Wnt signaling and hepatocarcinogenesis: the hepatoblastoma model.

The Wnt/ß-catenin pathway plays a key role in liver development, regeneration and tumorigenesis. Among human cancers tightly linked to abnormal Wnt/ß-catenin signaling, hepatoblastoma (HB) presents with the highest rate (50-90%) of ß-catenin mutations. HB is the most common malignant tumor of the liver in childhood. This embryonic tumor differs from hepatocellular carcinoma by the absence of viral etiology and underlying liver disease, and by distinctive morphological patterns evoking hepatoblasts, the bipotent precursors of hepatocytes and cholangiocytes. Recent studies of the molecular pathogenesis of hepatoblastoma have led to identify two major tumor subclasses resembling early and late phases of prenatal liver development and presenting distinctive chromosomal alterations. It has been shown that the molecular signature of Wnt/ß-catenin signaling in hepatoblastoma is mainly imposed by liver context, but differs according to developmental stage. Finally, the differentiation stage of tumor cells strongly influences their invasive and metastatic properties, therefore affecting clinical behavior.

Molecular biology of the hepatitis B virus and role of the X gene.

The hepatitis B virus (HBV) is a widespread human pathogen and a major health problem in many countries. Molecular cloning and sequencing of the viral DNA genome has demonstrated a small and compact structure organized into four overlapping reading frames that encode the viral proteins. Besides structural proteins of the core and the envelope, HBV encodes a DNA polymerase with reverse transcriptase activity, a secreted antigen of unknown function, and a transcriptional activator that is essential for viral replication. Major steps of the viral life cycle have been unraveled, including transcription of all viral RNAs from nuclear covalently closed circular DNA (cccDNA), followed by encapsidation of pregenomic RNA, a more-than-genome length transcript, and reverse transcription of pregenomic RNA leading to asymmetric synthesis of the DNA strands. Although HBV has been recognized as a human tumor virus, no direct transforming activity could be evidenced in different cellular and animal models. However, the transcriptional regulatory protein HBx encoded by the X gene is endowed with weak oncogenic activity. HBx harbors pleiotropic activities and plays a major role in HBV pathogenesis and in liver carcinogenesis.

[Fundamental and translational research on hepatocellular carcinoma in 2008: forces and priorities]. / Recherche fondamentale et translationnelle sur le carcinome hépatocellulaire en 2008 : avancées récentes et perspectives.

Hepatocellular carcinogenesis is usually the result of a muti-step process. It begins with an exposure to various risk factors; followed by the development of a chronic hepatitis and cirrhosis that is a pre-neoplastic step; and finally after the occurrence of an hepatocellular carcinoma (HCC), different molecular events control aggressiveness of the tumors. The aim of this work was to identify in the international context, forces and priorities of the fundamental and translational HCC research.

Physical and functional cooperation between AP-1 and beta-catenin for the regulation of TCF-dependent genes.

Stabilization of cytoplasmic beta-catenin is a hallmark of a variety of cancers. The stabilized beta-catenin is able to translocate to the nucleus, where it acts as a transcriptional activator of T-cell factor (TCF)-regulated genes. beta-Catenin may cross-talk with many signalling cascades to activate target genes. Whether beta-catenin cooperates with AP-1, another transcriptional complex activated during tumorigenesis is not fully clarified. We show that beta-catenin co-immunoprecipitates with c-Jun and c-Fos. GST pull-down experiments indicate a physical association of the armadillo repeat domain of beta-catenin with the DNA-binding domain of c-Jun and of the C-terminal domain of beta-catenin with the N-terminal domain of c-Fos. Promoter studies indicate that overexpression of AP-1 activates the transcription of two beta-catenin target genes, cyclin D1 and c-myc, by a mechanism independent of the AP-1 site, and fully dependent on the TCF-binding site. We further demonstrate that AP-1/beta-catenin synergism is involved during serum-induced cyclin D1 transcriptional activation. We identify a TCF-binding site on the cyclin D1 promoter which binds in vivo a complex induced by serum, containing beta-catenin, TCF4, c-Fos, c-Jun, JunB and JunD. This novel mechanism of interaction between two signalling cascades might contribute to the potentiation of malignancy.

Overexpression of regenerating islet-derived 1 alpha and 3 alpha genes in human primary liver tumors with beta-catenin mutations.

The Wnt/beta-catenin signaling pathway is activated in many human hepatocellular carcinomas (HCC). We tried to identify the genes involved in carcinogenesis and progression of HCC with beta-catenin mutations. We used PCR-based subtractive hybridization to compare gene expression between malignant and benign components of a human HCC occurring in pre-existing adenoma activated for beta-catenin. Two of the genes identified belong to the Regenerating gene (REG) family. They encode the Regenerating islet-derived 3 alpha (REG3A/HIP/PAP/REG-III) and 1 alpha (REG1A) proteins, both involved in liver and pancreatic regeneration and proliferation. Using siRNA directed against beta-catenin, we demonstrated that REG3A is a target of beta-catenin signaling in Huh7 hepatoma cells. The upregulation of REG3A and REG1A expression is significantly correlated to the beta-catenin status in 42 HCC and 28 hepatoblastomas characterized for their beta-catenin status. Thus, we report strong evidence that both genes are downstream targets of the Wnt pathway during liver tumorigenesis.

Preliminary results of interferon-alpha therapy on woodchuck hepatitis virus-induced hepatocarcinogenesis: possible benefit in female transgenic mice.

BACKGROUND: C-myc activation is a potent oncogenic event in hepatocarcinogenesis. The aim of this study was to test the preventive effect of interferon-alpha (IFN-alpha) on the development of dysplasia and subsequent hepatocellular carcinoma (HCC) in transgenic (Tg) mice overexpressing c-myc in the liver. METHODS: The WHV/c-myc Tg mice recapitulating woodchuck hepatitis virus-induced hepatocarcinogenesis were treated with IFN-alpha, starting early in life until sacrifice at pre-neoplastic or neoplastic stages. Transgene expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), hepatocyte proliferation was assessed by bromodeoxyuridine incorporation and RT-PCR for proliferating cell nuclear antigen, and apoptosis was assessed by in situ nick-end-labeling of DNA. RESULTS: C-myc expression and hepatocyte proliferation were significantly reduced in treated female mice, without modification of apoptosis, correlating with a lower severity of dysplasia in 9/12 treated animals at pre-neoplastic stages. At the neoplastic stage, 2/3 treated females neither exhibited carcinoma nor dysplasia, while all 6/6 untreated mice and 3/3 treated males developed carcinomas. CONCLUSIONS: Inhibition of c-myc and hepatocyte proliferation by long-term administration of IFN-alpha was associated with a decrease, or a delay, of oncogenesis in the mouse Tg HCC model. Whether c-myc and hepatocyte proliferation down-regulation could be relevant parameters of IFN-alpha efficiency for hepatocarcinogenesis prevention in cirrhotic patients should be established.

[Endobronchial and laryngeal metastasis. An unusual form of dissemination of the hypernephroma]. / Metástasis endobronquiales y laríngeas. Una forma poco habitual de diseminación del hipernefroma.

OBJECTIVE: To describe uncommon forms of dissemination of hypernephroma. METHODS: A case of hypernephroma that metastasized to the laryngeal vallecula and bronchi is presented. Our findings were compared with those reported in the literature. The diagnostic, radiological, clinical aspects and route of dissemination of some atypical sites of metastasis are discussed. RESULTS/CONCLUSIONS: It is important to be familiar with these atypical sites of metastasis since these lesions may appear at the same time or before the primary tumor is detected. A high index of suspicion will make a major impact on treatment and prognosis. The radiological findings are undoubtedly of enormous value, although histological confirmation is necessary in order to make the correct diagnosis.

Genetics of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the human cancers clearly linked to viral infections. Although the major viral and environmental risk factors for HCC development have been unravelled, the oncogenic pathways leading to malignant transformation of liver cells have long remained obscure. Recent outcomes have been provided by extensive allelotype studies which resulted in a comprehensive overview of the main genetic abnormalities in HCC, including DNA copy gains and losses. The differential involvement of the p53 tumor-suppressor gene in tumors associated with various risk factors has been largely clarified. Evidence for a crucial role of the reactivation of the Wnt/beta-catenin pathway, through mutations in the beta-catenin and axin genes in 30-40% of liver tumors, represents a major breakthrough. It has also been shown that the Rb pathway is frequently disrupted by methylation-dependent silencing of the p16INK4A gene and stimulation of Rb degradation by a proteosomal subunit. Presently, the identification of candidate oncogenes and tumor suppressors in the most frequently altered chromosomal regions is a major challenge. Great insights will come from integrating the signals from different pathways operating at preneoplastic and neoplastic stages. This search might, in time, permit an accurate evaluation of the major targets for therapeutic treatments.

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 beta-catenin were found in 25% of liver tumors and correlated with reduced tumor latency (P<0.05), confirming the important role of beta-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+/Delta mice harbored either a deletion of the wt p53 allele or a beta-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. Oncogene (2000).

Activation of beta-catenin in epithelial and mesenchymal hepatoblastomas.

Wnt/beta-catenin signaling is frequently activated in cancer cells by stabilizing mutations of beta-catenin or loss-of-function mutations of the APC tumor suppressor gene. We have analysed the role of beta-catenin in the pathogenesis of hepatoblastoma (HB), an embryonic liver tumor occurring mainly in children under 2 years of age. Sequence analysis of the beta-catenin NH2-terminal domain in 18 epithelial and mixed HBs revealed missense mutations in the GSK3beta phosphorylation motif or interstitial deletions in 12 tumors (67%). In the remaining cases, no truncating mutation of APC could be evidenced. Immunohistochemical analysis of beta-catenin in 11 HBs demonstrated nuclear/cytoplasmic accumulation of the protein in all tumors analysed, with predominant nuclear beta-catenin immunostaining in undifferentiated cells. Membranous beta-catenin localization was preserved only in fetal-type tumoral hepatocytes and was associated with E-cadherin expression. Moreover, we show that beta-catenin is aberrantly overexpressed in a large spectrum of tumor components, including hepatocyte-like cells at various differentiation stages and heterologous elements such as squamous, osteoid and chrondroid tissues, and in occasional other mesenchymally-derived cells. These data strongly suggest that activation of beta-catenin signaling is an obligatory step in HB pathogenesis, and raise the possibility that it interferes with developmental signals that specify different tissue types at early stages of hepatic differentiation.

Nuclear accumulation of mutated beta-catenin in hepatocellular carcinoma is associated with increased cell proliferation.

Inappropriate activation of the Wnt pathway resulting from beta-catenin gene alterations has recently been implicated in the development of hepatocellular carcinoma (HCC). To explore the in vivo effects of mutated beta-catenin, HCC specimens from 32 patients carrying one or several tumors were screened for somatic mutations in exon 3 of the beta-catenin gene, and the expression and subcellular localization of beta-catenin was studied by immunohistochemistry. Missense mutations or interstitial deletions in beta-catenin exon 3 were detected in 12 of 35 (34%) HCC samples. After immunostaining, most tumors exhibited increased membranous and/or cytoplasmic expression of beta-catenin compared with adjacent nontumoral liver. Strong nuclear accumulation of beta-catenin was observed either focally or uniformly in 15 of 35 (43%) tumor specimens, but not in cirrhotic nodules or dysplastic liver cells in adjacent liver. Aberrant nuclear expression of beta-catenin was significantly associated with the presence of mutations in the beta-catenin gene (P < 0.005). Moreover, nuclear beta-catenin staining correlated significantly with increased Ki-67 proliferative index in tumor (P < 0.001) and seemed to be associated with poor outcome in patients with HCC. In conclusion, our data indicate that activation of the Wnt/beta-catenin pathway in HCC results mainly from somatic mutations in the beta-catenin gene and may promote tumor progression by stimulating tumor cell proliferation.

Identification of three distinct regions of allelic deletions on the short arm of chromosome 8 in hepatocellular carcinoma.

The chromosome 8p is associated with a large number of allelic imbalances in epithelial tumors including hepatocellular carcinoma (HCC). However, no tumor suppressor gene has been identified so far in this particular region of the genome. To further clarify the pattern of allelic deletions on chromosome 8p in HCC, we have undertaken high-density polymorphic marker analysis of 109 paired normal and primary tumor samples using 40 microsatellites positioned every 2 cm in average throughout 8p. We found that 60% of the tumors exhibited loss of heterozygosity (LOH) at one or more loci at 8p with three distinct minimal deleted areas: a 13 cm region in the distal part of 8p21, a 9 cm area in the more proximal portion of 8p22 and a 5 cm area in 8p23. These data strongly suggest the presence of at least three novel tumor suppressor loci on 8p in hepatocellular carcinoma.

Pro-apoptotic effect of the hepatitis B virus X gene.

The hepatitis B virus (HBV) is a common human pathogen that causes acute and chronic liver disease. Persistent HBV infection is strongly associated with the development of hepatocellular carcinoma. The contribution of the viral regulatory protein HBx in liver oncogenesis has been supported by our recent studies in a transgenic mouse model, showing that HBx cooperates with c-myc by accelerating the onset of primary liver tumors. Here we show that liver expression of HBx is associated with increased rates of spontaneous apoptosis in liver cells from two different transgenic lines. In transient transfection assays, overexpression of HBx in the established hepatocyte cell line MMHD3 and in human hepatoma cells HepG2 was found to induce apoptosis in a dose-dependent manner. These data suggest that HBx might trigger an apoptotic process in HBV-infected hepatocytes, in turn possibly favoring liver regeneration and accumulation of genetic alterations, ultimately leading to liver cell transformation in chronically infected patients.

p53-independent apoptotic effects of the hepatitis B virus HBx protein in vivo and in vitro.

The hepatitis B virus protein HBx is a promiscuous transactivator implicated in both cell growth and death and in the development of hepatocellular carcinoma. We recently reported that HBx can potentiate c-myc-induced liver oncogenesis in a transgenic model where low level expression of HBx induces no pathology. To assess if HBx could affect the hepatocyte turnover, we investigated the HBx-elicited apoptotic responses in transgenic livers and in primary hepatocyte cultures. Here we show that transgenic expression of HBx is associated with a twofold increase of spontaneous cell death in the mouse liver. The finding that apoptosis was enhanced to similar extents in HBx mice carrying homozygous p53 null mutations implied that functionally intact p53 was not required to transduce the death signal. A direct, dose-dependent apoptotic function of HBx was demonstrated in transient transfections of liver-derived cell lines. We further show that stable expression of HBx at low, presumably physiological levels in primary hepatocytes, induced cellular susceptibility to diverse apoptotic insults, including growth factor deprivation, treatment with anti-Fas antibodies or doxorubicine and oxidative stress. HBx expression, but not p53 status profoundly affected the commitment of cells to die upon apoptotic stimuli. These data strengthen the notion that HBX may contribute to HBV pathogenesis by enhancing apoptotic death in the chronically infected liver.

Hepatitis B viruses and cancerogenesis.

Hepatitis B virus (HBV) is a small, enveloped DNA virus which primarily infects liver cells and causes acute and persistent liver disease. Chronic HBV infection, frequently associated with cirrhosis and eventually hepatocellular carcinoma (HCC), represents a major health problem in the world. HBV is the prototype member of the hepadnavirus family, which includes several related mammalian viruses also implicated in liver carcinogenesis in the host. Although epidemiological evidence has clearly linked HBV infection with HCC development, the precise role of the virus and the molecular mechanisms of liver cell transformation remain elusive. Here we discuss potential oncogenic strategies of HBV, ranging from indirect mechanisms related to chronic necroinflammatory disease and to the effects of viral gene products on cell proliferation and apoptosis, to direct insertional activation of cellular (onco)genes. Presently, vaccination of high risk populations represents a major way to prevent the development of HBV-related liver cancer.

Infection of WHV/c-myc transgenic mice with Moloney murine leukaemia virus and proviral insertion near the syndecan-4 gene in an early liver tumour.

The capacity of Moloney murine leukaemia virus (MoMLV) to infect neonatal hepatocytes and to accelerate liver carcinogenesis was examined in a transgenic mouse model. WHV/c-myc mice which are highly susceptible to the development of liver tumours were infected with MoMLV shortly after birth, when expression of the murine ecotropic retroviral receptor gene was still detectable in the neonatal liver. All MoMLV-infected transgenic mice and non-transgenic littermates succumbed to T-cell lymphomas within 2-9 months; during this period of time, three infected transgenic animals developed primary hepatocellular carcinomas. Remarkably, one of these liver tumours arose significantly faster than tumours from uninfected WHV/c-myc controls, and it harboured a unique MoMLV provirus. The provirus integration site was located 5.5 kb upstream of the first exon of the syndecan-4 gene, which encodes a heparan sulphate proteoglycan implicated in growth factor activation and protein kinase C distribution in focal adhesions. Our data provide evidence for clonal MoMLV provirus integration in a hepatocellular carcinoma, and indicate that parenchymal liver cells may be susceptible to MoMLV infection following neonatal inoculation.

Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading to liver cell transformation remain largely unknown. The Wnt-beta-catenin pathway is activated in colon cancers and some melanoma cell lines, but has not yet been investigated in HCC. We have examined the status of the beta-catenin gene in different transgenic mouse lines of HCC obtained with the oncogenes c-myc or H-ras. Fifty percent of the hepatic tumors in these transgenic mice had activating somatic mutations within the beta-catenin gene similar to those found in colon cancers and melanomas. These alterations in the beta-catenin gene (point mutations or deletions) lead to a disregulation of the signaling function of beta-catenin and thus to carcinogenesis. We then analyzed human HCCs and found similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations led to the accumulation of beta-catenin in the nucleus. Thus alterations in the beta-catenin gene frequently are selected for during liver tumorigenesis and suggest that disregulation of the Wnt-beta-catenin pathway is a major event in the development of HCC in humans and mice.

Woodchuck hepatitis virus enhancer I and enhancer II are both involved in N-myc2 activation in woodchuck liver tumors.

Direct activation of the N-myc2 oncogene by insertion of woodchuck hepatitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatocarcinogenesis. We previously reported that WHV enhancer II (We2), which controls expression of the core/pregenome RNA, can also activate the N-myc2 promoter in hepatoma cell lines. To better define the integrated WHV regulatory sequences responsible for N-myc2 promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the win locus in tumor 2260T1 and mapping approximately 175 kb 3' of N-myc2. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with We2 sequences and 1 with WHV sequence homologous to that of hepatitis B virus enhancer I (We1). In transient transfection assays in hepatoma-derived cells, the We2 activator was found to be fully effective only when inserted in close proximity to the N-myc2 promoter whereas the We1 element by itself was apparently devoid of activity. In contrast, the 2260T1 viral insert exhibited a potent enhancer capacity that depended both on multimerized We2 and on We1 sequences. In a survey of different woodchuck hepatomas, both elements were commonly found within integrated viral sequences involved in long-range N-myc2 activation.

[Hepatitis B virus and hepatocellular carcinoma]. / Virus de l'hépatite B et hépatocarcinome.

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.