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Endocrinology |
Department of Surgery, Stanford University, Stanford, California 94305
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estrogen exerts its effects through a nuclear ligand-activated
transcription factor, the
ER.4
An activated hormone-receptor complex transactivates genes by binding
to regulatory promoter elements called EREs (7)
. Two known
subtypes of ER exist, ER-
and ER-ß, that have distinct tissue and
cell patterns of expression (8)
. In this study, ER
specifically refers to ER-. ER expression is routinely used as a
clinical marker to predict the hormone responsiveness of breast tumors
(9)
. Compared with ER-negative breast tumors, a high
percentage of ER-positive tumors respond favorably to endocrine
treatment, are associated with a better prognosis, and have a
well-differentiated phenotype (10, 11, 12)
.
The marked physiological and phenotypic differences between the
ER-positive and ER-negative breast tumors have been hypothesized to be
due to differences in gene expression between these two tumor types.
Studies have demonstrated the differential expression of a number of
genes when comparing ER-positive and ER-negative breast carcinoma cell
lines (2
, 13, 14, 15, 16)
. Although most of the genes found to be
differentially expressed in ER-positive cancers were not estradiol
responsive, it is likely that estrogen induces the expression of a
repertoire of genes controlling cell growth. Using techniques to
isolate differentially expressed genes, several estradiol-inducible
mRNAs have been identified in ER-positive human breast cancer cell
lines. Genes known to be induced by estrogen treatment include
PR (9)
, pS2 (17)
,
cathepsin D (18)
, HSP27 (heat shock
protein 27,000; Ref. 19
), aldolase A,
dehydrogenase, -tubulin, and
glyceraldehyde-3-phosphate (reviewed in Ref.
20
). In addition to these genes, a number of
estrogen-induced mRNAs of unknown function have been isolated
(2
, 15)
.
The repertoire of genes responsible for the physiological effects of estrogen on hormone-responsive breast cancers has not been characterized completely (2) . In addition, well-characterized genes such as pS2 are only expressed in 30–60% of hormone-responsive tumors (21, 22, 23) , suggesting that their function is not necessary for the normal physiological response to estrogen. Therefore, to more completely understand hormone-responsive breast cancer, there is a pressing need to identify more estrogen-responsive genes and establish their role in tumorigenesis and progression.
In this study, efforts were made to delineate genes that are differentially expressed in response to estradiol treatment in MCF7, an ER-positive, hormone-responsive human breast carcinoma cell line. Using SSH (24) , a set of estrogen-responsive genes has been isolated that is also repressed by tamoxifen. Further screening has enabled us to focus on two estrogen-responsive genes that exhibit a distinct correlation with ER expression in breast carcinoma cell lines and primary breast carcinomas. The pattern of expression indicates a critical functional role of these genes in hormone-responsive tissues and cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
All cell lines were obtained from American Type Culture Collection
(Manassas, VA). Cell lines MCF7, T-47D, BT-20, MDA-MB-231, and HBL-100
were maintained in MEM (Life Technologies, Inc., Gaithersburg, MD);
ZR-75-1 and BT-474 were maintained in RPMI 1640 (Life Technologies,
Inc.); and MDA-MB-361 was maintained in Leibovitz’s L-15 medium (Life
Technologies, Inc.). Media were supplemented with 10% FCS (Gemini
Bio-Products, Calabasas, CA), 10 units/ml penicillin G (Life
Technologies, Inc.), 10 µg/ml streptomycin (Life Technologies, Inc.),
and 6 ng/ml bovine insulin (Sigma Chemical Co., St. Louis, MO). All
cells were incubated at 37°C in 5% CO2 except
MDA-MB-361 cells, which were maintained in a
CO2-free environment.
MCF7 cells induced with estradiol were grown under normal conditions as described above until they reached approximately 25% confluence. The media were subsequently changed to phenol red-free MEM (Life Technologies, Inc.) supplemented with 10% charcoal-stripped FCS (Gemini Bio-Products; certified to be ß-estradiol free), 10 units/ml penicillin G, 10 µg/ml streptomycin, and 6 ng/ml bovine insulin. MCF7 cells were treated with 1 x 10-8 M water-soluble ß-estradiol (Sigma Chemical Co.) and/or 1 x 10-6 M 4-hydroxytamoxifen (Sigma Chemical Co.) for periods of 6–72 h. Cycloheximide (Sigma Chemical Co.) was used at a concentration of 10 µg/ml.
mRNA Isolation.
Poly(A)+ RNA was isolated from cell lines using
Fast Track Kit 2.0 (Invitrogen Corp., Carlsbad, CA) according to the
manufacturer’s instructions.
SSH.
SSH (24)
was performed with the Clontech PCR-Select cDNA
Subtraction Kit (Clontech Laboratories Inc., Palo Alto, CA) as
described by the manufacturer. Starting material consisted of 2 µg of
poly(A)+ RNA from MCF7 cells grown in the absence
of ß-estradiol for 5 days and then treated with ß-estradiol for
72 h (referred to as the tester) and 2 µg of
poly(A)+ RNA from MCF7 cells grown in the absence
of ß-estradiol for 8 days (referred to as the driver). All PCR
products generated using SSH were subcloned into pCR 2.1 vector using
the Original TA Cloning Kit (Invitrogen).
Isolation of GREB1 cDNAs.
Approximately 1 x 106 plaques
from an oligodeoxythymidylic acid and a random-primed MCF7 cDNA
library were screened using the 600-bp GREB1 fragment isolated by SSH.
The 
phage plaques were grown in Escherichia coli
XL1-blue MRF' cells (Stratagene), and phages were transferred to
Optitran supported nitrocellulose (Schleicher & Schuell) using the
in situ plaque hybridization technique (25)
.
Phage lifts were prehybridized and hybridized in a solution containing
50% formamide (v/v), 5x SSPE, 5x Denhardt’s solution, 0.1% SDS
(v/v), and 100 µg/ml denatured salmon sperm DNA at 42°C. Fifty ng
of GREB1 cDNA was 32P-labeled by random priming
(Roche Biochemicals) using 50 µCi of
[32P]dCTP (3000 Ci/mmol; Amersham). After a
16-h hybridization, membranes were washed once in 2x SSC/0.1% SDS
(v/v) for 20 min at 42°C and twice in 0.2x SSC/0.1% SDS (v/v) for
20 min at 65°C before exposure to Kodak XAR-2 film overnight at
-80°C with intensifying screens. The phage plaques that
hybridized to 32P-labeled 600-bp GREB1 fragment
were isolated, and plaques were purified by secondary and tertiary
rounds of screening. Isolated plaques were processed to excise the cDNA
inserts from the vector to yield phagemids containing the inserts
of interest in the pBK-CMV vector (Stratagene).
The region of GREB1 cDNA that was not obtained from screening the
library was obtained by RT-PCR. The region between GREB1 and GREB2
(later determined to be the 3' end of GREB1 cDNA) was amplified using
two separate RT-PCR amplifications. One reaction used the primers
5'-GREB1/2 (5'-TGGACCTGGGATCCTTTGAGAAGGTGGACTTTC-3') and 3'-GREB1/2
(5'-ACGACGAGGTGACCGACACCTGGACGCTCCTCTG-3'), which generated a DNA
fragment of 1565 bp. The second reaction used primers ck5'-GREB1/2
(5'-CCCCAACATTGTGACACTTCACGTGACC-3') and ck3'-Greb1/2
(5'-CCAAGCAGGAGGAGTTTATCAATCGCAGG-3'), which generated a DNA fragment
of 720 bp. Both PCR reactions were cloned and had the identical
sequence for the intervening region between GREB1 and GREB2.
Northern Analysis.
One µg of mRNA was electrophoresed on a 1% agarose formaldehyde
denaturing gel in 1x 4-morpholinepropanesulfonic acid and then
transferred to a Nytran membrane (Schleicher & Schuell). Approximately
25–50 ng of each clone in pCR 2.1 or pBK-CMV was
32P-labeled by random priming (Roche
Biochemicals). As controls, 25–50 ng of cDNA for pS2, ER,
GAPDH, and ß-actin were also labeled. Northern blots were
prehybridized and hybridized in 50% formamide, 5x Denhardt’s
solution, 5x SSPE, 1% SDS, and 100 µg/ml denatured salmon sperm DNA
at 42°C. Blots were washed in 2x SSC/0.1% SDS (v/v) at 42°C for
20 min, followed by two washes in 0.2x SSC/0.1% SDS (v/v) at 65°C
for 20 min each. Northern blots were placed on film with an
intensifying screen at -80°C.
DNA Sequence Analysis.
Sequencing of the SSH fragments was performed on double-stranded
templates using the dideoxynucleotide chain-termination method with
[-35S]dATP (1000 Ci/mmol; Amersham) as a
label. Sequencing reactions were carried out using the Sequenase
version 2.0 T7 DNA polymerase DNA Sequencing Kits (USB
Corporation, Cleveland, OH) with the T7 promoter primer in the
pCR 2.1 vector.
The sequence for the GREB1a, GREB1b, and GREB1c cDNAs isolated from the MCF7 cDNA library was determined on both strands using the automated ABI 373 DNA sequencing system with the standard dye terminator AmpliTaq polymerase FS kit by the Stanford University Protein and Nucleic Acid facility (Stanford, CA). T3 and T7 promoter primers and custom sequence-specific primers were used, and all sequences reported were determined by sequencing both strands of DNA. The nucleotide sequence of the cDNA and the protein sequence of GREB1a, GREB1b, and GREB1c were compared against the National Center for Biotechnology Information databases using BLASTN and BLASTP, respectively.
RT-PCR from Primary Tumors.
Primary human breast tumor tissue was collected fresh from mastectomy
and biopsy specimens (kindly provided by Dr. Helen Feiner, Breast
Cancer Resource, New York University Medical Center) and snap frozen in
liquid nitrogen. Approximately 0.5 mg of tissue was homogenized, and
total RNA was isolated using Trizol reagent (Life Technologies, Inc.)
according to the manufacturer’s recommendations. One µg of RNA from
each sample was reverse transcribed using random hexamers with the
Advantage RT-for-PCR kit (Clontech) according to the manufacturer’s
recommendations. The 20-µl reverse transcription sample was diluted
to 100 µl with water, and then 2 µl of each sample were analyzed by
PCR for GREB1a, GREB1b, GREB1c, PDZK1, ER, and ß-actin messages using
the Advantage cDNA PCR kit (Clontech) with Advantage KlenTaq Polymerase
mixture and gene-specific primers designed across intron/exon
junctions. The common 5' primer for GREB1a, GREB1b, and
GREB1c was oGREB-5' (5'-GGACCAGCTTCAGTCACCTTTCCAGTGGTGGCC-3'). The 3'
primer for GREB1a was oGREB1a-3'
(5'-GGAAGATCTGCTCCAGGTCCTCCATCAAGGGC-3'), the 3' primer for GREB1b was
oGREB1b-3' (5'-CTCTTTTATGAATTGTCTGGTTTATACGTCCGG-3'), and 3' primer for
GREB1c was oGREB1c-3'
(5'-GGGAGTAAAGCTGGTGCCTGGGGCACAGGTCACG-3'). The fragments
generated for GREB1a, GREB1b, and GREB1c were 334, 339, and 325 bp,
respectively. Primers for PDZK1 were oPDZ-5'RT
(5'-GCTTCTCCTGCGAATTGAGAAGGACACCGAGG-3') and oPDZ-3'RT
(5'-GGTCCAAGTTTGCACACC TCCATTCATCACAGG -3'), which generated a PCR
fragment of 331 bp. Primers for ER were oER-1A-long
(5'-GTGCCCTACTACCTGGAGAACGAGCCCAGC-3') and oER-1B-long
(5'-AGCATAGTCATTGCACACTGCACAGTAGCG-3'), which generated a 195-bp DNA
fragment. Primers for ß-actin were oACTIN-5'
(5'-AGCAAGAGAGGCATCCTCACCCTGAAGTACC-3') and oACTIN-3'
(5'-CAGATTCTCCTTAATGTCACGCACGATTTCCC-3'), which generated a 471-bp DNA
fragment. Two-step PCR was used to amplify the genes and was performed
on a Perkin-Elmer 9600 DNA thermal cycler as follows: (a) 1
cycle of 94°C for 1 min; (b) 30 cycles of 94°C for
30 s and 68°C for 3 min; and (c) 1 cycle of 68°C
for 3 min. PCR samples were then analyzed for GREB1a, GREB1b,
GREB1c, PDZK1, ER, and ß-actin by electrophoresis on 1.5%
agarose gels.
After electrophoresis, the samples were transferred onto 0.2 µm
Nytran (Schleicher & Schuell) nylon membranes by Southern blotting.
Blots were prehybridized and hybridized in 20% formamide, 5x
Denhardt’s solution, 5x SSPE, 1% SDS (v/v), and 100 µg/ml
denatured salmon sperm DNA at 42°C. Southern blots were probed with
oligonucleotides that had been end-labeled using
[
-32P]ATP (6000 Ci/mmol; Amersham) and T4
polynucleotide kinase (New England BioLabs). The oligonucleotides were
designed internal to the sequence of each gene amplified. The four GREB
transcripts were detected using oGREBint
(5'-GGCTTGGCCTTGCATATTTTCAGC-3'). Other internal oligonucleotides were
oPDZK1/int (5'-CATATGCAGGTTGTGGATCTGG-3'), oERpro-86
(5'-ACCCTGGCGTCGATTAT CTGAATTTGGC-3'), and oActin-int
(5'-ATGACCCAGATCATGTTTGAGACC-3'). After 16 h of hybridization, the
blots were washed sequentially in 2x SSC/0.1% SDS (v/v) at 42°C for
20 min followed by two washes in 2x SSC/0.1% SDS (v/v) at 65°C for
20 min each and then placed on X-ray film with an intensifying screen
at for 10 min. Subsequently, the blots were placed on a Kodak Phosphor
screen (Molecular Dynamics, Sunnyvale, CA) and quantified using
ImageQuant software and a Molecular Dynamics PhosphorImager.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of Tamoxifen on Estrogen-responsive Genes.
Tamoxifen is a competitive antagonist of endogenous and exogenous
estradiol. Tamoxifen acts as an inhibitor of estrogen-induced responses
and also inhibits the effect of estradiol on cell proliferation and on
the regulation of specific genes. Thus, the effect of tamoxifen on the
15 estrogen-responsive genes was examined. Northern blot analysis of
MCF7 cells grown in the absence or presence of ß-estradiol and in the
presence of ß-estradiol and tamoxifen indicated that all of the 15
genes that were induced by ß-estradiol were repressed by tamoxifen
(Fig. 1)
. Expression of the estradiol-responsive gene pS2 was also
found to be repressed by tamoxifen, as has been reported previously
(26)
.
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Time-Course Study of Gene Induction by ß-Estradiol.
To study the temporal response to estrogen stimulation, a kinetic study
of gene induction of the 15 estrogen-responsive genes was undertaken in
which the expression of the genes at different time points after the
addition of ß-estradiol was examined. MCF7 cells grown in
estrogen-free media for 5 days were then supplemented with
ß-estradiol (1 x 10-8 M) for 6,
24, or 48 h before harvesting. A similar set of MCF7 cells grown
in the absence of ß-estradiol was used as a control. Northern blot
analysis revealed that 14 of the 15 genes examined responded early to
ß-estradiol treatment because gene expression was apparent within
6 h of treatment. These results suggest that these genes are
direct targets of activated ER. Gene expression was detected after
48 h for the one gene that did not respond early. Fig. 2
shows representative examples of early responders to the ß-estradiol
treatment.
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cDNA library that spanned the
region between these two clones (see below). Therefore, RT-PCR was used
to clone the region not represented by the clones. Two separate PCR
reactions were performed using primers in the 3' end of GREB1 and the
5' end of GREB2. Based on the size of the mouse gene, these PCR
reactions generated fragments of the expected size. Both PCR products
were cloned and sequenced to determine the region between GREB1 and
GREB2, thus confirming that these two clones are from the same mRNA.
Expression of Estrogen-responsive Genes on a Panel of Breast
Carcinoma Cell Lines.
We wanted to focus attention on genes whose pattern of expression
correlated with ER expression in breast cancer cell lines. To correlate
the estrogen responsiveness of the genes to ER expression in human
breast carcinomas, we examined the expression of the 14
estrogen-responsive genes in a panel of breast cancer cell lines. The
cDNA fragments isolated during SSH were used as probes on Northern
blots containing six ER-positive and two ER-negative human breast
carcinoma cell lines. Six of the 14 genes examined (PDZK1,
GREB1, and clones 3, 21, 76, and 138) were
expressed in some or all of the ER-positive breast carcinoma cell lines
and absent in all of the ER-negative breast carcinoma cell lines. Fig. 3
shows the results of the two estrogen-responsive genes,
GREB1 and PDZK1, with the best correlation to ER
expression. The expression pattern of pS2 is shown for comparison. In
addition to exhibiting an on/off response to ß-estradiol stimulation,
GREB1 was expressed in all five of the strongly ER-positive cell lines
and in none of the two ER-negative breast carcinoma cell lines or in
BT-20, which has low levels of ER. In addition, GREB1 is induced by
estradiol in ECC-1, an endometrial carcinoma cell line (data not
shown). As indicated in Fig. 3
, GREB1 shows a better correlation with
ER than is seen with pS2, a gene that is often used as a
marker for hormone-responsive breast cancer. PDZK1, which
has been associated with breast cancer but has not been related to
hormone responsiveness, was detected in three of the five ER-positive
breast cancers cell lines as well as in BT-20. Because the expression
pattern of these two genes had the best correlation with ER expression
in this panel of cell lines, we chose to focus additional experiments
on these two genes.
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. These were designated as GREB1a and GREB1b, respectively. Screening of
an oligodeoxythymidylic acid-primed MCF7 cDNA library yielded a third
clone, GREB1c, which had a poly(A) tail and also had 5' and 3' ends
that were divergent from those of GREB1a and GREB1b (Fig. 4A)
. The three clones were sequenced on both strands
and analyzed. Comparison of the sequences with the European Molecular
Biology Laboratory/GenBank databases using the BLASTN and BLASTP DNA
analysis programs did not yield any significant similarity with known
sequences in the database.
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. Using a
combination of our clones, the GenBank sequence of AB011147, and
the PCR-generated region between the two, the longest cDNA of
GREB1 gene was obtained (see Fig. 4A
). The GREB1a
mRNA is 8482 bases long [not including the poly(A)+ tail], which
agrees with the size obtained by Northern blot. This mRNA is predicted
to encode a protein of 1949 amino acids, which is shown in Fig. 4B
. However, the alternate transcripts from this gene may
encode smaller proteins with variable COOH termini. Comparison of
GREB1a, GREB1b, and GREB1c sequences using the Clustal V sequence
alignment program (27)
revealed that the 5' UTR was
dissimilar for all three cDNAs until 264 bases upstream of the putative
start site of the longest ORF predicted by DNA Strider version 1.2
(28)
, as exhibited in Fig. 4, A–C
. GREB1b
differed slightly in that it had a deletion of 13 bases prior to the
beginning of the sequence that matched for all three of the cDNAs.
Other shorter clones [GREB1b(i), 1b(ii), and 1d] with this same 5'
end did not contain this deletion (Fig. 4C)
. Sequence
homology between GREB1a and GREB1b continues for 1346 nucleotides
(indicated by 
) downstream of the putative start site beyond
which the predicted ORF of GREB1b continues for an additional 28
nucleotides. Divergence of sequence homology between GREB1a and GREB1c
occurs after 1159 nucleotides (indicated by
). The predicted stop codon for
the GREB1c ORF is 71 nucleotides beyond the sequence divergence from
GREB1a. GREB1b and GREB1c had putative ORFs of 457 and 409 amino acids,
respectively. In addition, both their 3' untranslated sequences were
different from each other and from that of GREB1a (Fig. 4D)
.
Within the region that was homologous, GREB1c had a single-nucleotide
change from an A to a C as compared with GREB1a and GREB1b. On
translation, this alteration would result in a conservative amino acid
change from asparagine (N) to threonine (T), as indicated with by
• in Fig. 4B
.
To establish that GREB1a, GREB1b, and GREB1c were genuine transcripts
and not chimeras, Northern blot analysis and RT-PCR were performed. In
addition, GREB1d, a transcript of 1 kb that was isolated during the
cloning process, was included for comparison. GREB1d had been sequenced
from both ends, and analysis revealed that sequences at the 3' end of
this clone matched those of a rRNA gene, whereas the 5' end was unique
and matched GREB1b. This seemed to imply that GREB1d could be a
chimera. Northern blot analysis using GREB1a, GREB1b, and GREB1c cDNAs
as probes confirmed differential expression of these transcripts.
However, Northern blot analysis using GREB1d showed hybridization to
the expected 8-kb band as well as to a 4.2-kb band that was not
differentially expressed with ß-estradiol treatment (data not shown).
Additionally, RT-PCR was performed on the four clones using 5' primers
that were common to all four transcripts and 3' primers that were
designed against the unique 3' region of each clone. As indicated in
Fig. 5
, a single band of the expected size could be seen for GREB1a, GREB1b,
and GREB1c in the control lanes (with cDNA from each clone) and also in
the lanes containing RNA from MCF7 cells grown in the presence of
ß-estradiol that had been reverse transcribed. Conversely, for
GREB1d, which was believed to be a chimera, no expression of the gene
was visible in the lane with reverse-transcribed MCF7 mRNA + ß-estradiol, confirming that it was chimeric. In all cases,
gene amplification was not visible in the negative control lanes (MCF7
mRNA without reverse transcriptase; Fig. 5
, Lane 3 of each
set). These data prove that the cDNAs of GREB1a, GREB1b, and GREB1c
represent the expression of different mRNAs with divergent 5'
untranslated exons.
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, GREB1 is expressed in the presence of cycloheximide. The slight
increase over estradiol alone is likely the result of stabilization of
mRNA known to occur with cycloheximide treatment. PDZK1, however, is
repressed by cycloheximide, indicating the need for new protein
synthesis for expression. This finding demonstrates that PDZK1 is a
secondary target for estradiol induction and that the PDZK1 promoter is
not likely to be dependent on an ERE. The pS2 gene is
expressed in the presence of cycloheximide, although the level of
expression is slightly reduced. This result suggests that there may be
additional mechanisms increasing expression of pS2 that require the
synthesis of new protein. The result with actin confirms the abundance
of mRNA in each lane.
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Preliminary experiments were performed in the absence of reverse
transcriptase to verify that the PCR amplification was due to reverse
transcription of mRNA present in the tumor samples and not due to
contamination with genomic DNA in the total RNA extract. Qualitative
analysis of the expression of the three genes is presented in Fig. 7A
. As the figure indicates, GREB1 and PDZK1 were
predominantly present in ER-positive breast tumors, with only a few
ER-negative tumors exhibiting gene expression above background levels.
Semiquantitative analysis was performed by determining signal intensity
using phosphorimaging. Values were normalized against ß-actin to
determine the relative expression of the different genes analyzed in
this study. As indicated in Fig. 7B
, GREB1 expression was
3.5-fold greater in ER-positive compared with ER-negative breast
tumors. Similarly, PDZK1 gene expression was 19-fold greater
in ER-positive breast tumors than in ER-negative breast tumors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and is only expressed in 30–60% of ER-positive tumors
(21, 22, 23)
. In addition, pS2 null/null mice have hyperplasia
of gastric mucosa but do not appear to have altered responses to
estrogen (34)
. More recently, attention has been focused
on cyclin D1 as an important mediator of the response of tumors to
estrogen (35)
. PR (9)
,
HSP27 (19)
, and TGF-ß
(36)
are other genes induced by estrogen in
hormone-responsive breast cancer that have been studied extensively.
Although several physiological functions have been ascribed to these
genes, the expression of the known estradiol-regulated genes fails to
adequately explain the effects of estrogen in hormone-responsive
tumors. For this reason, we have undertaken a study to identify
additional genes induced in hormone-responsive tumors in response to
estradiol treatment. Specifically, we were interested in identifying
genes that were induced by estradiol and repressed by tamoxifen and
that demonstrated a high degree of correlation with ER expression in
breast cancer cell lines and primary tumors. Two genes were
identified—PDZK1 and GREB1—that fulfill these
criteria. Several methods have been described to identify estrogen-regulated genes. Early research studying estrogen-induced gene expression used techniques such as double isotope protein labeling of induced proteins followed by analysis on SDS-PAGE (14) . The pS2 gene had been identified using the technique of differential library screening (17) . This technique is useful for identifying abundant genes but does not allow isolation of scarce mRNAs (15) . SSH has been recently described for the identification of differentially expressed genes and has several advantages over older methods (24) . SSH allows isolation of differentially expressed mRNAs of low abundance with a false positive rate of 30–80% (15 , 37) . SSH has not previously been applied to the isolation of estradiol-induced genes, and earlier studies suggested that there are many more estradiol-regulated genes than had been reported previously (15) . For these reasons, applying this new technique to identify estradiol-responsive genes was likely to result in the isolation of genes critical to hormone response.
Four of the estrogen-induced genes identified in this study were genes that had been reported previously. These genes include thombospondin, PDZK1, a mRNA coding for an unidentified protein (KIAA0575) from human brain (herein termed GREB2), and an immunoglobulin-like gene. None of these had previously been identified as being responsive to estradiol. Thrombospondin is a matrix-bound adhesive glycoprotein that is present in a variety of cells, including mammary epithelial cells and osteoblasts (38) . It has been shown to play a role in tumor angiogenesis and tumor cell proliferation (39 , 40) . A study of human osteoblast-like cells exhibited colocalization of thrombospondin with transforming growth factor ßI and insulin-like growth factor-I. These proteins were expressed in the extracellular matrix and were modulated by ß-estradiol (41) . However, in another study, thrombospondin was not found to be responsive to estrogen in ER-positive MCF7 breast carcinoma cells (40) . Our study established that although thrombospondin did not correlate with ER in a panel of breast carcinoma cell lines, there was an approximately 4-fold up-regulation of expression in MCF7 cells in response to ß-estradiol treatment.
One of the genes induced by estrogen identified in these studies was PDZK1. The PDZ domain was named for three proteins in which the domain was first recognized: (a) post-synaptic density protein PSD-95 (42) ; (b) Drosophila disc large tumor suppressor dlg (43) ; and (c) the tight junction-associated protein ZO-1 (44) . Proteins containing this domain have been reported to be involved in organizing proteins at the cell membrane (45) and are also involved in linking transmembrane proteins to the actin cytoskeleton (46) . Through these interactions, PDZ domain proteins regulate a diverse set of cell functions including control of signal transduction, determination of cell polarity, cell differentiation (47) , and ion transport (48) . PDZK1 was first isolated in a yeast two-hybrid screen designed to identify proteins interacting with MAP17, a membrane-associated protein involved in regulation of cell proliferation (49) . Subsequently, PDZK1 has been reported to interact with cMOAT(MRP2), which is a canalicular organic anion transporter associated with multidrug resistance (50) . Expression of PDZK1 is limited to epithelial cells, and overexpression of this protein has been reported in a variety of carcinomas (49) . PDZK1 contains four PDZ domains and could interact with a number of proteins simultaneously, thereby coordinating the interplay of multiple proteins at the cell membrane. The demonstration that PDZK1 is induced by estradiol in hormone-responsive breast cancer provides an important mechanism to explain membrane alterations such as formation of microvilli that occur with estrogen treatment (51) . It has recently been reported that estrogen induces expression of Na+-H+ exchanger regulatory factor (NHE-RF), the human homologue of the Na+/K+ exchange regulatory factor (52) . These results provide important clues related to the physiological effects of estrogen on cell membrane alterations.
The GREB1 gene demonstrated a striking correlation with ER expression in a panel of breast cancer cell lines. Expression of GREB1 was similarly associated with ER expression in primary breast cancers, although the correlation was not perfect. However, cancer specimens obtained from patients are not controlled for ligand exposure. Because expression of these genes is dependent on estrogen, it might be expected that tumors resected from postmenopausal women might not demonstrate expression of estrogen-induced genes. In addition, the use of estrogens and newer antiestrogens such as raloxifene may alter patterns of gene expression. However, these results do establish that these estrogen-regulated genes are expressed in primary breast tumors.
The structure of GREB1 is complex in that there are at least three different noncoding 5' exons. The expression of each of these transcripts is estrogen regulated, suggesting the presence of multiple estrogen-inducible promoters. The occurrence of multiple estrogen-inducible promoters is not unique. The PR gene has been shown to have multiple estrogen-regulated promoters that are controlled by distinct EREs. In the case of PR, these different transcripts encode two distinct PRs, PR A and B, which have different transcriptional activation ability (53) . The different 5' exons for GREB1 are noncoding, and each transcript uses the same initiation codon. However, there are various splicing patterns involving the 3' end of the gene that could result in proteins with different COOH termini. Recently, the mouse homologue of this gene was identified, but no function has been determined (54) . Although the function of this gene remains unknown, the pattern of expression and regulation by estrogen implies an important function in hormone-responsive breast cancer.
In conclusion, SSH was used to identify estradiol-regulated genes in hormone-responsive breast cancer. Of the 14 genes identified, 13 were induced early (6 h), and all were repressed with tamoxifen. In addition, SSH was used to compare the pattern of gene expression in the absence of estrogen and in the presence of estrogen together with tamoxifen. No significant differences were detected, suggesting that tamoxifen is a pure antiestrogen in breast tissue. These results indicate that the only effect that tamoxifen has on gene expression occurs through its interaction with ER. Two estrogen-regulated genes were identified: PDZK1 and GREB1. The pattern of expression of these genes suggests an important role for these proteins in the physiological response of tumors to estrogen.
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FOOTNOTES |
|---|
1 This work was supported by Department of the
Army Award DAMD17-99-1-9384 (to M. G. G.). This award was
administered by the United States Army Medical Research Acquisition
Activity (Fort Detrick, MD). D. A. T. was supported by funds from the
California Breast Cancer Research Program of the University of
California, Grant 5KB-0083. This work was also supported by NIH Grant
R0I-CA77350. R. J. W. was supported in part by a Clowes Career
Development Award from the American College of Surgeons. 
2 The nucleotide sequence data published in this
paper have been submitted to the GenBank/European Molecular Biology
Laboratory database at the National Center for Biotechnology
Information, and accession numbers AF245388, AF245389, and AF245390
have been assigned for GREB1a, GREB1b, and GREB1c, respectively. The
cDNA clones generated by SSH have been submitted to the
GenBank/European Molecular Biology Laboratory database and assigned the
following accession numbers: (a) GREB3, accession number
BE491961; (b) GREB18, accession number BE491962;
(c) GREB21, accession number BE491963;
(d) GREB65, accession number BE491964;
(e) GREB203, accession number BE491965;
(f) GREB76, accession number BE491966;
(g) GREB80, accession number BE491967;
(h) GREB89, accession number BE491968;
(i) GREB98, accession number BE491969;
(j) GREB138, accession number BE491970;
(k) GREB181, accession number BE491971;
(l) GREB199, accession number BE491972; and
(m) GREB227, accession number BE491973.
3 To whom requests for reprints should be
addressed, at MSLS Room P214, Stanford University School of Medicine,
1201 Welch Road, Stanford, CA 94305. Phone: (650) 723-9799; Fax:
(650) 724-3229; E-mail: ronald.weigel{at}stanford.edu
4 The abbreviations used are: ER, estrogen
receptor; PR, progesterone receptor; RT-PCR, reverse
transcription-polymerase chain reaction; SSH, suppression subtractive
hybridization; ERE, estrogen response element; poly(A)+
RNA, polyadenylated RNA; SSPE, saline-sodium phosphate-EDTA; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; ORF, open reading frame; UTR,
untranslated region.
Received 3/24/00. Accepted 9/13/00.
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.
Although other transcripts also begin sequence homology at
.
