Hepatocellular carcinoma in WHV/N-myc2 transgenic mice - Nature

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Oncogene (2000) 19, 2678 ± 2686 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc

Hepatocellular carcinoma in WHV/N-myc2 transgenic mice: oncogenic mutations of b-catenin and synergistic eect of p53 null alleles Claire-AngeÂlique Renard1, GenevieÁve Fourel1,4, Marie-Pierre Bralet2,5, Claude Degott2, Alix De La Coste3, Christine Perret3, Pierre Tiollais1 and Marie Annick Buendia*,1 1

UniteÂ de Recombinaison et Expression GeÂneÂtique (INSERM U163), Institut Pasteur, 28 rue du Dr. Roux, 75015 Paris, France; Laboratoire d'Anatomopathologie, HoÃpital Beaujon, Clichy, France; 3INSERM U129, ICGM, UniversiteÂ Paris V ReneÂ Descartes, Paris, France

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The intronless N-myc2 gene was originally identi®ed 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 b-catenin were found in 25% of liver tumors and correlated with reduced tumor latency (P50.05), con®rming the important role of b-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+/D mice harbored either a deletion of the wt p53 allele or a b-catenin mutation. These ®ndings provide direct evidence that activation of Nmyc2 and reduction of p53 levels act synergistically during multistage carcinogenesis in vivo and suggest that dierent genetic pathways may underlie liver carcinogenesis initiated by a myc transgene. Oncogene (2000) 19, 2678 ± 2686. Keywords: woodchuck; hepatocellular hepatitis B virus; N-myc; p53; b-catenin

carcinoma;

Introduction Primary liver cancer or hepatocellular carcinoma (HCC) is among the most prevalent human malignancies, and it ranks as the third most frequent cause of death by cancer worldwide (Murray and Lopez, 1997). Epidemiological evidence supports a predomi-

*Correspondence: MA Buendia Current addresses: 4CNRS UMR 49, Ecole Normale SupeÂrieure, Lyon, France; 5DeÂpartement de Pathologie, HoÃpital Henri Mondor, Creteil, France Received 23 February 2000; revised 20 March 2000; accepted 10 April 2000

nant role of hepatitis B virus (HBV) as a causal agent of liver cancer, but the precise role of this virus in liver cell transformation remains poorly de®ned (Buendia et al., 1998; Schafer and Sorrell, 1999). Studies of animal models for HBV and liver cancer have shown that activation of myc family genes plays a pivotal role in virally-induced hepatocarcinogenesis. Woodchucks persistently infected with the woodchuck hepatitis virus (WHV) develop liver tumors within 2 ± 4 years, with a life time risk approaching 100% (Popper et al., 1987). The higher oncogenicity of WHV compared with other hepadnaviruses may be related to the speci®c ability of this virus to integrate its DNA near myc family genes and to cis-activate transcription from the myc promoters. Recurrent integrations of WHV DNA were ®rst evidenced near the c-myc oncogene (Hsu et al., 1988), and the strong impact of these insertion events was demonstrated in a transgenic model which recapitulates virally-induced liver carcinogenesis (Etiemble et al., 1994; Liu et al., 1997). Further studies of WHV insertion sites have led to the isolation of the intronless N-myc2 gene, a N-myc-like retrotransposon found only in rodents of the Sciuridae family (Fourel et al., 1990; Quignon et al., 1996). We and others have shown that N-myc2 is activated by WHV insertion from short or long distances in a majority of woodchuck HCCs (Bruni et al., 1999; Fourel et al., 1994; Hansen et al., 1993; Wei et al., 1992). N-myc2 shares with other oncogenes of the myc family the ability to transform primary rat embryo ®broblasts in cooperation with activated ras, and to induce both proliferation and apoptosis in liver epithelial cell lines (Fourel et al., 1990; Ueda and Ganem, 1996; Yang et al., 1996). The transgenic mouse system provides a research tool for investigating the oncogenic pathways triggered by dierent cellular and viral genes and for uncovering collaborating (onco)genes. It has been shown that deregulated hepatic expression of c-myc is among the most potent oncogenic events recorded in hepatocarcinogenesis (Cartier et al., 1992; Etiemble et al., 1994; Sandgren et al., 1989). Search for myc collaborating genes has shown a strong synergistic interaction between c-myc and transforming growth factor (TGF) -a (Murakami et al., 1993; Sandgren et al., 1993). TGF-b1 and the HBV regulatory protein HBx also cooperate with Myc in accelerating HCC development (Factor et al., 1997; Terradillos et al., 1997). In contrast, coexpression of the hepatocyte growth factor (HGF) or the Bcl-2 anti-apoptotic gene inhibits the emergence of Myc-induced liver tumors (de La Coste et

WHV/N-myc2 transgenic mice C-A Renard et al

al., 1999; Santoni-Rugiu et al., 1996). Consistent with the notion that deregulated expression of c-myc is insucient to promote full malignancy, recent studies have revealed that the b-catenin oncogene is frequently mutated in murine HCCs induced by c-myc (de La Coste et al., 1998). Synergy between c-myc and tumor suppressor gene mutations in liver tumorigenesis has not been investigated thus far. The tumor suppressor gene p53 has been implicated in the pathogenesis of human HCC by the ®nding of p53 mutations and allelic deletions at chromosome 17p in a signi®cant percentage of tumors (reviewed in Nagai and Buendia, 1998). Notably, a hot-spot mutation at codon 249 has been associated with a¯atoxin B1 (AFB1) exposure (Bressac et al., 1991). Mice carrying a targeted disruption of the p53 gene or overexpressing a mutant p53 allele are highly prone to cancer, but the frequency of spontaneous or carcinogen-induced hepatic neoplasms is not increased in these animals (Donehower et al., 1992; Jacks et al., 1994; Lavigueur et al., 1989). However, loss of p53 function has been shown to potentiate liver oncogenesis in AFB1-treated HBV transgenic mice (Ghebranious and Sell, 1998). It has been reported that the HBV X protein can bind p53 in the cytoplasm and inhibit its tumor-suppressor functions, suggesting that functional inactivation of p53 by viral gene products may also contribute in hepatocellular transformation (Ueda et al., 1995). To better de®ne the oncogenic impact of WHV integration events in the woodchuck N-myc2 gene, we have generated transgenic mice carrying N-myc2 linked to WHV DNA sequences previously isolated from a woodchuck HCC (Fourel et al., 1990). WHV/N-myc2 mice were highly predisposed to liver cancer, and constitutive, low level expression of a minimally deregulated N-myc2 transgene was sucient for hepatocellular transformation. To investigate the tumorigenic pathways associated with N-myc2 activation, we evaluated the potential synergistic eect of p53 de®ciency and the prevalence of activating mutations of the b-catenin oncogene at neoplastic stages.

Results and discussion Generation of WHV/N-myc2 transgenic mice Cloning of the unique WHV integration site in woodchuck W204 HCC has revealed that WHV sequences were inserted into the woodchuck intronless N-myc2 gene (Fourel et al., 1990). In this tumor, a 1.4 kb viral linear subgenomic fragment spanning the WHV enhancer I and II sequences (nt positions 4921901 on the WHV map according to Galibert et al., 1982) was inserted 7 bp downstream of the N-myc2 translation termination codon (Figure 1). To generate WHV/N-myc2 transgenic lines, a genomic DNA fragment covering N-myc2 and adjacent WHV DNA was ligated to contiguous WHV sequences providing the viral polyA site (Figure 1). The 5.7 kb N-myc2WHV fragment of the resulting construct was microinjected into C57BL/66SJL/J one-cell mouse embryos. Of 55 pups derived from microinjected zygotes, only three were found to carry sequences of the transgene by Southern blot analysis of tail DNA. In one of the

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Figure 1 Genetic map of the 5.7 kb WHV/N-myc2 transgene. The genomic DNA fragment isolated from a woodchuck tumor included sequences of the intronless N-myc2 gene (grey box: coding region and open boxes: non coding regions) with 1.1 kb of 5' ¯anking woodchuck genomic DNA, and WHV DNA (strippled box) inserted 7 bp downstream of the N-myc2 stop codon. A detailed representation of the WHV insert is shown at the top left (S: viral surface gene; X, transactivator gene; C, core gene; e1 and e2, WHV enhancers 1 and 2). N-myc2 transcriptional start site is indicated by an arrow

founders (NM14), the intact transgene was stably integrated at a single site containing three head-to-tail copies per haploid genome. A second animal (NM7) carried a rearranged transgene lacking most N-myc2 coding sequences. These animals were bred to establish the NM14 and NM7 lines in which the transgene was transmitted in a Mendelian fashion. A third animal (NM50) carrying one copy of the transgene was a sterile male. Liver tumors in WHV/N-myc2 transgenic mice Transgenic animals of the NM14 line were apparently normal and developed no liver pathology during the ®rst year of life. In older mice, a wide spectrum of benign and malignant liver lesions was detected in 90% of cases (Table 1). Most animals (55%) developed HCCs between 11 and 30 months of age (Figure 2). Male and female animals developed tumors with similar frequency and latency (average: 20.1 versus 20.3 months). In about 50% of animals, two to ®ve distinct tumor masses were found in dierent locations, while single tumors were systematically localized on the right liver lobe. Histological examination revealed well dierentiated HCCs as described in WHV/c-myc mice (Etiemble et al., 1994) (Figure 3). Tumor architecture was mainly trabecular, sometimes pseudoglandular or compact; local invasion or metastasis was not detected. In addition, 16% of animals developed hepatocellular adenomas during the second year of life. Peritumorous tissues harbored a normal architecture with frequent periportal in¯ammatory in®ltrates. Dysplastic cells, mainly in mediolobular areas, and/or preneoplastic foci and nodules were present in the livers of one-third of tumor-bearing mice and in most tumor-free animals sacri®ced at 22 months of age (Figure 3). Other pathologies in this line included occasional lymphomas with extensive spleen enlargement (Table 1). No nontransgenic littermate or animal of the NM7 line (in which most N-myc2 sequences were deleted) developed any neoplasm over a 30 month observation period. The NM50 transgenic male succumbed at 2 months with three tumor masses (5 ± 17 mm in diameter) replacing most of the liver parenchyma. At microscopic examination, these tumors were diagnosed as well to Oncogene


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moderately dierentiated HCCs (Figure 3e). Clear cell alterations, acinar formations, focal extra-medullar hematopoiesis, and large areas of hemorragic necrosis were seen in dierent tumor zones. The adjacent liver carried multiple preneoplastic foci and a dramatic number of apoptotic bodies (Figure 3f). All other tissues examined were histologically normal. Thus, as previously shown for WHV/c-myc transgenic mice (Etiemble et al., 1994), animals carrying a N-myc2 transgene linked to WHV sequences develop liver tumors at a high frequency. These data provide direct evidence of the strong oncogenic impact of hepadnavirus integration into the woodchuck intronless N-myc2 gene.

WHV hybrid transcript were produced in all HCCs and hepatocellular adenomas examined, and in some non tumoral livers and in kidney. Faint signals were seen in normal heart, lung and brain tissues (Figure 4a). RT ± PCR analysis of liver RNA from threemonth-old mice, performed with a pair of primers speci®c for the WHV X gene, showed a band of the expected size in liver, heart, kidney, spleen, and lung (Figure 4b). Thus in this line, despite very low

Expression and methylation status of the WHV/N-myc2 transgene Expression of the transgene in a range of tissues and in tumors was ®rst assessed by Northern blot studies in the NM14 line. Analysis of total RNA from embryonic, neonatal and adult liver yielded no detectable signal using either a N-myc2 probe (Figure 4a) or a WHV DNA probe (data not shown). At neoplastic stages, high levels of the 4.6 kb N-myc2/ Table 1 Pathological lesions in WHV/N-myc2 transgenic mice with dierent p53 status

Pathology Liver HCC Adenoma Dysplasia/PF Angiosarcoma Angioma Metastasis* Lymphoid infiltrate No pathology Other tissues Lymphoma Sarcoma Other pathologies

Transgenic mouse lines NM14 NM14/p53+/D NM14/p53D/D (n=55) (%) (n=28) (%) (n=14) (%) 30 (55) 9 (16) 18 (33) ± 1 (2) ± 19 (35) 10 (18)

7 (25) 1 (3.5) 27 (96) ± ± 1 (3.5) 4 (14) 1 (4)

± ± 9 (64) 1 (7) ± ± 4 (28) 1 (7)

3 (5) ± ±

4 (14) 6 (22) ±

8 (57) 3 (21) ±

*A NM14/p53+/D mouse harbored intrahepatic metastasis of a neuroendocrine tumor. PF: preneoplastic foci; ±: not found

Figure 3 Histopathology of hepatic lesions. (a) Liver of a 10month-old NM14 transgenic mouse, showing normal hepatic structure with minimal dysplasia. (b) Liver of a 10-month-old NM14/p53+/D mouse with high grade dysplasia. Note the presence of dysplastic hepatocytes with hyperchromatic and greatly enlarged nuclei (arrows). (c) An hepatocellular adenoma compressing adjacent nonneoplastic liver in an 18-month old NM14 mouse. The tumor consists of hepatocytes arranged in distorted trabeculae no more than two cells thick. (d) A dierentiated hepatocellular carcinoma in a 21-month-old NM14 mouse. Note the macrotrabecular structure and the presence of mitotic ®gures. (e) Well dierentiated HCC with extramedullary hematopoietic foci in the NM50 mouse. Arrow: megacaryocyte. (f) NM50 nontumorous liver, with numerous apoptotic bodies (arrows). Magni®cation: a ± e, 6200; b/insert and f, 6400

Figure 2 Incidence of hepatocellular carcinoma and adenoma in WHV/N-myc2 transgenic animals of the NM14 line. The age and the number of animals presenting with benign or malignant liver tumors at sacri®ce are shown. Tumor-bearing WHV/N-myc2 transgenics on a p53+/+ background are depicted as grey bars and those on a p53+/D background as black bars Oncogene


Figure 4 Expression and methylated status of the WHV/N-myc2 transgene. (a) Northern analysis of total RNA from liver of NM14 animals at dierent embryonic and post-natal ages, and at neoplastic stages, from HCC and various tissues of NM14 animals and in the NM50 HCC. The woodchuck N-myc2 probe M13-13 does not recognize any endogenous sequence in the mouse. Equal loading in each lane was veri®ed by hybridization with an 18S RNA probe (lower panel). (b) RT ± PCR analysis of transgene expression in dierent tissues of a 3-month-old NM14 mouse. Primers bracketing the WHV X gene were used together with GAPDH primers. (c) Southern blot analysis of genomic DNA from dierent tissues of a 3-month-old NM14 mouse and a NM14 HCC (left part) and from the NM50 mouse (right part). DNA was doubly digested with HindIII and MspI or HindIII and HpaII, and the blot was probed with RsB which recognizes the Nmyc2 promoter

expression of WHV/N-myc2 transgene in liver and in a variety of tissues at pretumoral stages, tumors arose almost exclusively in the liver (Table 1). Signi®cantly, the onset of benign and malignant tumors was associated with a dramatic increase of transgene expression, as noted previously in several other oncomice (Etiemble et al., 1994; LandesmanBollag et al., 1998; Sandgren et al., 1993). Northern analysis of dierent tissues from the NM50 mouse showed abundant 4.5 kb transgene RNAs in HCC (Figure 4a) and in adjacent liver, kidney and

intestine, but not in other tissues examined (data not shown). Because extensive methylation of CpG residues within or near promoters has been associated with gene silencing (Kass et al., 1997), we next investigated the methylation status of WHV/N-myc2 regulatory sequences in NM14 mice. Genomic DNAs were sequentially digested with HindIII and either HpaII or MspI, two isoschizomers recognizing the sequence CCGG (HpaII is methylation-sensitive while MspI is not). Southern blot hybridization with a N-myc2 promoter probe showed one fragment in MspI digests, and partial resistance to HpaII digestion was demonstrated in various tissues and in HCC from NM14 transgenic mice (Figure 4c). Similar data were obtained in dierent NM14 osprings and when the blots were probed with WHV enhancer I sequences (data not shown), indicating that constitutive methylation of the transgene regulatory elements was maintained through generations in the NM14 line. In sharp contrast, transgene sequences in the NM50 mouse appeared to be constitutively undermethylated in a parallel MspI/ HpaII test performed with spleen and HCC samples (Figure 4c). The early post-natal appearance of liver cancer in the NM50 mouse opposed to the long tumor latency in the NM14 line strongly suggests that transgene DNA methylation might in¯uence tumor kinetics by modulating the level of N-myc2 expression. Indeed, in normal tissues from NM14 animals, low transgene expression correlated with hypermethylation of regulatory sequences. Notably however, reactivation of the transgene at tumoral stages did not apparently result from de novo demethylation, suggesting that the repressive eect of methylated DNA segments can be overcomed by some factors involved in the initial stages of tumor emergence. It has been shown previously that most transgenic lines harboring hepatic expression of c-myc develop liver tumors within 8 ± 18 months, and that stimulation of myc expression by appropriate regimens can eciently accelerate tumor onset (Cartier et al., 1992; Etiemble et al., 1994). Taken together, our present data demonstrate that the woodchuck N-myc2 intronless gene exhibits tumorigenic properties similar to that of the c-myc oncogene. Our ®ndings of a very low yield of viable WHV/N-myc2 transgenic pups (3 out of 55 animals born), as well as extensive apoptosis in the NM50 liver, con®rm previous studies showing that Nmyc2, like c-myc, is endowed with pro-apoptotic activity (Ueda and Ganem, 1996; Yang et al., 1996). Cytotoxic eects of N-myc2 overexpression might also explain why transgenic lines could be established only from founder animals carrying either hypermethylated (NM14) or deleted (NM7) WHV/N-myc2 sequences. As we found a low rate of apoptotic cells in tumors that strongly expressed the N-myc2 transgene, it seems probable that transformed hepatocytes acquired resistance to Myc-induced cell death through genetic or epigenetic events. Indeed, IGF-II, a survival factor known to counteract apoptosis triggered by N-myc2 (Ueda and Ganem, 1996; Yang et al., 1996), was strongly re-expressed in the NM50 HCC (data not shown). The question of whether viral proteins encoded by transgene sequences may contribute in some step of the

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tumorigenic process is an interesting issue. The WHV/ N-myc2 transgene has potential to express the regulatory protein WHx and the core protein WHc from independent promoters (see Figure 1). In Northern analyses, WHV DNA probes detected weak amounts of 0.8 kb X mRNAs in tumors and tissues harboring high levels of the 4.5 kb N-myc2/WHV cotranscript, but we obtained no evidence of the expression of a WHc-speci®c mRNA (data not shown). The X protein of WHV, as its HBV homologue, has been shown to participate as a co-factor in liver oncogenesis by accelerating the onset of liver tumors induced by diethylnitrosamine (Dandri et al., 1996; Slagle et al., 1996). Other studies in HBX transgenic mice have shown that this viral transactivator can either induce HCCs in a particular mouse strain, or potentiate c-myc-induced liver carcinogenesis (Kim et al., 1991; Terradillos et al., 1997). These data suggest that faint expression of the WHx regulatory protein in HCCs might play a role in tumor progression in the WHV/N-myc2 transgenic model. p53 deficiency accelerates liver tumor onset in WHV/N-myc2 mice Previous studies of the Myc/p53 interactions in transgenic mice have revealed contrasting eects in dierent models, with synergistic cooperation in lymphoma genesis, but not in mammary tumorigenesis (Blyth et al., 1995; Elson et al., 1995; Hsu et al., 1995; McCormack et al., 1998). To address the role of p53 in N-myc2-induced hepatocarcinogenesis, we crossed NM14 mice with p53 knock-out mice (p53D/D) (Jacks et al., 1994), and F1 osprings were subsequently intercrossed or backcrossed to p53D/D animals to generate an experimental cohort of transgenic and non transgenic animals on dierent p53 backgrounds. The latency of liver tumor onset was markedly reduced in transgenic animals carrying one p53 mutant allele (average 11.9+3.6 months versus 20.2+4.1 months for NM14 mice) (Figure 2), and this dierence was highly signi®cant (P=0.0001, Mann-Whitney test). Importantly, no metastasis or other type of cancer was observed in HCC-bearing animals. Histological examination of tumors showed that all were dierentiated HCCs or hepatocellular adenomas (Table 1), undistinguishable from those observed in NM14 mice carrying two normal p53 alleles (see Figure 3). Non tumorous livers were mainly characterized by occasional periportal lymphocytic in®ltration and by extensive dysplastic cells and preneoplastic foci which appeared earlier in life (around 4 months) than in the NM14 line (Figure 3b and Table 1). Among ten animals sacri®ced between 9 ± 10 months of age, only one carried a liver tumor, but all others had severe preneoplastic liver lesions. Other major dierences were the frequent occurrence of intrahepatic hematopoeitic foci and the marked hyperplasia of Ito cells in p53+/D mice carrying the myc transgene (data not shown). Otherwise, the predominant tumor types in Mycpositive, p53 heterozygous animals were sarcomas and lymphomas as previously reported for non transgenic p53+/D mice (Jacks et al., 1994). These tumors occurred between 10 ± 17 months of age in WHV/N-myc2/p53+/D mice, while only 28% of p53+/D animals had developed neoplastic disease at

Oncogene

17 months, suggesting that faint expression of N-myc2 might cooperate with reduction of p53 levels in various mouse tissues. In sharp contrast, the tumor spectrum and incidence were essentially similar in p53D/D mice either positive or negative for the N-myc2 transgene (see Table 1 and Jacks et al., 1994). All animals died with lymphomas and sarcomas between 3 ± 8 months (average 4.5 months in both groups). Importantly, no HCC was seen in any Myc-positive, p53D/D animal at autopsy, despite the presence of extensive liver cell dysplasia in most livers. Thus, lymphomas arising with extremely short latency precluded the evaluation of liver tumor latency. It suggests that loss of p53 function is the phenotypic determinant of tumorigenesis in this animal group, as also found in previous studies of Myc/p53 interactions in mammary tumorigenesis (McCormack et al., 1998). In human tumors, mutations in one p53 allele are generally associated with loss of the second allele. In contrast, complete inactivation of p53 is not required for cancer formation in mice, and one copy of the normal p53 gene is frequently retained in sarcomas and carcinomas arising in mice heterozygous for the p53 null mutation (Venkatachalam et al., 1998). We therefore examined the status of the remaining wild type (wt) p53 allele in tumors from seven N-myc2 positive, p53+/D mice. Southern blot analysis showed that the wt p53 allele was almost completely lost in three out of 10 HCC samples analysed (Figure 5 and Table 2). The apparent clonality of tumor cells for wt allele loss indicate that it occurred as an early step in liver tumorigenesis induced by the combined action of N-myc2 activation and p53 hemizygosity. As the loss

Figure 5 p53 status in HCCs from WHV/N-myc2 transgenic mice on p53+/D background. Loss of the wt p53 allele in tumors was monitored by Southern blotting of EcoRI ± StuI digested DNA and hybridization with p53 cDNA. NL, normal mouse liver control; T, tumor; NT, adjacent nontumorous liver

Table 2 Loss of wild type p53 allele and alterations in the b-catenin gene in liver tumors induced by N-myc2 on p53+/+ or p53+/D backgrounds Tumor F2-27 F2-12 F3-26T1{ F6-1 P2-26 P2-36 P2-45 P2-7 T1 P2-7 T2 P2-7 T3 P3-23

Genotype

p53 LOH*

b-catenin alteration

NM14 NM14 NM14 NM14 NM14/p53+/D NM14/p53+/D NM14/p53+/D NM14/p53+/D NM14/p53+/D NM14/p53+/D NM14/p53+/D

ND ND ND ND + + + 7 7 7 7

T41A (ACC?GCC) D15-51 D5-76 D5-76 no no no S33P (TCT?TTT) D5-76 S37P (TCT?TTT) D5-76

*LOH: loss of heterozygosity; ND: not determined; +: loss of wt p53 allele; 7: no LOH. {: Hepatocellular adenoma


of p53 function in heterozygous animals is known to occur essentially via a chromosomal LOH mechanism (Jacks et al., 1994; Venkatachalam et al., 1998), point mutations in the remaining p53 gene were not investigated in this study. These data provide the ®rst published evidence that deregulated expression of a myc gene and p53 de®ciency can act synergistically to promote the development of liver tumors. Our preliminary observations in WHV/c-myc transgenic mice also indicate a dramatic acceleration of HCC kinetics in c-myc positive, p53 de®cient animals (CA Renard and MA Buendia, unpublished results). The cooperative interaction between p53 de®ciency and deregulated Myc expression might be explained by dierent, non exclusive mechanisms. Reduction of p53 levels might relieve methylation-dependent N-myc2 silencing at preneoplastic stages, and loss of the wt p53 allele might confer resistance to N-myc2-induced apoptosis at early steps of malignant conversion. Alternatively, reduction of p53 dosage might induce genetic instability and increase the rate of chromosomal lesions (Venkatachalam et al., 1998). Future work will be aimed at investigating these dierent issues. Mutations of the b-catenin gene in N-myc2-induced liver tumors The stochastic development of tumors and the long latent period in the NM14 line strongly argues for additional mutational events. We have shown previously that about one half of HCCs in LPK/myc and WHV/c-myc transgenic mice carried activating mutations or deletions in the second exon of the bcatenin gene (de La Coste et al., 1998). We therefore screened a total of 27 HCCs from NM14 and NM14/ p53+/D mice for genetic alterations in the N-terminal domain of b-catenin by RT ± PCR analysis. As shown in Figure 6a, we found additional faster migrating bands corresponding to truncated b-catenin cDNAs in ®ve independent HCCs. Sequence analysis revealed deletion of the second exon or a smaller deletion of the GSK3-b phosphorylation motif (Table 2). Among other tumors, three carried a point mutation of one b-catenin allele changing one of the residues phosphorylated by GSK3-b (S33, S37 or T41). In one animal, three tumor nodules harbored distinct mutations or deletions of b-catenin, and in a second case, only one of two tumor samples carried an abnormal b-catenin allele, indicating that the development of tumors may be polyclonal in this model. Interestingly, a b-catenin mutation was detected in one of the adenomas analysed, con®rming previous studies of chemically-induced mouse liver tumors (Devereux et al., 1999). In summary, alterations of b-catenin were found in 4/16 (25%) of tumors in NM14 mice and in 4/11 (36%) of tumors from NM14/p53+/D animals. Tumors carrying b-catenin gene alterations appeared signi®cantly earlier than those with two normal b-catenin alleles in 16 NM14 mice analysed (mean age: 15.2+3.7 months vs 19+2.5 months; P50.05), but notably, the b-catenin gene was apparently normal in the early NM50 HCC. In the view that tumor development proceeds through a succession of genetic changes conferring dierent types of growth advantage (Hanahan and

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Figure 6 Deletions in the second exon of the b-catenin gene in N-myc2-induced tumors. (a) Analysis of RT ± PCR products revealed deletions of the N-terminal domain of one b-catenin allele together with the normal product in tumors from NM14 and NM14/p53+/D mice. (b) Western blot analysis of b-catenin expression in NM14 HCCs and corresponding nontumor livers. Cellular extract from a normal liver was used as control. WT bcatenin (92 KDa) is indicated by the arrow

Weinberg, 2000), it seems probable that Myc and bcatenin act in dierent pathways. Interestingly, most tumors that arose in NM14/ p53+/D mice harbored either b-catenin mutation or loss of the wt p53 allele (Table 2). It is conceivable that these two genetic events were caused by dierent mechanisms, and that the oncogenic pathways activated by the early occurrence of one or the other did not require the second one as a rate-limiting step to reach full malignancy. In line with this hypothesis, it has been reported recently that the frequency and patterns of b-catenin mutations vary greatly in rat hepatocarcinogenesis induced by exogenous and endogenous carcinogens (Tsujiuchi et al., 1999). In human HCCs, b-catenin mutations correlate with a low rate of LOH, suggesting alternative mechanisms which may (or not) activate the Wnt pathway (Legoix et al., 1999). Alternatively, and although the numbers are small, the two events might be mutually exclusive, implying that loss of p53 function and oncogenic deregulation of b-catenin act in the same pathway. However, there is no published evidence supporting the involvement of p53 in Wnt signaling and by contrast, it has been recently proposed that overexpression of bcatenin stimulates p53 activity, thereby imposing a selective pressure for p53 inactivation at late stages of colon cancer (Damalas et al., 1999). Further work in dierent transgenic models and in human HCCs is therefore necessary to better understand the relative contribution of b-catenin and p53 in liver carcinogenesis. b-catenin expression According to recent data, the stability of b-catenin in tumor cells is strongly enhanced by mutations or deletions aecting the GSK3-b phosphorylation sites and ubiquitination consensus sequences (Morin et al., 1997). Stabilized forms of b-catenin accumulate in the Oncogene


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cell cytoplasm, translocate to the nucleus and bind LEF/Tcf factors, thereby stimulating transcription of a number of cellular targets (reviewed in Morin, 1999). Recent investigations of b-catenin in human HCCs have shown a signi®cant correlation between the presence of stabilizing mutations of the gene and nuclear accumulation of the protein (de La Coste et al., 1998; Tran Van Nhieu et al., 1999). Western blot analysis of the b-catenin protein in WHV/N-myc2 tumors and corresponding nontumor livers showed a band of normal size in all cases, and a truncated protein in the tumors harboring a deletion of the second b-catenin exon (Figure 6b). Notably, b-catenin protein levels were not considerably enhanced in tumors compared to nontumorous adjacent livers. To determine the intracellular localization of b-catenin in N-myc2-induced tumors, we examined dierent mouse liver samples by indirect immuno¯uorescence, using monoclonal anti-b-catenin antibodies. As shown in Figure 7b, b-catenin expression in the normal liver was restricted to the cytoplasmic membrane of hepatocytes, with stronger signals on the sinusoidal face, and in sinusoidal lining cells and bile duct epithelium. In some tumors carrying b-catenin gene alterations, a weak to moderate expression of the protein could be detected in the cytoplasm of tumorous hepatocytes (Figure 7a), while in most cases, b-catenin labeling remained primarily membranous. We also failed to detect nuclear b-catenin staining using dierent anti-b-catenin antibodies and a variety of experimental protocols (data not shown). This result contrasts with previous studies in human liver tumors (de La Coste et al., 1998; Tran Van Nhieu et al., 1999), but others also failed to detect nuclear expression of mutated b-catenin in mouse HCCs (Devereux et al., 1999). The reasons for this discrepancy remain at present obscure. Nevertheless, the recent ®nding that modest cytosolic accumulation of b-catenin can induce neoplastic transformation of normal epithelial cells (Kolligs et al., 1999; Orford et al., 1999) supports the notion that dysregulation of bcatenin plays a crucial role in the development of murine HCC. In conclusion, this study showing that WHV/N-myc2 transgenic mice are highly predisposed to liver cancer, as previously shown for WHV/c-myc mice Etiemble et al. (1994) clearly establish the woodchuck intronless Nmyc2 gene as an oncogene in vivo. Our data also strengthen the important role of b-catenin activation in Myc-induced hepatocarcinogenesis and demonstrate a strong synergy between Myc and p53 in liver cell

transformation. Since overexpression of c-myc and mutations in the p53 and b-catenin genes are among the most commonly recorded alterations in human HCCs, our results may provide a ®rst basis for investigating the interplay between these dierent factors in the development of liver cancer. Materials and methods Constructs and transgenic mice A genomic DNA fragment encompassing the woodchuck Nmyc2 gene and WHV sequences integrated in the 3' noncoding region of N-myc2 was isolated from a genomic library of woodchuck 204 HCC (Fourel et al., 1990). The 4.1 kb SalI ± ApaI fragment including the 5' ¯anking region and coding sequences of N-myc2 together with the adjacent S gene of WHV was excised and ligated with the 1.7 kb ApaI ± BglII subgenomic fragment of WHV (Galibert et al., 1982), and the ligation product was subcloned into the SalI and BamHI sites of pBS (Stratagene). The resulting plasmid (pTR27) carried N-myc2 and viral sequences spanning the WHV S and X genes as found in the original woodchuck tumor, and additional sequences of the WHV C gene providing the viral polyadenylation site. To generate WHV/ N-myc2 transgenic mice, the 5.7 kb vector-free fragment of pTR27 was gel-puri®ed and injected into fertilized mouse oocytes derived from matings between C57BL/66SJL/J F1 animals. Successful transgene integration was monitored by hybridization of mouse tail DNA with cloned WHV DNA as described (Etiemble et al., 1994). Mice nullizygous for p53 (p53D/D) have been previously described (Jacks et al., 1994). These animals were derived from homologous recombination in 129/Sv murine stem cells and subsequently crossed onto a C57BL/6 background. WHV/N-myc2 transgenic mice (NM14 line) were mated with p53-null animals and the progeny was genotyped by PCR analysis of tail DNA as previously described (Jacks et al., 1994). Animals heterozygous for both the p53 mutation and the myc transgene were subsequently backcrossed onto p53 homozygous null mice or bred together to generate myc transgenic mice on a p53 +/+, p53 +/D and p53 D/D background. All animals were maintained in a pathogen-free facility and handled according to the guidelines of the MinisteÁre de l'Agriculture et de la PeÃche (France). Macroscopical and histological analyses Animals were checked weekly for tumor appearance and sacri®ced when presenting with abnormal clinical signs. At autopsy, livers and dierent mouse organs were examined for macroscopic lesions. Samples for histological examination were ®xed in Bouin's ¯uid. Five mm-thick paran-embedded sections were stained with hematoxilin and eosin.

Figure 7 Immunolocalization of b-catenin in HCC (a) and in nontumor liver (b) of a NM14 transgenic mouse by indirect immuno¯uorescence. A û-catenin monoclonal antibody detects membrane staining in normal hepatocytes, and intracytoplasmic accumulation, but no nuclear staining in tumoral cells harboring a missense mutation of the b-catenin gene. Magni®cation; 6400 Oncogene


DNA and RNA analysis Genomic DNA was prepared from frozen mouse tissues by a conventional proteinase K/phenol procedure. For methylation studies, DNA samples (20 mg) were sequentially digested by HindIII and MspI or HpaII, and analysed by Southern blotting using alkaline transfert on Hybond-N+ (Amersham, UK). Blots were probed with a 360 bp RsaI ± BglII (RsB) fragment covering the N-myc2 promoter (Fourel et al., 1992) or with a 600 bp ApaI ± NcoI subgenomic fragment of WHV spanning enhancer I sequences. For analysis of the p53 locus, Southern blots of EcoR1/StuI-restricted DNA were hybridized with a full length p53 cDNA (Soussi et al., 1988). Total cellular RNA was prepared from frozen tissues by a guanidinium isothiocyanate/phenol method (RNA PLUS, Bioprobe). For Northern analysis, RNA samples (30 mg per lane) were resolved by electrophoresis through 1% agarose gels in 2.2 M formaldehyde and blotted onto a Hybond N+ membrane in 0.05 N NaOH. Blots were hybridized as described in Church and Gilbert, 1984, using probes labeled by random priming. The probes used were M13.13, a 700 bp fragment of the N-myc2 coding region (Fourel et al., 1990), WHV DNA (Galibert et al., 1982), and a 0.2 kb PstI cDNA fragment of the 18S rRNA gene used as control for RNA loading (Valbiotech). For RT ± PCR analysis of transgene expression, total RNA was treated with RNAse-free DNAse (Promega), and cDNA was synthesized with 5 mg of total RNA, 300 ng of random hexamers (Pharmacia) and 200 U reverse transcriptase MMLV Superscript (GIBCO BRL) at 378C for 45 min. cDNA was then ampli®ed by PCR as described previously (Renard et al., 1998), using primers located at positions 1158 ± 1168 (forward) and 1478 ± 1498 (reverse) on the WHV genome. As control, a 900 bp fragment of GAPDH cDNA was ampli®ed simultaneously.

10% glycerol; dithiothreitol 100 mM; 0.1% bromophenol blue), boiled for 10 min, size fractionated on 8% SDSpolyacrylamide gels and then transferred to nitrocellulose membranes (Hybond C Extra, Amersham) by electroblotting. After blocking with 0.2% casein and 0.1% Tween-20 in PBS, Western blots were incubated with monoclonal anti-b-catenin antibodies (Transduction Laboratories) at a 1 : 5000 dilution. For detection, alkaline phosphatase-conjugated goat antimouse antibodies were used, followed by chemiluminescence using the Western-Star chemiluminescence detection system (Tropix, PE Applied Biosystems) according to the manufacturer's instructions.

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Indirect immunofluorescence Liver tissues were ®xed in 4% paraformaldehyde, embedded in paran, and 5 mm-thick sections were cut. After dewaxing, the sections were processed by a conventional microwave oven heating in 10 nM citric acid buer (pH 6.0), and incubated with bovine serum albumin in PBS for 15 min to block non-speci®c antibody binding. Incubation with anti-bcatenin monoclonal antibodies (Transduction Laboratories) at a 1 : 250 dilution or polyclonal antibodies (Sigma ± Aldrich) at a 1 : 2000 dilution for 1 h at room temperature was followed by treatment for 30 min with FITC-conjugated goat anti-mouse or anti-rabbit antibodies (Sigma ± Aldrich). Nuclei were lightly counterstained with hematoxylin. Statistical analysis The Mann-Whitney nonparametric test was used to compare the kinetics of tumor formation between dierent animal groups. Values less than 0.05 were considered to be statistically signi®cant.

Mutational analysis of b-catenin First strand cDNA was synthesized on total RNA from liver and tumor tissues using a random primer p(dN)6 (Pharmacia). Conditions and primers for PCR ampli®cation of bcatenin exons 1 to 3 have been described previously (de La Coste et al., 1998). Samples were analysed by agarose gel electrophoresis to detect truncated b-catenin cDNAs, and puri®ed PCR products were sequenced using the T7 sequenase PCR product sequencing kit (Amersham/USB) with the forward primer 5'-TGATGGAGTTGGACATGGCCATG-3' and the reverse primer 5'-CCCACTCATACAGGACTTGGGAGG -3'. Immunoblots Proteins were solubilized from mouse liver and tumor tissues by lysis in sample buer (60 mM Tris-HCl pH 6.8; 2% SDS;

Acknowledgments We are grateful to C Babinet for generating the WHV/Nmyc2 transgenic mice. T Jacks provided the P53 knock-out mice and the Centre de DeÂveloppement des Techniques AvanceÂes pour l'ExpeÂrimentation Animale (CNRS, OrleÂans, France) provided pathogen-free animals on C57/Bl6 background. We thank O Terradillos for help in immuno¯uorescence studies and J Tran van Nhieu for immunohistological analysis. This work was supported in part by the Association pour la Recherche sur le Cancer (ARC contract 5236). MA Buendia is an agent of the Centre National pour la Recherche Scienti®que, France.

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