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Coexpression of Estrogen Receptor and ß

Poor Prognostic Factors in Human Breast Cancer?¹

Departments of Medicine [V. S., A. T. P., D. S. W., S. L. A.] and Surgery [M. J. K., P. J. C., J. N. F.], University of Hull, Hull HU6 7RX, United Kingdom

	ABSTRACT

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The cloning of a second estrogen receptor (ER), ERß, has prompted a reevaluation of the role of ERs in breast cancer. The aim of this study was to determine the expression of both ER isoforms in normal (n = 23) and malignant (n = 60) human breast tissue by reverse transcription-PCR and correlate this information with known prognostic factors including tumor grade and node status. In normal breast tissue, expression of ERß predominated, with 22% of samples exclusively expressing ERß; this was not observed in any of the breast tumor samples investigated. Most breast tumors expressed ER, either alone or in combination with ERß. Interestingly, those tumors that coexpressed ER and ERß were node positive (P = 0.02; Fisher’s exact test) and tended to be of higher grade. Because antiestrogens are agonists when signaling through the AP1 element, overexpression of ERß in tumors expressing both ER subtypes may explain the failure of antiestrogen therapy in some breast cancer patients. Thus, ERß may be a useful prognostic factor in patients with breast cancer.

	Introduction

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Since the cloning of the ER³ in 1986 (1 , 2) , it was believed that only a single receptor (now termed ER) was responsible for mediating the effects of estrogens on target tissues. Recently, a second ER, referred to as ERß, has been identified in the rat, human, and mouse (3, 4, 5) . ERß is highly homologous to ER at the DNA (96%) and ligand binding (58%) domains, whereas the A/B domain, hinge region, and F region are not well conserved (4) . ERß binds estrogens with a similar affinity to ER and activates the expression of reporter genes containing estrogen response elements in an estrogen-dependent manner (3 , 6) . Curiously, although the discovery of ERß has prompted a reevaluation of the molecular basis for estrogen action, there have only been a few studies addressing a possible role for ERß in human breast cancer. This is surprising because alterations in ER signal transduction are believed to contribute to breast cancer progression and the development of a hormone-independent and more aggressive phenotype. Of the studies reported to date, ERß is expressed in human breast tumors (7 , 8) and in chemically transformed human breast epithelial cells (9) . ERß splice variants have also been described in some breast tumors (10 , 11) . Furthermore, the ratios of ER:ERß gene expression appear to alter during carcinogenesis, suggesting that ER- and ERß-specific pathways may have definitive roles in this process (12) . In addition to mediating gene transcription via the classic estrogen response element, ER subtypes can signal from an AP1 enhancer element that requires ligand and the AP1 transcription factors Fos and Jun for transcriptional activation (13) . Recent data show that ER and ERß signal in opposite ways from an AP1 element. When bound to ER, 17ß-estradiol activates transcription, whereas with ERß, transcription is inhibited (14) . However, when antiestrogens bind to ERß, they are potent transcriptional activators at an AP1 site, acting as agonists rather than antagonists (14) .

Clinically, around two-thirds of all breast cancer patients with ER⁺ tumors initially benefit from antiestrogen therapy. However, some of these patients eventually relapse. The mechanisms associated with the acquisition of an antiestrogen-resistant phenotype are incompletely understood. Because estrogens play an important physiological role in the normal breast and are involved in the pathophysiology of breast cancer, the aim of this study was to determine the expression of both ER isoforms in normal and malignant human breast tissue using RT-PCR and to correlate our observations with available clinical information. The use of RT-PCR rather than immunohistochemistry has been necessitated by the lack of any publication to date that has outlined the use of specific antibodies raised against human ERß.

	Materials and Methods

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Tumor Samples.
Sixty breast tumors were obtained from patients who presented sequentially for surgical removal of a clinically confirmed malignant breast lesion. All patients were staged by standard UICC criteria. Routine histological staging was performed by a consultant pathologist. Details are presented in Table 1 . As a source of normal breast tissue, samples were obtained from 23 patients undergoing reduction mammoplasty who had no previous history of breast disease (mean age, 35 years; range, 18–42 years). Ethical approval was obtained, and all patients gave informed consent.

PCR Amplification.
Primers were obtained from Life Technologies and designed from published gene sequences. Primers used to amplify ER were 5'-TGCCAAGGAGACTCGCTA-3' (nucleotides 894–912) and 5'-TCAACATTCTCCCTCCTC-3' (nucleotides 1139–1157), giving an amplified product of 263 bp. For ERß, primer sequences were 5'-TGTTACGAAGTGGGAATGTGA-3' (nucleotides 484–505) and 5'-TCTTGTTCTGGACAG-GGATG-3' (nucleotides 936–956), giving an amplified product of 472 bp. To check cDNA integrity, fragments of glyceraldehyde phosphate-3-dehydrogenase, a standard housekeeping gene, were amplified in parallel; this was consistently positive (data not shown). The PCR reaction contained 2 units of BIOTAQ; 10x PCR buffer containing 1.5 mM MgCl₂ (both from Bioline, London, United Kingdom); 0.5 µg of each oligonucleotide primer; 200 µM each of dATP, dCTP, dGTP, and dTTP; 1 µl of nascent cDNA; and sterile distilled water to bring the volume to 50 µl. As a positive control for ER, cDNA from the ER⁺ MCF-7 human breast cancer cell line was used; for ERß, human testis cDNA was used. Negative controls included the substitution of RNA or cDNA with distilled water or the substitution of cDNA with an irrelevant cDNA (synthesized from human tibialis anterior muscle). These were consistently negative. All transcripts were analyzed in parallel on at least two separate occasions in a thermal cycler (Hybaid OmniGene, Teddington, United Kingdom) with the following cycle: (a) a denaturation step of 94°C for 2 min; (b) 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and (c) a final primer extension step of 72°C for 5 min. PCR products were analyzed by electrophoresis through a 1.2% agarose gel and visualized by ethidium bromide staining under UV illumination.

Restriction Enzyme Digests.
Restriction digests were performed on representative PCR products to confirm their identity. Amplified product (5 µl ) was digested overnight at 37°C with either AvaII (ER) or HinfI (ERß), which yielded discrete fragments of 197 and 66 bp (ER) or 321 and 151 bp (ERß). Digested products were then electrophoresed through a 2% agarose gel and visualized as described above.

Statistical Analysis.
Statistical analysis was performed using the Arcus software package for Windows (Research Solutions, Cambridge, United Kingdom). Fisher’s exact test was used to test the difference between the groups. Results were considered to be significant at P 0.05.

	Results

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Expression of ER and ERß in Normal and Malignant Human Breast Tissues.
Sixty breast tumor samples and 23 samples of normal breast tissue were analyzed and compared for expression of both ER subtypes by RT-PCR. A significant difference in the expression of one or both receptor subtypes was observed between these tissue groups. As outlined in Table 2 , compared with normal breast tissue, a significantly higher proportion of breast tumors coexpressed ER and ERß (50%; P = 0.0002). Although 27% of breast tumors exclusively expressed ER, this was not statistically significant when compared with normal breast tissue, in which 13% of samples exclusively expressed this subtype. Expression of ERß alone was observed in a proportion of normal breast samples (22%; P = 0.0011 versus breast tumors). Expression of this subtype was not observed in any of the breast tumors. These either coexpressed ER and ERß or expressed only ER. A total of 52% of normal breast samples failed to express either ER subtype, compared with only 23% of breast tumors (P = 0.017 versus breast tumors). A representative gel showing transcripts for ER and ERß and their restriction-mapped products is illustrated in Fig. 1 .

View this table: [in this window] [in a new window]   Table 2 Expression of ER and ERß mRNA in normal breast and breast tumors

View larger version (137K): [in this window] [in a new window]   Fig. 1. Representative agarose gel showing RT-PCR products for ER (A) and ERß (B). Lane 1, 100-bp ladder; Lane 2, positive control; Lane 3, restriction-digested PCR products; Lanes 4–7, breast cancer samples; Lane 8, negative control. A, single and double arrowheads indicate restriction-digested products of 197 and 66 bp, respectively. B, single and double arrowheads indicate restriction-digested products of 321 and 151 bp, respectively. This confirms the identity of the PCR products.

	Discussion

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Our data show that compared with normal breast tissue, a significantly higher proportion of breast tumor samples expressed both ER subtypes. Furthermore, 50% of all breast tumors analyzed coexpressed ER and ERß. The only related study published to date has shown that expression of ERß does not correlate with that of ER (7) . The ability of both ER subtypes to form DNA-binding homo- and heterodimers (6 , 16 , 17) points to three possible pathways of estrogen signaling. In those tissues that exclusively express either ER or ERß, signaling would be via the specific receptor, whereas in those tissues expressing both subtypes, signaling would be mediated by ER/ERß heterodimers. Thus, coexpression of ER subtypes within the same breast tumor offers an intriguing possibility whereby ER and ERß proteins may interact, leading to differential responses to estrogens/antiestrogen. It has been shown that ER subtypes can signal not only from the classic estrogen response element but also from an AP1 enhancer element (13) . The downstream effects of AP1 signaling are both receptor and ligand specific. With ER, 17ß-estradiol activates transcription, whereas with ERß, the reverse is true (14) . However, when complexed with ERß, antiestrogens including tamoxifen, raloxifene, and the pure antiestrogen ZM 164384 are potent transcriptional activators at an AP1 site, acting paradoxically as agonists rather than antagonists (14) . Clinically, antiestrogens, particularly tamoxifen, are currently the first-line therapy for treatment of hormone-dependent breast cancer. Around 70% of all breast cancer patients with ER⁺ lesions initially benefit from antiestrogen therapy. However, most of these patients eventually relapse. Mechanisms associated with the acquisition of an antiestrogen-resistant phenotype are poorly understood. Our results indicate that coexpression of both ER subtypes is significantly higher in breast tumors. Furthermore, those tumors with the ER⁺/ERß⁺ phenotype were node positive (P < 0.02), and although only grade II tumors showed statistical significance, there was a trend toward an association with more poorly differentiated tumors. Both of these features point to a poorer prognosis. Speculatively, the overexpression/activation of ERß in tumors that express both ER subtypes and the subsequent interaction with ligand-bound AP1 elements may explain the failure of antiestrogen therapy in some breast cancer patients.

A distinct pattern of expression of ER subtypes was observed between normal breast tissue and breast tumors. Both tissue sets expressed ER. Although it appeared that a greater number of breast cancer samples expressed this subtype, this did not reach significance. The number of normal breast samples expressing ER (13%) is slightly higher than the 7% reported by immunohistochemistry (18) but may be a reflection of the higher sensitivity of the RT-PCR over immunohistochemistry. Exclusive expression of ERß was not observed in any of the breast tumor samples. Instead, this subtype was significantly associated with normal breast tissue and was found in 22% of these samples. This observation parallels a recent study by Leygue et al. (12) in which the relative expression of ER:ERß was determined using multiplex RT-PCR. These authors showed that ERß expression was reduced in tumor compared to adjacent normal breast tissue, and although this did not reach statistical significance, there was a suggestion of a trend. Although our RT-PCR conditions were qualitative rather than quantitative, we too noted that in those samples that coexpressed both receptor subtypes, expression of the ER product was always greater than that of ERß (see Fig. 1 ). High levels of ERß have recently been reported in the normal human breast epithelial cell line HBL100 and in chemically transformed human breast epithelial cells (9) . Although we did not observe expression of just the ERß subtype in breast tumors, this has been reported in both ER⁺ and ER^- breast cancer cell lines (7) . However, it is possible that the acquisition of an ERß⁺ phenotype may be an artifact of in vitro culture, which is not observed in primary tumors.

The age distribution of our study populations should also be considered. All normal breast samples were obtained from premenopausal women undergoing reduction mammoplasty for cosmetic purposes (mean age, 35 years; range, 18–42 years), whereas the majority of breast tumors were from postmenopausal patients. Although only six tumors were obtained from premenopausal subjects (mean age, 44 years; range, 32–51 years), four of these six subjects had ER⁺/ERß⁺ phenotype (with the remainder expressing ER). This somewhat contradicts previously reported findings, which show that in comparison with breast tumors from postmenopausal women, generally fewer premenopausal patients have ER⁺ lesions (19) . However, greater numbers of samples must be studied before any significance can be attributed to this observation.

In accord with all studies that evaluate gene expression, the detection of a gene transcript gives no indication of whether the transcript will go on to be transcribed and translated into an active peptide. Nevertheless, a positive relationship between ER mRNA and the ligand binding assay has been observed in this laboratory,⁴ whereas others have shown associations between ER mRNA and immunohistochemistry (20) . Trends toward associations between ERß status by ligand binding assay and RT-PCR have also been reported (12) . However, there have been no reports confirming that the expression of ERß mRNA parallels that of protein, which may reflect the difficulties in raising specific ERß antibodies to date.

In conclusion, overexpression of ERß in tumors expressing both ER subtypes and the subsequent interaction of ligand-bound receptors with AP1 elements may help explain the failure of antiestrogen therapy in some breast cancer patients. Current diagnostic procedures for breast cancer use immunological techniques designed to detect only ER. With the imminent development of ERß-specific antibodies, our results suggest that it may be relevant to determine the expression of both ER subtypes. This may be helpful in predicting the response of breast tumors to endocrine therapy. This information may permit the subsequent development of selective ER modulators to target tumors with a particular ER phenotype.

	ACKNOWLEDGMENTS

 
We thank N. B. Hart and P. O’Hare of Royal Hull Hospitals for providing samples of normal breast tissue.

	FOOTNOTES

 
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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Yorkshire Cancer Research (Harrogate, United Kingdom) and Royal Hull Hospitals NHS Trust.

² To whom requests for reprints should be addressed, at Department of Medicine, Medical Research Laboratory, University of Hull, Hull HU6 7RX, United Kingdom. Phone: 44-1482-466030; Fax: 44-1484-466033; E-mail: v.speirs{at}medschool.hull.ac.uk

³ The abbreviations used are: ER, estrogen receptor; RT-PCR, reverse transcription-PCR; UICC, Union International Contre le Cancer.

⁴ V. Speirs, unpublished observation.

Received 10/19/98. Accepted 12/14/98.

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				T. Otsuki, O. Yamada, J. Kurebayashi, T. Moriya, H. Sakaguchi, H. Kunisue, K. Yata, M. Uno, Y. Yawata, and A. Ueki Estrogen Receptors in Human Myeloma Cells Cancer Res., March 1, 2000; 60(5): 1434 - 1441. [Abstract] [Full Text]

				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]

				T. A. H. Jarvinen, M. Pelto-Huikko, K. Holli, and J. Isola Estrogen Receptor {beta} Is Coexpressed with ER{alpha} and PR and Associated with Nodal Status, Grade, and Proliferation Rate in Breast Cancer Am. J. Pathol., January 1, 2000; 156(1): 29 - 35. [Abstract] [Full Text] [PDF]

				V. Speirs, C. Malone, D. S. Walton, M. J. Kerin, and S. L. Atkin Increased Expression of Estrogen Receptor {{beta}} mRNA in Tamoxifen-resistant Breast Cancer Patients Cancer Res., November 1, 1999; 59(21): 5421 - 5424. [Abstract] [Full Text] [PDF]