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SHORT REPORTS
Searching for microsatellite mutations in coding regions in lung, breast,
ovarian and colorectal cancers
Eva Forgacs1, Jonathan D Wren2, Craig Kamibayashi1, Masashi Kondo1, Xie L Xu1,
Sanford Markowitz9, Gail E Tomlinson1, Carolyn Y Muller1, Adi F Gazdar1,3,
Harold R Garner4,5,6,7 and John D Minna*,1,4,8
1
The Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center at Dallas, Dallas,
Texas, TX 75390-8593, USA; 2Program of Genetics and Development, University of Texas Southwestern Medical Center at
Dallas, Dallas, Texas, TX 75390-8593, USA; 3Department of Pathology, University of Texas Southwestern Medical Center at
Dallas, Dallas, Texas, TX 75390-8593, USA; 4Department of Internal Medicine, University of Texas Southwestern Medical Center
at Dallas, Dallas, Texas, TX 75390-8593, USA; 5Department of Biochemistry, University of Texas Southwestern Medical Center
at Dallas, Dallas, Texas, TX 75390-8593, USA; 6McDermott Center for Human Growth and Development, University of Texas
Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 7Center for Biomedical Inventions, University of
Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 8Department of Pharmacology, University of
Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 9Howard Hughes Medical Institute, Case
Western Reserve University, Cleveland, Ohio, OH 44106, USA
RepX represents a new informatics approach to probe
the UniGene database for potentially polymorphic repeat
sequences in the open reading frame (ORF) of genes,
56% of which were found to be actually polymorphic.
We now have performed mutational analysis of 17 such
sites in genes not found to be polymorphic (50.03
frequency) in a large panel of human cancer genomic
DNAs derived from 31 lung, 21 breast, seven ovarian, 21
(13 microsatellite instability (MSI)+ and eight MSI7)
colorectal cancer cell lines. In the lung, breast and
ovarian tumor DNAs we found no mutations (50.03 ±
0.04 rate of tumor associated open reading frame
mutations) in these sequences. By contrast, 18 MSI+
colorectal cancers (13 cancer cell lines and ®ve primary
tumors) with mismatch repair defects exhibited six
mutations in three of the 17 genes (SREBP-2, TAN-1,
GR6) (P50.000003 compared to all other cancers
tested). We conclude that coding region microsatellite
alterations are rare in lung, breast, ovarian carcinomas
and MSI (7) colorectal cancers, but are relatively
frequent in MSI (+) colorectal cancers with mismatch
repair de®cits. Oncogene (2001) 20, 1005 ± 1009.
Keywords: microsatellite instability; mutation; repetitive DNA sequences
Repetitive DNA sequences such as microsatellites
consist of 1 to 10 nucleotides that are repeated a
variable number of times. These repeat sequences can
*Correspondence: JD Minna, Hamon Center for Therapeutic
Oncology, University of Texas Medical Center, 6000 Harry Hines
Blvd., NB. 8.206, Dallas, Texas, TX 75390-8593, USA
Received 25 September 2000; revised 28 December 2000; accepted 3
January 2001
be polymorphic, and such polymorphic microsatellite
markers have been used in a variety of genetic studies,
such as linkage analysis. When the repeat sequences
occur in the open reading frame, polymorphic amino
acid sequences can be generated. Several inherited
neurological disorders such as Friedreich's Ataxia and
Huntington's Disease have been linked to intergenerational locus-speci®c expansion of tri-nucleotide tracts
(Andrew et al., 1993; Bidichandani et al., 1998). The
mechanism of expansion or contraction of these
repetitive sequences is not fully understood, but is
believed to be associated with slipped-strand mispairing, uneven recombination, or a combination of both
(Jakupciak and Wells, 1999; Wells, 1996).
Somatically acquired microsatellite alterations occur
frequently in endometrial, gastrointestinal, testicular,
colorectal and ovarian carcinomas leading to characterizing these tumors as microsatellite instable
(MSI+) or not (MSI7) (Gurin et al., 1999; King et
al., 1997; Liu et al., 1995; Orth et al., 1994; Yamamoto
et al., 1997). These alterations are correlated with
mutations in mismatch repair (MMR) system genes
(hMSH2, hMLH1, hMSH6 etc.) (Bronner et al., 1994;
Herman et al., 1998; Liu et al., 1996, 1999; Malkhosyan et al., 1996; Nicolaides et al., 1994). Several
studies have shown that mismatch repair mutant
colorectal tumors also exhibit mutations in genes with
nucleotide repeats such as TGF b II and BAX
(Markowitz et al., 1995; Parsons et al., 1995; Rampino
et al., 1997).
We have developed informatics tools to search
DNA databases for sequences such as repeats that
could potentially be polymorphic (Fondon et al.,
1998; Wren et al., 2000). Currently our RepX
software has identi®ed over 30 000 expressed sequences with repetitive DNA elements predicted to
be polymorphic, providing a comprehensive list of
UniGene predicted polymorphisms (Wren et al.,
Alterations of coding microsatellite sequences in cancers
E Forgacs et al
1006
2000). Out of those, 104 genes were selected with
putative polymorphic repetitive regions for experimental validation, and 67% were found to be
polymorphic in a panel of 30 B-lymphocyte cell line
DNAs examined. Repeat sequences in the 5' untranslated region were polymorphic in 67% of the
cases; those in the 3' untranslated region varied in
77%, while those in the open reading frame were
polymorphic in 56% of the cases (Wren et al., 2000).
In the present study we have examined 20 genes with
repeat sequences in the open reading frames for
development of microsatellite alterations in human
cancers. Of these, 17 exhibited no germline polymorphisms for 60 chromosomes tested (Table 1). A
total of 80 cancer cell lines including 31 lung (15
NSCLC and 16 SCLC), 21 breast, seven ovarian, 21
Table 1
colorectal (13 MSI+ and eight MSI7) were screened
for mutations by single-stranded conformational
polymorphism (SSCP) analysis. Corresponding, normal EBV (Epstein-Barr Virus) immortalized B
lymphocyte DNAs (n=52) when available were also
analysed as controls. The list of genes tested,
characteristics of the repeat sequences, and alterations
found are given in Tables 1 and 2. In addition we
further tested ®ve MSI7 and ®ve MSI+ primary
colorectal cancer sample DNAs for alterations in
those genes (TAN1, SREBP-2, GR6) showing acquired
alterations in the tumor cell line screen.
We found microsatellite alterations in cancer cell
lines in four of the 20 genes which were classi®ed as
`non-polymorphic' in our previous study, using BL
(germline) DNA sample (Table 2, Figure 1).
List of genes tested for acquired alterations within the open reading frame repeat sequences in human cancers
Gene
TAN-1
T-type calcium channel alpha-1 subunit
Ankyrin G
Major centromere autoantigen CENP-B
Potassium voltage-gated channel, shaker-related subfamily
Transcriptional activation factor TAFII32
Insulin receptor substrate-1
Nck, Ash and phospholipase C gamma-binding protein NAP4
CIq/MBL/SPA receptor CIqR(p)-for phagocytosis
ALR mRNA
Sterol regulatory element binding protein-2 (SREBP)
CREB-binding protein (CBP)
Actin-binding protein(filamin)(ABP-280)
SRY(sex-determining regionY)-box11
Human pim-2 protooncogene
Homo sapiens putative GR6 protein
Extracellular matrix protein{
Polycomb 2 homolog (hPc2){
Putative transcription factor CA150{
MEKK1{
GenBank accession no.
Repeat
No. of repeats
Amino acid repeat*
M73980
AF051946
U13616
X05299
M55514
U21858
S62539
AB005216
U94333
AF010403
U02031
U47741
X53416
U23752
U77735
AF008192
AB011792
AF013956
AF017789
AF042838
acc
acc
acc
agg
agg
agt
cag
cag
cag
cag
cag
cag
agg
cag
gcctg
ga
agg
acc
aggccc
acc
6
11
18
12
14
10
7
7
8
9
12
18
7
10
5
9
21
15
22
8
valine
histidine
threonine
glutamic acid
glutamic acid
aspartic acid
serine
glutamine
leucine
glutamine
serine
glutamine
glutamic acid
serine
frameshift
frameshift
glutamic acid
histidine
glutamine, alanine
threonine
*Amino acid repeat sequence that would be altered or in the case of penta or dinucleotide repeats a frameshift would be introduced with
mutations. {Extracellular matrix protein was only studied in breast and ovarian cancers. {Indicates the genes with known germline
polymorphisms (Wren et al., 2000).
Table 2 Microsatellite alterations of gene regions in di€erent tumor cell line DNAs
Gene
Sterol regulatory element binding
protein-2 (SREBP)
Nck, Ash and phospholipase C
gamma-binding protein NAP4
Homo sapiens putative GR6
protein
TAN-1
Tumor cell line,
Accession no. Primary tumor Tissue type Nucleotide change
Codon
Effect
Acquired (Acq),
Germline (GL)
U02031
V6
colorectal
TGC insertion
codon 74
serine insert
Acq
AB005216
HCC1143
breast
CAG insertion
codon 56
glutamine insert
GL
AF008192
H2882
SW48
NSCLC
colorectal
CAG insertion
GA deletion
codon 56
glutamine insert
frameshift
GL
Acq
T8 (MSI+)
T10 (MSI+)
V481
V703
colorectal
colorectal
colorectal
colorectal
GA deletion
GA insertion
GTG deletion
GTG deletion
M73980
codon 1578 valine deletion
codon 1578 valine deletion
Acq
Acq
The following primers were used in the RT ± PCR reaction: SREBP-2; SREBP-2fw 5'-GAA CAG CTG TGT AGC TCC-3' and SREBP-2rev 5'CCT GCT GCT GAA TGG TGA CC-3' (Data not shown). TAN-1: TAN1fw 5'-GGT GAG ACC TGC CTG AAT GG-3' and TAN1rev 5'CCA CAC TCG TTG ACA TCC-3'
Oncogene
Alterations of coding microsatellite sequences in cancers
E Forgacs et al
1007
Figure 1 Single-stranded conformational polymorphism (SSCP) analysis of the various genes with repeats. (a) SSCP analysis of the
polyserine track in the SREBP-2 gene. The left panel shows insertion of a serine residue in colorectal cancer cell line V6. Right
panel: three amino acid insertion (glycine, serine, serine) in a normal control individual of Caucasian origin. (b) SSCP analysis of the
glutamine repeat in the NAP4 gene. Extra band in HCC1143BL shows the glutamine insertion. The corresponding tumor cell line
HCC1143 is missing the lower allele, and is homozygous for that change. HCC1007BL was heterozygous, and the upper allele was
missing in the corresponding tumor cell line HCC1007. (c) SSCP analysis of the GR6 gene in colorectal tumor DNAs. The arrows
indicate a GA insertion in T8 (MSI+), and GA deletions in T10 (MSI+), and in colorectal cancer cell line SW48. (d) Deletion of a
valine residue at codon 1578 of the TAN-1 gene in two MSI+ colorectal cancer cell lines V481 and V703. (e) Mutational analysis of
the putative transcription factor CA150 gene in MSI+(®rst 13) and MSI7 colorectal cell line DNAs. A panel of 81 genomic DNAs
were ampli®ed by polymerase chain reaction using the `touchdown' methodology with an initial denaturation step at 958C for
10 min. This was followed by 10 touchdown cycles of 30 s at 948C, 30 s at 708C (with a decrease in the annealing temperature by
18C each cycle), and 30 s at 728C. This was followed by 30 cycles of 30 s at 948C, 30 s at 608C and 30 s at 728C, with a ®nal
extension at 728C for 10 min. DNA (*50 ± 100 ng of genomic DNA) was ampli®ed in 20 ul reaction volumes containing: 50 mM
KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl2, 200 mM dNTPs, 1 mM of each primer, and 0.5 U Amplitaq Gold (Perkin Elmer Cetus),
and of 2 mCi of 32P-dCTP (Amersham). The samples were heat denatured, snap chilled, and run on a 0.56MDE gel, at 220 Volts,
overnight. The gels were dried and exposed overnight using BioMax ®lm (Kodak). For sequencing shifted SSCP bands were excised
from MDE gels, and the DNA was eluted with distilled water and re-ampli®ed using the original PCR primers. The PCR product
was run on a 2% agarose gel and puri®ed by Geneclean (BIO, CA, USA). Automated bi-directional sequencing was performed by
ABI 377 Dye Terminator cycle sequencing. Sequences were analysed and compared to wild type sequences with DNAStar software
(DNAStar Inc., Madison, WI, USA). Primer sequences for the SSCP analysis are available upon request. The following cancer cell
line DNAs were used: Lung cancer genomic DNAs: NCI-H128, H209, H289, H1184, H1339, H1450, H1607, H1672, H1770, H1819,
H1963, H2009, H2052, H2087, H2107, H2122, H2126, H2141, H2171, H2195, H2347, H2882, H2887, HCC15, HCC33, HCC44,
HCC78, HCC193, HCC366, HCC515, HCC827 (genomic DNAs of EBV immortalized `BL' cell lines are available for all of the
above DNAs). Breast cancer genomic DNAs: HCC38, HCC70, HCC202, HCC712, HCC1007, HCC1008, HCC1143, HCC1187,
HCC1395, HCC1419, HCC1428, HCC1500, HCC1569, HCC1599, HCC1739, HCC1937, HCC1954, HCC2157, HCC2185,
HCC1806, HCC2218 (the corresponding normal DNAs for the breast cancer cell lines were available). Ovarian cancer genomic
DNAs: 2008, UCI101, 2774, PAI, SCC60, UCI107, SW626. Colorectal cancer genomic DNA: MSI+: Vaco5, Vaco6, Vaco432,
Vaco481, Vaco457, Vaco670, Vaco703, RKO, HCT116, Lovo, LS180, HCT15, SW48. MSI7: Vaco8, Vaco206, Vaco394, Vaco451,
Vaco478, Vaco241, Vaco364, Vaco410
Oncogene
Alterations of coding microsatellite sequences in cancers
E Forgacs et al
1008
TAN-1, the human homologue of the Drosophila
Notch-1 gene, a common translocation site in Tlymphoblastic neoplasms (Ellisen et al., 1991), was
found to have a 3 bp deletion (Val1578), which appears
to be homozygous, in V481 and V703 MSI+ colorectal
cancer cell lines (Figure 1d). This change is somatically
acquired in V703 colon cancer cell line, as the
corresponding control DNA did not have the alteration. This alteration could not be detected when the
panel of 56 normal controls (individuals of di€erent
racial origin and with no family history of cancer) were
screened. In addition, with RT ± PCR we found
expression of the TAN-1 gene in V481 and V703 colon
cancer cells (data not shown, Table 2). This alteration
is in a region which was previously found to be a
tumor-associated proviral insertion site in mice (Girard
et al., 1996), and further studies are needed to
determine if this alteration a€ects the function of the
TAN-1 protein. No alterations in TAN-1 were found
when ®ve MSI7 and ®ve MSI+ colorectal primary
tumors were screened.
There was a TGC (Ser) insertion following codon 73
in the SREBP-2 gene (Sterol Regulatory Element
Binding Protein-2) (Hua et al., 1993) in an MSI+
colorectal cancer cell line (V6). Expression of SREBP-2
gene in the colon cancer cells was con®rmed by RT ±
PCR (data not shown, Table 2). The corresponding
normal DNA from patient V6 was not available.
Therefore DNAs from 56 normal controls were
analysed, and the insert Ser73 was not found. However
a previously unknown 9 bp deletion (Gly68, Ser69, Ser70)
change was detected in one Caucasian individual
(Figure 1a).
No alterations were found for SREBP-2 gene in ®ve
MSI7 and ®ve MSI+ primary colorectal tumors.
A CAG (Glu) insertion was detected in the NAP4
(Nck, Ash and phospholipase C) gamma-binding
protein (Matuoka et al., 1997) in a breast cancer
Table 3
Acquired microsatellite open reading frame alterations and
frequencies in the various tumors*
Tumor type
Lung
Breast
Colon(MSI+)
Colon(MSI7)
Ovarian
N=
Alterations
Frequency
31
21
18
13
7
0
0
6
0
0
50.03
50.05
0.33
50.13
50.13
P=0.000003. w2 for comparison of MSI+ colon cancers versus all
other tumors. *The ®ve MSI+ and ®ve MSI7 primary colorectal
cancer samples were only screened for TAN-1, SREBP-2 and GR6
alterations
Table 4
hPc2
CA150
MEKK1
Oncogene
lll
(HCC1143), and a non-small cell lung (H2882) cancer
cell line. The breast cell line alteration was found to be
homozygous while the change in the other cell line was
heterozygous. While we had not detected this polymorphism in our prior screen, we found this alteration
present in the corresponding B lymphocyte DNA for
HCC1143 and H2882, and thus, this represented a
germline polymorphism (Figure 1b).
The SW48 (MSI+) colorectal cancer line and a
MSI+ colorectal primary tumor (T10) had a GA
frameshifting deletion, and another MSI+ tumor (T8)
had a GA insertion in the GR6 gene, which is located
within the leukemia breakpoint region at 3q21
(Pekarsky et al., 1997). There were no alterations
found in our normal control population of 56
individuals. Table 3 summarizes the alteration frequencies for the various tumor cell lines and tumors tested.
To determine whether loci that exhibit polymorphisms could be mutated in cancer cells, or to see if a
cancer speci®c genotype exists, we examined three
genes (hPc2, CA150 and MEKK1) previously found to
exhibit germline polymorphisms (Wren et al., 2000) for
di€erent mutations in the tumor cell lines. The results
are summarized in Table 4. A CAC (His) insertion at
codon 398 of the histidine track of the hPc2 gene
(Satijn et al., 1997) was found in the V451(MSI7)
colorectal cancer cells. This genotype was not found in
our prior germline screen. We also found that the
CA150 gene has a signi®cantly higher frequency of
repeat expansions and contractions in MSI+ (6/13) as
compared with MSI7 (0/8) colorectal cancers (Figure
1e).
We have combined a new informatics approach
which identi®es repeat sequences with a propensity for
polymorphism in the open reading frames of genes in
the UniGene database with experimental testing for
alterations in these sequences in human cancers. For
this ®rst study we selected genes with known or
predicted function. As knowledge of the UniGene
dataset enlarges other candidates can be tested.
Our study indicates that coding region microsatellite
alterations are rare in lung, breast, ovarian cancers,
and MSI7 colon cancers, but are more frequent in
MSI+ colorectal carcinomas with known mismatch
repair mutations (P=0.000003 by w2 test, Table 4).
Human tumors with mismatch repair mutations are
known to have frequent repeat sequence alterations,
and our results (6/18 of MSI+ colorectal tumors with
mutations) would indicate that such alterations a€ecting the open reading frame are even more frequent
than previously thought, since they a€ect three of the
17 genes. Whether these mutations have functional
The rate of polymorphisms in normal vs cancer cell line DNAs in the genes with known open reading frame polymorphisms
BL cell lines
No. of alterations/no. of examined
Breast and lung cancer
No. of alterations/no. of examined
1/30 (0.03)
5/25 (0.2)
6/24 (0.25)
2/52 (0.04)
3/52 (0.058)
5/52 (0.097)
Colorectal cancer
MSI+
MSI7
3/13 (0.23)
6/13 (0.46)
5/13 (0.38)
1/8 (0.125)
0/8 (0.0)
2/8 (0.25)
Alterations of coding microsatellite sequences in cancers
E Forgacs et al
consequences requires further study. By contrast,
human tumors that do not exhibit the MSI phenotype
(0/67 tumors) appear to have rarely mutated repeat
sequences in the open reading frame. Thus, if an open
reading frame repeat sequence was found that was
frequently mutated in MSI7 tumors it would quite
likely be of functional importance. Finally, other
genome-wide informatics searches combined with
experimental wet lab validations of sequences predicted
to have mutations or polymorphisms need to be further
explored in understanding cancer pathogenesis.
Abbreviations
MSI, microsatellite instability; SCLC, small cell lung
cancer; NSCLC, non small cell lung cancer; SSCP, single-
stranded conformational polymorphism; RT ± PCR, reverse
transcriptase polymerase chain reaction; MMR, mismatch
repair.
1009
Acknowledgments
The authors thank James Lutterbaugh and Dr Donald
Lombardi for tumor cell lines, and Luc Girard for helpful
comments on this manuscript. This work has been
supported by the National Cancer Institute Lung Cancer
SPORE grant P50 CA70907, G Harold and Leila Y
Mathers Charitable Foundation. HR Garner is supported
by the Patrick O'Brien Montgomery Distinguished Chair. S
Markowitz is an associate investigator of the Howard
Hughes Medical Institute, and was also supported by PHSCA 67409.
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Oncogene