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Original Studies |
and ß in Human Breast Carcinoma1
Departments of Pathology (H.S., T.S., H.N.) and Obstetrics and Gynecology (Y.M., T.F.), Tohoku University School of Medicine; Department of Pathology (M.E.), Institute of Development, Aging and Cancer, Tohoku University 980-8575; and Department of Surgery (M.K.), Tohoku Kousai Hospital, Sendai, Japan 980-0803
Address all correspondence and requests for reprints to: Hironobu Sasano, M.D., Department of Pathology, Tohoku University School of Medicine, 2–1 Seiryou-machi, Aoba-ku, Sendai, Japan 980-0872. E-mail: hsasano{at}patholo2.med.tohoku.ac.jp
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Abstract |
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, to study
its localization and its possible biological significance in human
breast cancer. ER and ERß hybridization signals were both
detected, predominantly in carcinoma cells and in some stromal cells,
in 18 of 25 (72%) and 11 of 25 (44%) cases, respectively. The cases
in which more than 25% of carcinoma cells demonstrated mRNA
hybridization signals were 13 of 25 (52%) and 2 of 25 (8%) cases for
ER and ERß, respectively. Among the cases expressing ERß, 10 of
11 (91%) also expressed ER mRNA; and in these 10 cases,
coexpressing both ER and ß, the number of carcinoma cells
expressing ER was greater than that expressing ERß in 9 cases.
Eight cases demonstrated only ER mRNA hybridization signals in
carcinoma cells. These results indicate that ERß is coexpressed with
ER in most ERß-positive breast carcinoma cells, which suggests
that the expression of ERß depends on the presence of ER in the
great majority of human breast cancer. In addition, the number of
carcinoma cases and/or the ratio of carcinoma cells expressing ER
was much greater than those expressing ERß. The relative ratio of
ER and ERß expression in carcinoma cells may be related to various
estrogen-dependent biological features of human breast cancer. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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had been considered as the only receptor able to bind
estrogen with high affinity. ERß protein is smaller than ER
protein but has a similar high affinity for estradiol, as does the
receptor (6, 7, 8, 9). Both receptors demonstrate high conservation of amino
acid sequence in regions of the hormone binding domain known to be
important in contacting ligands (6, 7, 8, 9). However, tissue distribution
and relative expression levels of ERß have been demonstrated to be
different from those of ER (10, 11, 12), which suggests possible
different biological roles of ERß in mammalian estrogen-dependent
tissues.
Estrogen signal transduction plays very important roles in both normal
and neoplastic mammary tissue. Both Dotzlaw et al. (13) and
Vladusic et al. (14) very recently demonstrated the
expression of ERß messenger RNA (mRNA) in human breast cancer tissues
and breast epithelial cell lines, using RT-PCR. However, human breast
carcinoma is composed of stromal and carcinoma cells and is known to be
associated with diverse morphological features. Therefore, a more
thorough study of the tissue distribution of ERß in human breast
cancer, especially that of its localization, is essential to the study
of its function. Therefore, we performed mRNA in situ
hybridization of both ER and ERß in clinical specimens of human
breast carcinoma to examine the localization of these receptors,
especially whether they are coexpressed in the same cases and, if
coexpressed, whether they are expressed in the same cell types.
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Materials and Methods |
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Tissues from twenty-five cases of human breast carcinoma (specimens from mastectomies performed at the Department of Surgery, Tohoku Kousai Hospital) were obtained . The patients were all Japanese, and all carcinomas were invasive ductal carcinoma. Portions of carcinoma tissues were trimmed into tissue slices, measuring 0.8–1 cm in their greatest dimension, and were immediately fixed in 4% paraformaldehyde with 0.5% glutaldehyde at pH 7.4 for 18–36 h at 4 C. They were subsequently embedded in paraffin wax.
Oligonucleotide probes
The sequence of 30-base antisense oligodeoxynucleotide probes
for ERs employed for mRNA in situ hybridization were as
follows: ER: 5'-CAG CTC GTT CCC TTG GAT CTG ATG CAG TAG-3'; and
ERß: 5'-TGT TGG CCA CAA CAC ATT TGG GCT TCT GGT-3', which correspond
to nucleotides 332–361 and 76–105 of ER and ERß genes,
respectively. These two sequences had the same guanine, cytosine
(GC) content and were selected from the N-terminal A/B domain.
Corresponding sense oligonucleotide probes were used as negative
controls. The probes were synthesized with 3'-biotinylated tail
(Brigati tail;
5'-probe-biotin-biotin-biotin-TAG-TAG-biotin-biotin-biotin-3")
(15, 16, 17).
Both of these oligonucleotide probes demonstrated no homology with each other and other known human genes, including glucocorticoid, mineralocorticoid, or progesterone receptors, by a computer-assisted search.
mRNA in situ hybridization
In situ hybridization of ER mRNA was performed with the MicroProbe staining system (Fisher Scientific International, Inc., Pittsburgh, PA) using manual capillary actions, with modification of methods previously published (15, 16, 17). Tissue sections (3 µm, applied to Probe On Plus slides, Fisher Scientific International, Inc.) were rapidly dewaxed, cleared with alcohol, dehydrated with Tris-based buffer (pH 7.4) (Universal Buffer, Research Genetics, Inc., Huntsville, AL), and digested with pepsin (2.5 mg/mL, Research Genetics, Inc.) for 3 min at 105 C. Probe was applied in formamide-free diluent, and the slides were heated to 105 C for 3 min, cooled for approximately 1 min at room temperature, and allowed to hybridize at 45 C for 60 min. The sections were then washed twice with 2x standard saline citrate at 45 C (3 min per wash) and detected with alkaline phosphatase-conjugated streptavidin (Research Genetics, Inc.). After washing three times in AP (alkaline phosphatase) chromogen buffer (pH9.5; Research Genetics, Inc.) at room temperature, hybridization products were visualized with fast red for 10 min at room temperature in all the tissue sections. The slides were counterstained with hematoxylin, air dried, and cover slipped for microscopic examination.
Evaluation of mRNA hybridization signals
We determined the labeling index (i.e. the ratio of
cells with relatively strong mRNA hybridization signals) in each
carcinoma, according to Watanabe et al. (18), with some
modifications (19, 20, 21), as follows. Labeling index was determined by
thorough histological examination of the reacted tissue sections, not
by actual counting of the cells, because of cytoplasmic localization of
histochemical products. After completely reviewing the sections of each
carcinoma, two of the authors (H. Sasano and T. Suzuki) independently
divided the carcinomas into the following five groups; no hybridization
signals (-); 0–5% (±); 5–25% (+); 25–50% (++); and more than
50% (+++) of cells positive for ER and ERß. Interobserver
differences were 3 of 25 (12). These disconcordant cases between these
two observers were simultaneously reevaluated by the same two authors
above, using double-headed light microscopy (BH-2, Olympus Corp., Co. Ltd., Tokyo, Japan).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß mRNA hybridization signals, appearing red as a
result of fast red salt reaction, were detected predominantly in the
cytoplasm of carcinoma cells, and in some stromal cells, including
fibroblasts [Figs. 1 (A, B, and C) and
2A]. In negative controls using the
sense oligonucleotide probes, no significant accumulation of mRNA was
detected in carcinoma cells (Fig. 1D).
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mRNA hybridization signals were detected in 18 of 25 cases
(72%), whereas ERß signals were detected in 11 of 25 cases (44%).
The cases in which more than 25% of carcinoma cells demonstrated mRNA
hybridization signals were 13 of 25 for ER (52%) and 2 of 25 (8%)
for ERß.
Among 11 ERß-positive cases, 10 cases also demonstrated ER
hybridization signals (Fig. 1
, A and B). In these 10 cases coexpressing
both ER and ß, the great majority of ERß-positive carcinoma
cells also expressed ER mRNA but not vice versa, and the
number of carcinoma cells expressing ER mRNA was greater than that
expressing ERß in all but one case (case no. 16 in Table 1). Eight
cases demonstrated only ER mRNA hybridization signals, but not ERß
mRNA hybridization signals, in carcinoma cells (Fig. 2, A and B). Both
ER and ERß mRNA hybridization signals were detected in adjacent
nonneoplastic mammary ductal cells and some stromal cells, which were
available for examination in 7 cases (Fig. 3). However, ER-positive cells were
much more widely distributed than ERß-positive cells in these
nonneoplastic mammary ducts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we employed oligonucleotides derived from the sequence
of the A/B domain of the receptors, because A/B domains of ER and
ERß are markedly different, compared with other regions of the genes
(13, 14). It is well known that there are several splice variant forms
with deleted exons of ER mRNA in DNA and hormone-binding domains in
breast cancer (22, 23, 24). Several ERß mRNA variants also have been
reported (25, 26). The oligonucleotide probes employed in our study can
detect all of these mRNA species. Therefore, mRNA hybridization signals
detected in this study are considered to include both genuine and
variant forms of the ER and ERß mRNA. We also detected ERß mRNA
hybridization signals in epithelial cells of nonneoplastic breast
tissues adjacent to carcinoma. Very recently, Vladusic et
al. reported that mRNA coding for a variant of ERß was
coexpressed with wild-type ERß in breast carcinoma but was not
detected in nonneoplastic human breast tissue (14). Therefore, ERß
mRNA hybridization signals in nonneoplastic breast may represent
wild-type ERß mRNA, but it awaits further investigations for
clarification.
We then demonstrated both ER and ERß mRNA hybridization signals in
parenchymal or epithelial cells and some stromal cells of human breast
carcinoma, using biotinylated oligonucleotide probes and manual
capillary actions. The presence of ERß mRNA hybridization signals in
human breast carcinoma cells is consistent with the recent study of
Dotzlaw et al. (26) (who demonstrated ERß mRNA in both
human breast epithelial cell lines, growing in culture, and some
clinical specimens of breast cancer, using RT-PCR study) and that of
Enmark et al. (10) (who showed ERß mRNA hybridization
signals in some breast carcinoma cases). Dotzlaw et al. (13)
did not detect any correlation between ER and ERß expression; but
in our present study, the great majority of breast carcinoma cells with
ERß mRNA hybridization signals did express ER but not vice
versa. This finding is consistent with previous reports of tissue
distribution of ER and ERß in classical estrogen-dependent
tissues, i.e. ERß is expressed in an over-lapping but
nonidentical tissue distribution to ER, and the biological functions
of ERß may be dependent on the presence of ER in certain types of
cells, especially in classical estrogen-dependent tissues (2, 3, 12).
Both the number of the carcinoma cases and the ratio of carcinoma cells
expressing ER was much greater than those expressing ERß in
invasive ductal carcinomas of the breast examined in this study. We
also detected ER and ERß mRNA hybridization signals in a small
number of stromal cells in both neoplastic and nonneoplastic breast
tissue. This finding may be consistent with the recent report of Cooke
et al. (27), who demonstrated that ER of both epithelial and
stromal cells, possibly through paracrine actions, are required for
estradiol-induced uterine epithelial cell proliferation. However,
further investigations are required for clarifying the possible
biological significance of ER and ERß in stromal cells of human
breast.
Brandenberger et al. very recently reported marked decrease
of ERß mRNA levels in 10 cases of serous cystadenocarcinoma of the
ovary, which is also estrogen-dependent carcinoma, as in breast
carcinomas, using RT-PCR (28). Biological roles or functions of ERß
have not been established in both normal and pathological human
tissues. However, both ER and ERß are considered to interact with
similar DNA response element(s), through the course of estrogen
actions, although their A/B domains and activation function-1 regions
are quite different, which suggests that their transcriptional
activation of different estrogen-responsive genes may be different (29, 30). The mechanism of estrogenic or antiestrogenic actions is well
known to be very complicated, but the analysis of ER and ERß
expression in clinical specimens of breast cancer can provide new
insights into endocrinological characteristics of individual carcinoma
cases. However, it awaits further investigations, including the
correlation between the level of ERß expression and clinical outcome
and/or response to antiestrogens, to clarify biological significance of
ERß in human breast carcinoma.
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Acknowledgments |
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Footnotes |
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Received April 29, 1998.
Revised October 20, 1998.
Accepted October 25, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Endocrinology. 138:863–870. (ER-) and ß
(ER-ß) mRNA in the midgestational human fetus. J Clin Endocrinol
Metab. 82:3509–3512. (ER) and estrogen receptor-ß (ERß) messenger
rebonucleic acid in the wild-type and ER-knockout mouse. Endocrinology. 138:4613–4621. (ER-) and ß (ER-ß) mRNAs in normal ovary, ovarian
serous cystadenocarcinoma and ovarian cancer cell lines:
Down-regulation of ERß in neoplastic tissues. J Clin Endocrinol
Metab. 83:1025–1028.This article has been cited by other articles:
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  X. Li, J. Huang, P. Yi, R. A. Bambara, R. Hilf, and M. Muyan Single-Chain Estrogen Receptors (ERs) Reveal that the ER{alpha}/{beta} Heterodimer Emulates Functions of the ER{alpha} Dimer in Genomic Estrogen Signaling Pathways Mol. Cell. Biol., September 1, 2004; 24(17): 7681 - 7694. [Abstract] [Full Text] [PDF]
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 V. Speirs, I. P. Adams, D. S. Walton, and S. L. Atkin Identification of Wild-Type and Exon 5 Deletion Variants of Estrogen Receptor {beta} in Normal Human Mammary Gland J. Clin. Endocrinol. Metab., April 1, 2000; 85(4): 1601 - 1605. [Abstract] [Full Text]
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 S. Saji, E. V. Jensen, S. Nilsson, T. Rylander, M. Warner, and J.-A. Gustafsson Estrogen receptors alpha and beta in the rodent mammary gland PNAS, January 4, 2000; 97(1): 337 - 342. [Abstract] [Full Text] [PDF]
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 S. Matsuzaki, T. Fukaya, T. Suzuki, T. Murakami, H. Sasano, and A. Yajima Oestrogen receptor {alpha} and ß mRNA expression in human endometrium throughout the menstrual cycle Mol. Hum. Reprod., June 1, 1999; 5(6): 559 - 564. [Abstract] [Full Text] [PDF]
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