Dienes, H. Sirma, S. Urban, M. Beaugrand, D. Franco, P. Schirmacher, C. Bre´chot, P. Paterlini Bre´chot, Expression of mutated hepatitis B virus X genes in human hepatocellular carcinomas, Int. J. Cancer 80 (1999) 497–505. [29] H.R. Rosen, D.R. Gretch, Hepatitis C virus: current understanding and prospects for future therapies, Mol. Med. Today 5 (1999) 393–399. [30] Zs. Schaff, K. Lapis, D.E. Henson, Liver, in: D.E. Henson, J. Albores-Saavedra (Eds.), Pathology of Incipient Neoplasia (Second edition). Major Problems in Pathology. Vol. 28, W.B. Saunders Co, Philadelphia, London, Toronto, 1993, pp. 151–166. [31] Zs. Schaff, I. Sarosi, C.C. Hsia, A. Kiss, E. Tabor, P53 in malignant and benign liver lesions, Eur. J. Cancer 31A (1995) 1847– 1850. [32] Zs. Schaff, G. Lotz, G. Eder, R. Schulte-Hermann, Pathomorphology and apoptosis in viral hepatitis, in: R.F. Schinazi, J.P. Sommadossi, H. Thomas (Eds.), Therapies for Viral Hepatitis, Int. Med. Press Ltd., London, 1998, pp. 77–86. [33] J. Schnur, P. Nagy, A. Sebestye´n, Zs. Schaff, S.S. Thorgeirsson, Chemical hepatocarcinogenesis in transgenic mice overexpressing mature TGFb-1 in liver, Eur. J. Cancer 35 (1999) 1842–1845. [34] K. Shimotohno, Hepatitis C virus and its pathogenesis, Semin. Cancer Biol. 10 (2000) 233–240. [35] Y. Shintani, H. Yotsuyanagi, K. Moriya, H. Fujie, T. Tsutsumi, Y. Kanegae, S. Kimura, I. Saito, K. Koike, Induction of apoptosis after switch-on of the hepatitis B virus X gene mediated by
[36]
[37]
[38]
[39] [40]
[41]
[42]
the cre/IoxP recombination system, J. Gen. Virol. 80 (1999) 3257–3265. S. Simon, G. Lotz, F. Kury, B. Reipert, A. Steinkasserer, M.M. Eibl, Zs. Schaff, In situ localization of PCR-amplified hepatitis C virus RNA on human erythrocytes, in: R. Schinazi, C. Rice, J.-P. Sommadossi (Eds.), Frontiers in Viral Hepatitis, Int. Med. Press Ltd., London, 2000 (in press). H. Sirma, C. Giannini, K. Poussin, P. Paterlini, D. Kremsdorf, C. Bre´chot, Hepatitis B virus X mutants, present in hepatocellular carcinoma tissue abrogate both the antiproliferative and transactivation effects of HBx, Oncogene 18 (1999) 4848–4859. Q. Su, C.H. Schro¨der, G. Otto, P. Bannasch, Overexpression of p53 protein is not directly related to hepatitis B x protein expression and is associated with neoplastic progression in hepatocellular carcinomas rather than hepatic preneoplasia, Mutat. Res. 462 (2000) 365–380. E. Tabor, Hepatitis C virus and hepatocellular carcinoma, Proc. Am. Assoc. Cancer Res. 40 (1999) 749–750. L. Tornillo, V. Carafa, J. Richter, G. Sauter, H. Moch, E. Minola, M. Gambacorta, L. Bianchi, R. Vecchione, L.M. Terracciano, Marked genetic similarities between hepatitis B virus-positive and hepatitis C virus-positive hepatocellular carcinomas, J. Pathol. 192 (2000) 307–312. R. Weil, H. Sirma, C. Giannini, D. Kremsdorf, C. Bessia, C. Dargemont, C. Bre´chot, A. Israel, Direct association and nuclear import of the hepatitis B virus x protein with the NF-kB inhibitor IkBa, Mol. Cell. Biol. 19 (1999) 6345–6354. K. Werling, Z. Szentirmay, A´. Szepesi, Zs. Schaff, F. Szalay, Zs. Szabo´, L. Telegdy, K. Da´vid, G. Stotz, Z. Tulassay, Hepatocyte proliferation and cell cycle phase fractions in chronic viral hepatitis C by image analysis method. Eur. J. Gastroenterol. Hepatol. 13 (2001) 1–5.
Hepatitis viruses and hepatocarcinogenesis Ga´bor Lotz, Andra´s Kiss, Pa´l Kaposi Nova´k, Ga´bor Sobel, Zsuzsa Schaff* Second Department of Pathology, Semmelweis University of Budapest, U¨llo¨i street 93, H-1091 Budapest, Hungary
Abstract Hepatocellular carcinoma (HCC) is among the most frequent malignancies worldwide. Hepatitis viruses, such as the hepatitis B virus (HBV) and hepatitis C virus (HCV) are undoubtedly listed in the etiology of HCC. Studies show that, in the near future, viral hepatitis will carry increasing weight in the etiology of HCC. This review briefly discusses the known carcinogenic effects of HBV and HCV in the light of experimental and human studies. The data show that viral proteins may directly interfere with gene products responsible for cell proliferation and cell growth. Many other signal transduction cascades may be affected as well. Direct integration of HBV viral sequences into the host genome increases the genomic instability. The genomic inbalance allows the development and survival of malignant clones bearing defected genomic information. HBV and HCV infection induces indirect and direct mechanisms through cellular damage, increased regeneration and cell proliferation, therefore enhancing the development of HCC. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Hepatitis B virus; Hepatitis C virus; Hepatocellular carcinoma
Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide, representing 4% of all malignancies and being the seventh most frequent carcinoma in males and the ninth most frequent in females [6,12,19,23,26]. The death rate due to HCC has been increasing over the past 20–30 years. HCC shows a wide degree of variability in occurrence rates in different parts of the world [9]. These patterns of HCC occurrence are in close correlation with the prevalence of infection with both hepatitis B virus (HBV) and hepatitis C virus (HCV) [34]. HCCs associated with HBV infection are very frequent in Southeast Asia and sub-Saharan Africa, whereas HCCs associated with HCV are most prevalent in southern Europe and Japan [1,4,20,21,22]. In Italy, Spain and Japan, 50–70% of HCC cases are associated with HCV infection [21]. Long-term natural history studies have clearly demonstrated links between chronic HBV or HCV infection and HCC. Chronic infections by these viruses lead to slowly progressive liver disease that over a period of up to 30 years, may result in cirrhosis and perhaps HCC (Table 1). Patients with more active and severe liver disease seem to be at higher risk of developing cancer [1]. * Corresponding author. Tel./fax: +36-1-215-6921. E-mail address: schaff@korb2.sote.hu (Z. Schaff).
The mechanism of HBV and HCV infection is still not exactly clear [30]. Expression of viral proteins stimulates the host immune response and triggers liver inflammation [14,32]. The importance of cell death, in the form of apoptosis, is well known and recognized in association with HBV and HCV infection [32]. It is hypothesized that cycles of necroinflammatory lesions and hepatocellular regeneration might be prerequisites for genetic alterations resulting in malignant changes leading to HCC. Certain viral components, and insertion of the viral DNA into the liver cell genome, also directly interfere with cell proliferation and viability and induce genetic alterations [2,3,5,7,8,10,13, 14,16,18,20–22,25, 28,35,37,38,40,41]. Liver cell progression into the cell cycle can overcome DNA repair mechanisms in the presence of mutational events. This may induce fixed DNA mutations and chromosomal rearrangements which are major determinants of cell transformation. Thus, any pathological conditions associated with chronic stimulation of the entry of quiescent liver cells into the cell cycle should be viewed as a risk factor for liver cancer.
1. The role of HBV infection in hepatocarcinogenesis HBV is a wide spread human pathogen that causes acute and persistent liver disease. In a recent estimate,
0928-4257/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(01)00057-2
418
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
about 350 million people in the world suffer from chronic HBV infection [6] (Table 2). Prospective epidemiological studies have shown that the risk of developing HCC is increased by 100-fold in chronic HbsAg carriers [6]. HBV, the founding member of a small family of viruses called hepadna-viruses, is a small, enveloped, partially double stranded DNA virus [7–10,13]. With a size of 3.2 kb, the HBV genome is one of the smallest of
all animal virus genomes and shows a very compact organization. HBV replicates its genome by reverse transcription. The entire coding minus strand carries four overlapping open reading frames that encode seven different peptides such as: core, HBeAg, HBsAg (preS1, preS1, S), Polygene (DNA polymerase with RT activity). Finally the X gene encodes the viral transcriptional transactivator HbxAg which is required for infection of liver cells in vivo [2,8,10,13,18,37].
Table 1 Pathomechanism of virus induced HCCa
a
Based on Bre´chot et al. [8], Buendia [11], Feitelson [18], Feo et al. [19], Idilman et al. [22], Ozturk [26].
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
Mechanisms of HBV replication have led to the discovery of common features with the replication of retroviruses, especially a step of reverse transcription. The question whether HBV is directly involved in the multistep hepatocarcinogenesis remains to be answered. It is known that viral DNA integration into the cellular DNA is not necessary for viral replication but allows persistence of the viral genome in the cell. Viral DNA insertion as well as cellular DNA replication occur during liver-cell proliferation, secondary to the necrosis/apoptosis of adjacent hepatocytes. The HBVDNA sequences are integrated into cellular DNA in most (90%) liver tumor samples deriving from HBV positive patients. The data available to date have failed to demonstrate any preferential integration site for HBV-DNA (however, there is a trend towards a high level of integrants in chromosome 11) [8–11] (Table 3). It has been shown that integration of viral DNA into the cellular genome preceded development of HCC. At
the time of HCC development, tumor cells no longer allow viral DNA replication and do not express HBcAg, although HBsAg can be detected in approximately 20% of the cases. The consequences of HBV-DNA integration are chromosomal DNA instability, synthesis of X and truncated preS2/S proteins and insertional mutagenesis (cis-activation) [2,8–10,18]. The HBx gene is highly conserved among all mammalian hepadnaviruses and encodes a small polypeptide which is expressed at low levels during acute and chronic hepatitis. The observation that HBx transactivates cellular genes controlling cell growth suggested that HBx might participate in the transformation of hepatocytes by HBV [2,8]. The biological actions of HBx are complex; it activates several cytoplasmic and nuclear transduction cascades (majority being cytosolic), binds to cellular proteins (including p53, RB suppressor genes) and has
Table 2 The features of HBV and HCV, and the infection caused by these virusesa HBV
HCV
Viral family
Hepadnaviridae
Flaviviridae
Nucleic acid Viral DNA integration Reverse transcriptional activity Size Number of infected individuals
ds DNA, circular 3.2 kb Yes Yes 42 nm total of 2 billion people 350 million chronically (chronicity: 5%) 1 million/year Parental, sexual, perinatal, etc.
ss RNA 9.4 kb No No 28 nm total of 170 million people 140 million chronically (chronicity: 80%) ? Parental, ?
Number of death cases Transmission a
Based on Buendia [11], Chisari [14], Cohen [16].
Table 3 The features and consequences of HBV-DNA integrationa HBV DNA integration Consequences
Features
Chromosomal DNA instability Insertional mutagenesis (cis-activation) Production of incomplete viral proteins Increased production of HBx protein Alteration of cellular genes and proto-oncogenes
Present in 90% of HBV-associated HCCs Random Present previous to HCC development
a
Based on Feitelson [18], Idilman et al. [22], Sirma et al. [37].
Table 4 The role of HBxa Transactivation of cellular genes controlling cell proliferation (proto-oncogenes; c-jun, c-fos, c-myc) Deregulation of the cell cycle check points Tumor promotion Activation of cytoplasmic and nuclear signal transduction cascades Induces HCC in HBx-transgenic mice Binding and inactivation of proteins produced by tumor suppressor genes (p53, RB, etc.) a
419
Based on Andrisani and Barnabas [2], Bre´chot et al. [8], Feitelson [18], ldilman et al. [22].
420
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
an effect on both apoptosis and cell proliferation [2,8] (Table 4). P53 mutation and binding of p53 to HBx result intracellular accumulation of the protein, which is detectable by immunohistochemistry [38]. Studies (including ours) have shown that p53 mutation is associated with progression, rather than early development [31,38]. It has been suggested that the association of HBx and p53 may cause the functional inactivation of p53 [8,26]. Persistence of viral genomes in hepatocytes is a requirement for the development of HBV-related HCC, and is due to a combination of several factors. The study of certain growth factors as transforming growth factor alpha (TGFa) [24] and transforming growth factor beta (TGFb) [33] probably add to our understanding of the pathomechanism of tumor development. HBV may act at two complementary stages of liver cell transformation: it may exert a direct role through a combination of cis- and transactivating mechanisms and in addition, HBV may induce promotion and clonal expansion in initiated cells by inducing liver cell death and proliferation [8,17,22,26].
2. The role of HCV in hepatocarcinogenesis More recently chronic infection with HCV has been recognized as an important cause of HCC [1,4,12, 20,22,34,39]. HCV is an ss RNA virus within the Flaviviridae family, without any RT activity [15]. There is no evidence that HCV-RNA is integrated into the host genome. The structural proteins are encoded at the 5 end, followed by the non-structural proteins that have various functions, including a helicase, or protease (NS3) and an RNA polymerase (NS5). Different isolates of HCV show substantial nucleotide sequence variability distributed throughout the viral genome [15]. The low
fidelity of the RNA-dependent RNA polymerase is responsible for genetic heterogeneity, leading to the evolution of quasispecies, being a complex population of closely related, but distinct virions in a given host [29]. It is known that HCV has many genomic mutation. In fact, the mutation rate of the HCV base is about 106 times higher than that of human DNA [20]. The development of accurate molecular assays for assessing circulating HCV viraemia or ‘viral load’ has become increasingly important for confirming the diagnosis of HCV infection. HCV-RNA testing has been particularly useful in seronegative patients with chronic hepatitis, especially in immunocompromized individuals who might lack serological evidence of HCV infection despite clinical and molecular evidence of HCV. Our observation concerning the association of HCV to the erythrocytes might be important from this point of view [36]. Detection of PCR-amplified hepatitis C virus RNA on human erythrocytes suggests that the association might serve as an internal pool for the virus [36]. The lack of a practical system for the propagation of HCV in cultured cells has hampered our understanding of the life cycle of HCV. Recently, a novel hepatitis C virus-positive cell line has been developed from a chimpanzee with chronic HCV infection, which might help to solve the problem [27]. HCV is considered as a non-cytopathic virus. However, HCV proteins might modulate cell proliferation [34, 42] (Table 5). There are factors interacting with HCV proteins which might modify cell proliferation [34] (Table 5). The HCV core protein seems to be important in several viral functions (Table 5). The core protein acts as a transcriptional regulator of various viral and cellular genes including c-myc, c-fos, RB, p53, etc. All these functions suggest that the HCV core protein may directly modulate cellular signaling [34]. Full-length core protein (of 191 aa) is located in the cytoplasm, in the perinuclear region [5]; core 151 mainly
Table 5 Modulatory effects of HCV proteins on cell proliferationa HCV protein
Factors interacting with HCV proteins
Functions of HCV proteins
Core
Apolipoprotein All Interaction with p53 (?) TNF receptor 1 DEAD domain of RNA helicase
Modulates lipid metabolism Changes in p21/waf1 gene expression Modulates apoptosis, steatosis Inhibits host-cell mRNA translation Induces HCC in transgenic mice Steatosis in transgenic mice Represses p53 transactivation function Inhibits PKR functions Transforms NIH 3T3 Transforms NIH 3T3 Modulates interferon sensitivity Anti-apoptotic and oncogenic potential Represses PKR activity Transactivator
Lymphotoxin receptor E2 NS3 NS4B NS5A
a
Based on Shimotohno [34].
PKR p53 ? ? PKR
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
in the nucleus, core 173 in the nucleus and the perinuclear cytoplasm. It has been shown by deletion analysis that the subcellular localization of the core protein could be shifted from the cytoplasm to the nucleus. It is possible that the HCV core protein, in regenerating hepatocytes, enhances growth stimuli and repeated hepatocyte proliferation may cause disorders of the genes in the hepatocytes, thus causing HCC. The core protein can alter lipid metabolism. In vivo and in vitro studies have revealed lipid droplets to accumulate in core transfected cell lines and in hepatocytes infected with HCV [5]. It has been shown that the HCV core is a predominantly cytoplasmic protein, localized in part around lipid droplets in vitro in HCV core expressing cells, and in vivo in chimpanzees [5]. Studies have been performed on the mechanism underlying the development of liver steatosis in an in vivo transgenic murine model of HCV core protein expression [25]. It has been shown that transgenic mice expressing HCV core protein develop microvesicular steatosis and HCC, in the absence of an immune response [25]. This suggests that the HCV protein probably has direct cytopathic effects in the pathogenesis of HCC.
3. The ‘‘common’’ pathomechanism of HBV and HCV associated HCC The above discussed data suggest that both hepatotropic viruses are able to drive the hepatocytes in an accelerated cycle through increased virus-induced apoptosis and proliferation. Depending on the host immune response and the viral factors, chronic inflammation may develop varying in intensity and propensity. Viral proteins such as HBx and HCV core protein may directly interfere with gene products responsible for cell proliferation and growth. The genomic integration of HBV-DNA may increase the genomic instability which favors the development of clones bearing genetic defects. The increased growth stimulus may help the clonal expansion of the damaged cell, finally leading to hepatocellular carcinoma. HBV and HCV might therefore lead to the development of hepatocellular carcinoma, directly and in part indirectly through increased cell proliferation—by strengthening the impact of each mechanism.
References [1] Y. Aizawa, Y. Shibamoto, I. Takagi, M. Zeniya, G. Toda, Analysis of factors affecting the appearance of hepatocellular carcinoma in patients with chronic hepatitis C, Cancer 89 (2000) 53–59. [2] O.M. Andrisani, S. Barnabas, The transcriptional function of the hepatitis B virus X protein and its role in hepatocarcinogenesis, Int. J. Oncol. 15 (1999) 373–379.
421
[3] H. Aoki, J. Hayashi, M. Moriyama, Y. Arakawa, O. Hino, Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1, J. Virol. 74 (2000) 1736–1741. [4] A.M. Bisceglie, Hepatitis C and hepatocellular carcinoma, Hepatology 26 (1997) 345–385. [5] G. Barba, F. Harper, T. Harada, M. Kohara, S. Goulinet, Y. Matsuura, G. Eder, Zs. Schaff, M.J. Chapman, T. Miyamura, C. Bre´chot, Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets, Proc. Natl. Acad. Sci. USA 94 (1997) 1200–1205. [6] F.X. Bosch, Global epidemiology of hepatocellular carcinoma, in: K. Okuda, E. Tabor (Eds.), Liver Cancer, Churchill Livingstone, New York, 1997, pp. 13–28. [7] C. Bre´chot, R.H.P. Mario Rizzetto, J.L. Gerin, G. Verme, Molecular mechanisms of hepatitis B and C viruses related liver carcinogenesis, Viral Hepatitis and Liver Diseases (1998) 490–508. [8] C. Bre´chot, D. Gozuacik, Y. Murakami, P. Paterlini-Bre´chot, Molecular bases for the development of hepatitis B virus (HBV)related hepatocellular carcinoma (HCC), Semin. Cancer Biol. 10 (2000) 211–231. [9] M.A. Buendia, Hepatitis B viruses and hepatocellular carcinoma, Adv. Cancer Res. 59 (1992) 167–226. [10] M.A. Buendia, Hepatitis B viruses and cancerogenesis, Biomed. & Pharmacother. 52 (1998) 34–43. [11] M.A. Buendia, Genetics of hepatocellular carcinoma, Semin. Cancer Biol. 10 (2000) 185–200. [12] W.H. Caselmann, M. Alt, Hepatitis C virus infection as a major risk factor for hepatocellular carcinoma, J. Hepatol. 24 (1996) 61–66. [13] P.-J. Chen, D.-S. Chen, Hepatitis B virus infection and hepatocellular carcinoma: molecular genetics and clinical perspectives, Semin. Liver Dis. 19 (1999) 253–262. [14] F.V. Chisari, Viruses, immunity, and cancer: Lessons from hepatitis B, Am. J. Path. 156 (2000) 1118–1132. [15] B. Clarke, Molecular virology of hepatitis C virus, J. Gen. Virol. 78 (1997) 2397–2410. [16] J. Cohen, The scientific challenge of hepatitis C, Science 285 (1999) 26–30. [17] N. Fausto, Mouse liver tumorigenesis: models, mechanisms, and relevance to human disease, Semin. Liver Dis. 19 (1999) 243–252. [18] M.A. Feitelson, Hepatitis B virus in hepatocarcinogenesis, J. Cell. Physiol. 181 (1999) 188–202. [19] F. Feo, R.M. Pascale, M.M. Simile, M.R. DeMiglio, M.R. Muroni, D. Calvisi, Genetic alterations in liver carcinogenesis: implications for new preventive and therapeutic strategies, Crit. Rev. Oncogen 11 (2000) 19–62. [20] J. Hayashi, H. Aoki, Y. Arakawa, O. Hino, Hepatitis C virus and hepatocarcinogenesis, Intervirology 42 (1999) 205–210. [21] H. Huang, H. Fujii, A. Sankila, B.M. Mahler-Araujo, M. Matsuda, G. Cathomas, H. Ohgaki, b-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection, Am. J. Pathol. 155 (1999) 1795–1801. [22] R. Idilman, N. DeMaria, A. Colantoni, D.H. Van Thiel, Pathogenesis of hepatitis B and C-induced hepatocellular carcinoma, J. Viral Hepat. 5 (1998) 285–299. [23] M.C. Kew, M.C. Yu, M.A. Kedda, A. Coppin, A. Sarkin, J. Hodkinson, The relative roles of hepatitis B anc C viruses in the etiology of hepatocellular carcinoma in Southern African Blacks, Gastroenterology 112 (1997) 184–187. [24] A. Kiss, A´. Szepesi, G. Lotz, P. Nagy, Zs. Schaff, Expression of transforming growth factor-a in hepatoblastoma, Cancer 83 (1998) 690–697. [25] H. Lerat, K. Loesch, M. Honda, R. Gosert, G.L.H. Pin, A.B. West, S.A. Wienman, S.M. Semon, Transgenic mice expressing HCV proteins develop microvesicular steatosis and hepatocellular carcinoma (HCC), 6th Int. Symp. on Hepatitis C and Related Viruses. NIH, June 6–9, 1999, Bethesda, MD, p. 204.
422
G. Lotz et al. / Journal of Physiology - Paris 95 (2001) 417–422
[26] M. Ozturk, Genetic aspects of hepatocellular carcinogenesis, Semin. Liver Dis. 19 (1999) 235–242. [27] B. Pavlova, Zs. Schaff, G. Eder, Novel hepatitis C virus-positive cell line derived from a chimpanzee with chronic HCV infection, in: R. Schinazi, C. Rice, J.-P. Sommadossi (Eds.), Frontiers in Viral Hepatitis, Int. Med. Press Ltd., London, 2000 (in press). [28] K. Poussin, H. Dienes, H. Sirma, S. Urban, M. Beaugrand, D. Franco, P. Schirmacher, C. Bre´chot, P. Paterlini Bre´chot, Expression of mutated hepatitis B virus X genes in human hepatocellular carcinomas, Int. J. Cancer 80 (1999) 497–505. [29] H.R. Rosen, D.R. Gretch, Hepatitis C virus: current understanding and prospects for future therapies, Mol. Med. Today 5 (1999) 393–399. [30] Zs. Schaff, K. Lapis, D.E. Henson, Liver, in: D.E. Henson, J. Albores-Saavedra (Eds.), Pathology of Incipient Neoplasia (Second edition). Major Problems in Pathology. Vol. 28, W.B. Saunders Co, Philadelphia, London, Toronto, 1993, pp. 151–166. [31] Zs. Schaff, I. Sarosi, C.C. Hsia, A. Kiss, E. Tabor, P53 in malignant and benign liver lesions, Eur. J. Cancer 31A (1995) 1847– 1850. [32] Zs. Schaff, G. Lotz, G. Eder, R. Schulte-Hermann, Pathomorphology and apoptosis in viral hepatitis, in: R.F. Schinazi, J.P. Sommadossi, H. Thomas (Eds.), Therapies for Viral Hepatitis, Int. Med. Press Ltd., London, 1998, pp. 77–86. [33] J. Schnur, P. Nagy, A. Sebestye´n, Zs. Schaff, S.S. Thorgeirsson, Chemical hepatocarcinogenesis in transgenic mice overexpressing mature TGFb-1 in liver, Eur. J. Cancer 35 (1999) 1842–1845. [34] K. Shimotohno, Hepatitis C virus and its pathogenesis, Semin. Cancer Biol. 10 (2000) 233–240. [35] Y. Shintani, H. Yotsuyanagi, K. Moriya, H. Fujie, T. Tsutsumi, Y. Kanegae, S. Kimura, I. Saito, K. Koike, Induction of apoptosis after switch-on of the hepatitis B virus X gene mediated by
[36]
[37]
[38]
[39] [40]
[41]
[42]
the cre/IoxP recombination system, J. Gen. Virol. 80 (1999) 3257–3265. S. Simon, G. Lotz, F. Kury, B. Reipert, A. Steinkasserer, M.M. Eibl, Zs. Schaff, In situ localization of PCR-amplified hepatitis C virus RNA on human erythrocytes, in: R. Schinazi, C. Rice, J.-P. Sommadossi (Eds.), Frontiers in Viral Hepatitis, Int. Med. Press Ltd., London, 2000 (in press). H. Sirma, C. Giannini, K. Poussin, P. Paterlini, D. Kremsdorf, C. Bre´chot, Hepatitis B virus X mutants, present in hepatocellular carcinoma tissue abrogate both the antiproliferative and transactivation effects of HBx, Oncogene 18 (1999) 4848–4859. Q. Su, C.H. Schro¨der, G. Otto, P. Bannasch, Overexpression of p53 protein is not directly related to hepatitis B x protein expression and is associated with neoplastic progression in hepatocellular carcinomas rather than hepatic preneoplasia, Mutat. Res. 462 (2000) 365–380. E. Tabor, Hepatitis C virus and hepatocellular carcinoma, Proc. Am. Assoc. Cancer Res. 40 (1999) 749–750. L. Tornillo, V. Carafa, J. Richter, G. Sauter, H. Moch, E. Minola, M. Gambacorta, L. Bianchi, R. Vecchione, L.M. Terracciano, Marked genetic similarities between hepatitis B virus-positive and hepatitis C virus-positive hepatocellular carcinomas, J. Pathol. 192 (2000) 307–312. R. Weil, H. Sirma, C. Giannini, D. Kremsdorf, C. Bessia, C. Dargemont, C. Bre´chot, A. Israel, Direct association and nuclear import of the hepatitis B virus x protein with the NF-kB inhibitor IkBa, Mol. Cell. Biol. 19 (1999) 6345–6354. K. Werling, Z. Szentirmay, A´. Szepesi, Zs. Schaff, F. Szalay, Zs. Szabo´, L. Telegdy, K. Da´vid, G. Stotz, Z. Tulassay, Hepatocyte proliferation and cell cycle phase fractions in chronic viral hepatitis C by image analysis method. Eur. J. Gastroenterol. Hepatol. 13 (2001) 1–5.