Download Estrogen Receptor Expression in Human Breast

[CANCER RESEARCH 49, 145-148, January 1. 1989]
Estrogen Receptor Expression in Human Breast Cancer Associated with an
Estrogen Receptor Gene Restriction Fragment Length Polymorphism1
Steven M. Hill, Suzanne A. W. Fuqua, Gary C. Chamness, Geoffrey L. Greene, and William L. McGuire2
The University of Texas Health Science Center at San Antonio, Department of Medicine/Division of Oncology, San Antonio, Texas 7S284-7S84 [S. M. H., S. A. W. F.,
G. C. C., W. L. M.], and Ben May Laboratory for Cancer Research, University of Chicago, Chicago, Illinois 6063 7 [G. L. G.J
ABSTRACT
Estrogen receptor (ER) content is a well-known predictor of clinical
outcome in human breast cancer. The recent cloning of a human ER
complementary DNA has made possible the characterization of the ER
gene on a molecular level. We have examined in human breast cancers a
single, two-allele restriction fragment length polymorphism using the
restriction enzyme Pviill. Initial studies in human breast cancer cell lines
suggested a possible association between the absence of one alÃ-eleand
the absence of ER expression; subsequent analysis of alÃ-eledistribution
and frequency in 188 primary human breast tumor biopsies did indeed
show a significant but not complete correlation between the absence of
one alÃ-eleand the failure to express ER. Preliminary data suggest that
this restriction fragment length polymorphism is located within gene
sequences coding for the putative DNA or hormone-binding domains of
the ER.
INTRODUCTION
The proliferation of human breast cancer cells is often estro
gen responsive (1), and approximately one-third of breast cancer
patients respond to endocrine therapy (2). Estrogens, in com
mon with other steroid hormones, modulate cell proliferation
by regulating gene expression in target cells, via their interac
tion with specific binding proteins termed "receptors" (3). Cur
rently, ER3 content in tumors is used to predict those patients
who would benefit from endocrine therapy; significant levels of
ER are detected in 60% of human breast cancers, and the
majority of these tumors are responsive to endocrine therapy
(4). ER status also provides prognostic information (5).
In principle the ER can be examined at several levels: (a) at
the genomic level; (b) at the transcriptional level; (c) at the
translational level; and (d) at the functional level. At the ge
nomic level, recent advances in recombinant DNA technology,
including restriction enzyme analysis, have helped clarify de
fects in other genes causing several types of hemoglobinopathies
(6) and one form of growth hormone deficiency (7), and they
have identified genetic variations in the human insulin and
progesterone receptor genes (8, 9). Thus, restriction enzyme
analysis can be used as a tool for the diagnosis and understand
ing of genetic disorders and the organization of genes associated
with various forms of cancer. Restriction enzyme analysis can
detect RFLPs, which are a consequence of single nucleotide
substitutions or insertions or deletions of a DNA segment, in a
genomic sequence (10).
Several groups have identified RFLPs within steroid receptor
genes. An RFLP produced by the restriction enzyme Hindlll
has been described in the progesterone receptor gene (9), and
an RFLP has also been identified in the human ER gene using
the restriction enzyme Pvull (11). The latter was described as
a single, two-allele polymorphism consisting of fragments of
approximately 1.5 and 0.7 kilobases.
We have now examined this single, two-allele Pvull RFLP
in the ER gene of both primary human breast tumors and
peripheral blood leukocytes. One of these alÃ-elesis preferen
tially associated with the expression of ER in primary human
breast tumors.
MATERIALS
AND METHODS
Cell Lines. Five human breast cancer cell lines and one human breast
epithelial cell line were used in these studies. MCF-7 cells were kindly
provided by Dr. B. Katzenellenbogen (University of Illinois). T47D,
ZR-75-1, MDA-MB-231, and HBL-100 cell lines were obtained from
The American Tissue Culture Collection (Rockville, MD), and the BT20 cell line was purchased from Mason Research Inst. (Rockville, MD).
Cells were maintained as monolayer cultures in Eagle's minimal essen
tial medium with 10% fetal bovine serum. All cell lines were shown to
be free of Mycoplasma contamination.
Human Tissue Specimens. Human peripheral blood leukocyte DNA
was kindly provided by Dr. S. Naylor and Dr. F. Yang (University of
Texas Health Science Center at San Antonio). Frozen human breast
tumor specimens, stored at —70°C,
were obtained from the San Antonio
Breast Tumor Bank. This bank is derived from specimens originally
obtained for estrogen and progesterone receptor determination and
consists of tissue remaining after these assays have been performed.
DNA Isolation, Restriction Enzyme Digestion, and Southern Blot
Analysis. High-molecular-weight DNA from cell lines and tissues was
extracted as previously described (12) and quantitated by absorption
spectroscopy using a modification of the diphenylamine assay (13).
DNA was digested with 5 units of Pvull restriction enzyme per ng of
DNA for 4 h as recommended by the manufacturer (BoehringerMannheim). Ten ¿ig
of each DNA digest were separated by electrophoresis on a 1% agarose gel and transferred onto nitrocellulose (14). The
nitrocellulose filter was hybridized to a 32P-labeled (15) human ER
cDNA probe (XOR8) originally obtained from Dr. Geoffrey Greene.
Hybridization was performed for 48 h at 42°Cin 45% formamide, 4x
standard saline citrate, 0. l M sodium phosphate (pH. 6.5), 0.1 % sodium
pyrophosphate, 0.1% sodium dodecyl sulfate. Ix Denhardt's solution,
100 ¿ig/mlof denatured salmon sperm DNA, and 10% dextran sulfate.
Blots were washed in 0.1 x standard saline citrate-0.1 % sodium dodecyl
sulfate at 62°C.Autoradiography was at —70°C
for 1 to 3 days using
intensifying screens. Variations in loading and transfer were determined
by staining agarose gels with ethidium bromide before and after transfer
to nitrocellulose.
Region-specific Probes. Region-specific probes used in the localiza
tion studies were prepared by digestion of the 2.1-kilobase ER cDNA
Received 6/2/88; revised 6/19/88; accepted 9/27/88.
insert with Bgll. This restriction enzyme was chosen based upon a
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
restriction map from sequence analysis of XOR8 (16), showing that
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Bgll cuts OR8 at a single site producing 2 fragments. The two resulting
1Supported by NIH Grants CA30195, CAI 1378, and HD10202; The Robert
fragments of 1.3 and 0.7 kilobases comprise sequences coding for the
A. Welch Foundation: and the Susan G. Komen Foundation.
putative DNA and hormone-binding region and the remaining 5'2 To whom requests for reprints should be addressed.
3 The abbreviations used are: ER, estrogen receptor; RFLP, restriction frag
sequences, respectively. The region-specific fragments were separated
ment length polymorphism; PI alÃ-ele,alÃ-eleidentified by a 1.6-kilobase restriction
on
a 1% agarose gel, electroeluted, labeled by nick translation (17), and
fragment; P2 alÃ-ele,alÃ-eleidentified by a 0.6-kilobase restriction fragment; cDNA,
complementary DNA.
used in hybridization analysis.
145
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ESTROGEN RECEPTOR GENE RFLP IN BREAST CANCER
AlÃ-eleDistribution in Human Breast Cancer Cell Lines and a
Breast Epithelial Cell Line. To determine the relationship be
tween this RFLP and ER expression, we examined DNA from
five well-characterized human breast cancer cell lines and one
breast epithelial cell line (Fig. 2). The 3 breast cancer cell lines
classified as ER positive by ligand-binding assays (MCF-7,
T47D, and ZR-75-1) were homozygous for P2 alÃ-ele,while the
2 ER-negative breast cancer cell lines (BT-20 and MDA-231)
were homozygous for PI alÃ-ele.Only the human normal breast
epithelial cell line (HBL-100), which is ER negative, did not
follow this pattern. This initial experiment suggested that a
particular genotype may be associated with the absence of ER
expression.
RESULTS
Pvull Polymorphism. Fig. 1 is a representative Southern
hybridization analysis with Pvull as the restriction enzyme and
the ER cDNA as the radioactive probe. This figure shows the
restriction fragment patterns of genomic DNA from several
samples each of human peripheral blood leukocytes and pri
mary human breast tumor biopsies. Six invariant restriction
fragments hybridizing with the human ER cDNA probe were
observed at 13, 7.5, 5.9, 3.9, 3.5, and 1.0 kilobases. In addition,
a single, two-allele RFLP consisting of variant bands at 1.6
and/or 0.6 kilobases was also identified. Samples 1 and 2 in
the first panel, and 11 in the second panel, are homozygous for
the 1.6-kilobase alÃ-ele(PI alÃ-ele);Samples 3 and 5 in the first
panel, and 8 in the second panel, are homozygous for the 0.6kilobase alÃ-ele(P2 alÃ-ele);and Sample 4 in the first panel, and
6, 7, 9, 10, and 12 in the second panel, contain specimens
heterozygous for both the 1.6- and 0.6-kilobase alÃ-eles.
RFLP Frequency in Normal Leukocytes and Breast Tumor
Cells. To determine the frequency of the ER Pvull RFLP in
fresh human tissues, we examined DNA from peripheral blood
leukocytes of 53 individuals and from 188 primary human
breast tumor biopsies. The frequency of the two alÃ-elesin both
normal and breast tumor samples exhibited a typical Mendelian
distribution, as predicted by the Hardy-Weinberg equation, and
the alÃ-elefrequencies did not differ between normal and breast
tumor tissues (Table 1).
Human Blood
¡o O
«' OU
o
IsN
I
OÙ
Human Breast Tumor
» ---•«
» - - - «*«*
«
*
»•»«•ft*
~ » •»••«
.alÃ-ele PI
(1.6kb)
_allelePI
(1,6kb)
.alÃ-eleP2
(0.6kb)
Fig. 1. ER RFLP. Representative Southern blot of genomic DNA from human
peripheral blood leukocytes and human breast tumor specimens digested with the
restriction enzyme Pvull. Ten tig of digested DNA samples were electrophoresed
on a \% agarose gel and transferred to nitrocellulose. The blot was hybridized
with "P-labeled ER cDNA insert. The positions of the two Pvu\\ alÃ-elesare
indicated in kilobases. The additional band above the tumor PI alÃ-eleband
probably represents bacterial plasmid contamination of the tumor DNA and was
not observed in other tumor DNA preparations.
_alleleP2
(0,6kb)
Table 1 Distribution and frequencies of ER gene Pvull alÃ-eles
Tissue
typePeripheral
blood leu
S3)Breast
kocytes (n =
(%)PI
ofPl"0.45
P2/P219
/PI
P1/P2
53
28
(0.36-0.55)'
Fig. 2. ER RFLP in human breast cancer cell lines. A Southern blot of
genomic DNA from human breast cancer cell lines digested with A'ull. Approx
imately 10 )jg of digested DNA samples were electrophoresed on a 1% agarose
gel and transferred to nitrocellulose. The blot was hybridized with "P-labeled ER
cDNA insert. The positions of the two Pvull alÃ-elesare indicated in kilobases.
Apparent discrepancies as to the size of MDA-231 bands are the result of sample
overloading.
tumors (n =
0.46(0.41-0.51)
20
53
27Frequency
188)Genotype
" Calculations for alÃ-elefrequencies based on the Hardy-Weinberg equation.
Numbers in parentheses. 95% confidence intervals.
146
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ESTROGEN RECEPTOR GENE RFLP IN BREAST CANCER
AlÃ-eleFrequencies in Human Breast Tumors as a Function of
Receptor Status. When the distribution and frequencies of the
ER /VÃ-/IIalÃ-eleswere examined as a function of tumor ER
status as determined by ligand-binding assays, significant dif
ferences were observed (Table 2). The PI alÃ-elefrequency was
greater in ER-negative tumors (55%) than in ER-positive tu
mors (42%). x2 analysis showed this to be a significant differ
ence (P = 0.050).
Some tumor specimens appearing heterozygous for the Pvull
alÃ-elesdisplayed unequal alÃ-eleintensities. Dissimilar alÃ-ele
intensities may result from heterogeneous breast tumor speci
mens, containing both tumor and normal tissue, in which
normal tissues are heterozygous for the two alÃ-elesand the
tumor tissue has lost heterozygosity for the ER gene, resulting
in Pvull alÃ-elehomozygosity. Thus, accurate analysis of appar
ently heterozygous specimens is difficult. We have therefore
separately analyzed only those specimens which were clearly
homozygous (Table 3). Again, ER-positive tumors are more
frequently homozygous for the P2 alÃ-ele(67%) than for the PI
alÃ-ele(33%), and the converse is true for ER-negative tumors.
X2analysis showed this difference to be significant (P = 0.014).
_ alÃ-elePI
(1.6kb)
1.0kb
band
_alleleP2
These results confirm our observations in breast cancer cell
(0.6 kb)
lines and suggest that the presence of the PI alÃ-eleis associated
with a more frequent failure of the tumor to express ER, but
Fig. 3. Localization of the ER /'â„¢ll alÃ-eles.Genomic DNA from a human
not an absolute failure.
breasl tumor specimen heterozygous for the ER A'ull alÃ-eleswas digested with
Localization of the Pvull RFLP in the ER Gene. Region/'nilI. electrophoresed in duplicate on a 1% agarose gel. and transferred to
nitrocellulose. The blots were hybridized with either a "P-labeled ER probe
specific fragments of the ER cDNA were used for a preliminary
representing the S'-segment of the ER cDNA or a "P-labeled ER probe repre
localization of the Pvull RFLP within the ER gene. In Fig. 3, senting the hormone-binding segment of the ER cDNA. The positions of the two
a Pvull DNA digest from a breast tumor heterozygous for the Pvull alÃ-elesare indicated in kilobases. Only the segment of the blot below 2.0
kilobases is presented.
Pvull alÃ-eleswas hybridized with either a segment of the ER
cDNA coding for the DNA and hormone-binding regions of
the receptor, or ER sequences 5' to this region. Both alÃ-eles the predictions of the Hardy-Weinberg equation. However, our
within the Pvull RFLP hybridized only to the DNA and hor
preliminary studies in breast cancer cell lines suggested an
mone-binding region probe, so that the Pvull RFLP appears to association between the absence of the P2 alÃ-eleand the failure
be located within this region. More detailed analysis will be of breast cancer cells to express ER. The subsequent examina
required to definitively localize the Pvull RFLP.
tion of alÃ-elefrequencies in primary human breast tumor biop
sies as a function of ER status did reveal statistically significant
differences between ER-positive and ER-negative tumors,
DISCUSSION
showing that the P2 alÃ-eleis more frequently associated with
We have confirmed the presence of the reported single, twoER expression, while the PI alÃ-eleis more frequently associated
allele PvwII RFLP within the ER gene in human primary breast
with the absence of ER expression. The molecular basis of this
tumors. Early observations suggested that this Pvull RFLP was phenomenon is unknown, but it is tempting to speculate
a neutral polymorphism, in that alÃ-elefrequencies for normal
whether the polymorphism affects the proper splicing of ER
human peripheral blood leukocytes and primary human breast
mRNA. We do know that, at the level measurable by Northern
tumors were the same and the observed distribution followed blot analysis, we have not detected structural polymorphisms
in ER mRNA. Sequence analysis of ER mRNA from breast
Table 2 Distribution and frequencies of ER gene Pvull alÃ-elesas a function of
cancer patients may provide insight into the relationship be
tumor estrogen receptor status
tween the observed Pvull RFLP and ER expression.
(%)"PI/PI1528P1/P25354P2/P23218Frequency
Tumor
Specific chromosomal aberrations have been detected in a
PI*0.42
of
statusER+(n=
ER
number of cancer types (18, 19). A frequent alteration detected
(0.36-0.48)'0.55
123)ERin these tumors has been the loss of constitutional heterozygos
(n = 65)Genotype
ity. For example, loss of heterozygosity on chromosome 22 has
(0.46-0.63)
" X2 analysis was performed to compare alÃ-eledistribution between ER+ and
been seen in meningiomas (20), and loss of chromosome 3p
ER-, giving P= 0.050.
markers has been reported in small-cell lung cancer (21). Re
* Calculations for alÃ-elefrequencies based on the Hardy-Weinberg equation.
cently,
loss of heterozygosity was observed at several loci on
' Numbers in parentheses, 95% confidence intervals.
chromosome 11 in primary human breast tumors (22). Loss of
heterozygosity has not yet been reported for chromosome 6,
Table 3 ER gene homozygote frequency and tumor estrogen receptor status
which carries the ER gene, and at first glance, the data presented
ER genotype (%)"
Tumor
in Table 1 do not show evidence of loss of heterozygosity for
ER status
PI/PI
P2/P2
the ER gene. It must be remembered, however, that breast
ER+ (n = 58)
67
33
tumor biopsies are heterogeneous, containing normal breast
epithelium, stromal cells, and infiltrating lymphocytes, as well
ER- (n = 30)
60
40
°x2 analysis was performed to compare alÃ-elefrequencies between ER+ and
as tumor cells (23). The breast tumor specimens used in this
ER-. giving P= 0.014.
study were examined to confirm the presence of tumor cells in
147
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ESTROGEN RECEPTOR GENE RFLP IN BREAST CANCER
2. McGuire, W. L. Hormone receptors: their role in predicting prognosis and
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breast cancer. Breast Cancer Res. Treat., 10: 5-9, 1987.
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Flavell, R. A., Goossens, M., Sergeant, G. R., and Montgomery, D. The
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7. Phillips, J. A., Hjelle, B. L., Seeburg, P. H., Plotnick, L. P., Migeon, C. J.,
and Zachmann, M. Heterogeneity in the molecular basis of familial growth
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netics, 33: 52 A, 1981.
8. Rotwein, P., Chyn, R., Chirgwin, J., Cordell, B., Goodman, H. M., and
Permuti, M. A. Polymorphism in the 5 '-Hanking region of the human insulin
each sample, and specimens in which no tumor cells were
evident were excluded from the study. Nevertheless, the per
centage of tumor cells ranged from 6% to 75% (data not shown).
Thus, samples from some patients whose normal cells are
heterozygous for this RFLP may well be scored as heterozygous
even if the tumor cells have lost this heterozygosity. Indeed,
densitometric scanning of "heterozygous" tumor sample autoradiographs in our study (data not shown) revealed that the
relative intensity of the two alÃ-elessometimes appeared to be
unequal, consistent with such an interpretation. Experiments
are therefore currently under way to examine the ER gene in
both normal tissue and breast tumor biopsies from the same
individuals, to detect any differences in alÃ-elefrequencies or
possible loss of heterozygosity in breast tumors. It is interesting
to note, however, that the breast tumor cell lines examined were
all homozygous for the Pvull ER RFLP. The tumor cell lines
obviously are not contaminated with normal cells as are the
majority of breast tumor specimens examined. Therefore,
paired normal/tumor
specimens will be informative as to
whether there is a loss of ER gene heterozygosity in human
breast tumors.
The precise location of the Pvull RFLP has not been identi
fied at present. However, hybridization to a segment of the ER
cDNA coding for DNA and the hormone-binding regions of
the receptor suggests that the polymorphism lies within these
domains. The ER gene does not show polymorphism with a
number of other enzymes including BamHl, EcoRl, Hindlll,
Sstl, and Pstl (data not shown). This may well indicate that the
alteration within the gene is rather small, possibly a single
nucleotide substitution, rather than a large chromosomal re
arrangement or deletion. Since the complete genomic sequence
of the ER gene is not known and a detailed restriction map is
not available, more detailed analysis will be required to defini
tively localize the PvwII RFLP.
RFLP studies have proven useful in detecting the loss of
heterozygosity in hereditary cancers (20, 21) and in detecting
defective genes in some genetic diseases (24). Using this type
of analysis, we have here observed that the presence of a
particular ER Pvull alÃ-elein breast cancer cell lines and primary
human breast cancers is associated with more frequent failure
of the tumor to express ER. Additional studies may help to
determine why only certain breast tumors express the ER gene
and are hormone responsive.
gene and its possible relation to type 2 diabetes. Science (Wash. DC), 213:
1117-1120, 1981.
9. Law, M. L., Kao, F. T., Wei, Q., Hartz, J. A., Greene, G. L., ZaruckiSchultz, T., Conneely, O. M., Jones, C., Puck, T. T., O'Malley, B. W., and
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
ACKNOWLEDGMENTS
The authors thank Saundra Davis, Alda Saenz, Julia Delgado, and
Margaret Benedix for their excellent technical assistance.
23.
24.
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148
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Estrogen Receptor Expression in Human Breast Cancer
Associated with an Estrogen Receptor Gene Restriction
Fragment Length Polymorphism
Steven M. Hill, Suzanne A. W. Fuqua, Gary C. Chamness, et al.
Cancer Res 1989;49:145-148.
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