Molecular Diagnostics

British Journal of Cancer (2006) 95, 616–626. doi:10.1038/sj.bjc.6603295 www.bjcancer.com
Published online 1 August 2006

Expression of oestrogen receptor-bold italic beta in oestrogen receptor-alpha negative human breast tumours

G P Skliris1, E Leygue1, L Curtis-Snell2, P H Watson2 and L C Murphy1

  1. 1Department of Biochemistry & Medical Genetics, Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OV9
  2. 2Department of Pathology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OV9

Correspondence: LC Murphy, E-mail: lcmurph@cc.umanitoba.ca

Received 17 January 2006; Revised 30 May 2006; Accepted 29 June 2006; Published online 1 August 2006.

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Abstract

To analyse the phenotype of breast tumours that express oestrogen receptor-beta (ERbeta) alone tissue microarrays were used to investigate if ERbeta isoforms are associated with specific prognostic markers and gene expression phenotypes in ERalpha-negative tumours. ERalpha-negative tumours were positive for ERbeta1 in 58% of cases (n=122/210), total ERbeta in 60% (n=115/192) and ERbeta2/cx in 57% of cases (n=114/199). Oestrogen receptor-beta1 and total ERbeta were significantly correlated with Ki67 (r=0.28, P<0.0001, n=209; r=0.29, P<0.0001, n=191) and with CK5/6, a marker of the basal phenotype (r=0.20, P=0.0106, n=170; r=0.18, P=0.0223, n=158). ERbeta2/cx was strongly associated with p-c-Jun and NF-kappaBp65 (r=0.53, P<0.0001, n=93; r=0.35, P<0.0001, n=176). This study shows that a range of ERbeta isoform expression occurs in ERalpha-negative breast tumours. While expression of ERbeta1, total and ERbeta2/cx are correlated, individual forms show associations with certain phenotypes that suggest different roles in subsets of ERalpha-negative cancers. Based on our in vivo observations, ERbeta may have the potential to become a therapeutic target in the specific subcohort of ERalpha-negative breast cancers.

Keywords:

oestrogen receptor-beta isoforms, breast cancer, immunohistochemistry, proliferation, basal phenotype

Oestrogen receptor-alpha (ERalpha) is an important biomarker of response to endocrine therapy in breast cancer (Osborne, 1998). However, the definition of ER status in breast cancer is potentially more complex, since there are now two known ERs, ERalpha and ERbeta. Oestrogen receptor-beta is expressed in both normal and neoplastic human breast tissue (Leygue et al, 1998; Mann et al, 2001; Murphy et al, 2002; Fuqua et al, 2003; Skliris et al, 2003) but its role in either tissue remains unknown. Several isoforms of ERbeta have been identified, which are either exon deletions or products of alternative splicing which result in proteins that are truncated at the C-terminus and do not bind ligand (Lu et al, 1998; Ogawa et al, 1998; Fuqua et al, 1999; Leygue et al, 1999; Saunders et al, 2002). Thirty percent of breast tumours are classified as ER negative at the time of diagnosis and will be mostly resistant to endocrine therapy (Lapidus et al, 1998; Osborne, 1998). However, the previous assays used for ER measurement favoured the detection of ERalpha (Harvey et al, 1999; Brouillet et al, 2001) and we now know that some of these tumours express ERbeta (Murphy et al, 2003). Considering studies where ERbeta protein expression was determined, the pooled data sets were used to estimate the frequency of ERbeta and ERalpha status in breast cancers (Murphy et al, 2003). The most frequently occurring tumour type is ERalpha+/ERbeta+ (approx60%) with similar frequencies of the other three ER phenotypes (ERalpha+/ERbeta-; ERalpha-/ERbeta+; ERalpha-/ERbeta-) at 10–20% (Murphy et al, 2003). It is important to note that there are two groups of ERbeta-positive breast tumours, those with coexpression of ERalpha and those expressing ERbeta alone. The former is the most frequent and probably dominates the analysis of most previously reported correlative studies, and hence the positive association of ERbeta expression generally with good prognosis and good clinical outcome with respect to tamoxifen treatment (Mann et al, 2001; Omoto et al, 2001; Murphy et al, 2002; Iwase et al, 2003; Esslimani-Sahla et al, 2004; Fleming et al, 2004; Hopp et al, 2004; Myers et al, 2004; Nakopoulou et al, 2004). There is little data exploring tumours that express ERbeta alone. Under the current system of determining ER status, these are classified clinically as ER negative, and currently there are few markers for further subclassifying these ERalpha-negative cancers. Nevertheless recent data show that some invasive breast cancers expressing the basal cytokeratin CK5/6, may represent one ERalpha-negative subset, known as the basal epithelial phenotype and show a relatively poor prognosis (Perou et al, 2000; Sorlie et al, 2001, 2003; Nielsen et al, 2004). In the present study, we have investigated the level and frequency of expression of ERbeta in ER-negative tumours and its association with the basal phenotype and other established markers of prognosis, such as indicators of signal transduction pathways, proliferative and apoptotic markers.

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Materials and methods

Tissues

All invasive breast cancers used in the current study were obtained from the Manitoba Breast Tumour Bank (MBTB, Department of Pathology, University of Manitoba) (Watson et al, 1996), which operates with approval from the Faculty of Medicine, University of Manitoba, Research Ethics Board. All samples included in the MBTB are rapidly frozen at -70°C immediately after surgical removal. A portion of the frozen tissue from each case is then processed to create matched formalin-fixed paraffin-embedded and frozen tissue blocks.

Clinical–pathological characteristics of the patient cohort

Cases selected for this study were on the basis of (a) minimum patient follow-up of 36 months, (b) invasive components occupying more than 20% of the tumour section, while normal epithelial areas comprised no more than 10% of the epithelial content and (c) ER-negative status as defined by ligand binding analysis (LBA) of less than or equal to3 fmol mg-1 protein. The criteria for interpretation of the variables were as follows: (a) PR-positive status was defined as >15 fmol mg-1 protein by LBA; (b) grade, (Nottingham system), was assigned to low (scores 3–5), moderate (scores 6 and 7), or high (scores 8 and 9) categories; (c) tumour size, was assigned either small (less than or equal to2 cm) or large (>2 cm) categories; (d) tumour inflammation was assessed by a scale from 1 to 5 and then assigned to low (scores 1–3) or high (scores 4 and 5) categories. All patients were treated with surgery and for 29 patients this was the only treatment regimen. The remaining patients received a variety of additional treatments, hormonal therapy (28), chemotherapy (49) or radiotherapy (9) alone, or combination of radiation followed by hormonal therapy (8), hormonal and chemotherapy (16), hormonal and chemotherapy (19) or chemotherapy (46), and for 6 patients the treatment regime was unknown.

Tissue microarrays

The histopathology of all MBTB cases has been assessed and entered into a computerised database to enable selection based on composition of the tissue as well as clinical–pathological parameters. After selection, cases were rereviewed on H&E sections by a breast histopathologist (PHW). Tissue microarrays (TMAs) from a total cohort of 255 ERalpha negative (ERalpha–255TMA), primary invasive ductal breast carcinomas were constructed. Briefly, duplicate core tissue samples (0.6 mm diameter), were taken from selected areas of maximum cellularity for each tumour with a tissue arrayer instrument (Beecher Instruments, Silver Spring, MD, USA). Although the TMA consisted of 255 cases of ER-negative tumours as determined by LBA (ER+ >3 fmol mg-1 protein), 39 of these were subsequently found to be ERalpha+ by immunohistochemistry (IHC) and were excluded from the later analysis.

Immunohistochemical assay

Serial sections (5 mum) of the ERalpha–255TMA were cut, mounted on Fisherbrand Superfrost/plus slides (Fisher Scientific, USA) and stained using IHC with commercially available specific antibodies (Table 1). Further details of the three specific ERbeta antibodies are as follows: ERbeta1 (polyclonal, GC17/385P, Biogenex, CA, USA, raised to peptide containing amino acids 449–465) at 1 : 100 dilution; total ERbeta (monoclonal, 14C8, Genetex, TX, USA, raised to peptide containing amino acids 1–153) at 1 : 100; ERbeta2/cx (mouse monoclonal, clone 57/3, raised to synthetic peptide derived from the specific C-terminus of hERbeta2/cx isoform; Serotec, UK) used at 1 : 20. Briefly, sections were dewaxed in two xylene baths (5 min each), taken through a series of alcohols (100, 95, 70%), rehydrated in distilled water and then submitted to heat-induced antigen retrieval for 8 min in the presence of a citrate buffer (CC1 mild/standard, Ventana Medical Systems, AZ, USA) using an automated tissue immunostainer (Discovery Staining Module, Ventana Medical Systems, AZ, USA). The staining protocol was set to 'Mild and Standard Cell Conditioning' procedure for all antibodies. Primary antibodies were applied for 60 min (except for NF-kappaBp65 which were applied for 30 min) while secondary antibodies were incubated for 32 min. Initial dilutions quoted above were diluted further 1 : 3 with buffer dispensed onto the slide with the primary antibody. Primary antibodies were omitted for negative controls.


Total ERbeta IHC was performed manually; sections were microwaved in the presence of 0.01 M citrate buffer, pH 6.0, for 20 min at full power (Danby, ON, Canada, model DMW 1001 W, 800 W maximum output). Sections were blocked and then incubated using an ERbeta monoclonal antibody (14C8, Genetex, TX, USA) at 1 : 100 dilution in a humidified chamber at 4°C overnight, as previously described (Skliris et al, 2002, 2003; Fuqua et al, 2003). Following incubation with biotinylated goat anti-mouse antibody for 60 min at 1 : 200 (Jackson ImmunoResearch Laboratories, PA, USA) and with the Vectastain ABC kit (Vector Laboratories, CA, USA) for 45 min, total ERbeta protein was visualised with 3,3'-diaminobenzidine (DAB, Sigma-Aldrich, ON, Canada). Slides were scored semiquantitatively under a standard light microscope. Images were captured using Polaroid DMC-2 software (version 2.0.1, Polaroid, MA, USA).

Quantification technique and marker selection

The expression of ERbeta isoforms (full-length-ligand binding ERbeta1, total ERbeta and ERbeta2/cx) and other prognostic markers was assessed using semiquantitative scoring (H-scores). H-scores derive from a semiquantitative assessment of both staining intensity (scale 0–3) and the percentage of positive cells (0–100%), which when multiplied, generates a score ranging from 0 to 300. Tissue microarray staining was evaluated by two authors (GPS, PHW) independently and where discordance was found, cases were re-evaluated together to reach agreement. For the primary categorical analysis, staining and cutoff points to distinguish low from high expression for each marker were as follows: only nuclear staining was evaluated for ERbeta1, total ERbeta and ERbeta2/cx isoforms and since there is no agreement or clinical relevant cutoff IHC-scores for ERbeta isoforms reported in the literature, several IHC-score cut-points equivalent to absent staining, the 25th percentile and median IHC-score values were tested in statistical analysis. Ki67, caspase-3 (markers of proliferation and apoptosis, respectively) and CK5/6 (a marker of the basal phenotype) were scored as previously described (Perou et al, 2000; Wykoff et al, 2001; Foulkes et al, 2004; El-Rehim et al, 2005). Since NF-kappaB has been associated previously with more aggressive breast cancer (Biswas et al, 2004) and both NF-kappaB and AP-1 have been shown to interact differentially with ERalpha and ERbeta (Paech et al, 1997; An et al, 1999) we have also assessed the relationship of ERbeta to these pathways in ERalpha-negative tumours. For NF-kappaB/p65 nuclear staining was assessed and multiple H-score cutoffs were tested. P-c-Jun, a marker of AP-1 activity, was defined by nuclear staining and an H-score of >0.

Statistical analysis

Associations between ERbeta isoforms and other clinical–pathological variables were tested using contingency methods (Fisher's exact test). Correlations were assessed by the Spearman's rank correlation test (r). Mann–Whitney rank sum tests, two-sided were also used to evaluate variables. Survival analyses were perfomed using the log rank test to generate Kaplan–Meier curves. Overall survival was defined as the time from initial surgery to the date of death attributable to breast cancer. Relapse-free survival was defined as the time from initial surgery to the date of clinically documented local or distant disease recurrence or death attributed to breast cancer. GraphPad Prism 4.02 version statistics software (GraphPad, San Diego, CA, USA) was used to perform all analyses.

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Results

Validation of ERbeta antibodies

Three antibodies previously validated to detect ERbeta related proteins were used in this study (Fuqua et al, 1999; Leav et al, 2001; Saunders et al, 2002). GC17/385P (Leav et al, 2001) was raised to a C-terminal epitope of the wild-type ligand binding isoform of ERbeta, generally referred to as ERbeta1. 14C8 antibody (Fuqua et al, 1999) was raised to an N-terminal epitope which would be found in both ERbeta1 and multiple C-terminal truncated nonligand binding forms of ERbeta and therefore would detect multiple known ERbeta isoforms including ERbeta1 and ERbeta2cx. Hence we refer to it as detecting 'total' ERbeta. The antibody used to detect the nonligand isoform ERbeta2/cx (Saunders et al, 2002) has been previously validated by IHC and immunoblotting (Saunders et al, 2002). However, we have also validated the antibody further at the IHC level, by using MCF7 breast cancer cell lines, which have been engineered to overexpress ERbeta1 or ERbeta2/cx, after induction with the tetracycline analogue doxycycline (Murphy et al, 2005). Agar embedded cell pellets (Riera et al, 1999), formalin-fixed and paraffin-embedded (Adeyinka et al, 2002) from only the doxycycline treated cells expressing ERbeta2/cx but not ERbeta1 or controls were found to show nuclear staining with the specific ERbeta2/cx antibody under the same IHC conditions described above for the human breast tumours (Figure 1A).

Figure 1.
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(A) Validation of ERbeta2/cx antibody (mouse monoclonal, clone 57/3, Serotec, UK): (a) Serotec clone 57/3 antibody staining of section from cell pellet of doxycycline treated tet-on-MDA231 cells stably overexpressing ERbeta2/cx, magnification times 500; (b) same as (a), magnification times 1250; (c) Serotec clone 57/3 antibody staining of section from cell pellet of doxycycline treated tet-on-MCF7 cells stably overexpressing ERbeta2/cx, magnification times 500; (d) same as (c), magnification times 1250; (e) Serotec clone 57/3 antibody staining of section from cell pellet of a separate clone of doxycycline treated tet-on-MCF7-cells stably overexpressing ERbeta2/cx, magnification times 500; (f) same as (e), magnification times 1250; (g) Serotec clone 57/3 antibody staining of section from cell pellet of doxycycline treated tet-on-MCF7 vector alone control cells, magnification times 500; (h) same as (g), magnification times 1250; (i) Serotec clone 57/3 antibody staining of section from cell pellet of doxycycline treated MCF7 stably overexpressing ERbeta1 (Murphy et al, 2005), magnification times 500. (B) Expression of ERbetacx/2 in ERalpha-negative invasive tumours and normal breast tissue detected by IHC is demonstrated in representative panels. (a) Tumour core stained with the specific ERbetacx/2 antibody (high H-score, 270); (b) tumour stained for ERbetacx/2 (low H-score, 25); (c) tumour core showing negative staining for ERbetacx/2 H-score, 0); (d) normal breast tissue showing strong, nuclear ERbetacx/2 protein expression; (e) nuclear ERbetacx/2 expression in normal breast ducts; (f) negative control (omission of ERbetacx/2 antibody). Magnification times 500 for a, b, c, and times 1250 for d, e, f.

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ERbeta isoform expression in ERalpha-negative human breast tumours

Serial sections of the ERalpha–255TMA were stained with specific antibodies for ERbeta1, total ERbeta, and ERbeta2/cx using IHC. Nuclear staining could be observed with ERbeta1 and total ERbeta antibodies in epithelial cells in our series of invasive cancers (Figure 2). Strong nuclear staining in both normal and neoplastic breast tissues for ERbeta2/cx isoform was often observed (Figure 1B). Using the 25% percentile of IHC-scores to define positive status for ERbeta1, total ERbeta and ERbeta2/cx, we observed that 58% of ERalpha-negative tumours were positive for ERbeta1 (n=122/210), 60% positive for total ERbeta (n=115/192) and 57% of cancers were positive for ERbeta2/cx (n=114/199; Table 2).

Figure 2.
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Expression of ERbeta and Ki67 in ERalpha-negative tissue microarray cores. (A–C) ERalpha-negative tumour cores stained with the specific ERbeta1 antibody (GC17/385P) showing negative, medium and high expression (a–c; H-scores of 0, 150 and 225, respectively); (D–F) ERalpha-negative tumour cores stained with total ERbeta antibody (14C8) showing negative, low and high expression (H-scores of 0, 25 and 100, respectively); (G–I) ERalpha-negative tumour cores showing negative, medium and high expression for Ki67, a proliferation marker (% positive, 0, 60 and 90%, respectively). Magnification times 500.

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ERbeta1 was significantly correlated with both total ERbeta and ERbeta2/cx (r=0.28, P<0.0001, n=189; r=0.27, P=0.0002, n=196, respectively; Table 3). The same relationship was evident in categorical analysis using a variety of cutoff values for contingency analysis, where ERbeta1 was also significantly associated with ERbeta2/cx and total ERbeta (P=0.0083, >10; P=0.0016, 0.0391; >10, >25 respectively, Fishers exact test). Using a cut-point for ERbeta1 of either >10 or >25, median levels of total ERbeta expression were significantly higher in ERbeta1-positive vs -negative tumours (P=0.0026 and P=0.011, Mann–Whitney rank sum tests, two-sided). Similarly using the same two cut-points for ERbeta1 positivity median levels of ERbeta2/cx expression were significantly higher in ERbeta1-positive vs -negative tumours (P=0.0024 and P=0.022, respectively Mann–Whitney rank sum tests). These data suggest frequent coexpression of multiple ERbeta isoforms in breast tumours.


Relationship of ERbeta isoform expression with markers of proliferation and apoptosis in ERalpha-negative human breast tumours

ERbeta1 (r=0.28, P<0.0001, n=209) and total ERbeta (r=0.29, P<0.0001, n=191; Table 3) were positively correlated with Ki67, a marker of proliferation, which was detected in the nuclei of ERalpha-negative tumours (Figure 2). Contingency analyses also showed that ERbeta1 and total ERbeta were associated with Ki67 (data not shown). Using the median Ki67 IHC-score as a cutoff to define low Ki67 (less than or equal to25) and high Ki67 (>25), the median level of ERbeta1 expression was significantly lower in low Ki67 expressors (median ERbeta1=25) compared to high Ki67 expressors (median ERbeta1=50; P=0.0008, Mann–Whitney rank sum test). Similarly the median level of total ERbeta expression was significantly lower in low Ki67 expressors (median total ERbeta=20) compared to high Ki67 expressors (median total ERbeta=50; P=0.0008, Mann–Whitney rank sum test). No significant differences in ERbeta2/cx were found between the low and high Ki67 groups.

However, high proliferation in primary tumours prior to treatment, is often associated with high levels of apoptosis (Lipponen et al, 1994; Lipponen, 1999; Parton et al, 2002). Therefore, ERbeta expression was investigated with respect to a marker of apoptosis, active caspase-3 (Parton et al, 2002). No correlations were detected between ERbeta isoforms and caspase-3. However, Ki67 expression was significantly correlated (r=0.44, P<0.0001, n=211, Table 3) and associated (P<0.0001 Fisher's exact test; Mann–Whitney rank sum test) with caspase-3 in this breast tumour cohort. These data suggest that ERbeta expression in ERalpha-negative tumours is associated with markers of a high proliferative index.

Relationship of ERbeta expression to basal epithelial phenotype markers in ERalpha-negative human breast tumours

Invasive breast cancers expressing the basal epithelial phenotype, based on the consensus of the published literature from cDNA microarray and IHC analyses, are ERalpha negative (Perou et al, 2000; Sorlie et al, 2001; Vijver et al, 2002; Nielsen et al, 2004; El-Rehim et al, 2005), CK5/6 positive (Sorlie et al, 2001; Korsching et al, 2002; Nielsen et al, 2004; Collett et al, 2005) and/or CK14 (El-Rehim et al, 2005) positive. The basal phenotype has also been associated with mutated BRCA1 (Foulkes et al, 2003, 2004; Sorlie et al, 2003; Collett et al, 2005). We were therefore interested to determine the relationship of ERbeta expression in ERalpha-negative tumours to markers of the basal epithelial phenotype. ERbeta1 and total ERbeta expression were weakly correlated with CK5/6 (r=0.20, P=0.010; n=170; r=0.18, P=0.022, n=158; Table 3). No correlations were seen with ERbeta2/cx. These data support the conclusion that many ERalpha-negative tumours expressing ERbeta are associated with some markers of a basal epithelial phenotype in breast cancer.

ERbeta2/cx expression in ERalpha-negative human breast tumours

Despite the correlations and associations of ERbeta2/cx to ERbeta1 and total ERbeta described above, ERbeta2/cx was not correlated with Ki67 nor activated caspase-3. However, ERbeta2/cx was strongly correlated with p-c-Jun IHC-score (r=0.53, P<0.0001, n=93; Table 3). Contingency analyses for ERbeta2/cx and p-c-Jun positivity, identified a significant association of ERbeta2/cx with p-c-Jun (P<0.0001, Fisher's exact test). When p-c-Jun expression level was examined in relation to ERbeta2/cx status, p-c-jun IHC-score was significantly lower in ERbeta2/cx-negative tumours (median p-c-Jun=5) compared to high ERbeta2/cx expressors (median p-c-Jun=40; P<0.0001, Mann–Whitney rank sum test).

Similarly, ERbeta2/cx expression was also correlated with NF-kappaBp65 (r=0.35, P<0.0001, n=176; Table 3). Using either the 25% percentile (>0) or the median (>25) ERbeta2/cx IHC-score as cut-points to define negative and positive ERbeta2/cx status the median level of NF-kappaBp65 expression was significantly lower in negative/low ERbeta2/cx expressors (median NF-kappaBp65=50) compared to high ERbeta2/cx expressors (median NF-kappaBp65=100; P<0.0001, Mann–Whitney rank sum test). Similar but weaker relationships were found for total ERbeta. Using the median (>25) total ERbeta IHC-score as a cutoff to define negative and positive total ERbeta status the median level of NF-kappaBp65 expression was significantly lower in negative/low total ERbeta expressors (median NF-kappaBp65=75) compared to high total ERbeta expressors (median NF-kappaBp65=100; P<0.026, Mann–Whitney rank sum test). These data suggest that ERbeta2/cx expression is associated with AP1 and NF-kappaB activity in ERalpha-negative breast tumours. A relationship between total ERbeta and p-c-Jun and NF-kappaBp65 was also demonstrated, and is likely to reflect the influence of the ERbeta2/cx component of the total ERbeta signal.

ERbeta isoform expression in relation to clinical and pathological prognostic variables and survival

Only total ERbeta was associated with tumour grade (P=0.03). No other statistically significant associations between ER isoforms and established prognostic variables such as tumour size, age at diagnosis, node status, inflammation or progesterone receptor, were observed (Table 2, showing associations with cut-points equivalent to the 25th percentile).

Univariate survival analyses in relation to axillary nodal status, size, grade, Ki67, active caspase-3, or basal phenotype, showed a significant association only with nodal status (P=0.024) in this cohort of ERalpha-negative tumours. Furthermore no difference in disease outcome (overall survival and relapse-free survival) was found between low and high ERbeta1, total ERbeta or ERbeta2/cx (Figure 3).

Figure 3.
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Kaplan–Meier graphs for 'overall survival' and 'relapse-free survival-time to progression' with respect to expression of ERbeta1 (A and B), ERbeta2cx (C and D) and total ERbeta isoforms (E and F, respectively). ERbeta1 overall survival (A), n=210, low ERbeta1 events=47, high ERbeta1 events=60. ERbeta1 time to progression (B), low ERbeta1 events=48, high ERbeta1 events=60. ERbeta2cx overall survival (C), n=199, low ERbeta2cx events=44, high ERbeta2cx events=53. ERbeta2cx time to progression (D), low ERbeta2cx events=44, high ERbeta2cx events=53. Total ERbeta overall survival (E), n=192, low total ERbeta events=40, high total ERbeta events=55. Total ERbeta time to progression, low total ERbeta events=40, high total ERbeta events=56.

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Discussion

Several interesting observations have been made in the present study concerning ERbeta isoform expression in ERalpha-negative breast tumours. The first is that ERbeta1, total ERbeta and ERbeta2/cx isoforms are frequently expressed in this cohort of ERalpha-negative breast cancers. The second is that there is a significant correlation of ERbeta1 and total ERbeta with Ki67, a marker of proliferation, which is of particular interest. As this was not found when ERbeta2/cx expression was assessed, it is likely that the correlation with total ERbeta reflects the ERbeta1 component, although we cannot exclude the existence of other, as yet unknown variant isoforms. Indeed, the frequent expression of the ERbeta variant isoform, ERbeta5, in ERalpha-negative breast tumours has recently been described (Poola et al, 2005), however, we did not have access to specific antibodies to investigate this variant isoform in our breast tumour cohort. Our data confirm and extend an observation made by Jensen et al (2001), where the highest expression of either Ki67 and Cyclin A was found in tumours that only expressed ERbeta, indicating that ERbeta may be related to proliferation in breast cancer. Jensen's observation showing an association of ERbeta with Ki67, using an antibody that recognised total ERbeta (Jensen et al, 2001), also suggests that ERbeta isoforms are not only expressed in cells with the potential to cycle but also can be expressed in cells that are cycling. The existence of this relationship was reflected only in a very small subset of seven tumours in the ERalpha-negative/ERbeta-positive cohort in his study (Jensen et al, 2001), but a study by O'Neill et al (2004) published during the execution of our study confirmed his observation in a larger cohort (n=167). However, results from these latter studies came only from subset analysis of mixed cohorts of ERalpha-positive and -negative tumours. Our study is the only one so far exclusively focusing on ERalpha-negative cancers to address the issue of ERbeta expression. The cohort used in our study (n=216) is the largest so far and included tumours that were all selected to be ERalpha negative, both immunohistochemically and by LBA. Thus, the relationship of ERbeta1 alone expression in human breast cancer to Ki67, seems to be highly reproducible and therefore likely offers a new significant insight into the possible role of ERbeta1 in breast cancer. In contrast, this relationship is generally not seen in ERalpha-positive/ERbeta-positive breast tumours (Jarvinen et al, 2000; Mann et al, 2001; Omoto et al, 2001; Murphy et al, 2002; Fuqua et al, 2003; Iwase et al, 2003; Fleming et al, 2004; Hopp et al, 2004; Myers et al, 2004; Nakopoulou et al, 2004) and therefore our data together with two other studies support the conclusion that the role of ERbeta1 when expressed alone in human breast cancers in vivo is likely quite different to when it is coexpressed with ERalpha. Such data suggest that ERbeta1 may have a direct role in proliferation in ERalpha-negative breast cancers, but this is unproven.

The involvement of ERbeta isoforms in proliferation using cell line models is unclear. Most cell line models in which ERbeta1 has been stably expressed either inducibly or constitutively show that overexpression of ERbeta1 inhibits proliferation irrespective of whether it is coexpressed with ERalpha (Paruthiyil et al, 2004; Strom et al, 2004; Murphy et al, 2005) or not (Lazennec et al, 2001; Cheng et al, 2004). However, two studies using cell line models have been published in which stable constitutive overexpression of ERbeta1 resulted in increased proliferation (Tonetti et al, 2003; Hou et al, 2004) although in the former publication the short form of ERbeta1 (truncated by 45 amino acids from the N-terminus) was used. Both breast cancer cell lines used (MDA-MB-231 and MDA-MB-435) are typically ERalpha negative and therefore can be considered to represent the ERbeta alone expressing breast tumours cohort in vivo. However, in another constitutive ERbeta overexpression model based on the MDA-MB-231 cells, little or no effect on proliferation, positive or negative, was seen (Rousseau et al, 2004). Such data indicate that differences in potential cell line background, the type of ERbeta isoforms expressed and experimental variables including possibly clonal selection can influence the effect of ERbeta on proliferation. However, in other cancer cells types where ERbeta1 has been overexpressed, increased ERbeta1 is most often associated with inhibition of proliferation and/or increased apoptosis (Qiu et al, 2002; Cheng et al, 2004). It is unclear, however, whether the overexpression of ERbeta1 in experimental cancer cell line models, is relevant to the levels of ERbeta1 seen in tumours in vivo, especially since generally ERbeta1 expression is reduced in tumours compared to normal tissues in multiple cancers (Foley et al, 2000; Roger et al, 2001; Skliris et al, 2003) leading to the suggestion that ERbeta1 is a tumour-suppressor gene, and certainly would be consistent with the hypothesis that it is antiproliferative (Weihua et al, 2000; Forster et al, 2002; Paruthiyil et al, 2004). As well the possibility exists that ERbeta1 may be frequently mutated and/or altered post-translationally in breast cancers in vivo, although no published data as yet address this issue to our knowledge.

ERbeta1 and total ERbeta isoforms were also significantly correlated with CK5/6, a marker of the basal epithelial phenotype as defined from DNA microarray and IHC analyses, predominantly as ERalpha negative and CK5/6 positive (Sorlie et al, 2001; Korsching et al, 2002; Collett et al, 2005; El-Rehim et al, 2005). As ERbeta is found widely expressed in the basal myoepithelium (Murphy et al, 2002; Speirs et al, 2002) as well as in luminal epithelial cells in normal human breast tissues, it is possible that many ER-negative breast cancers expressing ERbeta are derived from a myoepithelial cell lineage, and that ERbeta is a marker of this lineage. Interestingly, a reduced myoepithelial cell layer is found in the lactating mammary gland of the ERbeta knockout mouse in contrast to the wild-type controls (Forster et al, 2002). This led to the hypothesis that ERbeta may be involved in regulating pathways, which are required for the differentiation of the myoepithelial cell lineage in the mammary gland (Forster et al, 2002).

While proliferation and the basal phenotype have been associated with poor survival, no differences in clinical outcome were identified between high and low Ki67 or any markers of the basal phenotype in our ERalpha-negative breast cancer cohort. It is possible that the lack of association of any of parameters investigated here (ERbeta isoforms, Ki67 and caspase-3) with clinical outcome (disease-free survival and overall survival) is confounded by the variety of treatments the patient cohort later received. In addition, most other studies where Ki67 has been examined as a prognostic factor have included both ER-positive and ER-negative tumours in their cohorts (Trihia et al, 2003). It should also be noted that ERalpha-negative status in our cohort was defined by negative IHC and ligand binding assay. This definition eliminated 15% of an initial ERalpha-negative series selected only on the basis of ligand binding assay. A similar number of ERalpha IHC-negative tumours have been found to be positive by ligand binding assay (Huang et al, 1997). The basis for discrepancy between these two ERalpha assays has been a subject of past discussion in the literature (Huang et al, 1997), but is likely to reflect biological variables rather than tissue selection or composition, because of the design of our tumour bank. Therefore, the current study used stringently defined ERalpha-negative tumours and so was enriched for a generally more aggressive group of breast tumours.

In comparison to ERbeta1, the role of its variant, ERbeta2/cx, is even more unclear. Transient expression studies using human ERbeta2/cx, have shown that human ERbeta2/cx is unable to bind ligand and when overexpressed sufficiently can inhibit ERalpha transcriptional activity (Ogawa et al, 1998; Peng et al, 2003) but has little if any effect on ERbeta1 activity. In breast cancer ERbeta2/cx has been identified at both the RNA and protein levels (Saji et al, 2002; Esslimani-Sahla et al, 2004), and now with another antibody we have also shown the presence of ERbeta2/cx in both normal and neoplastic breast tissue. Most studies previously published suggested that ERbeta2/cx is increased in breast tumours compared to normal breast tissue (Omoto et al, 2002; Palmieri et al, 2004) and the relative expression of the ERbeta2/cx to ERbeta1 is likely to change during breast tumourigenesis. However, no studies focusing only on ERalpha-negative tumours have been published. A few studies have suggested hypotheses as to ERbeta2/cx function due to observed correlations and association with other prognostic markers and clinical outcome with or without treatment (Omoto et al, 2002; Esslimani-Sahla et al, 2004; Palmieri et al, 2004). Esslimani-Sahla et al (2004) showed that ERbeta2/cx expression was correlated with total ERbeta, which is in agreement with our observation in our ERalpha-negative series. However, among these studies contradictory conclusions have often been reached (Saji et al, 2002; Esslimani-Sahla et al, 2004; Palmieri et al, 2004). Our data suggest that in ERalpha-negative tumours, ERbeta2/cx expression is significantly associated with both increased AP-1 and NF-kappaB expression and that ERbeta1 may not be associated with these activities. This suggests that the different ERbeta isoforms may be involved in regulation of distinct pathways in these tumours or alternatively there is differential regulation of ERbeta isoforms by distinct pathways in these tumours.

The absence of any significant correlations between ERbeta isoforms and particularly total ERbeta with either overall or relapse-free survival is also in agreement with some other published studies (Hopp et al, 2004) but disagrees with other studies where increased ERbeta has been associated with better survival (Nakopoulou et al, 2004) and when patients were treated with tamoxifen alone, where an association was shown with better response to tamoxifen therapy (Murphy and Watson, 2006). However, in these latter studies the majority if not all the tumours assessed were ERalpha positive and so represent a different context where ERbeta is coexpressed with ERalpha. In the current study we have hypothesised that the function of ERbeta expressed alone will be different to that when ERbeta is coexpressed with ERalpha, and therefore we have looked at a distinct cohort of patients where their tumours are ERalpha negative.

These data support the hypothesis that the role of ERbeta expression is different when expressed alone, to its role when coexpressed with ERalpha in human breast cancer. This is specifically reflected in the present study, by the confirmation of a strong relationship of ERbeta1 with Ki67 in ERalpha-negative tumours, such that it seems likely that the addition of an ERbeta1 antagonist could be a potentially useful therapy in specific subsets of breast cancer patients in a clinical setting.

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Acknowledgements

GS was funded by a Postdoctoral Fellowship from the Manitoba Health Research Council (MHRC) and previously from the CancerCare Manitoba Foundation (CCMF). The research is supported by Canadian Institutes of Health Research (CIHR), Canadian Breast Cancer Research Inititative (CBCRI), CCMF and USAMRMC operating grants. We acknowledge the strong support of the CCMF for our facilities at MICB. The authors have no known conflicts of interests either financial or personal between themselves and others that might bias the work.