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LABORATORY INVESTIGATION
Vol. 82, No. 3, p. 265, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Frameshift Mutations of Human Gastrin Receptor
Gene (hGARE) in Gastrointestinal Cancers with
Microsatellite Instability
Luigi Laghi, Guglielmina Nadia Ranzani, Paolo Bianchi, Antonio Mori,
Karl Heinimann, Ombretta Orbetegli, Marco Rondo Spaudo, Ombretta Luinetti,
Simona Francisconi, Massimo Roncalli, Enrico Solcia, and Alberto Malesci
Division of Gastroenterology (LL, OO, SF, A. Malesci) and Department of Pathology (PB, MR), Istituto Clinico
Humanitas, Rozzano-Milan, Departments of Genetics and Microbiology (GNR, A. Mori, MRS) and Human Pathology
(
OL, ES), University of Pavia, and IRCCS Policlinico S. Matteo, Pavia, and Departments of Pathology (MR) and
Internal Medicine (A. Malesci), University of Milan, Milan, Italy; Research Group in Human Genetics (KH), University
Hospital, Basel, Switzerland
SUMMARY: Gastrointestinal tumors with DNA mismatch repair (MMR) defects show microsatellite instability (MSI) and harbor
frameshift mutations in coding mononucleotide repeats of cancer-related genes (targets). We assessed MSI status in 233
sporadic gastrointestinal tumors. We classified as MSI-H (high-frequency microsatellite instability) 15 (10%) of 150 colorectal
cancers and 13 (16%) of 83 gastric cancers. We searched for frameshift mutations in a coding poly(T)8 tract within the gastrin
receptor gene (hGARE), which has a potential role in gastrointestinal carcinogenesis. To this purpose, we screened 43 unstable
tumors (including 15 hereditary nonpolyposis colorectal cancer cases previously classified as MSI-H), 98 stable tumors, as well
as 3 MMR-deficient and 4 MMR-proficient gastrointestinal cancer cell lines. We found mutations in 8 (19%) of the 43 MSI-H
tumors but in none of the 98 stable cancers. hGARE mutation frequency was similar in gastric (23%) and colorectal cancers,
including sporadic (13%) and hereditary (20%) cases. All mutated tumors proved to harbor frameshift mutations in other
cancer-related genes that are considered as targets in MSI tumorigenesis. The MMR-deficient and gastrin-sensitive LoVo
colorectal cancer cells also showed a hGARE heterozygous frameshift mutation, but expressed only the mutated allele. All
detected mutations can be predicted to generate a truncated protein carrying amino acid changes. On the basis of genetic
findings, we propose hGARE as a new candidate target gene in MSI tumorigenesis. Functional studies are warranted to elucidate
the mechanism by which the hGARE mutation might contribute to gastrointestinal carcinogenesis. (Lab Invest 2002, 82:265–271).
he cancers arising in most patients with heredi-
tary nonpolyposis colorectal cancer (HNPCC)
and a subset (10–20%) of sporadic gastrointestinal
tumors exhibit widespread genome instability that is
manifested by increases in mutation rates and by
alterations in the size of simple repeated sequences
and unbalanced allelic expression are involved in
hereditary tumors (Charbonnier et al 2000; Curia et al,
1999; Peltomaki and Vasen, 1997), whereas somatic
mutations and promoter hypermethylation play a ma-
jor role in sporadic tumors (Herman et al, 1998; Kane
et al, 1997; Liu et al, 1995).
MMR-deficient tumors frequently harbor insertions/
deletions in mononucleotide tracts within coding re-
gions of genes involved in the control of cell prolifer-
ation and survival. Frameshift mutations of cancer-
related genes (targets) have been proposed to be
under positive selective pressure during the evolution
of unstable tumors (Yamamoto et al, 1997). A number
of target genes have been implicated in the pathogen-
esis of MMR-deficient gastrointestinal cancers (Duval
et al, 2001) and some of these genes have been found
to be frequently mutated in these tumors. These
include the growth factor receptor genes TGF␤RII
T
(
Aaltonen et al, 1993; Ionov et al, 1993; Thibodeau et
al, 1993). This tumor phenotype, called MSI (from
microsatellite instability), is the result of alterations in
DNA mismatch repair (MMR) genes, which play a
crucial role in the maintenance of DNA-replication
fidelity (reviewed by Peltomaki, 2001). Different molec-
ular mechanisms have been found to inactivate MMR
genes: germ line mutations, genomic rearrangements,
Received September 7, 2001.
The first two authors contributed equally to this paper.
This work was supported by Consiglio Nazionale delle Ricerche (grants to
Alberto Malesci and ES), by Ministero Università e Ricerca (grant to
GNR), and by Ministero Sanità (grant to IRCCS Policlinico S. Matteo,
Pavia). MRS is a fellow of the Associazione Italiana Ricerca sul Cancro.
Address reprint requests to: Dr. Alberto Malesci, Division of Gastroenter-
ology, Istituto Clinico Humanitas, Via Manzoni, 56 - 20089 Rozzano
(Markowitz et al, 1995) and IGFIIR (Souza et al, 1996),
the proapoptotic gene BAX (Rampino et al, 1997), the
DNA-MMR genes hMSH3, hMSH6 (Malkhosyan et al,
1996), and MED1 (Bader et al, 1999; Riccio et al,
1999), the protease CASP-5 gene (Schwartz et al,
(
Milano), Italy. E-mail: alberto.malesci@humanitas.it
Laboratory Investigation • March 2002 • Volume 82 • Number 3 265

Laghi et al
1
1
999), the transcription factor TCF-4 gene (Duval et al,
999a), and the retinoblastoma protein interacting zinc
hGARE exon-5 sequence encompassing the (T)8 re-
peat. We analyzed polymerase chain reaction (PCR)
products either by sequencing gel or by capillary
electrophoresis. To avoid misclassifications, PCR
products from each tumor and corresponding normal
tissue were analyzed in parallel.
finger RIZ gene (Piao et al, 2000). The identification of
new targets might contribute to elucidating the pro-
gression pathway of MMR-deficient tumors and even
provide clues for cancer treatment (Boland et al,
1
998).
Among cancer cell lines, only MMR-deficient LoVo
cells showed an hGARE heterozygous mutation due to
a 1-bp deletion within the (T)8 repeat. Seven 1-bp
deletion and one 1-bp insertion mutations were found
in 8 (19%) of 43 MSI-H gastrointestinal tumors, but in
none of the 98 stable cancers. The frequency of
mutation was similar in gastric (3 of 13, 23%) and
colorectal cancers of both sporadic (2 of 15, 13%) and
hereditary (3 of 15, 20%) types. All tumors showing
frameshift mutations also showed a wild type allele.
This indicates that neoplastic cells were heterozygous
for the observed mutations and/or that non-neoplastic
cells contributing the normal allele were present in the
tissue sections used for DNA extraction. Figure 1
shows 1-bp deletion mutations as detected by capil-
lary (A) and gel electrophoresis (B).
DNA sequencing confirmed the deletion of 1 bp
within the (T)8 repeat of hGARE exon 5 in the LoVo
cells (Fig. 2) and in seven MSI-H tumors. This muta-
tion, leading to a premature stop at codon 377, is
predicted to generate a truncated protein carrying
amino acid changes. Sequencing also confirmed the
insertion of 1 bp, causing a stop mutation at codon
We used the GenBank database to search for
mononucleotide repeats within the coding regions of
growth factor and growth factor receptor genes pos-
sibly related to gastrointestinal tumorigenesis. We
found a (T)8 repeat in exon 5 of the human gastrin
receptor gene (hGARE, or CCKBR). The peptide hor-
mone gastrin represents a recognized growth factor
for gastrointestinal tumor cells (Iwase et al, 1997;
Kusyk et al, 1986; Seva et al, 1994; Singh et al, 1994;
Stepan et al, 1999). In addition, gastrin and CCK-B
receptor, the expression of which is low or absent in
normal colon (Monstein et al, 1996; Wank, 1998), have
been proposed to be involved in maintaining auto-
crine/paracrine growth pathways in gastrointestinal
cancer cells (McWilliams et al, 1998). To verify whether
unstable tumors harbor hGARE frameshift mutations,
we analyzed the (T) repeat of exon 5 in a panel of both
8
stable and unstable gastric and colorectal cancers, as
well as in MMR-deficient/proficient cell lines derived
from gastrointestinal tumors.
Results
3
50, in one gastric cancer. In a gastric cancer harbor-
We assessed MSI status in 233 sporadic gastrointes-
tinal tumors. On the basis of recently established
consensus criteria (Boland et al, 1998), we classified
as MSI-H 15 (10%) of 150 colorectal cancers and 13
ing a (T)7 repeat, DNA-sequencing revealed a mis-
sense mutation (G 3 A transition; Val 3 Ile) at codon
339. By cloning the PCR product from genomic DNA
and by sequencing different clones, we assessed that
frameshift and missense mutations were located on
different alleles (Fig. 3). The functional significance, if
any, of the missense mutation remains unclear.
Among the eight hGARE mutated tumors, six also
harbored a frameshift mutation of TGF␤RII gene, four
of BAX, three of hMSH3, four of hMSH6, and three of
IGFIIR.
To evaluate hGARE expression, we performed RT-
PCR experiments on LoVo and HCT116 MMR-
deficient colon cancer cell lines. By using 30 amplifi-
cation cycles and specific primers encompassing
hGARE exons 4 and 5, we could detect a single PCR
product in LoVo but not in HCT116 cells (Fig. 4a); a
PCR product was detectable in HCT116 cells only by
reaching 50 amplification cycles. Direct sequencing of
(
16%) of 83 gastric cancers.
We analyzed MSI-H tumors for insertions/deletions
within coding repeats of TGF␤RII, CASP-5, BAX,
hMSH3, hMSH6, and IGFIIR target genes. As ex-
pected, somatic mutations proved to occur in all of
these genes, although with different frequencies. The
prevalence of gene mutations are reported in Table 1.
We searched for insertion/deletion mutations of
hGARE gene in 43 unstable and 98 stable tumors, as
well as in 3 MMR-deficient and 4 MMR-proficient cell
lines derived from gastrointestinal tumors. Unstable
tumors included 13 gastric and 15 sporadic colorectal
cancers that we identified during this survey, as well
as 15 HNPCC cases previously classified as MSI-H.
Stable tumors included 28 colorectal and 70 gastric
cancers. Genomic DNA were used to amplify the
Table 1. Frameshift Mutations at Coding Repeats of Target Genes in High-Frequency Microsatellite Instability
Gastrointestinal Tumors
TGF␤RII
n (%)
CASP-5
n (%)
BAX
n (%)
hMSH3
n (%)
hMSH6
n (%)
IGFRII
n (%)
n
a
Colorectal cancers
Gastric cancers
30
12
24 (80)
9 (75)
16 (53)
nd
14 (46)
2 (16)
6 (20)
2 (16)
8 (26)
6 (50)
6 (20)
4 (33)
b
nd, not determined.
a
Includes: 15 sporadic and 15 hereditary tumors.
DNA from one case was not available.
b
2
66 Laboratory Investigation • March 2002 • Volume 82 • Number 3

hGARE Mutations in MSI Tumors
background of instability present in MMR-deficient
cells. In this respect, hGARE cancer-related gene is
likely to represent a new target, the mutation of which
may have functional significance.
On the other hand, the analysis of a number of
noncoding (intronic) mononucleotide tracts has dem-
onstrated that there is great variation in the prevalence
of mutations among repeated sequences (Zhang et al,
2001). Some of the investigated tracts proved to be
altered in less than 5% of the MMR-deficient tumors,
whereas other tracts were altered in nearly 50% of the
cases (Zhang et al, 2001). Very recently, it has been
found that a large number of noncoding microsatellite
mutations can accumulate in phenotypically normal
MMR-deficient cells, even before the occurrence of a
gatekeeper gene mutation, and in the absence of
abnormal clonal expansion (Vilkki et al, 2001). Taken
together these findings indicate that microsatellite
instability in MSI-H tumors is a complex mechanism
depending on different factors including size, nature,
and localization of sequences, as well as functional
significance. Therefore, the implication of a mutated
gene as a target may present a difficulty in the
absence of evidence that coding microsatellite muta-
tions are functionally relevant in tumorigenesis (Boland
et al, 1998; Zhang et al, 2001).
Figure 1.
hGARE frameshift mutations. T, tumor DNA; N, DNA from normal tissue. A,
Electropherograms of fluoresceinated PCR-products from colon cancer cell
lines (LoVo and HCT116) and from sporadic (CRC#136 and CRC#11) and
hereditary (HNPCC#10) colorectal cancers. Numbers indicate the length of the
amplicons (in bp). Frameshift mutations are present in LoVo cells and in tumor
DNA from samples CRC#136 and HNPCC#10. B, Gel electrophoresis of
radiolabelled PCR-products from gastric cancers (GC). A tumor sample
Frameshift mutations in the hGARE (T) repeat lead
8
to major changes in the highly conserved structure of
the CCKB receptor, a member of the superfamily of
G-protein–coupled receptors possessing seven trans-
membrane domains (Wank, 1998). The VI transmem-
brane domain is modified in its amino acid sequence,
whereas the VII domain and the intracellular
C-terminus are completely lost. It has been shown that
the truncation of the carboxyl terminus of a G-protein–
coupled receptor can result in constitutive activity
(
GC#5G) with a wild-type (T) repeat, and a tumor sample (GC#9GG) harboring
8
7
a (T) repeat are shown.
the product we obtained from LoVo cells demon-
strated the presence of only one of the two isoforms
that are known to be generated by exon 4 alternative
splicing (Fig. 4c) (Song et al, 1993). Interestingly, this
isoform corresponded with the hGARE frameshift-
(Matus-Leibovitch et al, 1995; Thomas et al, 1995). In
mutated allele and has been characterized by a (T)7
particular, a rat CCKB receptor mutant, lacking the 44
carboxyl-terminal amino acids and closely resembling
the putative receptor originating by hGARE frameshift
mutations, has recently been shown to retain binding
properties, G-protein coupling, and signal transduc-
tion activity (Pohl et al, 1997). Thus, the identified
hGARE mutations might lead to a gain rather than a
loss of the receptor function. Preliminary evidence in
support of this concept derives from expression stud-
ies we performed on LoVo and HCT 116 MSI-H cancer
cell lines. LoVo cells, which harbor an hGARE het-
erozygous mutation, are known to be responsive to
the gastrin peptides growth stimulatory effect,
whereas HCT 116 are nonresponsive (Stepan et al,
repeat in exon 5 (Fig. 4b).
Discussion
We identified frameshift mutations within the hGARE
exon 5 coding repeat in approximately 20% of the
MSI-H gastrointestinal tumors. This value is similar to
values obtained for coding repeats of hMSH3,
hMSH6, and IGFIIR target genes in colorectal cancers,
and for coding repeats of hMSH3, BAX, and TCF-4
target genes in gastric cancers (see Table 1, and Duval
et al, 1999b).
Mutation frequencies of coding repeats within
genes that are potentially implicated in human carci-
nogenesis have recently been evaluated in a large
series of MSI-H colorectal tumors. Using a statistical
method, Duval et al (2001) separated genes into two
groups, with a cut-off value for mutation frequency of
about 12%. Genes showing higher mutation frequen-
cies have been proposed to represent valid “targets,”
the mutation of which is selected for and plays a role
during tumorigenesis. Genes with lower mutation fre-
quencies have been proposed to represent “bystand-
ers,” the mutation of which is probably due to the high
1999). We easily detected hGARE mRNA in gastrin-
responsive LoVo cells, but not in HCT 116 cells. In
addition, LoVo cells proved to express only mRNA
molecules corresponding to the frameshift mutated
allele. Accordingly, LoVo have recently been shown to
express at the cell surface only a gastrin receptor
isoform of 40 kDa (Watson et al, 1998), a molecular
weight corresponding to that predicted for a truncated
protein originating from the frameshift mutated
hGARE gene.
Laboratory Investigation • March 2002 • Volume 82 • Number 3 267

Laghi et al
Figure 2.
8
DNA sequence of hGARE PCR products from LoVo and HCT116 cell lines. A frameshift mutation (1-bp deletion) within the (T) repeat is detectable in LoVo cells,
whereas a wild-type repetitive tract is present in HCT116 cells.
which are known to grow in response to the gastrin, is
consistent with a role of the putative truncated recep-
tor in mediating a trophic effect. Taken together, our
findings makes hGARE a new candidate target gene
playing a role in the tumorigenesis of a fraction of
unstable tumors.
Materials and Methods
Cell Lines and Tumor Samples
MMR-deficient cells from colon cancers (LoVo and
HCT116) and from gastric cancers (SNU1), as well
as MMR-proficient cells from colon cancers (HT29,
FINCO1, FINCO2, and SW480), were kindly pro-
vided by Dr. G. Marra (Institute for Medical Radio-
biology, Zurich, Switzerland) or purchased from
the American Type Culture Collection (Manassas,
Virginia).
Figure 3.
DNA sequences of hGARE cloned PCR products from GC#10G gastric cancer
sample. A (T)7 repeat is present in Clone A; a G 3 A transition at codon 339
is present with a (T)8 repeat in Clone B. The arrow indicates the G 3 A
transition detected at codon 339 in Clone B.
The cancer panel included 150 consecutive colo-
rectal cancers surgically resected at the Istituto
Clinico Humanitas of Milan between 1997 and 1999
and 83 gastric cancers referred for histologic diag-
nosis to the Department of Pathology of the Univer-
sity of Pavia. Fifteen colorectal cancers from
HNPCC patients, already classified as MSI-H, were
obtained from the Department of Human Genetics
of the University Hospital of Basel.
Although a large body of data exists, further studies
are needed on normal and tumor gastrointestinal
tissues to definitely assess the role of gastrin and
gastrin-receptors in both cell differentiation and pro-
liferation. This makes it difficult to precisely evaluate
the functional significance of the frameshift-mutated
hGARE gene in unstable tumors. However, the pres-
ence of only mutated mRNA molecules in LoVo cells,
2
68 Laboratory Investigation • March 2002 • Volume 82 • Number 3

hGARE Mutations in MSI Tumors
Figure 4.
hGARE mRNA expression in cancer cell-lines. A, A 404 bp hGARE RT-PCR product is detectable in LoVo, but not in HCT116 cells, whereas a 281-bp ␤-actin RT-PCR
product is present in both cell lines. RTϩ: , RT-: negative control. B and C, Direct sequencing of the LoVo hGARE RT-PCR product. As evident, only the exon 5 (T)7
mutated allele is transcripted (B), and only the short exon 4 splice variant isoform accounts for the hGARE expression (C).
MSI-H Assessment and Target Gene Mutation Analysis
primers. Accordingly, we analyzed PCR products
either by standard polyacrylamide DNA sequencing
gel or by capillary electrophoresis on an ABI PRISM
DNA was purified by standard procedures from cancer
cell lines and from paraffin sections of formalin-fixed
specimens or from frozen tissue fragments of gastroin-
testinal tumors and matched normal mucosa. We
searched for instability in tumor samples by screening a
standard panel of microsatellite loci (Boland et al, 1998)
according to the conditions reported by Dietmaier et al
3
10 Genetic Analyzer (PE Applied Biosystems, Fos-
ter City, California). PCR products from tumor and
corresponding control DNA were analyzed in paral-
lel. We followed the recently assessed criteria for
the definition of MSI-H (Boland et al, 1998).
Frameshift mutations at coding mononucleotide re-
peats within TGF␤RII, BAX, hMSH3, hMSH6, IGFIIR,
(
1997). We amplified microsatellite sequences by
using either radiolabeled or fluoresceinated specific
Laboratory Investigation • March 2002 • Volume 82 • Number 3 269

Laghi et al
and CASP-5 genes were investigated by PCR. Reac-
tions were carried out with either radiolabeled or
fluoresceinated specific primers (Malkhosyan et al,
and hGARE mRNA were coamplified. The ␤-actin
primers were: 5'-ACCAACTGGGACGACATGGA-3'
(forward); and 5'-GCATACCCCTCGTAGATGGG-3'
(backward). RT-PCR products were run on 2% aga-
rose gel and visualized by ethidium bromide staining.
The specific hGARE RT-PCR product was character-
ized by direct cycle sequencing.
1996; Markowitz et al, 1995; Rampino et al, 1997;
Schwartz et al, 1999; Souza et al, 1996). PCR prod-
ucts were analyzed as reported above.
hGARE Gene Mutation Analysis
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