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

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.

Figure 1

(A) Validation of ERβ2/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 ERβ2/cx, magnification × 500; (b) same as (a), magnification × 1250; (c) Serotec clone 57/3 antibody staining of section from cell pellet of doxycycline treated tet-on-MCF7 cells stably overexpressing ERβ2/cx, magnification × 500; (d) same as (c), magnification × 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 ERβ2/cx, magnification × 500; (f) same as (e), magnification × 1250; (g) Serotec clone 57/3 antibody staining of section from cell pellet of doxycycline treated tet-on-MCF7 vector alone control cells, magnification × 500; (h) same as (g), magnification × 1250; (i) Serotec clone 57/3 antibody staining of section from cell pellet of doxycycline treated MCF7 stably overexpressing ERβ1 (Murphy et al, 2005), magnification × 500. (B) Expression of ERβcx/2 in ERα-negative invasive tumours and normal breast tissue detected by IHC is demonstrated in representative panels. (a) Tumour core stained with the specific ERβcx/2 antibody (high H-score, 270); (b) tumour stained for ERβcx/2 (low H-score, 25); (c) tumour core showing negative staining for ERβcx/2 H-score, 0); (d) normal breast tissue showing strong, nuclear ERβcx/2 protein expression; (e) nuclear ERβcx/2 expression in normal breast ducts; (f) negative control (omission of ERβcx/2 antibody). Magnification × 500 for a, b, c, and × 1250 for d, e, f.

Figure 2

Expression of ERβ and Ki67 in ERα-negative tissue microarray cores. (A–C) ERα-negative tumour cores stained with the specific ERβ1 antibody (GC17/385P) showing negative, medium and high expression (a–c; H-scores of 0, 150 and 225, respectively); (D–F) ERα-negative tumour cores stained with total ERβ antibody (14C8) showing negative, low and high expression (H-scores of 0, 25 and 100, respectively); (G–I) ERα-negative tumour cores showing negative, medium and high expression for Ki67, a proliferation marker (% positive, 0, 60 and 90%, respectively). Magnification × 500.

Figure 3

Kaplan–Meier graphs for ‘overall survival’ and ‘relapse-free survival-time to progression’ with respect to expression of ERβ1 (A and B), ERβ2cx (C and D) and total ERβ isoforms (E and F, respectively). ERβ1 overall survival (A), n=210, low ERβ1 events=47, high ERβ1 events=60. ERβ1 time to progression (B), low ERβ1 events=48, high ERβ1 events=60. ERβ2cx overall survival (C), n=199, low ERβ2cx events=44, high ERβ2cx events=53. ERβ2cx time to progression (D), low ERβ2cx events=44, high ERβ2cx events=53. Total ERβ overall survival (E), n=192, low total ERβ events=40, high total ERβ events=55. Total ERβ time to progression, low total ERβ events=40, high total ERβ events=56.