Prostate cancer with a high Gleason grade is a highly aggressive disease and, currently, the major target for treatment is the androgen receptor (AR). Recent literature points towards a tumour-suppressive role of oestrogen receptor (ER) β, which has the potential to be exploited as a target for novel therapeutics and treatment of prostate cancer. ERβ was discovered by our group in 19961 and is expressed below a Gleason grade of 3 and then declines, but is re-expressed in high-grade prostate cancer. Therefore, it is a potential target at the initial stage of the disease as well as the late stage.
The effects of oestrogen on prostate cancer are mainly mediated via the ERs. The human prostate expresses two ERs, from two independent genes that are located in different chromosomes: ERα and ERβ.2 They belong to the large superfamily of transcription factors called nuclear receptors. ERα was cloned in 1985,3 the protein is 595 amino acids long and the molecular weight is 66 kDa.4 ERβ was cloned in 1996, the protein is 530 amino acids long and the molecular weight is 59.5 kDa.1 The two receptors are structurally similar and can be divided into six regions (A–F) and three domains [N-terminal domain (NTD), DNA-binding domain (DBD) and ligand-binding domain (LBD)]. They share ≈97% similarity in their DBD and 59% in their LBD, whereas the NTD is only 16% similar.5 The ERs demonstrate unique binding affinities to different ligands and specific oestrogen receptor modulators (SERMs). For example, ERα has higher affinity towards estradiol (E2) than ERβ, whereas ERβ has higher affinity towards genistein and other phytoestrogens.6–8 In addition, ERβ demonstrates a higher affinity towards the prostate- and brain-specific ligand 5α-androstane-3β,17β-diol (3β-Adiol) than ERα.9 These studies have led to the concept that ERs demonstrate unique functions depending on the ligands/SERMs they interact with. This is further complicated by the binding sites on DNA. For example, ERβ-E2 acting through AP1 represses target genes and ERα-E2 acting through AP1 activates target genes.10
Oestrogen receptor α is primarily expressed in the stromal compartment and ERβ in the basal and luminal epithelial compartments of normal prostate cells.11–15 However, in cases of prostate cancer, ERα is expressed in the prostate luminal epithelium.16 In cases of benign prostatic hyperplasia (BPH), primarily a disease involving stromal expansion of the prostate gland, overexpression of ERα is involved in its aetiology. Studies using ERα-knockout mice have shown that epithelial prostatic intraepithelial neoplasia (PIN) lesions, stromal hyperplasia and inflammatory cell infiltration are mediated by ERα.17 The use of the ERα antagonist toremifene has shown some promise in prevention of development of prostate cancer in men with high-grade PIN lesions.18
The expression of ERβ is low at birth and its expression is highest at puberty. During development, ERβ is expressed in the epithelium and stroma of the urogenital sinus and is involved in cell differentiation and morphogenesis.19 In addition, ERβ is expressed in cases of BPH and low Gleason grade prostate cancers, but is lost in cases with a Gleason grade above 3. There are reports that ERβ expression returns in metastatic prostate cancer,20,21 but this observation requires further investigation because of the presence of ERβ splice variants that demonstrate proliferative and metastatic ability.22,23 A recent study in Mongolian gerbils showed that ERβ is lost above 1 year of age and these animals spontaneously developed prostate cancer at 18 to 36 months.24
The Prostate Cancer Prevention Trial has further emphasized the importance of ERβ in prostate cancer. In this trial, the prevalence of prostate cancer was reduced by 25% among patients treated with the 5α-reductase inhibitor finasteride. However, the rate of cancers with a higher Gleason grade (7–10) was higher in the finasteride-treated group (37%) than in the placebo-treated group (22.2%).25 The possible explanation for this anomaly is that the inhibition of 5α-reductase by finasteride also caused a decrease in 3β-Adiol, a metabolite of dihydrotestosterone.26 It has now been shown in multiple studies that 3β-Adiol is an endogenous ligand of ERβ and its inhibition can abrogate the antiproliferative function of ERβ.27,28
Until 1996, all the actions of oestrogen were considered to be mediated via ERα, the only ER known at that time,3 but thereafter ERβ was discovered,1 and this has changed the way we construe oestrogen signalling. The ERβ gene is not an isoform of ERα, but is encoded by a distinct gene located on chromosome 14 (14q23.2) whose gene product is 530 amino acids long.2,29 ERβ was first cloned in a rat prostate and later it was found to be expressed in other reproductive areas such as the testicles, ovaries and endometrium.2 In humans, it is ubiquitously expressed, with maximum expression in the brain, lung, immune system, breast, prostate, ovary and testicles. ERβ is the predominant ER in the human prostate gland and its role is to maintain prostate epithelium in a differentiated state and to oppose the proliferative action of the AR.30,31 This concept was supported by the study of ERβ-knockout mice, in which the expression of differentiation markers is diminished on ERβ knockdown.32 ERβ-knockout studies in mice have shown that the absence of ERβ causes epithelial hyperplasia.31
Our knowledge of the role of ERβ in prostate cancer pathogenesis is still evolving, especially in the last few years, when a large number of mechanistic studies have demonstrated antiproliferative and pro-differentiation activities of ERβ in the prostate.22,31,33,34
Role of oestrogen receptor β as an antiproliferative factor
The first ERβ–/– mice were created in 1998 by insertion of a neomycin cassette in exon 3.35 The 5-month-old ERβ–/– mice showed prostatic hyperplasia and knockout mice older than 1 year showed distinct signs of PIN.31 This is the first of a series of observations made in mouse models and cancer cell lines, demonstrating an antiproliferative function of ERβ. Furthermore, treatment with 3β-Adiol decreased prostatic cell proliferation in wild-type but not in ERβ-knockout animals.9 Studies in the adenocarcinoma of the mouse prostate cancer model showed that dietary genistein reduced the number of cases of prostate cancer in wild-type mice but not in ERβ–/– mice.36
In a recently published study,22 we found that ERβ decreases the expression of proliferative and oncogenic factors such as p45Skp2, cMyc and cyclin E in PC3 and 22Rv1 prostate cancer cell lines. Furthermore, the expression of cell cycle inhibitors such as p21WAF1 [cyclin-dependent kinase (CDK) inhibitor 1A] and p27Kip1 (CDK inhibitor 1B) was increased substantially by ERβ expression. Proliferation studies using bromodeoxyuridine incorporation, MTT [3-(4,5-dimethylthiazol-2-yl)–2,5-diphenyltetrazolium bromide] assays and cell counting also pointed towards the antiproliferative function of ERβ in prostate cancer. Moreover, mouse xenografts with PC3 cells expressing ERβ showed a greater reduction in tumour burden than control cells. Microarray analysis showed a strong correlation with ERβ expression and reduction in proliferation genes such as p45Skp2 (SKP2), Cyclin A2 (CCNA2), CDK14, CDCA7, CDCA4 and CKS1B.22 Furthermore, overexpression of ERβ in lymph node carcinoma of the prostate (LNCaP) cells caused G1 cell cycle arrest.37 In another study, we found that ERβ up-regulates FOXO3a.38
A recent new study39 found that the phytoestrogens angolensin and cosmosiin, targeting specifically the ERα/β heterodimer, have an antiproliferative effect.
The discovery of the transmembrane protease, serine 2 (TMPRSS2):erythroblast transformation-specific (ETS) fusion gene, which is associated with a very aggressive form of prostate cancer, is considered to be a milestone in the field of prostate cancer biomarkers. This fusion gene is present in ≈80% of prostate cancer cases. TMPRSS2 is an enzyme whose expression is up-regulated by androgen acting through the AR.40 In certain forms of prostate cancer, TMPRSS2 translocates to the promoter region of ETS family of transcription factors, such as ERG and ETV1, through gene fusion. This chromosomal translocation followed by gene fusion contributes to the development of castration-resistant prostate cancer. Interestingly, the formation of TMPRSS2:ERG is opposed by ERβ, as has been shown in a large meta-analysis41 conducted on biopsy samples from 455 prostate cancer patients in the Swedish Watchful Waiting cohort (1987–99) and the US-based Physicians Health Study cohort (1983–2003). This observation was validated in NCI-H660 prostate cancer cells, showing that inhibition of TMPRSS2:ERG fusion is caused by an ERβ agonist, but not by an ERα agonist.41,42
Oestrogen receptor β and apoptosis
In a study using prostate cancer cell lines (PC3, LNCaP and DU145) treated with the DNA demethylating agent 5-AZAC and the histone deacetylase inhibitor trichostatin A, ERβ expression was increased followed by an increase in caspase activity and apoptosis.43 Adenovirus-mediated delivery of ERβ led to an increase in poly(adenosine diphosphate ribose) polymerase cleavage and increase in the pro-apoptotic factor Bax in DU145 cells. In addition, cleaved caspase 3 was increased on ERβ expression, promoting apoptosis.44 Recent data showed that raloxifene, a SERM, induces apoptosis in prostate cancer cells by decreasing antiapoptotic factor Bcl-2 and increasing expression of Par-4 and caspase 3 activity. This effect of raloxifene was observed in both androgen-dependent epithelial cells and androgen-independent cancer prostate epithelial cells (derived from prostate cancer specimens).45
A recently published paper46 used the ERβ-selective agonist 8β-VE2 for treatment of castration-resistant prostate cancer. Androgen deprivation therapy (ADT) is unsuccessful in prostate cancer after an initial response to therapy, which is partly because the basal epithelial cells of prostate harbour stem/progenitor cells are insensitive to AR signalling. The prostate initially responds to ADT by undergoing apoptosis, mostly in the secretory luminal cells, but the stem/progenitor cells cause prostatic regeneration. The study found that, after two rounds of treatment and recovery of castrated wild-type mice, 8β-VE2 inhibited the regeneration ability of the prostate whereas this effect was not detected in untreated mice.46 The staining of the basal cell marker p63 showed that the treatment with 8β-VE2 depleted the site of atrophy of p63-positive cells, implying that the basal cells are required for the regeneration of prostate post ADT. Moreover, flow cytometric analysis of single-cell suspension prepared from mouse prostate demonstrated that stem cell populations were significantly reduced by 8β-VE2 treatment.46
In another publication from the same laboratory,47 the authors reported that ERβ mediated apoptosis via the extrinsic pathway that involves caspase 8. Castration causes apoptosis in the luminal epithelial and stromal cells, but the basal epithelial cells remain untouched. They found that, in both wild-type and aromatase-knockout mice, the stromal and epithelial (luminal and basal) cells undergo apoptosis on treatment with an ERβ-specific ligand and this effect is androgen independent. This is in sharp contrast to ERα ligands that cause prostatic inflammation. The mechanism for apoptosis involves tumour necrosis factor (TNF) α, as apoptosis was not observed in TNFα-knockout mice. TNFα causes activation of caspase 8 following activation by ERβ, which further activates caspase 3.47
We found that ERβ induces apoptosis by up-regulation of FOXO3a, a forkhead transcription factor class-O family member. The mechanism involves an increase in pro-apoptotic factor p53 up-regulated modulator of apoptosis (PUMA), a direct target of FOXO3a, but is independent of p53. Using prostate cancer cells (LNCaP, PC3 and 22Rv1) and ERβ-specific ligands (3β-Adiol, 8β-VE2 and diarylproponitrile), we showed that ERβ transcriptionally up-regulates FOXO3a, and this pathway is independent of phosphoinositide-3-kinase/protein kinase B (Akt) signalling. A study with siRNA for ERβ and FOXO3a also validated the hypothesis that the ERβ regulation of apoptosis via PUMA requires FOXO3a.38 Furthermore, a study38 using ERβ-knockout mice showed that FOXO3a expression correlates with ERβ expression. Prostate cancers of a higher Gleason grade, which show progressive decrease in ERβ expression, also show diminished FOXO3a expression. All of these studies demonstrate potentially beneficial effects of ERβ, which can be exploited in the treatment of prostate cancer.
The role of oestrogen receptor β in invasion and epithelial mesenchymal transition
Adenovirus-mediated delivery of ERβ caused a strong decrease in invasiveness of DU145 cells compared with control transfected cells.44 Another study using xenograft of DU145 cells showed that 3β-Adiol activated ERβ-blocked metastasis of prostate cancer cells.49 The antimigratory phenotype observed was because of the up-regulation of E-box protein, E-cadherin, by ERβ.48
In a seminal paper by Mak et al.,28 it was reported that ERβ promotes the epithelial phenotype and opposes epithelial to mesenchymal transition (EMT) in the prostate. The mesenchymal phenotype was independent of AR status as both androgen-dependent and -independent cell lines exhibited this phenomenon. The authors hypothesized that the mesenchymal phenotype exhibited by a high Gleason grade prostate cancer is associated with decreased expression of ERβ, loss of E-cadherin and increase in N-cadherin/vimentin expression. Knockdown studies in prostate cancer cell lines have demonstrated that the pathway is ERβ dependent and is not affected by ERα status.28 Mak et al.28 suggested that transforming growth factor (TGF) β and hypoxia cause a decrease in ERβ expression and promote migration and invasion, which are features of EMT. Moreover, following treatment with the ERβ-specific ligand, 3β-Adiol, the cells exhibited a more epithelial phenotype and increased E-cadherin expression. Mak et al.49 further established that hypoxia-inducing factor-1α (Hif-1α), a key sensor of hypoxic state in a cell, is reciprocally regulated by ERβ through induction of prolyl hydroxylase 2, which increases proteasomal degradation of Hif-1α. Knockdown of ERβ increases Hif-1α expression, whereas hypoxia decreases ERβ expression, possibly via Hif-1α. ERβ also decreases the expression and secretion of VEGF-A, a Hif-1α target gene which has previously been shown to be involved in EMT via its binding partner, neuropilin-1. VEGF-A is a direct target of ERβ, as indicated by the presence of an oestrogen response element on its promoter. In addition, the VEGF-A promoter carries a Hif-1α binding site, thus providing two mechanisms of regulation of VEGF-A by ERβ. In addition, ERβ was found to regulate GSK-3β by inhibiting its phosphorylation via Akt signalling. GSK-3β decreases the translocation of Snail to the nucleus, a feature seen in EMT. Overall, this paper highlights the point that the Hif-1α/VEGF-A/Snail pathway required for the genesis of EMT is regulated by ERβ.28
We have reported that ERβ represses the expression of bone metastasis factors Runx2 and Dickkopf homologue 1 and the EMT factor slug. The study was conducted using overexpression of ERβ in prostate cancer cells (PC3 and 22Rv1) and in a PC3 cell xenograft. The study also found down-regulation of β-catenin transcription and activity by ERβ.22
An interesting study described that ERβ opposes cell migration and motility of DU145 cells following activation by 3β-Adiol, which regulates E-cadherin.50 This antimigratory effect of ERβ was opposed by TGFβ signal-derived reactive oxygen species (ROS) such as H2O2. The H2O2 produced in the reactive stroma cells by the action of cyclooxygenase-2 (COX-2) prevented the binding of ERβ to the promoter of E-cadherin. Agents neutralizing H2O2 and COX-2 could reverse the inhibition of ERβ by ROS.50
Research on hypoxia in tumour microenvironment is rapidly gaining attention as it has been found to be involved in regulating a large number of survival pathways such as energy metabolism, EMT, mTOR and angiogenesis.51
In a normoxic state, Hif-1α is degraded following hydroxylation by prolyl hydroxylases (PHDs), which target the degradation by promoting the interaction with von Hippel–Lindau, a component of an E3 ubiquitin–ligase complex.52 It has been found that ERβ specifically transcriptionally up-regulates PHD2, an isoform of PHD, which causes degradation of Hif-1α. This is an important finding with potential therapeutic value.49
Oestrogen receptor β as a potential drug target
A diet rich in soya is beneficial for prostate cancer prevention. Soya food contains phytoestrogens such as genistein, indole-3-carbinol and resveratrol. Studies have demonstrated that the beneficial effect of phytoestrogens is mediated through ERβ by increasing p21 and decreasing ARs.53,54 A study conducted in Australia reported that PSA levels were decreased in prostate cancer patients treated with a high-phytoestrogen diet compared with those consuming a low-phytoestrogen diet.55 These studies, in combination with the vast number of reports from our laboratory and from other authors reviewed above, provide compelling evidence that ERβ-specific ligands/agonists should be considered for prostate cancer management.