Yonago Acta medica 2001;44:107–114

Frequent Mutations in the β-Catenin Gene in Cholangiocarcinoma
Noriko Miyake, Kanenori Endo, Eiji Nanba* and Tadashi Terada
Second Department of Pathology, Tottori University Faculty of Medicine and *Gene ResearchCenter, Tottori University, Yonago 683-0826 JapanThe molecular pathogenesis of cholangiocarcinoma (CC) remains unclear. β-Cateninfunctions in both intercellular adhesion and signal transduction. As a signaling molecule,mutations in exon 3 of the β-catenin gene encoding the regions phosphorylated by glycoβ stabilize this protein in cytoplasm. Subsequently, accumugen synthase kinase (GSK)-3β
lated β-catenin protein translocates to nuclei and up-regulates the transcriptional activityof genes involved in oncogenesis. Recently, mutations in exon 3 of the β-catenin gene
were detected in various carcinomas. Using polymerase chain reaction (PCR)-singlestrand conformational polymorphism (SSCP) analysis, direct sequencing and subcloning-sequencing, we investigated mutations of exon 3 of the β-catenin gene in CC.
Mutations were found in 26 out of 33 (78.8%) CC tumor samples. All of the mutations
were heterozygous 1-base deletions at codon 15, resulting in a stop codon at codon 46.
This is the first study demonstrating the presence of β-catenin gene mutations in CC.
However, it was suggested that this mutation might not be involved in deregulation of
β-catenin signaling, because no correlation was observed between the β-catenin mutationand immunolocalization of β -catenin protein.
Key words: β-catenin; cholangiocarcinoma; deletion; exon 3; mutation

Cholangiocarcinoma (CC) is an intrahepatic
bile duct carcinoma, and is thought to arise
from cholangiocytes or liver stem cells (Sell etal., 1989). The molecular mechanism for its
carcinogenesis is poorly understood, althoughsome studies demonstrating mutations of p53 or
K-ras have been reported in CC (Tada et al.,
1992; Tsuda et al., 1992; Kang et al., 1999).
β-Catenin, the vertebrate homologue ofarmadillo protein in Drosophila, is a multifunctional protein involved in cell-cell adhesion
regulation coupled with E-cadherin (Ozawa etal., 1989) and the Wnt-Wingless developmental
signal-transduction pathway (Gumbiner, 1995).
Disruption of this pathway has been suggested
in tumorigenesis. In the absence of Wnt signaling, β-catenin is assembled with adenomatous
polyposis coli (APC), axin and glycogen synthase kinase (GSK)-3β and is phosphorylated atexon 3, coding NH2 terminal serine/threonine

residue by GSK-3β and is subjected to degradation by the ubiquitin-proteasome system(Aberle et al., 1997). Activation of the Wnt signal pathway inhibits GSK-3β and subsequently
induces β-catenin stabilization. Unphosphorylated β-catenin will not be subjected to ubiquitination and accumulate in cells. β-Catenintranslocates to the nucleus with T cell factor(Tcf)/lymphoid enhancer factor (LEF) family
members and activates transcription of target
genes in cell growth control and apoptosis suchas c-myc (He et al., 1998) and cyclin D1 (Tetsu
and McCormick, 1999). Mutations in the βcatenin gene have been reported in various
human tumors and cell lines. The first studies
demonstrated mutations at the GSK-3β phosphorylation site in colon cancers (Morin et al.,1997) and melanoma cell lines (Rubinfeld et al.,1997). In colon cancers, expression of the βcatenin protein in the nucleus increased in

Abbreviations: APC, adenomatous polyposis coli; CC, cholangiocarcinoma; GSK, glycogen synthasekinase; LEF, lymphoid enhancer factor; PCR, polymerase chain reaction; SSCP, single-strand conformational
polymorphism; Tcf, T cell factor

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N. Miyake et al.

tumors with either APC mutations or β-cateninmutations at the GSK-3β phosphorylation site,
because mutations of APC or β-catenin prevented the degradation of β-catenin (Morin etal., 1997; Sparks et al., 1998). Similar mutations in exon 3 of β-catenin have been demonstrated in other cancers and cancer cell lines,
including endometrial carcinoma (Kobayashi
and Sagae, 1999), thyroid carcinoma (GarciaRostan et al., 1999), medulloblastoma (Zurawel etal., 1998), hepatocellular carcinoma (Miyoshi
et al., 1998), hepatoblastoma (Koch et al., 1999)
and colorectal cell lines (Ilyas et al., 1997). Nuclear and cytoplasmic localization of β-catenin
was also reported in endometrial carcinoma(Kobayashi and Sagae, 1999), thyroid carcinoma (Garcia-Rostan et al., 1999), hepatocellular
carcinoma (Van Nhieu et al., 1999), and hepatoblastoma (Koch et al., 1999). Therefore it wassuggested that mutations in β-catenin were involved in carcinogenesis. However, mutationsin the β-catenin gene in CC have never been
investigated. The purpose of this study was to
examine mutations in exon 3 of the β-catenin
gene in CC.

Materials and Methods

DNA preparation
Genomic DNA was extracted from 33 formalinfixed, paraffin-embedded CC tissues. The ageof the 33 patients ranged from 19 years to 81
years with an average of 65 years, and the maleto female ratio was 6:5. All tissues of CC wereadenocarcinomas, and they were classified into
well-differentiated CC (14 patients), moderately-differentiated CC (12 patients) and poorlydifferentiated CC (7 patients). DNA was also
extracted from peripheral blood of 53 normal
Japanese volunteers. The paraffin sections
were deparaffinized, and tumor lesions andnontumor liver tissue adjacent to the tumor
were scratched up separately under dissecting
microscopy (Nikon, Tokyo, Japan). DNA wasextracted using standard proteinase K digestionand phenol/chloroform extraction.

PCR/SSCP analysis
Exon 3 of the β-catenin gene was amplified in
two polymerase chain reactions (PCRs) using
intron-based nested primers. The outsideprimers for the first reaction were 5'-CCA ATCTAC TAA TGC TAA TAC TG-3' and 5'-CTG
CAT TCT GAC TTT CAG TAA GG-3' (Iwaoet al., 1998); the inside primers for the second
reaction were 5'-TCT ACT AAT GCT AAT
ACT GTT TCG-3' and 5'-ATT CTG ACT TTC
AGT AAG GCA ATG-3' (Samowitz et al.,1999). The PCR was carried out in a total reaction volume of 20 µL containing genomic
DNA, 10 pmol of each primer, 250 µmol/L ofeach dNTP, 1.5 mmol/L MgCl2 and 0.5 units ofGene Taq polymerase (Nippon Gene, Tokyo).
The products of the first PCR were used as atemplate for the second PCR. Both reactions
were performed on an ATTO Zymoreactor II(ATTO, Tokyo) with an initial denaturation
step at 94˚C for 2 min, then 30 cycles at 94˚C
for 1 min, 58˚C for 2 min and 72˚C for 2 min,and finnaly 1 cycle at 72˚C for 10 min.
A mixture of 2 µL PCR products and 5 µL
denaturing solution (98% formamide, 20 mmol/
L EDTA and 0.001% bromophenol blue) was
heated at 95˚C for 5 min followed by rapid
cooling on ice and electrophoresed on 14%
polyacrylamide gels with 5% glycerol in 1 ×
TBE running buffer at room temperature. Afterelectrophoresis, bands were visualized by silver
staining (Sugano et al., 1993).

Sequencing
The PCR products with an aberrant band detected by SSCP were purified using the QIAquick
PCR Purification Kit (Qiagen, Hilden, Germany)
and were subjected to cycle sequencing using
the BigDye Terminator Cycle Sequencing FS
Ready Reaction Kit (Perkin-Elmer Applied Biosystems, Foster City, CA). Sequencing was performed in both directions and each sequencing
product was run on an ABI PRISM 310 Genetic
Analyzer (Perkin-Elmer Applied Biosystems).
Data were analyzed by SequencingAnalysis 3.0
software (Perkin-Elmer Applied Biosystems).

108

Mutation of β-catenin in cholangiocarcinoma

The PCR products with deletion detected bydirect sequencing were also subcloned into apGEM-T Vector (Promega, Madison, WI). The
DNA sequence of each clone was determinedusing an ALFred sequencer (Amersham
Pharmacia Biotech, Uppsala, Sweden) with a
Cy5 Thermo Sequenase Dye Terminator Kit(Amersham Pharmacia Biotech).

Reliability of the technique
We performed PCR/SSCP analysis repeatedly
starting with the first PCR in all CC samples. In
addition, to confirm the reliability of ourtechnique, we analyzed the β-catenin gene inthyroid carcinoma (n = 6) in which both frozen
and formalin-fixed materials were available,using our method. Moreover, we analyzed theβ-catenin gene in formalin-fixed colorectal carcinoma (n = 6), which was thoroughly studied
for β-catenin gene mutation, using our method.

ported (Ashida et al., 1998). In each β-catenin
immunostaining, β-catenin expression was
evaluated and the results were semiquantitatively categorized into the seven ranging from
+3 to –3 according to the percentage of positive
cells within a tumor area in comparison to a
nontumor area, as previously reported (Ashida
et al., 1998). A nuclear expression was considered positive when more than 10% of nuclei
was strongly stained for β-catenin, as previously reported (Terris et al., 1999).

Statistical analysis
Statistical analysis was performed using Fisher’s
exact test and Mann-Whitney’s U test with asignificant level of P < 0.05.

Results

Exon 3 of the β-catenin gene from CC tumor
and nontumor samples was amplified by PCR
Immunohistochemistry
and the resulting PCR products were screened
Immunostaining was performed on 3-µm paraffin by SSCP analysis. Representative SSCP gel
sections with β-catenin monoclonal antibody patterns of 3 tumor and nontumor tissues from(clone 14, immunoglobulin G1 class) purchased CC are shown in Fig. 1. Aberrant bands werefrom Transduction Lab (Lexington, KY) using detected in the majority of tumor samples. PCR
the antigen retrieval protocol, as previously re- products with aberrant bands detected by SSCP
analysis were subjected to direct sequencing. The results of direct
sequencing analysis suggested that
Case 1Case 2
Case 3the β-catenin gene from tumor samNT
T
NT
T
NT
T
ples with aberrant SSCP bands mightbear heterozygous deletion (Fig. 2a).
By subcloning-sequencing, it was confirmed that one allele of the β-catenin
gene had a 1-base pair deletion (A) at
codon 15 and the other was a wildtype (Fig. 2b). We detected this mutation in 26 of 33 (78.8%) CC tumor tissues. Other types of mutations were
not found in this study. This alteration
was reproducible by repeated analysis
starting with the first PCR. In both thefrozen and formalin-fixed thyroid carFig. 1. Single-strand conformational polymorphism analysis
cinomas used as controls, there wereof tumor (T) and nontumor (NT) from cholangiocarcinoma
no mutations in any cases. Moreover,samples. Aberrant bands are indicated by the arrow. Mutations
we did not detect any mutations inwere detected in the tumor tissues in cases 1 and 3.
109

N. Miyake et al.

formalin-fixed colorectal carcinoma. This deletion at codon 15 of the β-catenin gene caused
a frameshift that resulted in a stop codon 31
amino acids downstream from the mutation,
thereby producing a truncated peptide (Fig. 3).
Since this mutation was detected in neitherabnontumor liver tissue nor peripheral blood fromhealthy volunteers (Table 1), it was consideredthat this mutation was somatic and might berelated to CC. To the best of our knowledge,
this is the first study demonstrating the presenceof β-catenin mutations in CC.
Relationships between the βcatenin mutation and the available
clinicopathological data were demutation
termined (Table 2). No significant
difference was found between the
AGG CATGGCC TGGANCCGNCCNNAAAANCGGTTGTT304050
β-catenin mutation and the clinicopathological parameters.
Relationships between the βcatenin mutation and immunolocalization were also determined(Table 3). We previously reported
that membranous expression was
wild-type codon 15
unchanged or decreased in all
TGGCATGGCC TGGAACCAGACA GAAAAGCGGCTGTTA
cases (Ashida et al., 1998). In this
20
30
40
50
study, we reevaluated intracytoplasmic and nuclear expression in
CC. Cytoplasmic staining was observed in 8 cases (24%). Amongthem, 3 cases (9.1%) showed immunoexpression in the tumor cell nuclei. Membranous expression incaseswith mutation did not signifwild-type allele
icantly decrease compared withthat in cases without mutation. No
correlation was found between βcatenin mutation and immunohistochemical data.
C C A T G G A A C C A G

mutant allele

C C

A

T G

deletion (A) at codon 15

G

A

C

C

A

G

Fig. 2. Sequencing data of β-catenin exon 3 in cholangiocarcinoma (CC) samples.
a: Electropherograms by direct sequencing showing the sequence of tumor samples with aberrant bands
detected by single-strand conformational polymorphism analysis and wild-type sequence derived fromthe control sample. The possibility of heterozygous 1-base pair deletion is indicated by the arrow.
b: Electropherograms from subcloned polymerase chain reaction products of CC tumor tissues with mutation showing sequences of the wild-type allele and mutant allele. One-base pair (A) deletion was
detected at codon 15 in mutant allele.

110Mutation of β-catenin in cholangiocarcinoma

1-bp deletionat codon 15
mutantstop codon

6–DLMELDMAMDQTEKRLLVTGSNSLTWTLESILVPLPQLLL*–46

wild-type 6–DLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSG–48
GSK-3β sites

NH 2

COOH
exon 3Fig. 3. Schematic representation of the location of β-catenin mutations in cholangiocarcinoma. bp, base pair.

Table 1. Alterations in the β-catenin gene

Discussion
The most frequent mutations in various cancers
have been shown to result in amino acid substitutions at several serine or threonine phosphorylation sites and flanking residues in theregulatory degradation targeting region encoded by exon 3 (Ilyas et al., 1997; Miyoshi et al.,
1998; Garcia-Rostan et al., 1999; Kobayashi
and Sagae, 1999; Koch et al., 1999). Small deletions at the GSK-3β phosphorylation site orlarger deletions spanning the whole of exon 3
were also reported in some cancers and all ofthem were in-frame mutations (Ilyas et al., 1997;
Rubinfeld et al., 1997; Iwao et al., 1998; Miyoshi
et al., 1998; Koch et al., 1999; Van Nhieu et al.,1999). Therefore it was suggested that unphosphorylated β-catenin accumulated in cytoplasmsof cells and translocated to the nuclei. This isconsistent with the data of immunohistochem-

Frequency of deletionat codon 15(%)
Tumor tissues (CC)
Nontumor tissues (CC)
Blood (healthy volunteers)CC, cholangiocarcinoma.

78.7 [26/33]
0 [ 0/33]
0 [ 0/53]

ical studies in various carcinomas (Iwao et al.,
1998; Garcia-Rostan et al., 1999; Kobayashi and
Sagae, 1999; Koch et al., 1999; Van Nhieu et al.,1999). Mutations in most carcinomas were
found at a frequency of 10 to 30%, although βcatenin gene alterations in some cancers including thyroid carcinoma (Garcia-Rostan et al.,1999) and hepatoblastoma (Koch et al., 1999)
occurred at a frequency of 50 to 60%. However, the mutation detected in this study occurred more frequently (78.8%). Moreover, thisTable 2. Correlations between β-catenin mutation and clinicopathological data
Variable

Category

No mutation

Mutation

P value

4
2
1
5
2
5
2

10
10
6
22
4
12
14

NS

Tumor grade

Well-differentiated CC
Moderate-differentiated CC
Poor-differentiated CC
Vascular invasion
Present
AbsentMetastasis
Present
AbsentCC, cholangiocarcinoma; NS, not significant.

111

NS
NS

N. Miyake et al.
Table 3. Relationship between β-catenin mutation and expression of β-catenin protein0Membranous–1–2

Expression of β-catenin protein
Cytoplasmic
–3
0
+1
+2
+3

No mutation
3
2
2
0
4
2
0
Mutation
11
7
7
1
21
3
2
0, the same expression as that of non-cancerous bile ducts;–1, mildly weak expression relative to non-cancerous bile ducts;–2, moderately weak expression relative to non-cancerous bile ducts;–3, markedly weak expression relative to non-cancerous bile ducts;+1, mildly strong expression relative to non-cancerous bile ducts;+2, moderately strong expression relative to non-cancerous bile ducts;+3, markedly strong expression relative to non-cancerous bile ducts;+, nuclear positive expression;–, nuclear negative expression.

mutation is a rare frameshift in the coding region, although mutations resulting in a stopcodon were also detected in some genes, including E-cadherin in sporadic lobular breast cancer(Berx et al., 1996) and p53 in ovarian carcinoma (Milner et al., 1993). Although we haveused formalin-fixed tissues in this study, it wasreported that non-reproducible sequence alterations were detected with the use of formalinfixed tissues (Williams et al., 1999). This
alteration was reproducible by repeated analysis starting with the first PCR. To confirm thereliability of our technique, we analyzed the βcatenin gene in thyroid carcinoma and there
were no mutations in both fresh and formalinfixed specimens. In addition, there were no mutations in the β-catenin gene in formalin-fixedcolorectal carcinoma. Therefore, we considered that the technique we used was reliable. Although immunolocalization of β-catenin in CC
tissues has hardly been investigated, we previously demonstrated that membranous expression of β-catenin was unchanged or reduced in
CC specimens (Ashida et al., 1998). Since immunolabeling was performed using an anti-βcatenin monoclonal antibody, raised against the
carboxyl-terminal fragment of β-catenin, it wassuggested that normal β-catenin protein expressed in CC tumor tissues. In this study, we demonstrated cytoplasmic and nuclear expression
in some cases. However it was considered thataberrant expression of β-catenin protein was
not associated with the mutation.

10

Nuclear
+
–
1
2

6
24

In our study, there were no significant differences between β-catenin mutations andclinicopathologic data. The reason for this is
unclear now, and remains to be clarified in the
future. It is unclear whether this truncatedprotein is stable. Commercial antibody against
the N-terminal of β-catenin, which is useful for
immunohistochemistry, was not available inour study. We also could not perform Western
blot analysis because the corresponding frozen
tissues were not available. Moreover, we could
not make an antibody against the new protein
sequence. However, we thought that this protein might be unstable and would immediately
degrade, because it is a very small protein consisting of 46 amino acids. It would be interesting to analyze a more related tissue during embryonal development like pancreas carcinoma
as a control; however, it was impossible toobtain pancreas carcinoma tissues in the present
study. Because no metastatic lesions of CC
were available, no investigation of β-catenin inmetastatic foci was performed in our study. For
these subjects, further studies are required.
It was reported that loss of heterozygosity of
APC was detected in CC (23.4%) and the alteration of APC might play a role in the tumorigenesis of CC (Kang et al., 1999). The alteration of APC might be involved in β-cateninprotein accumulation because APC interactedwith β-catenin in the Wnt signal pathway. In
addition, other factors such as axin and GSK-βwhich were related to the degradation of β-112

Mutation of β-catenin in cholangiocarcinoma

catenin in the ubiquitin-proteasome systemwere considered to be associated with increased
β-catenin protein expression, since it was recently reported that the mutation in AXIN1, an
encoding axin, caused hepatocellular carcinoma (Satoh et al., 2000).
In summary, the present study shows for the
first time that somatic mutation in the β-catenin
gene occurs frequently in CC. However, no correlation was found between the β-catenin mutation and immunolocalization of β-cateninprotein. Therefore, it was suggested that deregulation of β-catenin signaling might notoccur in CC. Further investigations are required to elucidate the role of heterozygous mutation in CC.

Acknowledgments: We thank Ms. Kaori Adachi forher excellent technical assistance.
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Received February 26, 2001; accepted March 12, 2001
Corresponding author: Prof. Tadashi Terada