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Frequent Deletions and Mutations of the ß-Catenin Gene Are Associated with Overexpression of Cyclin D1 and Fibronectin and Poorly Differentiated Histology in Childhood Hepatoblastoma¹

Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717 [H. T., T. O., A. N.]; Japanese Study Group for Pediatric Liver Tumor [H. H., E. H., T. M., Y. H., Y. W., S. S., M. K., F. S., K. H., N. O., A. N.]; and Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo 060-8638 [K. F., M. T.], Japan

	ABSTRACT

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Hepatoblastoma (HBL) is the most common malignant liver tumor in young children. Recent reports have shown that the ß-catenin gene was frequently mutated or deleted in HBLs. To elucidate the role of ß-catenin abnormalities in HBLs, we searched for mutations of ß-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLs. The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of ß-catenin, all of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation. However, no loss of function mutation of the APC gene was detected by the yeast functional assay. Of interest, ß-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR. The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmic accumulation of ß-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene. The significant correlation between the ß-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed. Furthermore, the nuclear accumulation of ß-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor. Thus, our present results suggested that the gain of function mutation of ß-catenin played a crucial role in the malignant progression of HBL in vivo.

	INTRODUCTION

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HBL³ is the most frequent pediatric liver cancer and 70% of the tumors are diagnosed during the first 2 years of life (1) . HBL originates from the hepatic precursor cells and exhibits a morphological similarity to the immature hepatocytes of the developing liver. In contrast to the adult hepatocellular carcinoma, HBL is not accompanied with hepatitis virus infection (2) . HBL is an embryonal tumor and often occurs in the patients with Beckwith-Wiedemann syndrome, which is associated with loss of heterozygosity at chromosome 11p15 (3) . Expression of the insulin-like growth factor 2 (IGF2) gene, mapped to 11p15.5, has been found to be regulated by the genomic imprinting (4) . Several lines of evidence suggest that loss of imprinting of the IGF2 gene is involved in the progression of HBL (5, 6, 7) . However, the molecular basis of the pathogenesis and development of HBL is still poorly understood.

The incidence of HBL is significantly high in patients with FAP (8, 9, 10) . The patients with FAP have been shown to carry germ-line mutations in the APC tumor suppressor gene (11 , 12) . The APC gene, mapped to 5q21, encodes a large cytoplasmic protein that functions in the Wnt signaling pathway (13) . Biallelic inactivation of the APC gene has been reported in the FAP-associated as well as in sporadic HBLs (14 , 15) . Furthermore, frequent mutations or deletions of ß-catenin have recently been demonstrated in HBLs (16 , 17) . This suggests that the abnormal Wnt signaling is involved in the genesis or progression of HBL.

The APC protein functions by associating with ß-catenin and Axin, and regulates the cytoplasmic levels of ß-catenin (18 , 19) . Phosphorylation of ß-catenin by GSK3ß stimulates its ubiquitin-dependent degradation and reduces the amount of cytoplasmic ß-catenin (20 , 21) . Loss of function mutations of APC and/or gain of function mutations of ß-catenin can cause nuclear translocation of the ß-catenin-Tcf/Lef complex and stimulate transcription of the target genes, which include E-cadherin (22) , connexin 43 (23) , ultrabithorax (24) , siamois (25) , cerberus (26) , and nodal-related 3 (27) . Recently, c-myc (28) , cyclin D1 (29) , and fibronectin (30) have also been identified to be possible target genes for the APC/ß-catenin pathway. Overexpression of c-myc and/or cyclin D1 is frequently observed in various human cancers (31 , 32) , and fibronectin plays an important role in the cancer metastasis (33) .

In the present study, we searched for mutations of ß-catenin and APC in primary HBLs found in Japan and examined the intracellular localization of ß-catenin. We found that the mutated ß-catenin was accumulated in the nuclei of the HBL cells, and the accumulation was observed in the tumor cells with poorly differentiated histology. Furthermore, mutations of ß-catenin were significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, in primary HBLs.

	MATERIALS AND METHODS

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Tissue Specimens.
Tumor tissues and their corresponding normal liver tissues were obtained at surgery, immediately frozen, and stored at -80°C until use. The tissues were also fixed in formalin and embedded in paraffin for histopathological analysis. The frozen tumor tissues were obtained from 68 patients with HBL, and the corresponding normal liver tissues were available from 20 patients. All of the specimens used in this paper were given from the Tissue Bank of the JPLT (34) . The patients were treated at various hospitals or institutions under the framework of JPLT between 1991 and 1999. Preparation of DNA was performed by the standard proteinase K-phenol-chloroform extraction, as reported previously (35) . Total RNA was prepared by a conventional guanidine thiocyanate-phenol-chloroform procedure (36) .

Mutation Analyses for ß-Catenin.
For the detection of mutations and deletions of the ß-catenin gene, genomic DNA derived from each tumor and the corresponding liver tissue was amplified by PCR using a primer pair specific for the exon 3. The sequence of the forward primer (ex3–2f) was 5'-AAAAATCCAGCGTGGACAATGG-3', and that of the reverse primer (ex3–2r) was 5'-TGTGGCAAGTTCTGCATCATC-3'. To search for mutations/deletions in the entire coding region of ß-catenin, RT-PCR was performed using five primer sets shown in Table 1 . Each reverse transcriptase mixture contained 5 µg of total RNA, 200 units Superscript II reverse transcriptase (Life Technologies. Inc., Gaithersburg, MD), and 160 pmol of random primers (TAKARA, Ohtsu, Japan) in a total volume of 30 µl. After denaturing at 65°C for 15 min, the reaction mixture was incubated at 42°C for 90 min. Then, the reverse transcriptase product was subjected to PCR amplification in a reaction mixture containing 1 µM each of primer, 200 µM dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH8.3), 1.5 mM MgCl₂, and 1 unit of Taq polymerase (Boehringer Mannheim, Mannheim, Germany). PCR was performed with thermal cycling parameters of 94°C for 15 s, 60°C for 15 s, and 72°C for 45 s, for 35 cycles using a Perkin-Elmer Corp. 9600 thermal cycler, automated. The identities of PCR products were analyzed by a 2% agarose gel electrophoresis. To detect interstitial deletions ranging from 12 to 33 bp, PCR products were size-fractionated by 5% polyacrylamide gels. PCR products with aberrant migration pattern were gel-purified, and their nucleotide sequences were determined by DNA sequence analysis.

To identify clonal missense mutation in the 42 sporadic HBL samples, at least four plasmids per sample were rescued from independent white colonies. Sequence analysis was performed between codons 1211 and 1541 of the APC gene that included the mutation cluster region (codons 1296 to 1513; Ref. 38 ).

PCR-SSCP Analysis.
For SSCP analysis, genomic DNA encoding exon 3 of ß-catenin was amplified using the following fluorescence-labeled primer pair: 5'-GCTTTTCTTGGCTGTCTTTC-3' (ex3–3f) and 5'-TCCACAGTTCAGCATTTACC-3' (ex3–3r). The 10-µl reaction mixture contained 200 nM each primer, 250 nM dNTPs, 1x reaction buffer, and 0.2 unit of TaKaRa Taq polymerase. Thirty-five cycles of amplification were performed under the following conditions: denaturation for 15 s at 94°C, annealing for 15 s at 55°C, and extension for 45 s at 72°C. PCR products were then analyzed by ALFexpress DNA sequencer (Amersham Pharmacia Biotech AB, Uppsala, Sweden). To confirm the data obtained by the above analysis, we carried out the standard PCR-SSCP analysis as described previously (39) . PCR products were purified from agarose gel using QIA Quick Gel Extraction kit (Qiagen, Inc.), and their nucleotide sequences were determined by direct sequencing using an Applied Biosystem 377 DNA sequencer (Perkin-Elmer Corp.).

Quantitative Real-Time RT-PCR.
The primer sets for amplification of the cyclin D1, fibronectin, and c-myc genes were shown in Table 2 . TaqMan ß-actin control reagents (Perkin-Elmer/Applied Biosystems) were used for the amplification of ß-actin as recommended by the manufacturer. PCR was performed using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems). Two µl of cDNA was amplified in a final volume of 25 µl containing 1x TaqMan PCR reaction buffer, 200 µM each dNTP, 5.5 mM MgCl₂, 0.9 µM each primer, 200 nM TaqMan probe, and 0.25 units of AmpliTaq Gold. The optional thermal cycling condition was as follows: 40 cycles of a two-step PCR (95°C for 15 s, 60°C for 60 s) after the initial denaturation (95°C for 10 min). Experiments were carried out in triplicate for each data point.

Statistical Analysis.
Statistical analyses were performed using Welch’s t test. A P of less than 0.05 was considered significant.

	RESULTS AND DISCUSSION

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The exon 3 of ß-catenin encodes a highly conserved, NH₂-terminal phosphorylation motif for GSK3ß and has been shown to be a target for mutation in various human cancers including HBL (16 , 17 , 40, 41, 42) . Mutation analysis was performed by focusing on the exon 3 of ß-catenin in 68 primary HBLs, using the standard PCR-SSCP procedure followed by subsequent DNA sequencing. Forty-four (65%) of 68 HBLs had mutations or deletions (Table 3) . The deletions were more frequent than the missense mutations in HBLs, as reported previously (16 , 17) . The interstitial deletions varying from 12 to 659 bp were found in 35 primary HBLs. All of them lost the whole or a part of exon 3, and some extended the deleted region to a part of exon 2 and/or exon 4. The DNA sequence analysis revealed that these mutants were in-frame deletions and might cause the generation of mutant ß-catenin proteins with a small molecular mass that lacked all or a part of NH₂-terminal phosphorylation sites required for their own degradation. On the other hand, the missense mutations were found in 9 tumors. They were Asp(GAC)32Gly(GGC) (case 11), Ser(TCT)33Pro(CCT)(cases 12 and 33), Gly(GGA)34Arg(AGA) (cases 5, 6, and 51), Gly(GGA)34Glu(GAA) (case 71), Ile(ATC)35Ser(AGC) (case 28), and Thr(ACC)41Ala(GCC) (case 38). Missense mutations detected in cases 12, 33, and 38 resulted in the replacement of Ser or Thr residue, which affected the phosphorylation by GSK3ß (20) . The other mutations, except that in case 28, led to the amino acid substitutions adjacent to Ser at codon 33 and may have affected the stability of ß-catenin (21) . The mutation in case 28 generated an alternative Ser at codon 35. We examined whether the alterations found in the ß-catenin gene were somatic or germ line by using the matched tumor and normal tissues, and we demonstrated that all of the mutations and deletions detected were somatic (data not shown).

To examine the expression and localization of ß-catenin in the tumor, we performed immunohistochemical analysis using 15 primary HBL tissues and their adjacent normal liver tissues. Intriguingly, the staining pattern of ß-catenin was different and heterogeneous within the tumor. HBL usually had both well-differentiated (fetal type) and poorly differentiated (embryonal type) cell components with varied percentage in the same tumor. ß-catenin was immunohistochemically stained in the cell membrane of the well-differentiated cell components at moderate levels (Fig. 1C) as observed in normal hepatic tissues (Fig. 1A) , whereas it was strongly stained in the nuclei of the poorly differentiated cell components (Fig. 1D) . Histologically, 15 HBL specimens were divided into four groups (Table 4) : 4 poorly differentiated HBLs, 5 mixed-type HBLs with predominant poorly differentiated components, 4 mixed-type HBLs with predominant well-differentiated components, and 2 well-differentiated HBLs. As shown in Table 4 and Fig. 1 , strong nuclear accumulation of ß-catenin was detected either focally or diffusely in 13 of 15 tumor specimens (Fig. 1, B and D) , whereas no significant nuclear staining was observed in the adjacent normal hepatic tissues (Fig. 1A) . Nine of 13 tumors with positive nuclear staining had mutations or deletions of the ß-catenin gene, which suggested that the mutated ß-catenin was accumulated and transported into the nuclei. However, four tumors (cases 19, 20, 26, and 65) that had the nuclear accumulation had no mutation of ß-catenin. There might be some explanations for the results. It could be possible that the tissue sample was heterogeneous, and only the well-differentiated region was subjected to mutation analysis of the ß-catenin gene. The other possibility is that they might have an alteration of other gene(s) in the APC/ß-catenin (or Wnt/Wingless) signaling pathway such as APC, GSK3ß, and Axin, which could be responsible for the nuclear accumulation of ß-catenin. However, the tumors from cases 22 and 26 had no loss of function mutation in the APC gene by yeast color assay. The tumor with well-differentiated histology from case 58 had the ß-catenin mutation, but there was no nuclear accumulation, the reason for which is unclear. Thus, the nuclear accumulation of ß-catenin was mostly caused by its own mutation or deletion and was strongly associated with poorly differentiated histology. In addition, aberration of the ß-catenin gene was a somatic event. These data suggested that the ß-catenin mutation could play a role in malignant progression of the HBL cells. The analysis using microdissection of each cell components should help to confirm it. The intratumorous heterogeneity of ß-catenin staining was also reported in other human tumors (41 , 42) .

View larger version (150K): [in this window] [in a new window]   Fig. 1. Immunohistochemical staining of ß-catenin and cyclin D1 in normal liver and HBLs with or without the ß-catenin abnormality. Shown were panels of tissues, including normal liver (A); HBL tissue from case 23, which carried ß-catenin mutation (deletion type; B); HBL tissues from case 22 without ß-catenin mutation (C and E); and HBL tissues from case 25 with ß-catenin mutation (deletion type; D and F). Each tissue was treated with an anti-ß-catenin monoclonal antibody (A–D) or an anticyclin D1 monoclonal antibody (E and F) and was visualized with biotin-conjugated secondary antibody. A, the plasma membrane of the normal hepatocytes were positive for ß-catenin. B, only the poorly differentiated components (right side) but not the well-differentiated components (left side) were positive for ß-catenin staining in the HBL tissue with ß-catenin mutation. C, like normal hepatocytes, the plasma membrane was positive for ß-catenin in the HBL tissue without the mutation. D, the nuclei were strongly positive for ß-catenin in the HBL tissue with the mutation. The cytoplasm of the tumor cells was also weakly stained. E, The staining of cyclin D1 was negative in the HBL tissue without the ß-catenin mutation. F, cyclin D1 was strongly positive in the nuclei of the tumor cells with ß-catenin mutation.

Characteristic nuclear localization of ß-catenin in primary HBLs prompted us to examine the ß-catenin-mediated transactivation of possible target genes. We performed quantitative real-time RT-PCR analysis to measure the mRNA levels of c-myc, cyclin D1, or fibronectin in 48 primary HBL tissues. Fourteen and 34 tumor samples were derived from patients without and with ß-catenin mutations/deletions, respectively. The adjacent normal liver tissues were available from eight patients and were used as the controls. The values of expression of the target genes were normalized by those of ß-actin. As shown in Fig. 2 , expression of cyclin D1 and fibronectin, but not c-myc, was significantly higher in HBL tissues with the ß-catenin mutation/deletion than in those without the abnormalities [cyclin D1: 14.8 ± 2.8 (n = 24) versus 4.6 ± 0.8 (n = 14), P = 0.028; fibronectin: 4.5 ± 1.0 (n = 24) versus 2.1 ± 0.4 (n = 14), P = 0.028]. However, the significance of fibronectin as a target of ß-catenin/TCF/Lef complex was still unclear because the difference between the levels of its expression in normal liver tissues and those in HBLs with ß-catenin mutations was not statistically significant. The elevated expression of cyclin D1 in HBL with ß-catenin mutation was also confirmed immunohistochemically (Fig. 1, E and F) . The tumor from case 25, which carried the ß-catenin mutation, exhibited the nuclear accumulation of ß-catenin as well as the increased levels of cyclin D1 expression (Fig. 1, D and F) . In contrast, the tumor from case 22 without ß-catenin mutation did not show positive immunostaining for either ß-catenin or cyclin D1 (Fig. 1, C and E) . The immunohistochemical study of cyclin D1 in 14 of 15 HBLs listed in Table 4 showed that 10 of 12 tumors with nuclear accumulation of ß-catenin had positive staining for cyclin D1, whereas 2 tumors without accumulation of ß-catenin showed no staining for it (data not shown). Thus, in vivo, mutation and nuclear accumulation of ß-catenin appeared to induce the target genes to enhance the HBL progression. However, there was a tendency that expression of cyclin D1 or fibronectin was decreased in HBLs without ß-catenin mutation/deletion, as compared with the adjacent normal liver tissues. In the previous reports, Iolascon et al. (44) showed that cyclin D1 expression is reduced in HBLs as compared with normal liver, whereas Kim et al. (45) reported the overexpression of both cyclin D1 and cdk4 in HBLs. The controversy between the data from the two independent groups may be attributable to the lack of the status of ß-catenin mutation in their samples. The reason why expression of both target genes was decreased in HBLs without ß-catenin mutation as compared with normal liver is unclear. Like cyclin D1 and fibronectin, c-myc was also reported as a target for the APC/ß-catenin pathway (28) . However, our present real-time RT-PCR analysis revealed that the expression levels of c-myc were reduced in HBLs irrespective of ß-catenin status as compared with adjacent normal tissues, although it was not significant. These results suggested that c-myc did not contribute to the progression of HBL to a great extent.

View larger version (16K): [in this window] [in a new window]   Fig. 2. mRNA expression of cyclin D1, fibronectin, and c-myc in HBL tissues and the adjacent normal liver measured by quantitative real-time RT-PCR. Total RNA was prepared from 8 normal liver tissues adjacent to the tumor, 14 HBL tissues without ß-catenin mutation, and 34 HBL tissues with ß-catenin mutation. The expression levels of the cyclin D1, fibronectin, and c-myc genes were determined by quantitative real-time RT-PCR analysis (see "Materials and Methods"). The values of expression of each gene was normalized by ß-actin. Results are expressed as the means; bars, SE (Welch’s t test).

	ACKNOWLEDGMENTS

 
We are grateful to Shigeru Sakiyama for valuable discussions and reading the manuscript and to Tomotane Shishikura for skillful technical assistance. We thank Eriko Isogai, Aiko Morohashi, and Naoko Sugimitsu for preparing RNA and sequencing analysis.

We also thank Drs. H. Kenmotsu (Division of Surgery, Ibaraki Children’s Hospital), O. Ijichi (Department of Pediatrics, Kagoshima University School of Medicine), H. Ikawa (Department of Pediatric Surgery Kanazawa Medical University), H. Nakadate (Department of Pediatrics, Kitasato University School of Medicine), H. Hosoi (Department of Pediatrics, Kyoto Prefectural University of Medicine), T. Noda (Department of Pediatrics, Kochi Municipal Central Hospital), H. Fujita (Department of Pediatrics, Juntendo University School of Medicine), S. Hasegawa (Division of Pediatrics, Nagoya Memorial Hospital), M. Iwafuchi (Department of Pediatric Surgery, Niigata University School of Medicine), E. Ito (Department of Pediatrics, Hirosaki University School of Medicine), H. Ayukawa (Department of Pediatrics, Yamaguchi University School of Medicine), Y. Tsuchida (Gunma Children’s Hospital Medical Center), J. Yokoyama (Department of Surgery, Keio University School of Medicine), A. Hayashi (Division of Surgery, Tokyo Metropolitan Kiyose Children’s Hospital), M. Miyake (Department of Pediatrics, Osaka Medical College), T. Matsuyama, Tetsushi Sugito (Department of Pediatrics, Nagoya First Red Cross Hospital), and H. Kurosawa (Department of Pediatrics, Dokkyo University School of Medicine) for providing the HBL tissue samples to JPLT.

	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 Supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a New Comprehensive 10-Year Strategy for Cancer Control of Japan and by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.

² To whom requests for reprints should be addressed, at Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuoh-ku, Chiba 260-8717, Japan. Phone: 81-43-264-5431; Fax: 81-43-265-4459; E-mail: akiranak{at}chiba-cc.pref.chiba.jp

³ The abbreviations used are: HBL, hepatoblastoma; APC, adenomatous polyposis coli; FAP, familial adenomatous polyposis; GSK3ß, glycogen synthase kinase 3ß; RT-PCR, reverse transcription-PCR; SSCP, single-strand conformation polymorphism; JPLT, Japanese Study Group for Pediatric Liver Tumor.

Received 11/ 6/00; revised 1/ 2/01; accepted 1/ 2/01.

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