Japanese Journal of Clinical Oncology Advance Access originally published online on October 11, 2007
Japanese Journal of Clinical Oncology 2007 37(11):820-828; doi:10.1093/jjco/hym114
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© 2007 Foundation for Promotion of Cancer Research
Expression of Estrogen Receptor β Wild-type and its Variant ERβcx/β2 is Correlated with Better Prognosis in Breast Cancer
1 Oncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya
2 Breast and Endocrine Surgery, Kumamoto University, Kumamoto, Japan
For all reprints and correspondence: Hiroko Yamashita, Oncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya 467-8601, Japan. E-mail: hirokoy{at}med.nagoya-cu.ac.jp
Received May 7, 2007; accepted July 23, 2007
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Abstract |
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 TOP Abstract INTRODUCTIONPatients and methodsRESULTSDISCUSSIONReferences |
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Background: The clinical value of estrogen receptor (ER) β and its variants has been investigated. However, reported results have frequently been discordant.
Methods: We investigated mRNA and protein expression of ERβ wild type (ERβ1) and its variant, ERβcx/β2, by quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry in 150 breast cancer tissues, and analyzed association between their expression and clinicopathological factors and prognosis.
Results: ERβ1 mRNA expression was significantly correlated with progesterone receptor expression and low histological grade. ERβ1 protein expression was significantly correlated with small tumor size, negative lymph node status and low histological grade. ERβcx/β2 protein expression was significantly correlated with ER
expression and low histological grade. Patients with high expression of ERβ1 or ERβcx/β2 had a significantly better disease-free and overall survival than those with low expression. In multivariate analysis, ERβcx/β2 mRNA expression was identified as a prognostic factor in disease-free and overall survival.
Conclusion: Our results indicate that ERβcx/β2 mRNA expression is an independent prognostic factor in breast cancer. ERβ expression status, both wild-type and the variant cx/β2, might represent significant predictors of breast cancer prognosis.
Key Words: breast cancer • estrogen receptor β • prognosis
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPatients and methodsRESULTSDISCUSSIONReferences |
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Estrogens are important for growth and development of normal human mammary tissues. However, they are also thought to promote the growth of breast tumors through their mitogenic effects on breast cancer cells. This concept led to the development of selective estrogen receptor (ER) modulators, such as tamoxifen and toremifene, as endocrine therapy for breast cancer. These modulators bind to the estrogen receptor alpha (ER
), a member of the nuclear receptor superfamily, and thereby inhibit estrogenic action. For many years only one gene for ER had been recognized, but in 1996 a second ER subtype, estrogen receptor beta (ERβ), was identified in rats (1), mice (2) and humans (3). ERβ shows strong homology with ER in the DNA-binding domain (96%) and in the ligand-binding domain (53%), but little elsewhere (4). In particular it binds specifically to 17β-estradiol with a high affinity (5). More recently, variants of ERβ have been identified with, for example, exon deletion (6,7), insertion (7–9), or C-terminal splicing (5,10). ERβcx/β2 is one such variant that has been characterized; it is C-terminal splicing, and has a poor binding affinity for 17β-estradiol (5,10). However, ERβcx/β2 forms heterodimers with ER, as well as wild-type ERβ (ERβ1), and mediates significant dominant negative activity against ER transactivation (5). This suggests that ERβ1 and its variants, such as ERβcx/β2, have different functions and behave in a manner different from ER in breast cancer.
Several groups have investigated the expression of ERβ and its variants using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique (6,7,11–19) and immunohistochemistry (IHC) or western blotting (12–14,16,18–21). However, the results have been rather inconsistent. In a previous study, we used IHC techniques to show that breast cancer patients with ERβ1-positive tumors tended to have a better prognosis than patients who were ERβ1-negative. Using the RT-PCR method, some groups reported that ERβ expression was increased in ER-negative tumors (7,15). More recently, expression of ERβ1 and its variants has been examined separately by several investigators. One group showed that ERβ1 mRNA expression was correlated with tumor grade, and that the variants ERβcx/β2 and ERβ5 were up-regulated during breast tumorigenesis and tumor progression (22). Using real-time RT-PCR, another group has reported that ERβ mRNA was down-regulated during breast carcinogenesis and that changes in the relative proportions of ERβ variants were implicated in this process (15). On the other hand, other investigators also showed that ERβ expression was not associated with ER (16–18).
In this study, we investigated the expression of ERβ1 and ERβcx/β2 mRNA using quantitative real-time RT-PCR and the respective protein expression using IHC in 150 breast cancer patients and examined its relationship with clinicopathological factors and prognosis.
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Patients and methods |
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Patients and Tumor Samples
Primary invasive breast carcinoma specimens were obtained by surgical excision from 150 female patients at Nagoya City University Hospital between 1993 and 2000. Informed consent was obtained from all patients before surgery. The study protocol was approved by the institutional review board and conformed with the guidelines of the 1975 Declaration of Helsinki. The samples in this study were chosen from the continuous series of invasive carcinoma tissues. The median age of the patients was 53 years (range 34–88 years). The patients' tumors were classified with the International Union Against Cancer (UICC) staging system as follows: 39 cases were classified as stage I, 93 cases as stage II, and 16 cases as stage III. Patients were graded histopathologically according to the modified Bloom and Richardson method proposed by Elston and Ellis (23). As postoperative adjuvant treatment, tamoxifen was given to patients with ER- and/or progesterone receptor (PgR)-positive tumors. One of the following three chemotherapy regimens was administered, depending on tumor stage: oral 5-fluorouracil (5-FU 300 mg/m2), CMF (cyclophosphamide 100 mg p.o., days 1–14; methotrexate 40 mg/m2 i.v., days 1 and 8; 5-FU 500 mg/m2 i.v., days 1 and 8), or CEF (cyclophosphamide 500 mg/m2 i.v., epirubicin 60 mg/m2 i.v., 5-FU 500 mg/m2 i.v., every 3 weeks). Since 1995, postoperative treatment has been done with reference to the recommendation of St Gullen. After surgery, 42 patients (28%) received no additional therapy. Of the remaining 108 patients, 55 (37%) received systemic therapy consisting of endocrine therapy alone, 23 (15%) received chemotherapy alone and 30 (20%) received combined endocrine therapy and chemotherapy. Patients were observed for disease recurrence and death at least once every 6 months for 5 years after surgery and yearly thereafter. The median follow-up period was 58 months (range, 22–90 months). Samples were snap-frozen in liquid nitrogen and stored at –80°C until RNA extraction.
In 19 of the 150 cases, there was a recurrence, including lymph node metastasis and distant metastasis, and these patients received endocrine therapy, such as tamoxifen or a luteinizing hormone-releasing hormone (LHRH) analog with tamoxifen. All these patients had ER and/or PgR positive tumors. Recurrence of their disease occurred either after adjuvant tamoxifen had been stopped, in which case they were re-challenged with tamoxifen, or while they were taking adjuvant tamoxifen. Patients were assessed monthly for clinical response, which was defined according to World Health Organization criteria as complete response, partial response, no change (stable disease) or progressive disease. Clinical benefit was recorded in seven out of 19 recurrent cases: complete response (one patient), partial response (four patients) and stable disease continuing over 24 weeks (two patients). These patients were classified as ‘responders’ to endocrine therapy. The remaining 12 patients with recurrent tumors did not respond to endocrine therapy, and these were classified as ‘nonresponders’.
Isolation of Total RNA and Reverse Transcription
Total RNA was extracted from approximately 500 mg of frozen breast cancer tissue using TRIZOL reagent (Life Technologies Inc., Tokyo, Japan) according to the manufacturer's instructions. Reverse transcription reactions were performed as previously described (24). Briefly, each 20 µl cDNA synthesis reaction contained 1 µg of total RNA, buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2), 0.1 M dithiothreitol (DTT), 1 mM each of deoxynucleotide triphosphates (dNTP), 25 units of RNAguard RNase inhibitor (Amersham Pharmacia Biotech Inc., Tokyo, Japan), 200 units of Superscript II reverse transcriptase (Life Technologies Inc.) and 100 ng of pd(N)6 random hexamer (Amersham Pharmacia Biotech Inc.).
Primers and Probes
Blast searches (GenBank) were used to confirm the specificity of nucleotide sequences chosen for the primers and probes and to confirm the absence of DNA polymorphism. To avoid detection of contaminating genomic DNA, the primers were targeted to the junction between different exons. The sequences of the primers were as follows: sense primer, 5'-GAT GCT TTG GTT TGG GTG AT-3' (1401–1420); antisense primer, 5'-CTT GTT ACT CGC ATG CCT GA-3' (1492–1511) for ERβ1, and sense primer, 5'-GAT GCT TTG GTT TGG GTG AT-3' (2578–2597); antisense primer, 5'-GTT TGA GAG GCC TTT TCT GC-3' (2683–2702) for ERβcx/β2. The donor probe for ERβ1, fluorescein-labeled at its 3' end, was 5'-TCT CCT CCC AGC AGC AAT CCA-3'; the acceptor probe for ERβ1, with LC Red 640 at its 5' end, was 5'-GCG CCT GGC TAA CCT CCT GAT-3'. The donor probe for ERβcx/β2 was 5'-CAA TCC ATG CGC CTG GCT AAC-3'; and the acceptor probe for ERβcx/β2 was 5'-TCC TGA TGC TCC TGT CCC ACG-3'. The close proximity of the two probes hybridized to adjacent internal sequences for each specific primer allows specific amplicons to be detected, which provides sequence-specific detection of the amplified target sequence.
To ensure the fidelity of mRNA extraction and reverse transcription, all samples were subjected to PCR amplification with oligonucleotide primers and probes specific for the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and normalized. GAPDH primers were as follows: forward primer, 5'-AAA TCA AGT GGG GCG ATG CTG-3' and reverse primer 5'-GCA GAG ATG ATG ACC CTT TTG-3'. The sequences of GAPDH probes for LightCycler real-time PCR were 5'-AGA AGG CTG GGG CTC ATT TGC AGG G-3' and 5'-GTC CAC TGG CGT CTT CAC CAC CAT G-3'. All primers and probes were purchased from the Japanese Gene Institute (Saitama, Japan).
Real-time PCR with Hybridization Probes
Real-time PCR was performed using a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) as previously reported (25). The PCR reaction for ERβ1 and ERβcx/β2 was carried out in a final volume of 20 µl containing 2.4 µl of 25 mM MgCl2; 0.5 µl of 20 pmol/µl sense primer and antisense primer; 0.4 µl of 10 pmol/µl donor and acceptor probe; 2 µl of PCR master mix; 1.5 µl of cDNA, and made up to 20 µl with water. After an initial denaturation step at 95°C for 60 s, temperature cycling was initiated. Each cycle consisted of denaturation at 95°C for 0 s, hybridization at 57°C for 5 s, and elongation at 72°C for 6 s. The fluorescence signal was acquired at the end of the hybridization step. A total of 45 cycles were performed. The cycling conditions for GAPDH were as follows: initial denaturation at 95°C for 60 s, followed by 50 cycles at 95°C for 0 s, 60°C for 5 s and 72°C for 8 s.
Standard Curves and Presentation of Results
For each PCR run, a standard curve was constructed from serial dilutions of cDNA obtained from COS-7 cells transfected with either expression plasmid encoding ERβ1 or ERβcx/β2 (kindly donated by Masami Muramatsu, Saitama, Japan) for detection of ERβ1 and ERβcx/β2, respectively. The levels of expression of ERβ1 and ERβcx/β2 were given as relative copy numbers normalized against GAPDH mRNA. A nontemplate negative control was included in each experiment. All of the nontemplate negative controls, the standard cDNA dilutions from the COS-7 cells and the tumor samples were assayed in duplicate.
Generation of Specific antibodies for ERβ Proteins
To detect specific ERβ1 and ERβcx/β2 proteins, rabbit polyclonal antibodies were generated against synthesized peptides of the C-terminal region of ERβ1 (CSPAEDSKSKEGSQNPQSQ) and ERβcx/β2 (MKMETLLPEATMEQ), according to the method of Ogawa et al. (5) and purified on affinity columns bound with each synthetic peptide as described previously (26). To confirm the specificity of these polyclonal antibodies, immunoblot analysis was performed using COS-7 cells transfected with either expression plasmid encoding ERβ1 or ERβcx/β2 (kindly donated by Masami Muramatsu, Saitama, Japan) as previously described (26). Immunoblotting with specific anti-ERβ antibodies demonstrated that the polyclonal antibody for ERβ1 detected a specific band at 60 kDa only in the lysates of COS-7 cells transfected with an ERβ1 expression plasmid but not in those transfected with an ERβcx/β2 expression plasmid. Conversely, the polyclonal antibody for ERβcx/β2 detected a specific band at 55 and 51 kDa only in the lysates of COS-7 cells transfected with an ERβcx/β2 expression plasmid but not in those transfected with an ERβ1 expression plasmid, as described previously by Ogawa et al. (5).
Immunohistochemical Staining of ER, PgR, ERβ1, and ERβcx/β2
Immunohistochemical staining of ER, PgR, ERβ1 and ERβcx/β2 was done using monoclonal mouse antihuman ER antibody (1D5, DAKO) at 1:100 dilution for ER, monoclonal mouse antihuman PgR antibody (636, DAKO) at 1:100 dilution for PgR, polyclonal rabbit anti-ERβ1 antibody at 1:10 000 dilution for ERβ1 and polyclonal rabbit anti-ERβcx/β2 antibody at 1:2000 dilution for ERβcx/β2 as previously described (26). The expression of proteins was estimated in accordance with the procedure of Allred et al. (27). In brief, a proportion score represented the estimated proportion of tumor cells staining positive, as follows: 0 (none); 1 (<1/100), 2 (1/100 to 1/10), 3 (1/10 to 1/3), 4 (1/3 to 2/3) and 5 (>2/3). Any brown nuclear staining in invasive breast cancer cells counted towards the proportion score. An intensity score represented the average intensity of the positive cells, as follows: 0 (none), 1 (weak), 2 (intermediate) and 3 (strong). The proportion and intensity scores were then added to obtain a total score, which could range from 0 to 8. Staining status by immunohistochemistry was then assessed as negative (scores of 0 and 2) or positive (scores of 3–8) for ER and PgR, and assessed as negative (scores of 0, 2 and 3) or positive (scores of 4–8) for ERβ1 and ERβcx/β2; the cut-off level was set between a score of 3 and 4, in order to detect the most significant difference between patient groups, which will be described in the Results section.
Statistical Analysis
Correlation between mRNA expression of ERβ1 and ERβcx/β2 and correlation between mRNA and protein expression of ERβ1 and ERβcx/β2 were evaluated using the Spearman rank correlation test. Correlations between the expression of ERβ1, ERβcx/β2 and clinicopathological factors were evaluated using the 
2 test. The Mann–Whitney U-test was used for the statistical analyses of the correlation between IHC scores and ERβ mRNA expression levels, and of the correlation between ERβ mRNA expression levels and response to tamoxifen. Disease-free and overall survival curves were generated by the Kaplan–Meier method and verified with the Breslow–Gehan–Wilcoxon test. Cox's proportional hazards model was used for univariate and multivariate analyses of prognostic values. Differences were considered significant when a P < 0.05 was obtained.
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RESULTS |
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TOPAbstractINTRODUCTIONPatients and methodsRESULTSDISCUSSIONReferences |
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Evaluation of ERβ1 and ERβcx/β2 mRNA Expression in Breast Cancer Tissues
The amount of ERβ1 mRNA in the tissue samples from 150 patients ranged from 12 to 846 relative copy numbers (mean, 104.2), whereas the amount of ERβcx/β2 mRNA ranged from 0 to 298 relative copy numbers (mean, 27.1). To identify a clinically meaningful cutoff point for levels of ERβ1 and ERβcx/β2 mRNA expression that could be used in disease prognosis analysis, various levels of ERβ1 and ERβcx/β2 mRNA expression were tested using the Kaplan–Meier method and verified by the Breslow–Gehan–Wilcoxon test. When analyzing disease-free and overall survival, the cutoff points for the levels of ERβ1 and ERβcx/β2 mRNA were set at 95 and 16, respectively. Interestingly, ERβ1 mRNA expression was strongly correlated with ERβcx/β2 mRNA expression (P < 0.0001; Fig. 1).
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Immunohistochemical Staining of ERβ1 and ERβcx/β2 Protein
Staining with IHC techniques was used to investigate the expression of ERβ1 and ERβcx/β2 proteins. Various patterns of the proportion and intensity of positive nuclear staining were observed in the tissue samples from the 150 breast cancer patients. In addition, in some samples, normal mammary duct cells were also stained with ERβ1 and ERβcx/β2 polyclonal antibodies. However, because many samples in the study did not contain any normal tissue part at all, it is not possible to evaluate the significance of this staining of normal mammary duct (Fig. 2). ERβ1 protein expression was strongly correlated with ERβ1 mRNA expression (P = 0.0016). ERβcx/β2 protein expression was also significantly correlated with ERβcx/β2 mRNA expression (P = 0.035).
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Staining status by IHC was assessed as negative (scores 0, 2 and 3) or positive (scores 4–8) for ERβ1 and ERβcx/β2 using a cut-off level that allowed us to obtain the most significant difference between patient groups in disease-free and overall analyses. Out of these 150 samples, 103 (69%) and 76 (51%), respectively, stained positive for ERβ1 and ERβcx/β2 polyclonal antibodies.
Relationship between mRNA and Protein Expression of ERβ1 and ERβcx/β2 and Clinicopathological Factors
ERβ1 mRNA expression was significantly correlated with positive PgR status (P = 0.0004) and low histological grade (P = 0.037; Table 1). Whereas ERβ1 protein expression was strongly correlated with small tumor size (P = 0.029), negative lymph node status (P = 0.03) and low histological grade (P = 0.007). ERβcx/β2 protein expression was significantly associated with positive ER status (P = 0.040) and low histological grade (P = 0.007), although ERβcx/β2 mRNA expression did not correlate with clinicopathological factors (Table 1).
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Patients whose Tumor Expressed Higher Levels of ERβ1 and ERβcx/β2 Expression had Better Disease-free and Overall Survival
The correlation between ERβ1 and ERβcx/β2 mRNA expression and prognosis is shown in Fig. 3. Patients with high expression of ERβ1 mRNA experienced significantly longer disease-free survival (Fig. 3a) and overall survival (Fig. 3b) than those with low expression (P = 0.02 and P = 0.04, respectively). Similarly, high expression of ERβcx/β2 mRNA was also significantly associated with better disease-free survival (Fig. 3c) and overall survival (Fig. 3d; P = 0.005 and P = 0.005, respectively).
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The correlation between ERβ1 and ERβcx/β2 protein expression and prognosis is shown in Fig. 4. Patient samples staining positive for ERβ1 protein expression experienced significantly longer disease-free survival (Fig. 4a) and overall survival (Fig. 4b) than those with negative expression (P = 0.01 and P = 0.002, respectively). Patients who were positive for the expression of ERβcx/β2 protein had significantly better overall survival than negative patients (Fig. 4d, P = 0.03), although there was no benefit in favor of ERβcx/β2-positive patients with respect to disease-free survival (Fig. 4c).
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ERβcx/β2 mRNA Expression is an Independent Prognostic Factor of Disease-free and Overall Survival in Breast Cancer
Univariate analysis of the impact of clinicopathological parameters on disease-free survival revealed that ERβ1 and ERβcx/β2 mRNA expression status, PgR status, lymph node status and histological grade were all statistically significant prognostic factors, although tumor size and ER
status were not significant (Table 2). Moreover, multivariate analysis identified ERβcx/β2 mRNA expression (P = 0.002) as well as PgR and lymph node status as a statistically significant prognostic factor for disease-free survival (Table 2). For overall survival, univariate analysis (Table 3) showed significant associations between overall survival and ERβcx/β2 mRNA expression (P = 0.01), PgR status (P = 0.0002), lymph node status (P = 0.001) and histological grade (P = 0.0005). In multivariate analysis, patients with high ERβcx/β2 mRNA expression (P = 0.03), positive PgR status and negative lymph node status had significantly increased overall survival (Table 3). We concluded from these analyses that ERβcx/β2 mRNA expression is an independent prognostic factor for disease-free and overall survival in breast cancer.
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Tamoxifen Response and ERβ1 and ERβcx/β2 mRNA Expression
There were 19 patients with recurrent breast cancer who received tamoxifen for metastatic breast cancer; seven (36.8%) patients responded. We analyzed whether ERβ1 and ERβcx/β2 mRNA expression in the primary breast tumors affected the response to tamoxifen. No significant differences were found between ‘responders’ and ‘nonresponders’ in the levels of expression of either ERβ1 mRNA (P = 0.44) or ERβcx/β2 mRNA (P = 0.86; Fig. 5).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPatients and methodsRESULTSDISCUSSIONReferences |
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In the present study, we showed that elevated levels of expression of both ERβ1 and ERβcx/β2 were significantly associated with superior disease-free and overall survival. These results were in agreement with our earlier work, where we showed that patients with ERβ1-positive cancers tended to have a better prognosis (12,28). Palmieri et al. also showed that patients whose tumors expressed ERβcx/β2 had a longer survival time (29). Furthermore, in multivariate analysis, we found that a high level of expression of ERβcx/β2 mRNA was a significant prognostic factor, indicating that it is a superior prognostic factor in breast cancer than ER
. It is not clear why ERβcx/β2 is a better prognostic factor than ERβ1 in our analysis, although ERβ1 expression is strongly correlated with ERβcx/β2 expression. Functional differences between ERβ1 and ERβcx/β2 in breast cancer, such as response to endocrine therapy, may affect the prognosis. Our data suggest that, in breast cancer, ERβ expression is correlated with low malignancy. Roger et al. reported that the ERβ/ER ratio in breast cancer was lower than that in benign disease, and that the ratio in breast benign disease was lower than that in breast normal tissue. This led them to propose that the expression of ERβ markedly decreases in the early stage of mammary carcinogenesis (30). It is hypothesized that both ERβ1 and ERβcx/β2 might act as repressors of ER function, and that this would result in the suppression of hormone-dependent tumor growth. We showed that, while high expression of ERβcx/β2 protein was correlated with superior overall survival benefits, it was not associated with superior disease-free survival. This could suggest that the patients with high protein expression ERβcx/β2 might respond to endocrine therapy after recurrence in order to further improve their survival rate.
Zhao et al. recently reported that ERβcx/β2 negatively regulates the transactivation of ER in human breast cancer cells and that expression of ERβcx/β2 significantly correlated with ER-negative phenotype in human breast cancer samples, although their study only included a total of 37 individual human breast cancer samples (31). Our results also showed that ERβcx/β2 protein expression positively correlated with ER expression in 150 breast cancer samples, although no correlation was found between ERβcx/β2 mRNA expression and ER status. On the other hand, because ERβ is expressed not only ER-positive phenotype but also ER-negative phenotype, ERβ may have the potential to become a therapeutic target in the specific subcohort of ER-negative breast cancers (32).
We analyzed the correlation between the expression of ERβ and response to endocrine therapy in breast cancer. In this study we analyzed 19 cases treated by tamoxifen, but there was no significant correlation between ERβ expression and response to tamoxifen. Hopp et al. showed that high levels of ERβ predict an improved survival in patients treated with adjuvant tamoxifen therapy (33). Another study showed that low ERβ level was an additional independent marker to predict tamoxifen resistance (34). Furthermore, from previous data of 11 evaluated cases, we showed that tamoxifen responders tended to express higher level of ERβ protein than nonresponders (35). However, we reported in another study that protein expression of ERβ1 and ERβcx/β2 did not affect response to endocrine therapy in 75 metastatic breast cancer patients (27). Other groups have reported that ERβ expression was not predictive of response to antiestroge therapy (36,37). Murphy and Watson recently reported that there were two ERβ expressing groups, one in which ERβ was co-expressed with ER and the other where ERβ was expressed alone, and that reports supported the hypothesis that increased expression of ERβ was associated with increased likelihood of response to endocrine therapy (38). However, the association between presence of ERβ and response to endocrine therapy is still controversial, and therefore further studies are required.
In conclusion, we carried out quantitative real-time RT-PCR and IHC to examine the expression of ERβ1 and ERβcx/β2 and have demonstrated that breast cancer patients with positive expression of ERβ1 and ERβcx/β2 had a better prognosis than those with negative expression. Moreover, our study suggests that the expression of ERβcx/β2 mRNA, in particular, is a strong candidate for consideration as a positive prognostic factor, together with confirmed factors such as ER expression, lymph node status and histological grade. However, the significance of ERβ status is still controversial and further analysis of the role it plays in the pathogenesis of breast cancer is required. A better understanding of its function will ultimately allow for more accurate treatment for patients with this disease.
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Acknowledgments |
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We are grateful for the technical assistance of Mrs Mariko Nishio, Second Department of Surgery, Nagoya City University Medical School, Japan. This work was supported in part by a grant-in-aid (project numbers 14370362) for scientific research from the Ministry of Education, Sports, Science and Technology of Japan.
Conflict of interest statement
None declared.
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Footnotes |
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Abbreviations: ER, estrogen receptor; IHC, immunohistochemistry; PgR, progesterone receptor; RT-PCR, reverse transcriptase-polymerase chain reaction.
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References |
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1 Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA (1996) 93:5925–30.
2 Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Mol Endocrinol (1997) 11:353–65.
3 Mosselman S, Polman J, Dijkema R. ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett (1996) 392:49–53.[CrossRef][ISI][Medline]
4 Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, Muramatsu M. The complete primary structure of human estrogen receptor beta (hER beta) and its heterodimerization with ER alpha in vivo and in vitro. Biochem Biophys Res Commun (1998) 243:122–6.[CrossRef][ISI][Medline]
5 Ogawa S, Inoue S, Watanabe T, Orimo A, Hosoi T, Ouchi Y, Muramatsu M. Molecular cloning and characterization of human estrogen receptor betacx: a potential inhibitor of estrogen action in human. Nucl Acids Res (1998) 26:3505–12.
6 Vladusic EA, Hornby AE, Guerra-Vladusic FK, Lupu R. Expression of estrogen receptor beta messenger RNA variant in breast cancer. Cancer Res (1998) 58:210–14.
7 Lu B, Leygue E, Dotzlaw H, Murphy LJ, Murphy LC, Watson PH. Estrogen receptor-beta mRNA variants in human and murine tissues. Mol Cell Endocrinol (1998) 138:199–203.[CrossRef][ISI][Medline]
8 Chu S, Fuller PJ. Identification of a splice variant of the rat estrogen receptor beta gene. Mol Cell Endocrinol (1997) 132:195–9.[CrossRef][ISI][Medline]
9 Hanstein B, Liu H, Yancisin MC, Brown M. Functional analysis of a novel estrogen receptor-beta isoform. Mol Endocrinol (1999) 13:129–37.
10 Moore JT, McKee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne EL, et al. Cloning and characterization of human estrogen receptor beta isoforms. Biochem Biophys Res Commun (1998) 247:75–8.[CrossRef][ISI][Medline]
11 Tong D, Schuster E, Seifert M, Czerwenka K, Leodolte S, Zeillinger R. Expression of estrogen receptor beta isoforms in human breast cancer tissues and cell lines. Breast Cancer Res Treat (2002) 71:249–55.[CrossRef][ISI][Medline]
12 Omoto Y, Kobayashi S, Inoue S, Ogawa S, Toyama T, Yamashita H, et al. Evaluation of oestrogen receptor beta wild-type and variant protein expression, and relationship with clinicopathological factors in breast cancers. Eur J Cancer (2002) 38:380–6.[CrossRef][ISI][Medline]
13 Saunders PT, Millar MR, Williams K, Macpherson S, Bayne C, O'Sullivan C, et al. Expression of oestrogen receptor beta (ERbeta1) protein in human breast cancer biopsies. Br J Cancer (2002) 86:250–6.[CrossRef][ISI][Medline]
14 Jarvinen TA, Pelto-Huikko M, Holli K, Isola J. Estrogen receptor beta is coexpressed with ERalpha and PR and associated with nodal status, grade, and proliferation rate in breast cancer. Am J Pathol (2000) 156:29–35.
15 Iwao K, Miyoshi Y, Egawa C, Ikeda N, Noguchi S. Quantitative analysis of estrogen receptor-beta mRNA and its variants in human breast cancers. Int J Cancer (2000) 88:733–6.[CrossRef][ISI][Medline]
16 Speirs V, Parkes AT, Kerin MJ, Walton DS, Carleton PJ, Fox JN, Atkin SL. Coexpression of estrogen receptor alpha and beta: poor prognostic factors in human breast cancer? Cancer Res (1999) 59:525–8.
17 Dotzlaw H, Leygue E, Watson PH, Murphy LC. Expression of estrogen receptor-beta in human breast tumors. J Clin Endocrinol Metab (1997) 82:2371–4.
18 Miyoshi Y, Taguchi T, Gustafsson JA, Noguchi S. Clinicopathological characteristics of estrogen receptor-beta-positive human breast cancers. Jpn J Cancer Res (2001) 92:1057–61.[CrossRef][ISI]
19 Skliris GP, Carder PJ, Lansdown MR, Speirs V. Immunohistochemical detection of ERbeta in breast cancer: towards more detailed receptor profiling? Br J Cancer (2001) 84:1095–8.[CrossRef][ISI][Medline]
20 Mann S, Laucirica R, Carlson N, Younes PS, Ali N, Younes A, Li Y, Younes M. Estrogen receptor beta expression in invasive breast cancer. Hum Pathol (2001) 32:113–18.[CrossRef][ISI][Medline]
21 Fuqua SA, Schiff R, Parra I, Friedrichs WE, Su JL, McKee DD, et al. Expression of wild-type estrogen receptor beta and variant isoforms in human breast cancer. Cancer Res (1999) 59:5425–8.
22 Leygue E, Dotzlaw H, Watson PH, Murphy LC. Expression of estrogen receptor beta1, beta2, and beta5 messenger RNAs in human breast tissue. Cancer Res (1999) 59:1175–9.
23 Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology (1991) 19:403–10.[ISI][Medline]
24 Toyama T, Iwase H, Watson P, Muzik H, Saettler E, Magliocco A, et al. Suppression of ING1 expression in sporadic breast cancer. Oncogene (1999) 18:5187–93.[CrossRef][ISI][Medline]
25 Zhang Z, Yamashita H, Toyama T, Sugiura H, Ando Y, Mita K, et al. ATBF1 – a messenger RNA expression is correlated with better prognosis in breast cancer. Clin Cancer Res (2005) 11:193–8.
26 Yamashita H, Nishio M, Kobayashi S, Ando Y, Sugiura H, Zhang Z, et al. Phosphorylation of estrogen receptor alpha serine 167 is predictive of response to endocrine therapy and increases postrelapse survival in metastatic breast cancer. Breast Cancer Res (2005) 7:R753–64.[CrossRef][ISI][Medline]
27 Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol (1998) 11:155–68.[ISI][Medline]
28 Omoto Y, Inoue S, Ogawa S, Toyama T, Yamashita H, Muramatsu M, et al. Clinical value of the wild-type estrogen receptor beta expression in breast cancer. Cancer Lett (2001) 163:207–12.[CrossRef][ISI][Medline]
29 Palmieri C, Lam EW, Mansi J, MacDonald C, Shousha S, Madden P, et al. The expression of ER beta cx in human breast cancer and the relationship to endocrine therapy and survival. Clin Cancer Res (2004) 10:2421–8.
30 Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochefort H. Decreased expression of estrogen receptor beta protein in proliferative preinvasive mammary tumors. Cancer Res (2001) 61:2537–41.
31 Zhao C, Matthews J, Tujague M, Wan J, Strom A, Toresson G, et al. Estrogen receptor {beta}2 negatively regulates the transactivation of estrogen receptor {alpha} in human breast cancer cells. Cancer Res (2007) 67:3955–62.
32 Skliris GP, Leygue E, Curtis-Snell L, Watson PH, Murphy LC. Expression of oestrogen receptor-beta in oestrogen receptor-alpha negative human breast tumours. Br J Cancer (2006) 95:616–26.[CrossRef][ISI][Medline]
33 Hopp TA, Weiss HL, Parra IS, Cui Y, Osborne CK, Fuqua SA. Low levels of estrogen receptor beta protein predict resistance to tamoxifen therapy in breast cancer. Clin Cancer Res (2004) 10:7490–9.
34 Esslimani-Sahla M, Simony-Lafontaine J, Kramar A, Lavaill R, Mollevi C, Warner M, et al. Estrogen receptor beta (ER beta) level but not its ER beta cx variant helps to predict tamoxifen resistance in breast cancer. Clin Cancer Res (2004) 10:5769–76.
35 Iwase H, Zhang Z, Omoto Y, Sugiura H, Yamashita H, Toyama T, Iwata H, Kobayashi S. Clinical significance of the expression of estrogen receptors alpha and beta for endocrine therapy of breast cancer. Cancer Chemother Pharmac (2003) 52((Suppl 1)):S34–8.
36 Murphy LC, Leygue E, Niu Y, Snell L, Ho SM, Watson PH. Relationship of coregulator and oestrogen receptor isoform expression to de novo tamoxifen resistance in human breast cancer. Br J Cancer (2002) 87:1411–16.[CrossRef][ISI][Medline]
37 Cappelletti V, Celio L, Bajetta E, Allevi A, Longarini R, Miodini P, et al. Prospective evaluation of estrogen receptor-beta in predicting response to neoadjuvant antiestrogen therapy in elderly breast cancer patients. Endocr Relat Cancer (2004) 11:761–70.
38 Murphy LC, Watson PH. Is oestrogen receptor-beta a predictor of endocrine therapy responsiveness in human breast cancer? Endocr Relat Cancer (2006) 13:327–34.
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