Geneviève Fourel

Hepatocellular carcinoma in WHV/N-myc2 transgenic mice: oncogenic mutations of beta-catenin and synergistic effect of p53 null alleles.

The intronless N-myc2 gene was originally identified as the major target of hepatitis virus insertion in woodchuck liver tumors. Here we report that transgenic mice carrying the N-myc2 gene controlled by woodchuck hepatitis virus (WHV) regulatory sequences are highly predisposed to liver cancer. In a WHV/N-myc2 transgenic line, hepatocellular carcinomas or adenomas arose in over 70% of mice, despite barely detectable expression of the methylated transgene in liver cells. Furthermore, a transgenic founder carrying unmethylated transgene sequences succumbed to a large liver tumor by the age of two months, demonstrating the high oncogenicity of the woodchuck N-myc2 retroposon. Stabilizing mutations or deletions of β-catenin were found in 25% of liver tumors and correlated with reduced tumor latency (P<0.05), confirming the important role of β-catenin activation in Myc-induced tumorigenesis. The ability of the tumor suppressor gene p53 to cooperate with N-myc2 in liver cell transformation was tested by introducing a p53-null allele into WHV/N-myc2 transgenic mice. The loss of one p53 allele in transgenic animals markedly accelerated the onset of liver cancer (P=0.0001), and most tumors of WHV/N-myc2 p53+/Δ mice harbored either a deletion of the wt p53 allele or a β-catenin mutation. These findings provide direct evidence that activation of N-myc2 and reduction of p53 levels act synergistically during multistage carcinogenesis in vivo and suggest that different genetic pathways may underlie liver carcinogenesis initiated by a myc transgene.

Cellular and viral trans-acting factors modulate N-myc2 promoter activity in woodchuck liver tumors

Activation of the N-myc2 oncogene by integration of woodchuck hepatitis virus (WHV) DNA is a central event in woodchuck liver oncogenesis. In this study, we have evaluated the influence of several cellular and viral trans-acting factors and mediators of inflammation on N-myc2 promoter activity in hepatoma cell lines. Ets oncoproteins, including Ets1, Ets2 and PEA3 efficiently activated a chimeric N-myc2 promoter/luciferase reporter gene. By electrophoretic mobility shift assays, we show that Ets1 and Ets2 proteins can efficiently bind two consensus Ets sites located within a 59 bp sequence upstream of the N-myc2 transcription start site. Site-directed mutagenesis of these Ets-binding motifs abolished transactivation of the N-myc2 promoter by Ets proteins. Addition of interleukin-6 (IL-6) induced a weak but reproducible activation of the N-myc2 promoter, while IL-1 was ineffective. We further show that the N-myc2 promoter can be transactivated by the hepadnavirus X protein, and that distal promoter sequences are required for both IL-6 and X responsiveness. Similar effects of these factors were observed in the context of the N-myc2 promoter activated by WHV cis-regulatory elements. In view of the high-level expression of the N-myc2 oncogene in most woodchuck liver tumors, the Ets oncoproteins, inflammation-associated cytokine IL-6 and the viral X transactivator might play important roles in hepadnavirus-associated tumorigenesis.

Integration of hepatitis virus DNA near c-myc in woodchuck hepatocellular carcinoma

SummaryA total of 33 hepatocellular carcinomas, induced in woodchucks by chronic infection with woodchuck hepatitis virus (WHV), a virus closely related to the human hepatitis B virus, were analyzed for the state of viral DNA, the expression of viral genes and of different cellular proto-oncogenes. Low levels of viral replication and presence of integrated viral forms including sequences of the enhancer element, appeared as a general rule in these tumors. Enhanced expression of one or more of the nuclear protooncogenes: c-myc, N-myc, c-fos, c-jun and jun-B was frequently observed. In two hepatomas, elevated expression and allelic alterations of c-myc were subsequent to integration of WHV DNA near the c-myc coding domain. The viral strategy for insertional activation of c-myc in these tumors appeared basically identical to that of mammalian retroviruses in T-cell lymphomas of mice and rats. Whether insertional mutagenesis of different oncogenes may be more generally linked to liver oncogenesis induced by WHV and hepatitis B viruses remains to be determined.

Integrated Viral Sequences may Contribute in cis and in trans to Hepatocarcinogenesis in Woodchuck Hepatitis Virus-infected Woodchucks

Our recent studies of woodchuck liver tumors have shown that induction of the oncogenic process can be achieved by insertional mutagenesis of the myc protooncogenes by woodchuck hepatitis virus (WHV) DNA. The N-myc2 intronless gene represents a major target for viral insertion. Activation of N-myc2 expression resulting from a cis-acting effect of the viral enhancers on the N-myc2 promoter is strongly associated with the development of liver tumors in woodchuck. Further studies of the expression of integrated WHV sequences in liver tumors, by Northern blotting and cDNA cloning, have revealed abnormal WHV transcripts and viral-host co-transcripts carrying truncated X gene sequences. In one case, the 3′ truncated X gene fused to cellular sequences retained the ability to transactivate a heterologous promoter. These data suggest that, like in hepatitis B virus-related hepatocellular carcinoma, integrated viral sequences in woodchuck tumors may contribute to alteration of normal cell growth control through deregulated expression of the WHV X trans-activator.

The Role of Integrated Hepadnavirus Sequences in Hepatocellular Carcinoma

In woodchuck hepatomas induced by woodchuck hepatitis virus (WHV) infection, the predominant role of viral integration has been pointed out. In more than 50% of cases, integrated viral sequences have been shown to interrupt myc family genes including c-myc, N-myc and a woodchuck specific retroposon called N-mycl. In a manner similar to that observed in retroviral insertions into myc genes in murine T lymphoma, activated expression from the myc promoters resulted from nearby integration of the viral enhancer. In ground squirrel hepatomas related to past or ongoing ground squirrel hepatitis virus (GSHV) infection, c-myc is amplified in about 50% of the tumors analyzed. In these tumors, integration of viral DNA in the host genome was not apparently implicated. Transgenic mice carrying a mutated c-myc gene and adjacent WHV sequences were constructed. Virtually all mice from two different strains developed hepatocellular carcinoma with a mean latency period of 8–12 months. A direct correlation was established between the level of c-myc expression in the liver and the onset of liver tumors. Woodchuck c-myc mRNA driven by the normal P1 and P2 promoters and WHV-specific transcript encoding viral surface antigens were produced in a strictly coregulated fashion during development and tumorigenesis, indicating a predominant regulatory influence of the viral enhancer.