| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Variant Messenger RNAs between Normal Human Breast Tissue and Primary Breast Carcinomas1
Division of Experimental Therapy [M. A. J. v. D., L. J. v. V.], and Departments of Radiotherapy [A. A. M. H.] and Pathology [L. J. v. V.], Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
(hER) variant mRNA between 21 normal breast
tissues and 41 primary breast carcinomas using a functional assay in
yeast for the hER. First, we found that the presence of wild-type hER,
relative to the total amount of hER, differs markedly
(P < 0.0001) between normal breast
tissue (median, 85% wild-type hER) and breast tumors (median, 74%
wild-type hER). Second, the hER variants with altered function that are
present in normal breast tissue are mainly one-exon deleted splicing
variants (median, 100%), whereas in breast tumors only half of all
variants lack just one single exon (median, 50%;
P < 0.0001). Our results suggest that
hER-dependent estrogen responsiveness of breast tissue may change
during tumor outgrowth, indicating that specific hER variants may play
a role in breast cancer development or progression. |
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
(hER)3
is a hormone-activated nuclear transcription factor that is an
important regulator of growth and differentiation in
estrogen-responsive cells (1, 2, 3)
. In the absence of
estrogen, hER is thought to be associated in the cytoplasm with heat
shock proteins such as HSP90 (2)
. Upon estrogen binding,
hER dissociates from this complex and binds in the nucleus to a
specific DNA sequence, the estrogen response element, located in the
promoter region of target genes (1
, 2
, 4)
. By binding this
estrogen response element, the receptor activates transcription of
target genes in a hormone-dependent way. Tamoxifen, an antiestrogen
widely used for the treatment of breast cancer, binds to hER in a
manner similar to that of estrogen. However, unlike estrogen
binding, tamoxifen binding represses the transcriptional
activation of most of the target genes by hER, thereby inhibiting the
growth of breast tumor cells. Clinical studies have shown that for
postmenopausal patients with hER-positive breast tumors, adjuvant
tamoxifen treatment is associated with significant improvement in both
recurrence rate and overall survival (reviewed in Ref. 5
).
However, a major problem in treatment of these patients with tamoxifen
is that 
40% of all patients with immunohistochemical hER-positive
breast tumors are nonresponsive to tamoxifen, as was shown in patients
with metastatic breast cancer (reviewed in Ref. 6
). Many hER splicing (related) variants have been identified in breast cancer specimens and breast cancer cell lines, whereas point mutations in hER are rare (reviewed in Ref. 7 ). The majority of these splicing variants lack one or more exons from the hER mRNA and have aberrant functional activity because they are either dominant negative, dominant active, or nonfunctional. Although their contribution to breast cancer development and, in particular, tamoxifen resistance is likely, their full significance in this respect is not yet clear because reports on the presence of hER variants in normal breast tissue are incomplete (8, 9, 10, 11, 12) . These studies that have reported hER variants in normal tissue involved either only one specimen (8 , 9) or a pool of normal breast tissue specimens (10) or used a method by which the presence of all hER variants among wild-type hER mRNA within one tissue specimen could not be evaluated (11 , 12) .
In this study we report the results of the hER-FASAY assay (13) that we used for analysis of the prevalence and functional activity of all variant receptors among wild-type hER in 21 normal human breast tissue specimens and 41 primary breast carcinomas. Our results show that hER variants are indeed prevalent in normal breast tissue but at a lower frequency and with a different molecular structure than in breast cancer.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Histopathological Reviewing of Tissue Sections.
Histopathological reviewing of all tissue specimens was performed on
H&E-stained cryostat sections. H&E sections were reviewed by a
pathologist and all normal breast tissue specimens were confirmed to be
normal (i.e., nonmalignant and containing normal ducts and
lobules) and all primary breast carcinoma specimens were confirmed to
be invasive carcinomas. The mean percentage of tumor cells in the
breast cancer specimens was 89% (SD, 18%; as a percentage of all
hER-expressing cells).
mRNA Isolation.
mRNA was isolated from 5-µm cryostat sections (five sections per
normal tissue specimen; three sections per breast tumor specimen) using
Dynabeads oligo-d(T)25 (Dynal, Oslo, Norway). The
cryostat sections used for mRNA isolation originated from tissue
consecutive to that used for histopathological analysis.
Reverse Transcription-PCR Amplification and hER-FASAY.
Reverse transcription-PCR and hER-FASAY were performed as we have
described (13)
except for the use of a different sense
primer, 5'-CCCGCCGGTTTCTGAGCCTTCTGCCC (bp 227–252, with bp counting as
in Ref. 14
). Two independent hER-FASAY experiments were
performed per tissue specimen (both starting from the same mRNA).
Automated Sequencing of hER Variants.
hER cDNA from all yeast colonies showing a negative (DB) or dominant
active (AF-2) phenotype in the hER-FASAYs of primary breast tumor
specimens were analyzed by automated sequencing. hER plasmid isolation
from yeast and DNA sequencing analysis was performed as described
before (13)
. From each tumor specimen, all yeast colonies
with AF-2 phenotype and a maximum of 10 colonies with DB phenotype were
analyzed.
DIG Dot-Spot Analysis of hER Splicing Variants.
hER cDNA from all yeast colonies showing an aberrant phenotype in the
hER-FASAYs of normal-CBR and normal-TCB specimens was analyzed for exon
deletions using DIG dot-spot analysis. With this assay, the hER
variants were analyzed for deletion of exons 2, 3, 4, 5, or 7 using
digoxigenin (DIG)-labeled probes. A probe for detection of exon 6 was
not made because deletion of exon 6 in hER mRNA from breast carcinomas
has never been found (DNA sequencing data in this paper and in Ref.
15
). Specific probes for detection of each exon were
synthesized by PCR (primer sequences available upon request) using 10
ng pMD11 containing full length hER cDNA, 10 pmol of each primer, and
the PCR DIG probe synthesis mix (all DIG reagents were obtained from
Boehringer Mannheim, Germany). Fragments were amplified and
labeled in 20 cycles of 20 s at 92°C, 1 min at 60°C (probes
exons 2, 3, and 4) or 55°C (probes exons 5 and 7), and 2 min at
72°C and then 7 min at 72°C and a cooling down to 15°C in a
PTC-200 PCR apparatus (MJ Research, Waltham, MA). Probes vary in
length from 70 to 110 bp.
hER cDNA present in the hER-FASAY yeast colonies was amplified by PCR using two hER-specific primers located in exons 1 and 8 [5'-CGGTCAGACCGGCCTCCCCTAC, bp 511–532 (bp counting as in Ref. 14 ) and 5'-ATGAGGGTAAAATGCAGCAG, bp 2115–2134]. PCR was performed in a total volume of 100 µl containing 10 mM Tris-HCl (pH 8.8), 25 mM KCl, 3.5 mM MgCl2, 200 µM of each deoxynucleotide triphosphate (Pharmacia Biotech, Inc., Uppsala, Sweden), 50 pmol of each primer, 3% DMSO, 8.3 units Taq polymerase (Life Technologies, Inc.), and 0.83 units Pyrococcus furiosus DNA polymerase (Stratagene, La Jolla, CA). A matchhead of yeast and 3 µl Triton X-100 were added to the PCR mixture. The mixture was preheated for 10 min at 95°C and amplified in 40 cycles of 30 s at 92°C, 45 s at 64°C, and 3 min at 72°C and then 7 min at 72°C and a cooling down to 15°C in a PTC-200 PCR apparatus (MJ Research). PCR products were heated for 5 min at 100°C and 1 µl of each PCR product was spotted on five separate N+ nylon membranes (Boehringer Mannheim). Membranes were dried and DNA was cross-linked to filters by exposure to UV light for 90 s. Filters were prehybridized in DIG Easy Hyb (30 min), hybridized in DIG Easy Hyb containing a DIG-labeled probe (2 h at 42°C), washed twice with 2 x SSC + 0.1% [5 min at rt] and twice with 0.5 x SSC + 0.1% SDS (15 min at 68°C), blocked with 1x blocking reagent (30 min at rt), hybridized with anti-DIG 1:10,000 in 1 x blocking reagent (30 min at rt), washed twice with 0.3% Tween 20, 0.1 M maleic acid, 0.15 M NaCl (pH 7.5; 15 min at rt), and soaked in detection reagent (2 min at rt). Finally, filters were soaked in CDP-star and diluted 1:100 in detection reagent. Hyperfilms (Amersham Corp., Arlington Heights, IL) were exposed to the filters for 10–20 min.
Analysis of hER 
22bpE7 by PCR.
Deletion of the first 22 bp of exon 7 of hER (22bpE7) was determined
by a triple-primer PCR, for which two primers were located 5' and 3' of
the deletion and one primer was located within the location of the
deletion (primer sequences available upon request). PCR was performed
with 1 µl of the same PCR product that was used for DIG dot-spot
analysis in a total volume of 50 µl containing 10 mM
Tris-HCl (pH 8.3), 25 mM KCl, 1.5 mM
MgCl2, 200 µM of each
deoxynucleotide triphosphate (Pharmacia Biotech, Inc.), 50 pmol of each
primer, 3% DMSO, and 5 units Taq polymerase (Life
Technologies). DNA was amplified in 35 cycles of 20 s at 92°C, 1
min at 56°C, and 2 min at 72°C and then 7 min at 72°C and a
cooling down to 15°C in a PTC-200 PCR apparatus (MJ Research).
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Functional Assay for hER.
A functional assay in yeast, the hER-FASAY, was used to determine the
prevalence and functional activity of hER in all breast tissue
specimens. The hER-FASAY is a relatively fast and simple screening
method for the presence and functional activity of hER
(13)
. In one experiment, it allows for the determination
of the relative abundance and functional activity of all variant and
wild-type hER mRNA present in a tissue specimen. With the hER-FASAY,
the functional activity of individual hER cDNA molecules is tested in
yeast by their ability to activate transcription of a reporter gene
from an estrogen response element-containing GAL1 promoter. The
assay can discriminate among wild-type hER, constitutively active hER
[transcription activation function 2 (AF-2) mutant] and inactive hER
[DNA binding (DB) mutant].
In Fig. 1
the hER-FASAY results of the three groups of tissue are shown. Fig. 1A
shows that all of the 14 normal-CBR specimens have a
percentage of wild-type hER 
75%, whereas that is true for 6 of the 7
normal-TCB specimens and only 20 of the 41 breast cancer specimens. In
the normal-CBR specimens the median percentage of hER that is wild-type
is 85% (SD, 5%), and in the normal-TCB specimens this percentage is
88% (SD, 10%). In contrast, in breast tumors the median percentage of
wild-type hER is 74% (SD, 17%; data not shown). In Fig. 1B
one can see that all of the 14 normal-CBR specimens have a percentage
of DB mutants 
20%, whereas that is the case for 5 of the 7
normal-TCB specimens and only for 15 of the 41 breast cancer specimens.
The percentage of AF-2 mutants is generally low in all three groups
(Fig. 1C)
.
|
between normal-CBR
specimens and breast tumor specimens are significant. In normal tissue,
the percentage of wild-type hER is proven to be generally higher than
in breast cancer (normal-CBR versus breast cancer,
P < 0.0001; Mann-Whitney test), whereas the
percentage of DB mutants is lower (normal-CBR versus breast
cancer, P < 0.0001; Mann-Whitney test). No
differences are found regarding the percentage of AF-2 mutants
(normal-CBR versus breast cancer, P = 0.25; Mann-Whitney test). Furthermore, although the number of
normal-TCB specimens is too low to draw definite conclusions,
comparison of the hER-FASAY data of normal-CBR specimens with those of
normal-TCB specimens did not show any apparent difference.
Analysis of hER Variants.
For 18 of the 41 primary breast carcinomas, the molecular structure of
the hER functional variants that were detected by the hER-FASAY was
determined by automated sequencing (data summarized in Fig. 2
, black
bars). In these carcinomas, only splicing variants and no point mutations
were detected. Most frequently, splicing variants were observed which
lack one or more complete exons. The only other splicing variant that
is detected more than once in these breast carcinomas is 22bpE7,
which is caused by the use of an alternative splicing acceptor site in
exon 7. For detection of the exon-skipping splicing variants in the
normal breast tissue specimens, we developed a "DIG dot-spot
analysis" with exon-specific probes. Besides this, 22bpE7 was
detected with a specific PCR test.
|
,
white and hatched bars respectively). These results are shown as the
expression of a specific variant relative to the total amount of
variant hER of each specimen. The variants present in normal-CBR
specimens are mostly one-exon deleted splicing variants (median, 100%;
SD, 11%; Fig. 2A
), which in most cases are variants lacking
exon 7 (median, 68%; SD, 28%; Fig. 2B
). In contrast, in
breast tumors a much more heterogeneous set of hER splicing variants is
present. The median percentage of one-exon deletions in breast tumors
is only 50% (SD, 22%; Fig. 2A
) and the median percentage
of variants lacking exon 7 is only 35% (SD, 21%; Fig. 2B
).
This difference in presence of one-exon deleted hER splicing variants
between normal-CBR tissue and breast cancer cannot be attributed to
chance (P < 0.0001; Mann-Whitney test on
one-exon deleted splicing variants as a percentage of observed
variants). Moreover, no apparent differences are seen when the hER
variants found in normal-CBR specimens are compared with those found in
normal-TCB specimens.
All hER variants with aberrant functional activity that were analyzed
by DIG dot-spot analysis or PCR in this study turned out to be splicing
variants lacking one or more complete exons or 22bpE7. The chance of
missing a splicing-related variant that is combined with an exon
deletion is small, a percentage that is on average 4% of all variants
detected in breast carcinomas (data not shown).
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
;
P < 0.0001). When the molecular structure of
the hER variants present in each specimen is evaluated in more detail,
even more differences are seen. In the normal-CBR group, 9 of the 12
specimens express only one-exon deleted hER variants, whereas that was
the case in none of the 18 tested breast tumors (data not shown). The
only additional variant that was seen in the remaining three normal-CBR
specimens was hER 22bpE7. In contrast, in breast tumors two-exon
deletions (nine tumors) and three-exon deletions (three tumors) were
also observed (Fig. 2A)
.
The differences in the amounts and molecular structures of hER variants
between normal tissue and breast cancer implicates that the
hER-dependent estrogen responsiveness of breast tissue might change
during tumor development. It also suggests that breast cells that
contain high levels of hER variants (Fig. 1)
or those containing
variants lacking multiple exons (Fig. 2)
may infer a selective
advantage during breast cancer development or progression.
Interestingly, despite the age differences and the presence or absence
of cancer, there are no apparent differences in amounts or molecular
structures of hER variants between normal-CBR and normal-TCB.
Apparently, the higher amount of hER variants and the different
molecular structure of the variants detected in breast cancer are
specific for tumor cells and not for the complete breast with malignant
disease.
hER E7 is the predominant variant in normal breast tissue.
Fig. 2B
shows that the relative amount of this variant
(i.e., the presence of hER E7 compared with the total
amount of variant hER) is much higher in normal breast tissue than in
tumors. Nevertheless, the absolute amount of hER E7
(i.e., the presence of hER E7 compared with the total
amount of variant and wild-type hER) is not found to be higher in
normal-CBR (mean, 9.9%; SD, 5.8%) or normal-TCB (mean, 9.9%; SD
5.8%) than in breast tumors (data not shown) [mean 10.8%, SD 9.1%;
data not shown; ANOVA, P = 0.96 (confidence
interval, -5.1 to +6.8%)]. This may indicate that the
presence of hER E7 should be considered normal in human breast
tissue and that this variant may even play a physiological role in
normal breast tissue. hER E7 has been shown to have a dominant
negative function (i.e., not only inactive itself but also
preventing the function of wild-type hER) in yeast but was
nonfunctional in HeLa cells (16
, 17)
. Because the
functional activity of hER E7 in breast tissue is not yet clear, we
can only speculate on its function in normal breast tissue. It recently
was shown that during specific embryonic stages hER variants lacking
exon 3 or 4 or both are expressed in the rat pituitary gland where they
may be involved in pituitary gland development (18)
.
Analogous to this finding we could hypothesize that hER E7 may play
a physiologically relevant role as a regulator of estrogen-dependent
transcription in the mammary gland. On the other hand, hER E7
expression also could be the remains of a development-specific
expression of this hER variant, as is also seen for hER E3, E4,
and E3,4 in the adult rat pituitary gland (18)
.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by Grant NKB NKI 96-1268 from the
Dutch Cancer Society. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Netherlands Cancer Institute,
Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
3 The abbreviations used are: hER, human estrogen
receptor ; hER-FASAY, functional analysis of separated alleles in
yeast of hER; normal-CBR, normal breast tissue from cosmetic breast
reduction; normal-TCB, normal breast tissue from tumor-containing
breast; AF-2, transcription activation function 2; DB, DNA binding;
DIG, digoxigenin; 22bpE7, mRNA splicing variant lacking the first 22
bp of exon 7; E, mRNA splicing variant lacking the indicated exon;
31bpE1, mRNA splicing variant lacking the last 31 bp of exon
1; rt, room temperature.
Received 10/18/99. Accepted 12/10/99.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
isoforms during ontogeny of the pituitary gland. Endocrinology, 140: 2781-2789, 1999.This article has been cited by other articles:
|
|
 |
|
  J. P. Venables Aberrant and Alternative Splicing in Cancer Cancer Res., November 1, 2004; 64(21): 7647 - 7654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Simon, P. Boutouyrie, J.M. Simon, B. Laloux, C. Tournigand, A.I. Tropeano, S. Laurent, and P. Jaillon Influence of Tamoxifen on Carotid Intima-Media Thickness in Postmenopausal Women Circulation, December 3, 2002; 106(23): 2925 - 2929. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Pasquali, V. Rossi, D. Esposito, C. Abbondanza, G. A. Puca, A. Bellastella, and A. A. Sinisi Loss of Estrogen Receptor {beta} Expression in Malignant Human Prostate Cells in Primary Cultures and in Prostate Cancer Tissues J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2051 - 2055. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), 6 normal-TCB specimens
(
), and 18 breast cancer specimens (
), as determined by DIG
dot-spot analysis and automated sequencing. The expression of a
specific variant is shown relative to the total amount of hER variants
of each specimen. Error bars indicate the SD. A, All hER
variants detected in normal breast tissue and breast cancer.
B, The category "1-exon deletions" (shown in
A) is subdivided in the individual exon deletions. The
category "2-exons deleted" (shown in A) consists of