Estrogen receptor beta2 and beta5 are associated with poor prognosis in prostate cancer, and promote cancer cell migration and invasion

Abstract

Estrogens play a pivotal role in the development and progression of prostate cancer (PCa). Their actions are mediated by estrogen receptors (ERs), particularly ERbeta in the prostate epithelium. With the discovery of ERbeta isoforms, data from previous studies that focused principally on the wild-type ERbeta (ERbeta1) may not be adequate in explaining the still controversial role of ERbeta(s) in prostate carcinogenesis. In this study, using newly generated isoform-specific antibodies, immunohistochemistry (IHC) was performed on a tumor microarray comprised of 144 specimens. IHC results were correlated with pathological and clinical follow-up data to delineate the distinct roles of ERbeta1, ERbeta2, and ERbeta5 in PCa. ERbeta2 was commonly found in the cytoplasm and was the most abundant isoform followed by ERbeta1 localized predominantly in the nucleus, and ERbeta5 was primarily located in the cytoplasm. Logistic regression analyses demonstrated that nuclear ERbeta2 (nERbeta2) is an independent prognostic marker for prostate specific antigen (PSA) failure and postoperative metastasis (POM). In a Kaplan-Meier analysis, the combined expression of both nERbeta2 and cytoplasmic ERbeta5 identified a group of patients with the shortest POM-free survival. Cox proportional hazard models revealed that nERbeta2 predicted shorter time to POM. In concordance with IHC data, stable, ectopic expression of ERbeta2 or ERbeta5 enhanced PCa cell invasiveness but only PCa cells expressing ERbeta5 exhibited augmented cell migration. This is the first study to uncover a metastasis-promoting role of ERbeta2 and ERbeta5 in PCa, and show that the two isoforms, singularly and conjointly, have prognostic values for PCa progression. These findings may aid future clinical management of PCa.

Figure 1

Specificity of ERβ isoform-specific antibodies. Western blot analyses: HEK293 cells expressing ERβ1–5 lysate were tested against ERβ pan antibody ((A) Santa Cruz H150), ERβ2-specific antibody (B), and ERβ5-specific antibody (C). Lane 1 was loaded with control cell lysate (HEK293 cells only), whereas lanes 2–6 were loaded with HEK293 cell lysate expressing ERβ1–5 respectively. M stands for protein marker (Invitrogen SeeBlue Plus2 protein ladder). The expected protein band of the ERβ isoforms 2 and 5 was labeled by an arrow in the middle and right panel respectively. Immunohistochemical (IHC) analyses: IHC analyses of benign prostatic tissue were carried out using ERβ2- (D–I) and β5-specific antisera (J–O). ERβ1-, ERβ2-, ERβ3-, ERβ4-, and ERβ5-specific peptides were applied to the IHC analyses. Figures showing IHC analyses with or without ERβ isoform-specific peptide are arranged as follow: D and J (no peptide); E and K (+ERβ1 peptide); F and L (+ERβ2 peptide); G and M (+ERβ3 peptide); H and N (+ERβ4 peptide); I and O (+ERβ5 peptide).

Figure 2

Immunohistochemical analyses of ERβ isoforms in benign prostate glands (A–F) and prostate adenocarcinoma in PCa specimen (G–M). PCa specimens from cancers with Gleason score 6 (G–M) and its adjacent normal region (A–F) were immunostained with ERβ1-, ERβ2- and ERβ5-specific antibodies/antisera. See Materials and methods for the experimental conditions. Figures D–F and J–M are the magnified view (×630) of a region (marked by a rectangle) in Figure A–C and G–I (×100) respectively. In ERβ1 immunostaining, positive signals were found mostly in the nuclei of basal epithelial cells in benign foci (A and D) and in the nuclei of PCa cells (G and J) despite sparse nuclear staining in stromal cells (indicated by light blue arrow heads in figures D and J). ERβ1 staining was lost in some PCa foci (K). ERβ2 staining is highly cytoplasmic in both benign (basal and luminal epithelial cells, B and E) and PCa foci (H and L). Immunopositive signals of ERβ5 were observed in the cytoplasmic region of the epithelial cells in adjacent normal foci (C and F) as well as in PCa cells in Gleason score 6 cancer (I and M).

Figure 3

Subcellular localization of ERβ isoforms in PC3 cells. Vectors carrying ERβ1 (E–H), ERβ2 (I–L), and ERβ5 (M–P) were transiently transfected into PC3 cells, and expressed in the form of fusion protein with an N-terminal YFP tag. Cells transfected with YFP only serve as a control (A–D). Cells were counterstained with nucleus-specific (DAPI, B, F, J, and N) and mitochondria-specific fluorescent dyes (Mito-tracker red, C, G, K, and O). Subcellular localization of each isoform was determined by fluorescence microscopy. Extended focus was applied to average and combine three optical sections of each signal. Merged images of YFP, DAPI, and Mito-tracker red signals are shown in D, H, I, and P.

Figure 4

Typical IHC results of ERβ immunostaining on TMAs. TMAs with 144 specimens were immunostained with ERβ1 (A–C), ERβ2 (D–E), and ERβ5 (G–I) antibodies/antisera. The Allred scoring system (Allred score=signal score+intensity score) was used to grade the immunostaining signals. Typical low Allred-scored sections (Allred=0+1 for nuclear positivity; Allred=1+2 to 1+3 for cytoplasmic positivity) are shown in A, D, and G. Higher Allred-scored sections (Allred=3+5 for both nuclear and cytoplasmic positivity) with strong nuclear and cytoplasmic staining are shown in B, E, and H. C, F, and I represent a magnified view (×630) of a region (marked by a rectangle) in B, E, and H respectively. Negative nuclear staining in C, F, and I is marked by solid arrows.

Figure 5

Distribution of ERβ isoforms in this prostate cancer TMA. The immunopositivity signal of each isoform was graded according to the Allred scoring system. Only an Allred score greater than cutpoint (i.e. Allred>3) was considered positive. The number of patients showing positive immunostaining signals in different cellular compartments was determined and analyzed. The correlations between two ERβ isoform expression were determined based on Spearman correlation test. P<0.05 was considered to be statistically significant. An asterisk represents a specific group of patients with a higher possibility of showing the worst clinical outcome.

Figure 6

Evaluation of ERβ isoforms as a predictor for time to PSA failure and time to metastasis by the Kaplan–Meier (KM) plot with the log-rank test. The Allred cutpoint (>3 or ≤3) for each ERβ isoform was used to determine ‘positive’ and ‘negative’ expression. (A) KM plot of nuclear ERβ2 versus recurrence/PSA-free survival (P<0.01). (B) KM plot of nuclear ERβ2 versus metastasis-free survival (P<0.01). (C) KM plot of cytoplasmic ERβ5 in metastasis-free survival (P=0.03). (D) KM plot of nuclear ERβ2 and cytoplasmic ERβ5 versus metastasis-free survival (P<0.01). Patients were further stratified into four groups according to status of ERβ isoform expression: i) both nuclear ERβ2 and cytoplasmic ERβ5 positive (n=9); ii) nuclear ERβ2 positive and cytoplasmic ERβ5 negative (n=12); iii) nuclear ERβ2 negative and cytoplasmic ERβ5 positive (n=46); and iv) both nuclear ERβ2 and cytoplasmic ERβ5 negative (n=68). P<0.05 was considered to be statistically significant.

Figure 7

Tumor metastasis of PC3 expressing ERβ isoforms. (A) Results of wound-healing assay. Migration distance of PC3 expressing LacZ (as a control), ERβ1, ERβ2, and ERβ5 was recorded and calculated after 24 h of wound introduction. Experiments were carried out in triplicate and were repeated with three independent sets. Student's t-test was used to compare the mean distance migrated of each ERβ isoform-expressing cell line versus LacZ control cells (average±s.e.m., *P<0.05). (B) Results of Matrigel-based invasion assay. Fixed number (5×10⁴) of cells was set up in a 24-well plate according to the manufacturer's recommendation. The cells that crossed the matrigel membrane were stained and counted under a microscope. Cell numbers were normalized by the MTS method. The fold invasiveness was calculated relative to the number of cells invaded in LacZ control. Experiments were carried out in triplicate and were repeated with three independent sets. Student's t-test was used to compare the fold invasiveness of each cell line versus LacZ control cells (average±s.e.m., *P<0.05).