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. 2012;7(7):e40732.
doi: 10.1371/journal.pone.0040732. Epub 2012 Jul 10.

Estrogen receptor β activation impairs prostatic regeneration by inducing apoptosis in murine and human stem/progenitor enriched cell populations

Shirin Hussain  1 , Mitchell G Lawrence, Renea A Taylor, Camden Yeung-Wah Lo; APC BioResource; Mark Frydenberg, Stuart J Ellem, Luc Furic, Gail P Risbridger
Affiliations

Estrogen receptor β activation impairs prostatic regeneration by inducing apoptosis in murine and human stem/progenitor enriched cell populations

Shirin Hussain et al. PLoS One. 2012.

Abstract

Androgen depletion is the primary treatment for prostate disease; however, it fails to target residual castrate-resistant cells that are regenerative and cells of origin of prostate cancer. Estrogens, like androgens, regulate survival in prostatic cells, and the goal of this study was to determine the advantages of selective activation of estrogen receptor β (ERβ) to induce cell death in stem cells that are castrate-resistant. Here we show two cycles of short-term ERβ agonist (8β-VE2) administration this treatment impairs regeneration, causing cystic atrophy that correlates with sustained depletion of p63+ basal cells. Furthermore, agonist treatment attenuates clonogenicity and self-renewal of murine prostatic stem/progenitor cells and depletes both murine (Lin(-)Sca1(+)CD49f(hi)) and human (CD49f(hi)Trop2(hi)) prostatic basal cells. Finally, we demonstrate the combined added benefits of selective stimulation of ERβ, including the induction of cell death in quiescent post-castration tissues. Subsequent to castration ERβ-induces further apoptosis in basal, luminal and intermediate cells. Our results reveal a novel benefit of ERβ activation for prostate disease and suggest that combining selective activation of ERβ with androgen-deprivation may be a feasible strategy to target stem cells implicated in the origin of prostatic disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Two cycles of short term ERβ agonist administration impair ventral prostate regenerative capacity.
(A) Schematic of cyclic castration (Cx) and testosterone replacement over the course of 2 cycles and representative micrographs showing haemotoxylin and eosin (H&E) staining for histology of intact (day 0), 1 cycle (day 24) and 2 cycle (day 48) Cx-recovery tissue. Second row of images in this panel shows lack of mucinous Periodic Acid-Schiff’s (PAS) staining in castrate-recovery (Cx) tissue (B) Schematic of cyclic 8β-VE2 treatment over the course of 2 cycles along with representative micrographs showing H&E staining for histology of vehicle treated (day 0), 1 cycle (day 24) and 2 cycle (day 48) 8β-VE2-recovery tissue. CA denotes areas of cystic atrophy. Second row of micrographs depicts mucinous PAS positive staining in the secretions within the lumens of ERβ agonist treated recovery tissue. (Scale bar for all images is 20 µm). (C) Stereological analysis of percent atrophy (n = 4–5 animals/group, One-Way ANOVA with Tukey’s post-hoc analysis, aP<0.001 vs. corresponding castrate tissue, bP<0.05 vs. cycle 1 tissue 8β-VE2). (D) Frequency of p63 positive cells per 100 luminal cells (n = 7 animals/group, One-Way ANOVA with Tukey’s post-hoc analysis, aP<0.001 vs. corresponding castrate tissue, bP<0.001 vs. non-atrophic region). # indicates values from (8).
Figure 2
Figure 2. ERβ attenuates the clonogenicity and self-renewal of murine prostatic stem cells.
(A) Representative micrographs of ERβ immunohistochemistry of primary and secondary colonies extracted from Matrigel (Inset; isotype negative control, Scale = 50 µm) (B–D) Formation of primary spheroids from mouse prostate cells cultured in Matrigel for 7 days with vehicle or 6 µM 8β-VE2. (B) Images of primary colonies extracted from Matrigel on day 7 (Scale = 250 µm). (C) The median size (µm) of primary colonies (***P<0.0001, Mann Whitney test, n = 60). (D) Relative number of primary spheroids formed with vehicle or 8β-VE2 treatment (n = 7 mice **P<0.01, T test). (E) Relative number of secondary spheroids arising from pooled primary colonies replated for 7 days without treatment (n = 7 mice, **P<0.01, T test). (F) Median number of secondary spheroids derived from individually passaged primary spheres (**P<0.01, Mann Whitney test, n = 19).
Figure 3
Figure 3. ERβ induces apoptosis in regenerative murine and human prostatic basal cells.
(A) Representative flow cytometry plots showing live, dead and apoptotic cells following vehicle or 8β-VE2 treatment of digested mouse prostate. (B) Percent apoptotic cells following vehicle (grey bar) or 8β-VE2 (open bar) treatment of digested mouse prostate (n = 3 with 3 mice/group/experiment). (C) Representative flow cytometry plots showing gating for regenerative murine LSC cells. (D) Changes in the regenerative murine LSC population following vehicle (grey bar) or 8β-VE2 treatment (open bar) (n = 3 with 3 mice/group). (E) Representative flow cytometry plots following vehicle or 8β-VE2 treatment of human prostatic epithelial cells. (F) Changes in the regenerative CD49fhiTrop2hi population following vehicle (grey bars) or 8β-VE2 treatment (open bars). (n = 3 patients), **P<0.01 by students t-test.
Figure 4
Figure 4. ERβ treatment after castration induces a second wave of apoptosis in castrate-resistant cells, including basal, luminal and intermediate cells.
(A) Time course of total levels of prostatic apoptosis after 14 days of Cx followed sequentially by 3 days of vehicle (circles) or 8β-VE2 (triangles). Following treatment, groups are coloured green (vehicle) or red (8β-VE2) for clarity (n = 7/group). Representative micrographs show basal cell apoptosis (black arrows indicate apoptag+ staining) in 8β-VE2 treated tissues that is absent in vehicle controls. Breakdown of apoptotic levels in individual cell types following sequential treatment (B–D). (B) Stereological analysis (n≥7/group) showing levels of apoptosis in individual cell types in the prostates of mice treated with 14 day Cx +3 day vehicle (14dCx+3dV) or 14 day Cx +3 day 8β-VE2 (14dCx+3dβ). (C) Representative micrograph of immunofluorescent dual labeling in mouse ventral prostate with CKHMW (basal cell marker, green) and CK18 (luminal cell marker, red) used to identify intermediate cells (dual stained in yellow and indicated by white arrow) for quantitation in sequentially treated tissues. (D) Relative numbers of intermediate cells following 14 days of Cx and 3 days of vehicle (grey bars) or 8β-VE2 (open bars) (n≥5/group). (nd; non-detectable, *P<0.05, **P<0.01, ***P<0.005 by Mann Whitney test (B) or students t-test (D)). (Scale A = 20 µm, C = 10 µm).
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