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. 1998 Jul 21;95(15):8847-51.
doi: 10.1073/pnas.95.15.8847.

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

A de La Coste  1 , B Romagnolo, P Billuart, C A Renard, M A Buendia, O Soubrane, M Fabre, J Chelly, C Beldjord, A Kahn, C Perret
Affiliations

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

A de La Coste et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Analysis of β-catenin in hepatocarcinomas developed in transgenic mice. (A) Immunoblot analysis for β-catenin in L-PK/c-myc transgenic mice. The expected 92-kDa band for β-catenin was detected in normal tissue (N, lanes 2, 4, 6, and 8–11) surrounding the tumor, whereas various truncated proteins were found in tumor nodules (T, lanes 3, 5, 7, 9, and 10). T1 and T2 (lanes 9 and 10) are two independent nodules from the same animal. L, sample from a nontransgenic mouse (lane 1). (B) RT-PCR strategy used to study the β-catenin mRNA. The diagram shows the β-catenin functional domains. The N-terminal domain is involved in the posttranslational stabilization via the GSK-3β phosphorylation site and in binding to α-catenin. The GSK-3β site includes the S33, S37, T41, and S45 phosphorylable residues. The 13 armadillo repeats are shown. Pairs of primers were designed to amplify overlapping PCR products that covered the whole ORF of β-catenin (F1-R1, F2-R2, F3-R3, F4-R4, and F5-R5, see Materials and Methods for the sequence). (C) Southern analysis of RT-PCR showing deleted β-catenin mRNA in L-PK/c-myc transgenic mice. mRNAs obtained from tumor tissue (lanes 2, 4, and 6) together with their nontumor counterparts (lanes 1, 3, and 5) were analyzed by RT-PCR with primers F1-R2, followed by Southern analysis.
Figure 2
Figure 2
Point mutations in the β-catenin gene in tumors developed in L-PK/c-myc mice. (A) Sequence analysis showing the D32Y and S37Y mutations. (B) Digestion of the RT-PCR products with appropriate restriction enzyme. Undigested wild-type amplified cDNA fragment (lane 1). Wild-type amplified cDNA fragments digested with XmnI (lane 2) and RsaI (lane 4). Digested amplified cDNA fragments from tumor sample showing a β-catenin gene mutation, the S37Y mutation led to the absence of digestion by the XmnI enzyme (lane 3), and the D32Y mutation led to the appearance of a RsaI restriction site (lane 5) on the mutated allele.
Figure 3
Figure 3
β-catenin in human hepatoma cell lines. (A) β-catenin immunolocalization. Cultured human hepatoma cells were immunolabeled with rabbit polyclonal anti-β-catenin antibody. (a) PLC/PRF/5 hepatoma cells containing wild-type β-catenin and showing only membrane staining. Hepatoma cells with activated β-catenin showed nuclear staining. (b) Huh6 cells. (c) HepG2 cells. There was no labeling when the first anti-β-catenin antibody was omitted (not shown). (B) Western blot analysis. Truncated β-catenin in HepG2 cells (lane 1). Full-length β-catenin in PLC/PRF/5 and Hep3B cells (lanes 2 and 3). The G34V β-catenin mutation identified in the Huh6 cells led to accumulation of the protein (lane 4).
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