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Associate with ErbB-2 Amplification and Affect Sensitivity to Topoisomerase II Inhibitor Doxorubicin in Breast Cancer
§
From the Laboratory of Cancer Biology,*
University and
University Hospital of Tampere, and the Department of
Oncology,
Tampere University Hospital,
Tampere, Finland; the Departments of Medical Biochemistry and
Gyneacology and Obstetrics,
University and
University Hospital of Turku, Turku, Finland; and the Department of
Oncology,§
University of Lund, Lund, Sweden
 |
Abstract |
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(topoII) is a key enzyme in DNA replication
and a molecular target for many anti-cancer drugs called topoII
inhibitors. The topoII gene is located at chromosome band
17q12-q21, close to the ErbB-2 oncogene
(HER-2/neu), which is the most commonly
amplified oncogene in breast cancer. Because of the physical proximity
to ErbB-2, copy number aberrations may also occur in the
topoII gene. These topoII gene copy number aberrations may be
related to the altered chemosensitivity to topoII inhibitors that
breast cancers with ErbB-2 amplification are known to have. We used
fluorescence in situ hybridization to study copy number
aberrations of both topoII and ErbB-2 in nine breast cancer cell
lines and in 97 clinical breast tumors, which were selected for
the study according to their ErbB-2 status by Southern blotting.
TopoII-protein expression was studied with Western blot and
sensitivity to doxorubicin (a topoII inhibitor) with a 96-well
clonogenic in vitro assay. Two of the five cell lines
with ErbB-2 gene amplification (SK-BR-3 and UACC-812) showed
amplification of topoII. In MDA-361 cells, ErbB-2
amplification (14 copies/cell) was associated with a physical deletion
of topoII (four copies of chromosome 17 centromere and two copies of
topoII). The topoII amplification in UACC-812 cells was
associated with 5.9-fold-increased topoII protein expression and
2.5-fold-increased sensitivity to the topoII inhibitor,
doxorubicin, whereas the deletion in MDA-361 leads to decreased
protein expression (45% of control) and a 2.4-fold-increased
chemoresistance in vitro. Of 57 ErbB-2-amplified primary
breast carcinomas, 25 (44%) showed ErbB-2-topoII
coamplification and 24 (42%) showed a physical deletion of the
topoII gene. No topoII copy number aberrations were found in 40
primary tumors without ErbB-2 amplification. TopoII gene
amplification and deletion are common in ErbB-2-amplified breast cancer
and are associated with increased or decreased sensitivity to topoII
inhibitors in vitro, respectively. These
findings may explain the altered chemosensitivity to topoII inhibitors
reported in ErbB-2-amplified breast cancers.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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(topoII inhibitors), in many
clinical trials.4-13
Most studies have linked ErbB-2 to
chemoresistance to topoII inhibitors,4-9
but there are
also clinical trials reporting either no association10-12
or even a higher likelihood for a response in ErbB-2-amplified
tumors.13,14
According to in vitro studies,
ErbB-2 amplification and overexpression associate exclusively with
resistance to cytotoxic drugs.15-17
Biological mechanisms that explain the association between ErbB-2
amplification and altered sensitivity to topoII inhibitors are not
known. Experiments with mouse and human cells have indicated that the
in vitro-induced ErbB-2 protein overexpression does not
alter the chemosensitivity of cancer cells to the topoII
inhibitor.16,17
Neither is the ErbB-2 protein as a
transmembrane growth factor receptor known to interact physically with
topoII inhibitors. Although the ErbB-2 oncogene is considered to be the
target gene for 17q12-q21 amplification, the amplicon harbors other
closely located genes, such as v-erbA/thyroid hormone
receptor (THRA)18
; retinoic acid receptor
19
; MLNs 50, 51, 62, and 6420,21
;
and topoII.19,22-24
Among the coamplified genes,
topoII is particularly interesting in breast cancer, because it is
the molecular target for topoII inhibitors.25,26
In
vitro studies with different experimental designs have established
that sensitivity to topoII inhibitors is dependent on the expression
level of topoII in target cancer cells.27-33
The cells
with a low concentration of topoII protein are less sensitive to
topoII-inhibiting drugs than cells containing a high concentration of
topoII because they contain less of the molecular target enzyme of
topoII inhibitors, topoII, than cells with a high concentration of
topoII.27-33
Studies with only a few primary breast carcinomas indicate that ErbB-2
and topoII can both be amplified simultaneously in breast
cancer.19,22,23
In accordance with coamplification,
immunohistochemically detectable topoII expression is significantly
correlated with ErbB-2 overexpression in breast cancer.34
We studied ErbB-2 and topoII gene copy number aberrations in breast
cancer cell lines and primary breast carcinomas and determined the
association between topoII copy number aberrations, protein
expression, and sensitivity to doxorubicin, a widely used topoII
inhibitor.
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Materials and Methods |
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Breast cancer cell lines BT-474, DU-4475, MCF-7, MDA-157, MDA-361, SK-BR-3, UACC-812, UACC-893, and ZR-75-1 were obtained from the American Type Culture collection (ATCC, Rockville, MD) and were cultured in recommended conditions. The confluent cultures were harvested to obtain interphase nuclei from the cells that were predominantly in the G1 phase of the cell cycle. The cells were fixed in Carnoy’s fluid (75% methanol, 25% acetic acid) and dropped on microscope slides.35 Primary breast tumors (97) were derived from the tumor bank of the University of Lund, Sweden. The primary tumors were selected from the set of tumors that had been studied previously for ErbB-2 amplification by Southern blotting.36 The primary tumors were freshly frozen and stored at -70°C. Imprint touch preparations were prepared for FISH by lightly pressing a semithawed frozen tumor piece onto Superfrost Plus microscope slides (Menzel, Germany).37
Probes for FISH
A PAC clone for ErbB-2 (RMC17P077) was obtained from Resource for
Molecular Genetics (Berkeley, CA), and a P1 probe for topoII was
obtained by a polymerase chain reaction (PCR)-based screening of a P1
library (Genome Systems Inc., St. Louis, MO).38,39
A
chromosome 17 pericentromeric probe (p17H8) was used as a reference
probe to determine the overall copy number of chromosome
17.38,39
The specificity of the large-insert-size genomic DNA probes was
confirmed by PCR with primers amplifying the sequences for ErbB-2 and
topoII and with a probe DNA as a template. The PCR conditions were
optimized for each primer pair, using PTC-100 thermocycler (MJ Research
Inc., Watertown, MA). Approximately 100 ng of each template probe and
25 pmol/L of corresponding primers were used in a 25-µl reaction
volume in a standard reaction mixture recommended with Dynazyme II
thermostable DNA polymerase (Finnzymes Oy, Espoo, Finland). The PCR
analysis showed that the ErbB-2 probe did not recognize sequences from
topoII and vice versa (data not shown).
FISH
Two-color FISH was done as previously described.34,36,37 Before hybridization, imprint touch specimens were fixed at room temperature with 50%, 70%, and 100% Carnoy’s fluid (10 minutes each). The probes were labeled with biotin-14-deoxyadenosine triphosphate and digoxigenin-11-deoxyuridine triphosphate by standard nick translation. The hybridization was carried out overnight at 42°C in a hybridization mixture35,37 containing 5 ng of pericentromeric probe, 20 ng of gene-specific probe, and 10 µg of human placental DNA. After hybridization, the slides were washed with 0.4x standard saline citrate (2 minutes at 74°C) and 2x standard saline citrate (1 minute at room temperature). Hybridization was detected immunohistochemically with avidin-fluorescein isothiocyanate (for biotin-labeled probe) and anti-digoxigenin rhodamine. The slides were counterstained with 0.2 mm 4,6-diamidino-2-phenylindole (DAPI) in an antifade solution (Vectashield, Vector Laboratories, Burlingame, CA).35,37
Hybridizations were evaluated using an Olympus BX50 epifluorescence
microscope equipped with a 63x oil-immersion objective (numeric
aperture 1.4). A dual band-pass fluorescence filter (Chromotechnology,
Brattleboro, VT) was used to visualize the fluorescein isothiocyanate
and rhodamine signals simultaneously. At least 80 nonoverlapping nuclei
with intact morphology (based on DAPI counterstaining) were scored to
determine the number of hybridization signals for each topoII,
ErbB-2, and 17 centromere probe. Control hybridizations to normal
lymphocytes were done to ascertain that the probes recognized a
single-copy target and that the hybridization efficiencies were
sufficient. Both absolute copy numbers and the relative copy number
ratio (ratio between mean number of ErbB-2 or topoII and mean number
of chromosome 17 centromere signals) were determined. The amplification
of ErbB-2 and topoII was defined if the copy number ratio was 1.5 or
more. TopoII was defined as deleted if the ratio was 0.7 or less.
Assays for Sensitivity to Doxorubicin
The chemosensitivity of cancer cell lines to doxorubicin was determined by the 96-well clonogenic assay and growth rate experiments, essentially as described previously.40,41 A 96-well clonogenic assay was selected, because it is considered the most reliable method for assessing cell killing after genotoxic stress.42 Briefly, cells in the midlogarithmic growth were used for the experiments. Variable concentrations (0–32 ng/ml) of doxorubicin (Adriamycin, Farmitalia Carlo Erba AB, Nerviano, Italy) were added to the plates for the incubation period of 4 weeks. Plating efficiency (PE) was calculated using the formula PE = -ln(number of negative wells/total number of wells)/number of cells plated per well. The fraction of surviving cells as a function of the doxorubicin dose was fitted with a linear quadratic equation. The comparison of drug sensitivity was made using 50% inhibitory concentration (IC50) values, corresponding to 50% inhibition of the surviving fraction, which were obtained from the fitted dose-response curves.40,41
Owing to the slow growth rate of the UACC-812 cell line, its sensitivity was determined by growth rate experiments (using MDA-361 cell line as an internal control to standardize the two assays).40,41 The cell density of the suspension used for plating was adjusted to 15,000 cells/ml for the MDA-361 cell line and to 25,000 cells/ml for the UACC-812 cells. Multiple 24-well plates were prepared to establish replicates for each drug concentration and time point studied. The cell number was presented as a function of time on the semilogarithmic scale.40,41 Values obtained from the clonogenic assay and growth rate experiments are expressed as a mean ± SD of six (n = 6) separate experiments.
Western Blotting
Nuclear extracts were prepared from cancer cell lines as described
previously.43
Whole cell extracts of primary breast tumors
were prepared for hormone receptor analysis and frozen at -70°C
until used in Western blots.36
Protein concentrations were
determined by the Bio-Rad protein assay (Bio-Rad, Richmond, CA).
Nuclear extracts and cytosols with equal protein concentrations were
electrophoretically separated on 8.0% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gel and subsequently
transferred to nitrocellulose membranes (Hybond-C Extra, Amersham,
Arlington Heights, IL). Immunostaining was carried out using
either topoII-specific monoclonal antibody Ki-S1 (Boehringer
Mannheim, Mannheim, Germany; dilution 1:1000),34,44-46
or
a monoclonal antibody to cyclin B1 (Neomarkers, Union City, CA;
dilution 1:300).47
A topoII-specific monoclonal
antibody, Ki-S1, is specific for a COOH-terminal epitope of human
topoII and does not cross-react with
topoIIß.34,44-46
The specificity of the antibody used
for the detection of topoII (Ki-S1) was also confirmed by doing a
Western blot with another topoII-specific monoclonal antibody, Ki-S4
(a kind gift from Dr. Udo Kellner, University of Kiel, Kiel,
Germany).46
The two topoII antibodies gave identical
bands from the same control samples (data not shown). Both topoII
and cyclin B1 antibodies were detected with the rabbit anti-mouse IgG
secondary antibody conjugated to horseradish peroxidase (dilution
1:1000) (Dakopatts, Glostrup, Denmark). The peroxidase-catalyzed
reaction was visualized with enhanced chemiluminescence (Amersham Life
Sciences, Arlington Heights, IL).
For quantitative determination of topoII protein expression, the
immunoreactive bands were quantitated with a densitometer. Because the
expression of topoII is
proliferation-dependent25,26,34,44-46
and the
proliferation rates of the cell lines are not equal, the topoII band
intensities were adjusted with band intensities of cyclin B1, which has
a cell cycle-specific (G2/M-phase) expression
pattern identical to that of topoII.47
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and ErbB-2 Gene Copy Numbers in Breast Cancer Cell Lines
Five of nine studied breast cancer cell lines (MDA-361, SK-BR-3,
UACC-812, BT474, UACC-893) showed high-level amplifications of the
ErbB-2 oncogene (Figure 1)
. Two of these
ErbB-2-amplified cell lines, UACC-812 and SK-BR-3, showed increased
gene copy numbers for topoII. SK-BR-3 was defined to have a
low-level topoII amplification (9 copies of topoII and 6 copies
of chromosome 17 centromere), whereas a high-level topoII
amplification was found in UACC-812 cells (27 copies of topoII gene
and 4 copies of chromosome 17 centromere; Figure 1
). The MDA-361 cells
showed ErbB-2 amplification (mean, 14 copies/cell) and, surprisingly, a
physical deletion of the other allele of topoII. On average, four
copies of chromosome 17 centromere were found, compared with only two
copies of topoII (mean relative gene copy number 0.46; Figure 1
).
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Gene Copy Number and Protein
Expression
TopoII protein expression of UACC-812, SK-BR-3, and MDA-361
cell lines was compared with that of BT-474 (ErbB-2-amplified) and
MCF-7 (no ErbB-2 gene amplification) cell lines, which do not show copy
number alterations of topoII (normal copy number of topoII gene).
Only one immunoreactive band was seen in Western blots at 170 kd, which
is the known molecular mass of topoII. Strong bands were
identified for UACC-812 and SK-BR-3 cells and only the weak band in
MDA-361 cells (Figure 2)
. To rule out the
possibility that the differences in the topoII protein expression
resulted from differences in the proliferation rates of the breast
cancer cell lines, the bands were quantitated after they were adjusted
for the variation in cell proliferation (amount of the cell population
in the G2/M phase of the cell cycle, where
topoII is expressed). This was determined by a parallel Western blot
of the cyclin B1 gene (Figure 2)
. After the adjustment, the UACC-812
and SK-BR-3 cells showed 5.9- and 2.0-fold-increased topoII protein
expression when compared with that of control cell lines (mean of
BT-474 and MCF-7; Table 1
). The MDA361
cell line, with a physical deletion of the topoII gene, showed
decreased topoII expression (45% of controls; Table 1
).
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Aberrations and Sensitivity to TopoII Inhibitor
Doxorubicin
Among the cell lines studied, MDA-361 (with topoII deletion and
the lowest protein expression) was the most resistant to doxorubicin
(IC50 24 ± 2 ng/ml, six different
experiments). The IC50 values for the control
cell lines BT-474 and MCF-7 were 8 and 12 ng/ml, respectively. The
UACC-812 cell line grew too slowly to be analyzed by the 96-well
clonogenic assay. Therefore, its IC50 value was
determined from growth inhibition experiments with various
concentrations of doxorubicin (1–32 ng/ml). MDA-361 cells served as an
internal control to confirm the similarity of the
IC50 values by the two methods. UACC-812 cells
were hypersensitive to doxorubicin. The use of 5, 10, or 30 ng/ml of
doxorubicin caused almost complete inhibition of growth. The
concentration of 1 ng/ml doxorubicin suppressed the growth
only slightly, whereas the dose of 3 ng/ml doxorubicin caused a growth
inhibition of 36% in the UACC-812 cells. Thus, it can be estimated
from these growth inhibition curves that the IC50
value of UACC-812 for doxorubicin is slightly less than 4 ng/ml (Table 1)
.
TopoII Gene Copy Number and Protein Expression in Clinical
Breast Tumors
Because topoII aberrations were found only in cell lines with
ErbB-2 amplification, we studied topoII copy numbers in 57 breast
tumors whose ErbB-2 amplification had previously been determined by
Southern blot36
and was now confirmed by FISH (Figure 1)
.
In this set of 57 ErbB-2-amplified primary breast tumors,
ErbB-2-topoII coamplification was seen in 25 tumors (44%) and
topoII deletion in 24 tumors (42%; Figure 1
; Table 3
). The relative topoII copy number
was unaltered only in eight tumors with ErbB-2 amplification (14%;
Table 3
). The average number of topoII gene copies in amplified
tumors (n = 25) was 12.9 ± 6.4. Seventeen
of the topoII amplifications (68%) were classified as high-level
amplifications (more than 10 gene copies/cell, over threefold relative
copy number increase, or both; Table 4
). The average gene copy numbers
of amplified ErbB-2 (by FISH) were not associated with topoII copy
number. The ErbB-2 gene copy number was 19.3 ± 10.3 in topoII
amplified tumors (n = 25), 18.9 ± 10.4 in
topoII deleted tumors (n = 24), and 21.9
± 12.2 in tumors with a normal topoII copy number
(n = 8; Table 4
).
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gene aberrations associate with ErbB-2
amplification also in vivo, we studied the topoII gene
aberrations in 40 primary tumors with normal ErbB-2 gene status (no
amplification by FISH and/or Southern blot). No topoII gene copy
number aberrations were found in these 40 primary tumors (Table 3)
.
Whole-tissue extracts, which were left over from hormone receptor
analysis,36
were available only from nine ErbB-2 amplified
primary breast cancers. Five of these had topoII amplification, two
had deletion, and two had normal copy numbers of topoII. All five
tumors with topoII amplification showed a strong immunoreactive band
for topoII in Western blot (Figure 2)
. The sensitivity
of Western blot using whole-tissue
cytosol samples was not sufficient to detect the topoII protein in
tumors with the normal or deleted topoII gene copy number.
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gene is present in
almost 90% of ErbB-2-amplified breast cancers. Together with our
in vitro chemosensitivity experiments, these findings may
explain at least partly the reported associations between ErbB-2
amplification and the altered sensitivity of these tumors to topoII
inhibitors.4-13
The topoII aberrations are most likely
driven by ErbB-2 oncogene amplification, because none of the cell lines
or primary tumors without ErbB-2 amplification showed gene copy number
aberrations of topoII.
ErbB-2 amplification was most frequently associated with topoII
amplification. TopoII amplification has previously been described in
the SK-BR-3 cell line19,24
and in a few clinical tumor
samples studied.19,22,23
We confirmed topoII
amplification in the SK-BR-3 cells and defined it as a low-level
amplification (nine copies of topoII and six copies of chromosome
17). The UACC-812 cells, in turn, showed a mean of 27 copies of the
topoII/cell, which is comparable to that in many clinical tumor
samples. Thus, the UACC-812 cell line may provide a clinically relevant
model to study the effects of topoII gene amplification. In clinical
tumors, topoII was amplified in 25 of 57 tumors (44%) with ErbB-2
amplification. Thus, calculating from the 20 to 30% prevalence of
ErbB-2 amplification, one can estimate that topoII is amplified in
as many as 5 to 15% of all breast cancers.
The amplification of the target genes of the cytotoxic drugs is a
common and a well-known mechanism by which cancer cell lines confer
resistance to cytotoxic drugs in vitro.48
For
example, the cytotoxic actions of such agents as coformycin and
methotrexate can be overcome by the amplification of the genes
(adenylate-deaminase 2 and dihydrofolate-reductase), which metabolize
these drugs, in cancer cell lines.48
However, the target
genes of the cytotoxic drugs have not been implicated to induce
resistance to chemotherapy in human tumors until
recently.49-52
We showed several years ago that hormone
refractory prostate cancer cells circumvent the androgen blockade
therapy by amplification of the androgen receptor gene, and thereby
adapt to the lower levels of circulating androgens.49,50
After our novel finding,49
the gene copy number
aberrations of other target genes have been implicated in causing
resistance to different forms of chemotherapy.51,52
In
this study, we present completely opposite evidence, the amplification
of the target gene induced hypersensitivity to the certain class of
chemotherapy. We identified that the amplification of topoII, a
target enzyme for topoII inhibitors, causes an increased sensitivity to
topoII inhibitors by inducing the overexpression of the topoII
protein. This may be a very important finding, because topoII
amplifications were seen in clinical tumors with the amplification of
the ErbB-2 oncogene. The amplification of the ErbB-2 oncogene, in turn,
is known to be associated with shortened disease-free and overall
survival.1-3
In addition, in two large trials it
was reported that patients with ErbB-2 amplification derived the
greatest benefit from topoII inhibitor chemotherapy13,14
and it was speculated in these studies that the effect was due to the
simultaneous overexpression of topoII.3,14
Because we
found topoII amplification in almost 50% of the ErbB-2-amplified
breast tumors, the preliminary speculations may indeed be true. We may
have identified a mechanism (gene amplification of topoII) by which
these patients with poor prognosis may obtain a favorable response to
conventional chemotherapy with topoII inhibitors.
Physical deletion of the other allele of the topoII gene was
unexpected and has not been described previously in breast cancer. We
found topoII deletions in one cell line and in more than 40%
(23/57) of primary tumors with an ErbB-2 amplification. The type of
topoII deletion in MDA-361 cells (four copies of chromosome 17 and
two copies of topoII) was the same as in most of the clinical tumors
that showed topoII deletion. The multiplied gene copy numbers for
both topoII and 17 centromere indicate that the deletion had
occurred before the tumor became aneuploid (tetraploid for chromosome
17). The evidence from tumor suppressor genes indicates that a deletion
of the other allele of the gene is a typical finding in breast cancer
and is sufficient to cause a biologically relevant reduction in the
function of the deleted gene (eg, p53; reviewed in Reference 53). The
molecular mechanisms underlying the adjacent ErbB-2 amplification and
topoII gene deletion are unknown, although a pathway for identical
chromosomal rearrangement has recently been presented in
vitro.54
Like topoII deletion, telomeric deletion
has also been documented to follow cyclin D1 amplification at 11q13 in
breast cancer.37,55
The chromosomal breakpoints and the
size of the deleted chromosomal segment (including topoII) are
currently not known.
Although topoII amplification and deletion appear to be consequences
of ErbB-2 amplification, topoII aberrations may clinically be highly
relevant, because topoII is the molecular target for several
important anti-cancer drugs, termed topoII inhibitors. Numerous
in vitro studies have established that chemosensitivity to
topoII inhibitors is dependent on the cellular concentration of
topoII in cancer cells.25,27-33,56
High concentrations
of the topoII protein are associated with increased
sensitivity, whereas low levels lead to decreased drug
sensitivity.27-33
Unfortunately, the regulation of
the topoII expression in cancer cells is largely
unknown.34,56
Mutations of topoII are known to
alter the activity of the topoII protein, but they are rare and,
thus, not considered to have any diagnostic value (reviewed in
Reference 56).
Our in vitro results showed that the topoII gene
amplification and deletion have opposite effects on both
topoII protein expression and on sensitivity to the topoII
inhibitor, doxorubicin. The UACC-812 cells with topoII amplification
were hypersensitive to doxorubicin, whereas the MDA-361 cells
with the topoII deletion were the most resistant. These results are
unlikely to be biased by differences in the proliferation rates,
because the most resistant cell line (MDA-361) was the fastest growing,
which is commonly considered a sign of increased chemosensitivity. In
Western blots, the confounding effect of cell proliferation on
topoII protein expression measurements was eliminated, because
topoII expression was adjusted by that of cyclin B1, which has an
identical (G2/M phase) cell cycle-specific
expression pattern as topoII.47
Although we do not have direct evidence to link topoII gene copy
number aberrations to drug sensitivity to topoII inhibitors in a
clinical material, topoII amplification was associated with protein
overexpression also in clinical tumors. This suggests that
topoII-amplified tumors might be more sensitive to topoII inhibitors
also in vivo. The topoII deletion, in turn, may be a
marker of tumors that are resistant to doxorubicin. This finding is
supported by data reported from an in vitro model of lung
cancer30,31
in which topoII deletions that were
acquired during exposure to doxorubicin led to decreased topoII
messenger RNA expression and, ultimately, to increased chemoresistance
to the topoII inhibitor in lung cancer cell lines.30,31
The association of the topoII deletion with decreased protein
expression needs to be established in a clinical material with a more
sensitive method, because our old whole-tissue extracts did not allow
sufficient sensitivity to detect low amounts of topoII. However, the
topoII deletion has to be considered as a very interesting
resistance mechanism to topoII inhibitors, especially at the moment,
when P-glycoprotein-mediated resistance to anthracyclines has been
seriously questioned.57
P-glycoprotein is a
transmembrane transporter protein that has been implicated in inducing
chemoresistance to a wide variety of cytotoxic drugs, including topoII
inhibitors, by transporting these drugs out of the cancer
cells.57
However, a recent study showed conclusively that
neither P-glycoprotein nor its messenger RNA is expressed in untreated
or anthracycline-treated breast cancer samples.57
Therefore, P-glycoprotein probably does not determine chemosensitivity
to anthracycline chemotherapy in breast cancer nor contribute to
the development of resistance to topoII inhibitors in breast
cancer.57
So far, clinical studies have highlighted the role of ErbB-2 as a
predictive factor for topoII inhibitor chemotherapy. Most studies have
linked ErbB-2 to chemoresistance to topoII inhibitors,4-9
but there are also clinical trials reporting either no
association10-12
or a tendency for higher response rates
among ErbB-2-amplified breast tumors.13,14
In
vitro studies, in turn, have correlated ErbB-2 amplification and
overexpression exclusively to chemoresistance to topoII
inhibitors.16
Despite an increased number of clinical
findings linking ErbB-2 to altered drug sensitivity, the molecular
mechanisms behind these associations have not been defined.
Sophisticated experiments in mouse and human in vitro models
transfecting the ErbB-2 complementary DNA have shown no direct effects
of ErbB-2 protein overexpression on chemosensitivity to topoII
inhibitors.16,17
Neither is the ErbB-2 protein as a growth
factor receptor known to interact with topoII inhibitors
physically.58
Thus, some authors have recently suggested
that ErbB-2 overexpression may be just a surrogate for altered
topoII activity.3,14
Our present findings support this
hypothesis. However, the relationship between ErbB-2 amplification and
chemosensitivity is probably more complex than previously thought,
because ErbB-2 amplification was associated equally often with
topoII amplification and deletion, which may have opposite effects
on chemosensitivity in vivo. Thus, our results encourage us
to correlate the response to topoII inhibitors directly with topoII
gene amplification and deletion.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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gene
amplification and deletion in primary breast cancers with ErbB-2
oncogene amplification with larger patient material.59
In
addition, we have shown that topoII and ErbB-2 genes are separately
amplified in breast cancer.59
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Acknowledgements |
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Footnotes |
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Supported by the Finnish Academy of Sciences, Finnish Cancer Society (Urho Känkänen Foundation), Duodecim Research Foundation (the Finnish Medical Foundation), Farmos Research and Science Foundation, Ida Montin and Maud Kuistila Foundation, Pirkanmaa Cancer and Cultural Foundation, Finnish Cultural Foundation, Emil Aaltonen Foundation, and Tampere University Hospital Research Foundation (to J. I., M. T., and T. J.). T. J. gratefully acknowledges a young investigator stipend from the Finnish Cancer Institute and M. T. acknowledges a fellowship-stipend from the Sigrid Juselius Foundation.
Accepted for publication October 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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and other prognostic factors for epirubicin chemotherapy in advanced breast cancer. Br J Cancer 1998, 72:2267-2273
co-amplification with erbB2 in human primary breast cancer, and breast cancer cell lines: relationship to m-AMSA and mitoxantrone sensitivity. Oncogene 1993, 8:933-938[Medline]
and retinoid acid receptor genes in breast cancer and allelic loss at topoisomerase I on chromosome 20. Eur J Cancer 1993, 29A:1469-1475
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