c-myc overexpression in hepatocarcinogenesis

Human PATHOLOGY VOLUME 35

November 2004

NUMBER 11

Editorial c-myc Overexpression in Hepatocarcinogenesis Hepatocellular carcinoma (HCC) is a devastating tumor that is diagnosed in more than 250,000 people worldwide each year.1 HCC has many etiologies, but the most common are chronic infections with hepatitis B or C viruses. There are an estimated 350 million people worldwide who are chronically infected with hepatitis B virus (HBV), and there are 170 million who are chronically infected with hepatitis C virus (HCV); both populations are at high risk for the development of hepatitis, cirrhosis, and finally HCC.2,3 Early HCC is usually asymptomatic, so that by the time of diagnosis, most patients present with multinodular tumors within the liver, which is not amenable to potentially curative surgical resection. The fact that the survival of untreated HCC is ⬍3% over 5 years underscores the need to identify key characteristics of chronically infected patients who are progressing toward tumor development. The identification of such early markers will improve the detection of patients with early tumors that are amenable to surgical resection and a chance for a cure. The development of HCC, like other tumor types, is a multistep process. For example, in the majority of chronic HBV and HCV infections, the elevated synthesis and accumulation of extracellular matrix in response to chronic hepatitis results in fibrosis, and later, in the development of cirrhosis. In many livers, there is extensive replacement of the liver parenchyma by connective tissue during the development of cirrhosis. The remaining hepatocytes in cirrhotic nodules often have elevated levels of proliferation in attempts to compensate and maintain normal levels of liver function. It is within these nodules that dysplastic hepatocytes appear, and it is within dysplastic nodules that foci of HCC appear.4 The progressive development of altered hepatic foci, adenomas, and HCC nodules has also been observed in woodchucks that are chronically infected with the HBV-related woodchuck hepatitis virus (WHV) in the absence of cirrhosis.5 HCC also develops in some

© 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2004.09.012

human HBV carriers on a background of chronic hepatitis instead of cirrhosis. HBV also encodes the “X” protein, which contributes significantly to hepatocellular transformation.6,7 In selected strains of X-transgenic mice,8,9 and in mice transgenic for transforming growth factor alpha (TGF␣)-c-myc10 or transforming growth factor beta (TGF␤1)-c-myc11, animals develop altered hepatic foci, adenomas, and then HCC nodules with age, also suggesting the multistep nature of hepatocarcinogenesis in vivo. In this case, HCC appears in the absence of intrahepatic inflammatory and fibrotic responses.8,9 These models provide opportunities to dissect the molecular pathways that contribute to hepatocarcinogenesis and to understand the temporal changes in which alterations in specific pathways result in an altered hepatocellular phenotype. Cancer is often characterized by the up-regulated expression of 1 or more oncogenes. However, early analyses did not show any oncogenes to be constitutively overexpressed in HBV-associated HCC,12-14 suggesting that HBV encodes 1 or more proteins that do the work of cellular oncogenes. However, the finding that HBV (and WHV) DNA fragments are integrated into the cellular DNA of carriers15-17 suggests that viral integration at a single or multiple sites in host DNA may result in the deregulated expression of oncogenes or tumor suppressor genes. This model appears to hold true for WHV-mediated carcinogenesis, in which cis activation of N-myc and c-myc oncogenes by insertionpromotion of viral DNA in or around these genes appears to be a common feature.18-20 It is important to note that virtually 100% of WHV carriers with altered myc expression develop HCC. Construction of transgenic mice by using a fragment of woodchuck HCC DNA containing WHV sequences adjacent to a mutated c-myc gene also resulted in a high frequency of HCC,21 suggesting that myc overexpression may be a central feature of hepatocarcinogenesis. In HCCs associated with ground squirrel hepatitis virus (GSHV) infections (GSHV is another HBV-like virus that naturally infects ground squirrels), overexpression of the c-myc gene was

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also observed but was associated with gene amplification and not with insertion-promotion.22 In contrast, the apparently random patterns of HBV integration into host DNA makes it unlikely that cis-acting mechanisms are common features of HBV-associated hepatocarcinogenesis. Instead, integrated viral sequences make the X protein that stimulates ras/raf/mitogenactivated protein kinase (MAPK) signaling23 and transcriptionally up-regulates expression of c-myc.24 Hence, up-regulated expression of c-myc may play an important role in HCC associated with chronic HBV and related virus infections, although whether constitutively elevated myc expression contributes significantly to the cause of HCC or is elevated as a consequence of tumor formation is not clear. Elevated myc expression has also been observed in HCV-associated HCC. It is significant that HCV coretransgenic mice develop steatosis, preneoplastic nodules, and finally HCC,25,26 implicating that core expression is important for tumor development. Further work has shown that HCV core trans-activates c-myc27 and insulin-like growth factor II28 and appears to cooperate with ras in the transformation of at least some cell lines.29-31 These observations suggest that HCV core may contribute to transformation by targeting up-regulated expression of components in several oncogenic signaling pathways, including c-myc. Independent observations have shown that the overexpression of c-myc in transgenic mice results in the development of HCC.32 Tumorigenesis is accelerated in double-transgenic mice who are overexpressing c-myc ⫹ TGF␣10 or c-myc ⫹ TGF␤1,11 suggesting that c-myc activation is an early step in multistep hepatocarcinogenesis. In TGF␣-c-myc transgenic mice,33 constitutive activation of ras/raf and of PI3K/Akt result in the phosphorylation of I␬B kinase, which inactivates I␬B. The latter was associated with the activation of nuclear factor kappa-B (NF-␬B) before tumor development, suggesting that these were early events in hepatocarcinogenesis. Overexpression of TGF␤1 in TGF␤1-c-myc transgenic mice may promote tumor development by inhibiting the growth of hepatocytes adjacent to HCC cells, the latter of which are resistant to the growth inhibitory effects of TGF␤1, probably because growthstimulatory signaling pathways (eg, phosphoinositol 3 kinase and NF-␬B) are constitutively activated in tumor compared to nontumor cells. In viral hepatocarcinogenesis, HBV X antigen and HCV core ⫹ nonstructural protein (NS) 5A proteins constitutively activate antiapoptotic pathways that block the negative growth-regulatory properties of TGF␤1.31,34,35 If this occurs in dysplastic cells or in cells from early HCC, it would also promote tumor development. Although the transgenic mice and tissue culture models above suggest that the constitutive overexpression of c-myc is an important feature of hepatocarcinogenesis, the results of microarray and other analyses do not paint such a clear picture. Some reports find upregulated expression of c-myc or c-myc-related factors in tumor compared with in nontumor liver.36,37 The mechanisms of c-myc up-regulated expression in HCC

include hypomethylation,38 point mutation,39 or gene amplification.36,40 The latter appears to be a late event in tumor progression.41 On the other hand, independent observations have reported a down-regulation of c-myc in HCC,42 whereas many other studies show no changes at all.43-45 c-myc expression was not elevated in a murine model of chemical hepatocarcarcinogenesis46 but was amplified in a rat model of HCC.47 In addition, c-myc is a transcriptional target of the oncogene ␤-catenin, which is mutated and constitutively active in roughly 20% of HCCs,48 but it is not clear whether activation of ␤-catenin is always associated with upregulated expression of c-myc.49-51 Hence, elevated cmyc expression appears to contribute significantly to hepatocarcinogenesis, but whether it is rate limiting early, late, or throughout many steps in the pathogenesis of this tumor type is still a major question. In this context, a study published in this issue of HUMAN PATHOLOGY52 convincingly addresses the clinicopathological significance of elevated c-myc expression in HCC and adjacent nontumor liver samples from more than 400 HCC patients, most of whom were HBV carriers. In this work, high-throughput tissue microarrays were analyzed for c-myc expression by fluorescence in situ hybridization and independently, by immunohistochemical staining. c-myc amplification was present in about 30% of HCC cases evaluated but was present in none of the corresponding nontumor liver samples that were analyzed. Despite this, HCC nodules demonstrated lower nuclear and cytoplasmic staining for c-myc (ie, lower c-myc activation) compared to adjacent nontumor samples from the same patients. These observations suggest that c-myc gene amplification does not lead to elevated c-myc expression in established HCC nodules and that elevated c-myc expression is important (possibly rate limiting) in preneoplastic liver. These observations are consistent with the hypothesis that the activation of c-myc in nontumor liver may occur through hypomethylation of the c-myc promoter, by point mutation, or by mechanisms that alter expression of the wild-type product. Although hypomethylation and point mutations in the c-myc have been documented during tumor progression and correlate with invasiveness and metastases,53,54 they do not provide an explanation for the activation of myc in preneoplastic liver. Given that HBV X antigen is strongly expressed in peritumor tissue compared to HCC cells,55 that c-myc is a transcriptional target of HBxAg,56 and that Wnt (␤catenin) signaling is activated in X antigen-expressing cells,57 suggest that X antigen may be responsible for the overexpression of wild-type c-myc in preneoplastic liver cells from which transformed cells arise. The fact that HCV also expresses and replicates at considerably higher levels in liver compared to tumor58 is consistent with the hypothesis that HCV also activates c-myc in preneoplastic liver and, perhaps to a lesser extent, in tumor. A key insight provided by this article derives from the observation that the amplification of the myc gene in HCC, which has been reported by many groups, does not correspond with increased c-myc expression. In addition, this work suggests that elevated

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c-myc expression is likely to be an early marker that signals elevated risk for tumor development. In addition, the inverse correlation between tumor aggressiveness (as measured by the extent of venous penetration into the tumor and the absence of tumor encapsidation) and nuclear c-myc expression suggests that activated c-myc does not contribute significantly to tumor progression. This work also helps to put into perspective the data from microarrays, which are not performed with large numbers of samples and often are not validated by immunohistochemical staining (for myc in particular). In addition, the results of this work help to distinguish which of the animal models and tissue culture systems described in this editorial are likely to be relevant for further dissecting the mechanisms whereby c-myc is activated in the early stages of tumorigenesis. The activation of myc in preneoplastic tissue is also consistent with the insertion-promotion model in chronic WHV infection outlined in this article, in which constitutively high levels of myc expression in chronically infected woodchucks is associated with 100% tumor incidence within 2-3 years of WHV infection. Finally, the observation that the expression of amplified c-myc is silenced in HCC may have several explanations. Given the fact that the overexpression of myc alone triggers apoptosis59 but that HBV and HCV activate multiple signaling pathways that rescue cells from apoptosis, when virus gene expression and replication is reduced or absent in tumor cells, there is a parallel decrease in myc expression in the surviving tumor cells. In addition, as tumors develop and mutations accumulate, it may be that elevated myc is no longer rate limiting for hepatocellular survival and that there is no longer any selective pressure to maintain elevated myc expression during tumor progression. MARK A. FEITELSON, PHD Department of Pathology, Anatomy, and Cell Biology and Department of Microbiology and Immunology Kimmel Cancer Center, Thomas Jefferson University Philadelphia, PA REFERENCES 1. Feitelson MA: HBV and cancer, in Notkins AL, Oldstone MBA (eds): Concepts in Viral Pathogenesis, vol II. New York, NY, SpringerVerlag, 1986, pp 269-275 2. Beasley RP, Hwang LY: Epidemiology of hepatocellular carcinoma, in Vyas GN, Dienstag JL, Hoofnagle JH (eds): Viral Hepatitis and Liver Disease. New York, NY, Grune and Stratton, 1984, pp 209-224 3. Purcell RH: Hepatitis viruses: Changing patterns of human disease. Proc Natl Acad Sci U S A 91:2401-2406, 1994 4. Lencioni R, Caramella D, Bartolozzi C, et al: Long-term follow-up study of adenomatous hyperplasia in liver cirrhosis. Ital J Gastroenterol 26:163-168, 1994 5. Fu XX, Su CY, Lee Y, et al: Insulin-like growth factor II expression and oval cell proliferation associated with hepatocarcinogenesis in woodchuck hepatitis virus carriers. J Virol 62:3422-3430, 1988 6. Feitelson MA, Duan LX: Hepatitis B virus x antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma. Am J Pathol 150:1141-1157, 1997 7. Henkler F, Koshy R: Hepatitis B virus transcriptional activa-

tors: Mechanisms and possible role in oncogenesis. J Viral Hepat 3:109-121, 1996 8. Ueda H, Ullrich SJ, Gangemi JD, et al: Functional inactivation but not structural mutation of p53 causes liver cancer. Nat Genet 9:41-47, 1995 9. Kim CM, Koike K, Saito I, et al: HBx gene of HBV induces liver cancer in transgenic mice. Nature 351:317-320, 1991 10. Murakami H, Sanderson ND, Nagy P, et al: Transgenic mouse model for synergistic effects of nuclear oncogenes and growth factors in tumorigenesis: Interaction of c-myc and transforming growth factor alpha in hepatic oncogenesis. Cancer Res 53:17191723, 1993 11. Factor VM, Kao CY, Santoni-Rugiu E, et al: Constitutive expression of mature transforming growth factor ␤1 in the liver accelerates hepatocarcinogenesis in transgenic mice. Cancer Res 57: 2089-2095, 1997 12. Gu JR: Molecular aspects of human hepatic carcinogenesis. Carcinogenesis 9:697-703, 1998 13. Lee HS, Rajagopalan MS, Vyas GN: A lack of direct role of hepatitis B virus in the activation or ras and c-myc oncogenes in human hepatocellular carcinogenesis. Hepatology 8:1116-1120, 1988 14. Varmus HE: Do hepatitis B viruses make a genetic contribution to primary hepatocellular carcinoma?, in Vyas GN, Dienstag JL, Hoofnagle JH (eds): Viral Hepatitis and Liver Disease. New York, NY, Grune and Stratton, 1984, pp 411-414 15. Shafritz DA, Rogler CE: Molecular characterization of viral forms observed in persistent hepatitis infections, chronic liver disease and hepatocellular carcinoma in woodchucks and humans, in Vyas GN, Dienstag JL, Hoofnagle JH (eds): Viral Hepatitis and Liver Disease. New York, NY, Grune and Stratton, 1984, pp 225-243 16. Rutter WJ, Ziemer M, Ou J, et al: Transcription units of HBV genes and structure and expression of integrated viral sequences, in Vyas GN, Dienstag JL, Hoofnagle JH (eds): Viral Hepatitis and Liver Disease. New York, NY, Grune and Stratton, 1984, pp 67-86 17. Rogler CE, Summers J: Cloning and structural analysis of integrated WHV sequences from a chronically infected liver. J Virol 50:832-837, 1984 18. Hsu TY, Moroy T, Etiemble J, et al: Activation of c-myc by woodchuck hepatitis virus insertion in hepatocellular carcinoma. Cell 55:627-635, 1988 19. Fourel G, Trepo C, Bougueleret L, et al: Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours. Nature 347:294-298, 1990 20. Moroy T, Marchio A, Etiemble J, et al: Rearrangement and enhanced expression of c-myc in hepatocellular carcinoma of hepatitis virus infected woodchucks. Nature 324:276-279, 1986 21. Etiemble J, Degott C, Renard CA, et al: Liver specific expression and high oncogenic efficiency of a c-myc transgene activated by woodchuck hepatitis virus insertion. Oncogene 9:727-737, 1994 22. Transy C, Fourel G, Robinson WS, et al: Frequent amplification of c-myc in ground squirrel liver tumors associated with past or ongoing infection with a hepadnavirus. Proc Natl Acad Sci U S A 89:3874-3878, 1992 23. Benn J, Schneider RJ: Hepatitis B virus HBx protein activates ras-GTP complex formation and establishes a ras, raf, MAP kinase signaling cascade. Proc Natl Acad Sci U S A 91:10350-10354, 1994 24. Balsano C, Avantaggiati ML, Natoli G, et al: Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the c-myc protooncogene at the transcriptional level. Biochem Biophys Res Commun 176:985-992, 1991 25. Moriya K, Fujie H, Shintani Y, et al: The core protein of hepatitis C virus induced hepatocellular carcinoma in transgenic mice. Nat Med 4:1065-1067, 1998 26. Moriya K, Yotsuyanagi H, Shintani Y, et al: Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J Gen Virol 78:1527-1531, 1997 27. Ray RB, Lagging LM, Meyer K, et al: Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein. Virus Res 37:209-220, 1995 28. Lee S, Park U, Lee YI: Hepatitis C virus core protein transactivates insulin-like growth factor II gene transcription through acting concurrently on Egr1 and Sp1 sites. Virology 283:167-177, 2001 29. Ray RB, Lagging M, Meyer K, et al: Hepatitis C virus core

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protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype. J Virol 70:4438-4443, 1996 30. Chang J, Yang SH, Cho YG, et al: Hepatitis C virus core from two different genotypes has an oncogenic potential but is not sufficient for transforming primary rat embryo fibroblasts in cooperation with the H-ras oncogene. J Virol 72:3060-3065, 1998 31. Tsuchihara K, Hijikata M, Fukuda K, et al: Hepatitis C virus core protein regulates cell growth and signal transduction pathway transmitting growth stimuli. Virology 258:100-107, 1999 32. Terradillos O, Billet O, Renard CA, et al: The hepatitis B virus X gene poteniates c-myc induced liver oncogenesis in transgenic mice. Oncogene 14:395-404, 1997 33. Factor V, Oliver AL, Panta GR, et al: Roles of Akt/PKB and IKK complex in constitutive induction of NF-␬B in hepatocellular carcinomas of transforming growth factor ␣/c-myc transgenic mice. Hepatology 34:32-41, 2001 34. Kato N, Yoshida H, Kiolo ONS, et al: Activation of intracellular signaling by hepatitis B and C viruses: C-viral core is the most potent signal inducer. Hepatology 32:405-412, 2000 35. He Y, Nakao H, Tan SL, et al: Subversion of cell signaling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and p85 phosphatidylinositol 3-kinase. J Virol 76:92079217, 2002 36. Takeo S, Hiroshi A, Kusano N, et al: Examination of oncogene amplification by genomic DNA microarray in hepatocellular carcinomas: Comparison with comparative genomic hybridization analysis. Cancer Genet Cytogenet 130:127-132, 2002 37. Shirota Y, Kaneko S, Honda M, et al: Identification of differentially expressed genes in hepatocellular carcinoma with cDNA microarrays. Hepatology 33:832-849, 2001 38. Shen L, Fang J, Qiu D, et al: Correlation between DNA methylation and pathological changes in human hepatocellular carcinoma. Hepato-gastroenterology 45:1753-1759, 1998 39. Pascale RM, Simile MM, Feo F: Genomic abnormalities in hepatocarcinogenesis: Implications for a chemopreventive strategy. Anticancer Res 13:1341-1356, 1993 40. Wong N, Lai P, Lee SW, et al: Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: Relationship to disease stage, tumor size, and cirrhosis. Am J Pathol 154:37-43, 1999 41. Wang Y, Wu MC, Sham JS, et al: Prognostic significance of c-myc and AIB1 amplification in hepatocellular carcinoma: A broad survey using high-throughput tissue microarray. Cancer 95:23462352, 2002 42. Delpuech O, Trabut JB, Carnot F, et al: Identification, using cDNA macroarray analysis, of distinct gene expression profiles associated with pathological and virological features of hepatocellular carcinoma. Oncogene 21:2926-2937, 2002 43. Okabe H, Satoh S, Kato T, et al: Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: Identification of gene involved in viral carcinogenesis and tumor progression. Cancer Res 61:2129-2137, 2001

44. Paradis V, Bieche I, Dargere D, et al: Molecular profiling of hepatocellular carcinomas (HCC) using a large-scale real-time RTPCR approach: Determination of a molecular diagnostic index. Am J Pathol 163:733-741, 2003 45. Goldenberg D, Ayesh S, Schneider T, et al: Analysis of differentially expressed gene in hepatocellular carcinoma using cDNA arrays. Mol Carcinog 33:113-124, 2002 46. Anna CH, Iida M, Sills RC, et al: Expression of potential beta-catenin targets, cyclin D1, c-Jun, c-myc, E-cadherin, and EGFR in chemically induced hepatocellular neoplasms from B6C3F1 mice. Toxicol Appl Pharmacol 190:135-145, 2003 47. Motiwala T, Ghoshal K, Das A, et al: Suppression of the protein tyrosine phosphatase receptor type O gene (PTPRO) by methylation in hepatocellular carcinoma. Oncogene 22:6319-6331, 2003 48. Miyoshi Y, Iwao K, Nagasawa Y, et al: Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3. Cancer Res 58:2524-2527, 1998 49. Prange W, Breuhahn K, Fischer F, et al: Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genes. J Pathol 201:250-259, 2003 50. Cui J, Zhou X, Liu Y, et al: Alterations of beta-catenin and Tcf-4 instead of GSK-3beta contribute to activation of Wnt pathway in hepatocellular carcinoma. Chin Med J 116:1885-1892, 2003 51. Shang XZ, Zhu H, Lin K, et al: Stabilized beta-catenin promotes hepatocyte proliferation and inhibits TNFalpha-induced apoptosis. Lab Invest 84:332-341, 2004 52. Chan KL, Guan XY, Ng IOL: High-throughput tissue microarray analysis of c-myc activation in chronic liver diseases and hepatocellular carcinoma. HUM PATHOL 35:1324-1331, 2004 53. Shen L, Fang J, Qiu D, et al: Correlation between DNA methylation and pathological changes in human hepatocellular carcinoma. Hepatogastroenterology 45:1753-1759, 1998 54. Pascale RM, Simile MM, Feo F: Genomic abnormalities in hepatocarcinogenesis: Implications for a chemopreventive strategy. Anticancer Res 13:1341-1356, 1993 55. Wang W, London WT, Feitelson MA: HBxAg in HBV carrier patients with liver cancer. Cancer Res 51:4971-4977, 1991 56. Balsano C, Avantaggiati ML, Natoli G, et al: Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the c-myc protooncogene at the transcriptional level. Biochem Biophys Res Commun 176:985-992, 1991 57. Cha MY, Kim CM, Park YM, et al: Hepatitis B virus X protein is essential for the activation of Wnt-␤-catenin signaling in hepatoma cells. Hepatology 39:1683-1693, 2004 58. Takeda S, Shibata M, Morishima T, et al: Hepatitis C virus infection in hepatocellular carcinoma. Cancer 70:2255-2259, 1992 59. Ueda K, Ganem D: Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II. J Virol 70:1375-1383, 1996

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