Estrogen receptor-beta activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNFalpha mediated

Abstract

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor-beta (ERbeta) in BPH and PCa. ERbeta agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFalpha knock-out mice fail to respond to ERbeta agonist, demonstrating the requirement for TNFalpha signaling. In human tissues, ERbeta agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133(+) enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERbeta causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERbeta agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERbeta agonist for treatment of PCa and/or BPH with or without androgen withdrawal.

Fig. 1.

Effect of selective ERβ agonist on prostatic apoptosis in ArKO (A) and wild-type (wt) (B) mice. (A) Apoptosis (%) in total tissue at 3 days in ArKO Control (Con), Castrate (Cx), ERβ agonist (ERβ), or ERα agonist (ERα)–treated mice. Apoptosis (%) in total tissue was subdivided into epithelial (further subdivided into luminal and basal epithelium) and stromal components. (B) Percentage apoptosis in total tissue at 3 days from wt Control (Con), Castrate (Cx), ERβ agonist (ERβ), or ERα agonist (ERα). Apoptosis (%) in total tissue subdivided into epithelial (luminal or basal) and stromal components. Values are mean ± SEM, n = 5 mice/group. nd, not detectable.*P < 0.05 vs. control.

Fig. 2.

ERβ agonist–induced apoptosis is androgen independent (A) and involves TNFα signaling (B). (A) Apoptosis (%) in total tissue subdivided into epithelial (luminal or basal) and stromal components at 3 days from control (Con), Castration (Cx), or ERβ agonist (ERβ)–treated ArKO mice. Cx+T, castrated mice receiving T supplementation; ERβ+T, ERβ mice receiving T supplementation; and Cx+ERβ, Cx mice treated with ERβ to maintain serum T levels. (B) Apoptosis (%) in wild-type mice (open bar) and TNFαKO mice (solid bar), after 3 days of vehicle (Con), Castrate (Cx), or ERβ agonist (ERβ). Values are mean ± SEM. Different superscripts indicate groups that are significantly different. P < 0.05; n = 5 mice/group.

Fig. 3.

Apoptotic pathways activated by ERβ agonist and castration in wt mice. (A) Expression and quantification of cleaved caspase-8 (open bars) and -9 (solid bars) in ERβ tissues. (B) Expression and quantification of cleaved caspase-8 (left) and -9 (right) in castrated tissues. In micrographs, arrows indicate cells positive for cleaved caspase-8 or -9. (Inset) Negative control. Values are mean ± SEM n = 5 mice/group. nd, not detectable. (Scale bar, A, 25 μm).

Fig. 4.

ERβ agonist–induced apoptosis in human xenograft tissues and cells. (A) Quantification of caspase positive cells in BPH-1 and RWPE-1 cells treated with vehicle (open bars) or ERβ agonist (closed bars). (B) Apoptosis (%) in epithelial (open bars) and stromal (solid bars) BPH tissue xenografts (n = 4 patients) after 3 days treatment with control (Con), castration (Cx), or ERβ agonist (ERβ). (C) ApopTag staining of ERβ–treated BPH xenografts. (D) Morphologically identifiable apoptosis in CKH-positive basal cells. (E) Percent apoptosis in unsorted BPH-1 cells treated with ERβ agonist-treated (solid bars) or vehicle-treated (open bars) controls. After further fractionation to enrich for α2β1^hi (basal) and α2β1^lo (luminal) populations, α2β1^hi cells were sub fractionated into CD133⁺ and CD133⁻ subpopulations (figure representative of two individual experiments; brackets show percent cells per fraction). (F) Quantification of caspase positive cells in PC3 and DU145 cells treated with ERβ agonist (solid bars) or vehicle control (open bars). (G) Quantification of apoptosis (%) in human PCa xenografts from three patients (P1, P2, and P3), treated with vehicle (Con; open bars), castration (Cx; gray bars), or ERβ agonist (ERβ; solid bars). (H) Caspase 8 in ERβ-treated PCa; inset negative control. Values are mean ± SEM; n = 4 replicates per group except in G, where n = 3. nd, not detectable. Analyses by Student’s t test (A, E, and F) or ANOVA (B and G). *P < 0.05 vs. control; ***P < 0.005 vs. control. (Scale bar, C, D, H, and I, 25 μm; F, 100 μm; and G, 200 μm.)