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ß-Catenin Mutations and Protein Accumulation in All Hepatoblastomas Examined from B6C3F1 Mice Treated with Anthraquinone or Oxazepam

Laboratories of Molecular Carcinogenesis [C. H. A., T. R. D.] and Experimental Pathology [R. C. S., J. F. F., P. S. S., T-V. T.], National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

	ABSTRACT

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The molecular pathogenesis of hepatoblastomas in the B6C3F1 mouse is unclear but may involve alterations in the ß-catenin/Wnt signaling pathway as was recently described for chemically induced hepatocellular neoplasms and human liver cancers. The objective of this study was to characterize the mutation frequency and spectrum of ß-catenin mutations and the intracellular localization of ß-catenin protein accumulation in chemically induced hepatoblastomas. In this study, ß-catenin mutations were identified in all 19 anthraquinone-induced hepatoblastomas and all 8 oxazepam-induced hepatoblastomas examined. Although several hepatoblastomas had multiple deletion and/or point mutations, the pattern of mutations in the hepatoblastomas did not differ from that identified in hepatocellular neoplasms. In a majority of the hepatoblastomas (six of seven) examined by immunohistochemical methods, both nuclear and cytoplasmic localization of ß-catenin protein were detected, whereas in hepatocellular adenomas, carcinomas, and normal liver only membrane staining was observed. Our data suggest that ß-catenin mutations and the subsequent translocation of ß-catenin protein from the cell membrane to the cytoplasm and nucleus may be critical steps in providing hepatocellular proliferative lesions with the growth advantage to progress to hepatoblastomas.

	INTRODUCTION

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An increased incidence of highly malignant hepatoblastomas has been observed in mice following treatment with certain chemicals in National Toxicology Program carcinogenesis studies (1 , 2) , whereas these tumors are rare in untreated mice. It has been hypothesized that these tumors arise from or are a rare variant of hepatocellular carcinomas (2) . Diwan et al. (3) found that hepatoblastomas in mice developed after exposure to promoters such as phenobarbital in initiation-promotion protocols. Few studies of molecular analyses on chemically induced hepatoblastomas have been reported, although H-ras activation and p53 mutation have not been found (4 , 5) .

Hepatoblastomas are the most frequent malignant liver tumor found in young children (6) . There is also an increased incidence of these tumors in patients with APC² who carry a germline mutation in the APC gene, and recently it was discovered that there is a high frequency of ß-catenin mutations and ß-catenin protein accumulation in sporadic hepatoblastomas (7) .

ß-catenin is a central and critical molecule in the Wnt signaling pathway. The APC gene product and the glycogen serine-threonine kinase-3ß together target ß-catenin for degradation and modulate its expression (8 , 9) . In some cancers, including colon and hepatocellular tumors, mutations in either APC or ß-catenin lead to ß-catenin protein accumulation and up-regulation of Wnt signaling, resulting ultimately in cell proliferation and inhibition of apoptosis (10) . During this process, cytoplasmic ß-catenin may form a complex with Tcf transcription factors, enter the nucleus, and target genes such as c-MYC for transcription (9, 10, 11) .

ß-catenin mutations have been identified at high frequency in human hepatocellular carcinomas (12) and in hepatocellular adenomas and carcinomas of mice exposed to certain chemicals (13 , 14) . In this study, we examined hepatoblastomas, which developed in mice treated with anthraquinone or oxazepam for 2 yr, for mutations in ß-catenin. Anthraquinone is used in the manufacture of dyes and pigment and in the pulp and paper industry, and oxazepam is a commonly prescribed tranquilizer. We were particularly interested in whether the ß-catenin mutation frequency or spectrum was chemical specific and whether the mutations in hepatoblastomas differed from those of hepatocellular adenomas or carcinomas. Therefore, we also assessed the mutation frequency and profile in hepatocellular neoplasms from B6C3F1 mice treated with anthraquinone. Laser capture microdissection was used to collect cells from some of the smaller hepatoblastomas to avoid contamination with surrounding normal tissue or adjacent hepatocellular tumors. In addition, we compared the protein expression and intracellular localization of ß-catenin protein in hepatoblastomas and hepatocellular neoplasms.

	MATERIALS AND METHODS

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Tumor Samples and DNA Isolation.
Oxazepam treatment, liver tumor collection, tumor incidence data of B6C3F1 mice, and H-ras oncogene mutation analysis for the studies have been reported previously (4) . The incidence of hepatoblastomas in male mice was 0%, 4%, 40%, and 24% at 0 ppm, 125 ppm, 2500 ppm, and 5000 ppm oxazepam, respectively; and for female mice, 0%, 2%, 16%, and 14%, respectively. For collection of liver tumors from the anthraquinone study B6C3F1 mice were fed diets containing 0, 833, 2500, or 7500 ppm anthraquinone for 105 weeks. The incidence of hepatoblastomas in male mice was 2%, 10%, 22%, and 43% at 0, 833, 2500, and 7500 ppm anthraquinone, respectively; and for female mice, only 2% at the 7500 ppm dose. A subset of the hepatocellular neoplasms and a small number of hepatoblastomas with surrounding tissue were frozen in liquid N2 and stored at -70°C. The remainder of the liver tissues was preserved in 10% neutral buffered formalin, trimmed, and embedded in paraffin. Sections 5–6 µm were used for staining with H&E. Normal liver, hepatoblastomas, and surrounding hepatocellular neoplasms (adenomas and carcinomas) were either hand-microdissected or laser capture microdissected (4 of 19 of the anthraquinone-induced hepatoblastomas) from 2–5 serial 10-µm paraffin sections for mutation analysis. A PixCell-I (30-µm spot size and about 500 hits/sample) LCM System (Arcturus Engineering Inc., Mountain View, CA) was used for the laser capture microdissection, as described previously (15 , 16) . Microdissected samples were digested overnight in 500 µl of digestion buffer [50 mM Tris (pH 8.5), 0.5% Tween 20, 1 mM EDTA, and 0.5 mg/ml proteinase K] at 55°C. DNA was obtained by boiling the digested samples for 5 min. In addition, most of the mutation analysis on the hepatocellular neoplasms was performed with DNA isolated from frozen tumor tissue, as described previously (4 , 16) .

ß-Catenin Mutation Screening and Identification.
SSCP analysis was carried out on PCR products of exon 2 (corresponds to exon 3 in human) of the mouse ß-catenin gene (12) , which contains the glycogen serine-threonine kinase-3ß targeted phosphorylation sites within residues 33–45. The sequences of the intronic PCR primers flanking the borders of exon 2 were: BCAT-1F, 5'-TACAGGTAGCATTTTCAGTTCAC-3'; and BCAT-2R, 5'-TAGCTTCCAAACACAAATGC-3 (12) . Inner primers were BCAT-7F, 5'-TAACATACTCTGTTTTTACAGCTG-3', and BCAT-8R, 5'-ACATCTTCTTCCTCAGGGTTG-3'. For DNA from frozen samples primers 1F x 2R were used for amplification. For DNA from fixed sections nested PCR was used with primers 1F x 2R for the outer reactions and both 1F x 8R and 7F x 8R for the inner reactions. [³³P]-dATP was incorporated into the inner PCR reactions for SSCP analysis, and two gel conditions were used to detect mutations: 6% acrylamide gels with 10% glycerol were electrophoresed at 40 W for 6 h at 5°C, and 0.5x mutation detection enhancement (AT Biochem, Malvern, PA) gels were electrophoresed at 3 W for 17 h at room temperature. DNA from normal liver and no-DNA controls were included with all amplification experiments to confirm no cross-contamination of PCR products.

Point mutations and some small deletions were identified by reamplifying samples with altered bands on SSCP gels. Other deletions were identified by excision and boiling of the altered band, followed by a fresh amplification. The amplified bands were gel purified on Qiagen columns (Qiagen, Valencia, CA) before sequencing with a [³³P]-Thermo-Sequenase kit (Amersham, Cleveland, OH) The amplification primers also served as sequencing primers. Mutation identification was confirmed with at least two amplification reactions from original DNA.

Immunohistochemistry.
Tumor tissues were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Localization of ß-catenin protein expression was investigated using a polyclonal goat anti-ß-catenin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:100 on serial 6-µm sections. Slides were deparaffinized and rehydrated through xylene and ethanol washes into 1x Automation buffer (Biomeda Corp., Foster City, CA). Antigen unmasking was accomplished by heating in 200 ml of citrate buffer in a microwave oven at 50% power for 5 min. Following a 1-min break, the cycle was repeated, and the slides were then allowed to cool for 20 min. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 15 min. After rinsing in 1 x Automation buffer, sections were blocked with 5% normal goat serum for 30 min. The primary ß-catenin antibody was then applied to sections for 1 h at room temperature. Nonimmune rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as the negative control at equivalent conditions in place of the primary antibody. The bound primary antibody was visualized by streptavidin-biotin-peroxidase detection using the goat Immunocruz staining system (Santa Cruz Biotechnology), according to the manufacturer’s instructions, and with 3,3'-diaminobenzidine as the color-developing reagent. Slides were counterstained with Harris hematoxylin, dehydrated through a graded series of ethanol washes to xylene, and coverslipped with Permount (Fisher, Springfield, NJ).

Western Analysis for ß-Catenin Protein Expression.
A small number (5) of frozen tumor samples containing hepatoblastomas from the anthraquinone study were obtained for this study. The samples were sectioned with a cryostat, and a single section was quick stained to identify the hepatoblastomas. Then, the unstained hepatoblastomas were dissected away from the rest of the liver tumor and normal tissue to isolate protein. Frozen samples of normal liver, hepatocellular adenomas, and carcinomas from this study were used for comparison. Cellular protein was extracted from these samples for Western blot analysis of protein expression, as described previously (13) .

	RESULTS

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Mutation Analysis.
A high incidence of malignant hepatoblastomas resulted from anthraquinone or oxazepam treatment of B6C3F1 mice, whereas this tumor type is found only rarely in control mice. In this study, we examined hepatoblastomas induced by these two chemicals for mutations in exon 2 (corresponds to exon 3 in humans) of the ß-catenin gene. Mutations were identified in all 19 anthraquinone-induced hepatoblastomas and all 8 oxazepam-induced hepatoblastomas examined (Table 1 and Fig. 1 , A and B). Multiple point mutations and/or deletions in exon 2 were detected in many hepatoblastomas from both chemical treatment groups.

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. ß-catenin mutation identification in hepatoblastomas and normal liver from B6C3F1 mice treated with anthraquinone. A, SSCP analysis. Shown is a 6% acrylamide/10% glycerol gel with samples amplified with primers 1F x 8R. Lanes 1–8, DNA samples from anthraquinone-induced hepatoblastomas, as shown in Table 1 : tumor samples 3, 4a, 7, 9, 11, 19a, 19b (a and b represent different parts of the same tumor), and 6; Lane 9, no DNA control; Lane 10, normal control B6C3F1 mouse liver DNA; Lanes 11–15, DNA samples from five different normal livers from the high-dose anthraquinone group of mice. B, cycle sequencing of ß-catenin within exon 2 in four tumor samples. The arrows point to mutations. Sequence 1, hepatoblastoma 4a with deletion of codons 31–38; sequence 2, hepatoblastoma 4b (different part of the same tumor) with mutation at codon 32, GAT to AAT (Asp to Asn); sequence 3, hepatoblastoma 12 with mutation at codon 33, TCT to TTT (Ser to Phe; a different smaller PCR product of this sample had a deletion mutation of codons 5–7, data not shown); sequence 4, hepatocellular carcinoma 43 with no mutation.

Among the point mutations, any of the three bases of the codon could be mutated, and specific patterns of G to A or G to T mutations were not apparent for either chemical treatment. There did not appear to be a chemical signature mutation pattern in that the spectrum of ß-catenin mutations did not differ between the hepatoblastomas from the two different chemicals. Moreover, the pattern of point mutations did not differ from those found in hepatocellular neoplasms induced by chronic treatment with other chemicals (13) .

In National Toxicology Program studies, hepatoblastomas are often found within a hepatocellular adenoma or carcinoma. It has been hypothesized that this tumor may represent a more malignant hepatocellular neoplasm. We, therefore, examined six samples from anthraquinone-treated mice in which a hepatocellular adenoma or a hepatocellular carcinoma was microdissected from the same tissue area as a nearby hepatoblastoma. Two of three adenomas and two of three carcinomas had ß-catenin mutations in exon 2 as compared with nearby hepatoblastomas that all had mutations. However, all of the point mutations or deletions in the hepatoblastomas were different from those found in the corresponding hepatocellular neoplasms (data not shown). These results suggested that mutations found in hepatoblastomas may be different and lead to a more malignant phenotype than those in the hepatocellular tumors.

To further address this question, we examined DNA from 63 frozen hepatocellular neoplasms for mutations in ß-catenin. Ten of 32 adenomas (31%) and 13 of 31 carcinomas (42%) exhibited ß-catenin mutations (Table 2 ). Although there were more deletions and more multiple mutations in the hepatoblastomas than in the hepatocellular neoplasms, the spectrum of mutations was not extensively different among the different tumor histotypes. We also analyzed six normal liver tissue samples from mice in the high-dose anthraquinone group and found no mutations in these samples (Fig. 1 ). The frequency of mutations in the hepatoblastomas (27 of 27) is statistically higher (P < 0.0001 by Fisher’s exact test) than in hepatocellular neoplasms (23 of 63 anthraquinone induced and 18 of 41 oxazepam induced (4) , providing evidence that hepatoblastomas evolve from preexisting hepatocellular neoplasms with ß-catenin mutations.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Expression of ß-catenin protein in anthraquinone-induced mouse liver tumors by Western blot analysis. Equivalent amounts of protein from total homogenates of each sample were electrophoresed and immunoblotted, as described in "Materials and Methods." The blot was cut horizontally and developed with anti-ß-catenin (top) and anti-actin (bottom). Lane 1, normal liver; Lanes 2–6, hepatocellular neoplasms; Lanes 7–11, hepatoblastomas 2, 5, 13, 6, and 14a.

View larger version (179K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Immunohistochemical analysis of ß-catenin expression in anthraquinone-induced hepatoblastomas and surrounding liver tumors and tissue. A, hepatoblastoma 14b (not analyzed for ß-catenin mutations) within a hepatocellular carcinoma; H&E (x40). B, the same hepatoblastoma reacted with ß-catenin antibody (x40). Note marked membrane and cytoplasmic staining, compared with surrounding hepatocellular cells of carcinoma with only weaker membrane staining. C, higher magnification of hepatoblastoma in B (x132). D, hepatoblastoma 2 with strong nuclear and cytoplasmic staining (x50). E, high magnification of area in hepatoblastoma showing some cytoplasmic and nuclear staining (x100). F, high magnification of hepatoblastoma showing marked nuclear staining (x200).

	DISCUSSION

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To date, the pathogenesis of hepatoblastomas in mice has not been well defined. Hepatoblastomas occur only rarely in untreated mice of certain strains or following treatment only with tumor initiators such as N-nitrosodiethylamine (19) . However, following chronic treatment of B6C3F1 mice with promoters such as phenobarbital, a high incidence of hepatoblastomas has been observed (20 , 21) . Hepatoblastomas are usually observed within hepatocellular carcinomas and may evolve as a variant or more malignant form of adenoma or carcinoma (3 , 21) . The finding of 31% of anthraquinone-induced adenomas, 42% of carcinomas, and 100% of hepatoblastomas with ß-catenin mutations suggests that this molecular event may be critical for the development of hepatoblastomas within existing benign and malignant hepatocellular neoplasms. Previously, we showed that hepatocellular neoplasms in untreated mice have only a low frequency of ß-catenin mutations (13) . Thus, the accumulation of ß-catenin protein at the cell membrane in the chemically induced hepatocellular neoplasms with subsequent translocation to the nucleus, and leading ultimately to transactivation of growth promoting genes, may provide the oncogenic potential to develop into hepatoblastomas.

We first hypothesized that the ß-catenin mutations in the malignant hepatoblastomas would be different from and/or more severe than those in the hepatocellular neoplasms, thus leading to the different phenotype. However, whereas the hepatoblastomas sometimes had multiple ß-catenin mutations and many of these were deletions, most of the specific mutations were found in each of the tumor types. Moreover, it could be argued that because of the high incidence of spontaneous hepatocellular neoplasms that lack ß-catenin mutations, some of which are found among the tumors in the treated mice, the frequency of ß-catenin mutations in the chemically induced hepatocellular neoplasms is actually higher than that observed. These findings suggest that another molecular event is needed for the development of the hepatoblastoma histotype or that multiple mutations lead to a different or more severe phenotype. E-cadherin at the cell membrane and the APC protein in the cytoplasm are known to compete for ß-catenin binding (22) . The accumulation of ß-catenin along the cell membranes in the hepatocellular neoplasms suggests that at least some ß-catenin mutations lead to binding of mutant ß-catenin to E-cadherin. It is possible that most of the ß-catenin point mutations and some of the deletions in these hepatocellular tumors lead to decreased binding efficiency of APC but not E-cadherin. In contrast, the hepatoblastomas had prominent cytoplasmic and nuclear localization of the ß-catenin protein, suggesting that E-cadherin expression may be decreased in these tumors. E-cadherin is a putative tumor suppressor protein for some human cancers (23) and is lost during progression in several human tumor types (24 , 25) . However, another possibility is that some ß-catenin mutations (especially larger deletions) alter or remove E-cadherin binding sites from the protein. This could permit unbound truncated, but potentially still active ß-catenin protein to move from the cell membrane into the cytoplasm and nucleus. Ultimately, following the binding of cotranscription factors such as Tcf-4 to the mutant ß-catenin protein and transactivation of genes such as c-Myc (11) , this cascade of events may result in enhanced cell proliferation. Most studies on human tumors that contain ß-catenin mutations have demonstrated cytoplasmic and nuclear staining of ß-catenin protein (12 , 26 , 27) , and the mouse hepatoblastomas seem to follow this pattern.

The dominant nature and high frequency of ß-catenin mutations identified in the mouse hepatoblastomas in this study suggest that alteration in the stability and regulation of ß-catenin expression is an important event in the formation of these tumors in the B6C3F1 mouse. Moreover, the ß-catenin mutations are the same as those found in human hepatoblastomas, suggesting that similar carcinogenic pathways exist in the two species. Studies in our laboratory are now investigating the consequences of ß-catenin protein accumulation and interaction with other proteins in the development of hepatocellular neoplasms and hepatoblastomas to further understand the mechanisms of chemical induction of liver carcinogenesis in this model.

	ACKNOWLEDGMENTS

 
We thank Drs. David Malarkey and James R. Hailey for critical reading of the manuscript and helpful comments.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at MD D4-04, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. Phone: (919) 541-3241; Fax: (919) 541-7784; devereux@niehs.nih.gov.

² The abbreviations used are: APC, adenomatous polyposis coli; SSCP, single-strand conformational polymorphism.

Received 9/ 3/99. Accepted 4/ 4/00.

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