Selective Estrogen Receptor Modulators for BPH: New Factors on the Ground

REVIEW Selective estrogen receptor modulators for BPH: new factors on the ground

M Garg1, D Dalela1, D Dalela1, A Goel1, M Kumar1, G Gupta2 and SN Sankhwar1

As the current management of BPH/lower urinary tract symptoms by traditionally involved pharmacological agents such as 5alpha- reductase inhibitors and a1-adrenoceptor antagonists is suboptimal, there is definite need of new therapeutic strategies. There is ample evidence in literature that suggests the role of estrogens in BPH development and management through the different tissue and cell-specific receptors. This article reviews the beneficial actions of selective estrogen receptor modulator (SERM) and ERb- selective ligands, which have been demonstrated through in vitro studies using human prostate cell lines and in vivo animal studies. SERMs have anti-proliferative, anti-inflammatory and pro-apoptotic mechanisms in BPH, and also act by inhibiting various growth factors, and thus represent a unique and novel approach in BPH management directed at estrogen receptors or estrogen metabolism.

Prostate Cancer and Prostatic Disease (2013) 16, 226–232; doi:10.1038/pcan.2013.17; published online 18 June 2013 Keywords: selective estrogen receptors modulators; BPH; estradiol; estrogen receptor b

INTRODUCTION relevant articles were selected for the discussion of role of SERMs in BPH in BPH is a common urological problem of aging males. It occurs in this review. about 10% of men below the age of 40 and increases to about 80% by 80 years of age.1 Clinical BPH is a benign enlargement of the prostate, which ESTROGENS AND ITS RECEPTORS: ROLE IN BPH results in voiding urinary difficulties that may, sometimes, Though BPH typically manifests in later stage of life when 2 significantly impair the quality of life in older patients. androgen levels are declining, androgens are thought to be pre- Currently, the favored treatment option for lower urinary tract requisites for the development of BPH.4,5 With advancing age, symptoms due to BPH/BPE is medical therapy with a-blockers that plasma androgen levels decrease gradually while estrogen levels help in alleviating the symptoms by reducing the stromal tone, remain constant or decrease slightly, resulting in increased ratio of and androgen suppressors that counteract the static component. estrogen to androgen in plasma.4–6 In elderly men, the ratio of These two approaches give modest improvement in objective free estradiol (E2) to free testosterone (T) was observed to increase parameters such as peak flow rate and prostate volume regression by up to 40%.7,8 Although estrogen level remains fairly constant in but on discontinuation of these agents, both disease as well as the epithelium during the entire age range, they get increasingly symptoms tends to recur. As the present medical therapies are accumulated in the stroma with advancing age.9 This could be of focused mostly on relief of lower urinary tract symptoms and have patho-physiological importance for BPH development. some impact on benign prostatic enlargement, there is a definite Various animal studies have highlighted that BPH occurs with the need to find novel therapies that affect the basic pathophysiology stimulation of the prostate by estrogens.10,11 An animal model of of BPH. BPH demonstrated that in castrated dogs, treatment with estradiol Estrogens and selective estrogen receptor modulators (SERMs) alone without androgens led to development of squamous prostate have been shown to affect the prostatic cellular and stromal epithelial metaplasia,12 whereas estradiol when given along with 3 proliferation. The current review intends to examine the potential androgens resulted in glandular prostatic hyperplasia.10,12 These role of SERMs in management of BPH. studies clearly indicated that estrogens might exert a synergistic effect with androgens in inducing prostatic hyperplasia. Climent et al.13 investigated the direct effects of estradiol on the proliferation of BPH-derived prostate cells in culture. Estradiol À 7 À 6 METHODS (10 and 10 M) moderately increased the proliferation of A PubMed search was conducted with the keywords ‘estrogens in BPH’; stromal cells in culture without affecting the proliferation of ‘selective estrogen receptor modulators’; ‘tamoxifen in BPH’; ‘raloxifene’; epithelial cells. Symptomatic BPH patients reveal a 33% increase in and ‘phytoestrogens’ to identify published articles on role of estrogens in stromal volume compared with healthy controls.14 These findings BPH. The terms ‘SERM in prostate diseases’; ‘BPH pathogenesis’; ‘estrogen receptors’; ‘apoptosis in BPH’; and ‘inflammation in BPH’ were also support the fact that estrogens may have an important role in the searched out in PubMed. In addition, the ‘related articles’ search option pathogenesis of BPH predominantly for the stromal component. on PubMed and references of relevant articles were also looked for Estrogens exert their effect on target gene expression by with knowledge of experimental study done on same subject at our binding to specific intracellular estrogen receptors (ERs). These research center was utilized. At the end of literature research, the most receptors, which act as hormone-inducible transcription factors,

1Department of Urology, King George Medical University, Lucknow, India and 2Division of Endocrinology, Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow, India. Correspondence: Dr M Garg, Department of Urology, King George Medical University, Lucknow 226003, India. E mail: [email protected] Received 21 February 2013; revised 11 May 2013; accepted 15 May 2013; published online 18 June 2013 Selective estrogen receptor modulators M Garg et al 227 are of two distinct types ERa and ERb. By mediating estrogen activation of ERa, as demonstrated by using tissue recombination signaling and transcription, these receptors drive growth, technology. Using intact aromatase knockout mice, a study proliferation, differentiation and many other cellular processes. demonstrated that the administration of an ERb-specific agonist ERa and ERb have specific expression patterns and distinct abrogates existing hyperplastic prostatic pathology.26 phenotypes, leading to their different biological functions. In the In our study,3 we found that the viability and proliferation of normal prostate and BPH, ERa is localized to stromal cells, and human BPH-derived stromal cells was reduced dose dependently estrogen mediates its effects on the prostate epithelium through by SERMs at concentration ranging from 0.625 to 10mM. paracrine pathways.13,15,16 ERa acts as a mediator of cell 4-Hydroxytamoxifen most effectively and significantly reduced proliferation while ERb acts in opposite way by inhibiting ERa- stromal cell viability followed by DL-2-[4-(2-piperidinoethoxy) mediated proliferation.17–23 phenyl]-3-phenyl-2H-1-benzopyran (BP) and tamoxifen, which were almost equipotent. On the other hand, all these SERMs were nearly equipotent in reducing the proliferation of BPH ERb-MODULATION IN BPH: POTENTIAL BENEFICIAL EFFECTS stromal cells. 39 SERMs are generally nonsteroidal compounds that affect ER In a study by Yang et al., raloxifene antagonized the estrogen- signaling in the tissue-specific way. The SERMs exert selective stimulated proliferation in cultured prostate stromal cells. Estradiol effects on various tissues that are estrogen sensitive, including the (E2), when used alone, induced the proliferation of the WPMY-1 prostate. These SERMs may act as either agonists or antagonists (stromal cell line) by 31% (Po0.05). However, tamoxifen, depending upon the site of action, unlike the estrogens, which are raloxifene and finasteride antagonized the proliferation of these uniformly agonists, and the pure anti-estrogens, which are cells when used along with E2 by 16, 17 and 25% respectively uniformly antagonists.24 There is sufficient evidence which (Po0.05), showing that SERMs can have additive effects over and suggests that the beneficial effects of estradiol are mediated above the one exerted by finasteride. by ERb,25–27 while ERa might be responsible for aberrant proliferation. Therefore, it would be highly desirable to have Inhibition of growth factors agents that are capable of neutralizing the ill-effects of increased Various growth factors such as transforming growth factor-b1, estradiol signaling in the aged prostate by upregulating the ERb fibroblast growth factor and insulin-like growth factor-1 (IGF-1) are expression and downregulating ERa receptors. the key regulators of cell proliferation and extracellular matrix Different natural and synthetic ERb-selective ligands have been 40 28,29 turnover. These agents are all subject to alteration with age and identified, which provide the beneficial effects of estrogens are potential triggers for the initiation of stromal hyperplasia. while avoiding their unwanted side effects. The b-selective SERMs Prostatic IGF-1 gene expression has been correlated positively significantly increased the transcript and protein levels of ERb 41 3 with both BPH and prostate cancer, while epidermal growth and markedly reduced those of ERa in BPH stromal cells. Hence, factor receptor (EGFR) protein has been localized predominantly in SERMs have the potential of effectively controlling prostate BPH tissues.42 We found that ormeloxifene (a SERM) significantly hyperplasia through favorable modifications in intraprostatic downregulated IGF-1 expression, whereas hydroxytamoxifen and stromal–epithelial cross-talk by increasing signaling through ERb 3 3 tamoxifen also reduced EGFR level in BPH stromal cells. However, and simultaneously decreasing signaling through ERa. BP not only decreased IGF-1 and EGFR transcript levels, but also Various studies have suggested that this targeted approach for concurrently increased the transcripts of growth regulatory treating BPH through the use of SERMs, and more specifically ERb 26,30 peptide TGF-b1, whose impaired function has been directly agonist, would be of benefit. correlated with pathogenesis of BPH.43 Thus, SERMS may have Many epidemiological and experimental researches have shown potential to directly affect the levels of growth factors. that dietary estrogens are beneficial to men’s health.29,31,32 This may be evident from the fact that men living in Western nations have higher incidence of prostate cancer and BPH, plausibly Anti-inflammatory effects because of their lower dietary phytoestrogen intake, as compared Prostatic inflammation may be one of the important factors in the with their counterparts in Asian countries.33–35 Many of these pathogenesis of human BPH.44,45 BPH nodules are thought to be phytoestrogens are found to display ERb receptor selectivity, associated with chronic inflammatory infiltrates, mainly composed especially compounds whose core structures have isoflavone or of activated T-cells and macrophages.46–48 These inflammatory flavones group. Genistein, a naturally occurring SERM which cells produces cytokines (IL-2 and IFNg) that may support shows 22-fold selectivity for ERb, is an isoflavone usually found in fibromuscular growth in BPH.49 While ERa have been shown to soy.36 Liquiritigenin (component of an herbal extract) is another induce inflammation, ERb has been suggested to have anti- natural ERb-selective (20-fold) phytoestrogen, and the new ERb- inflammatory effects. Fispemifene,50 a novel SERM, showed anti- selective ligands may be synthesized using its pharmacophore.37 inflammatory action in the dorsolateral prostate by decreasing the number and density of inflamed acini. As reported by Sharma et al.,51 Sprague-Dawley rats on soy diet were found to have Different types of ERb-selective agonists have been classified as reduced risk of chronic prostatitis and this mechanism may be responsible for the lower incidence of BPH in Asian men 1. Agents which have higher binding affinity to ERb receptors as consuming soy.52,53 Though there are sufficient data that the 38 compared with a-receptors, for example, ERB-041 (WAY-202041) regular consumption of soy prevents the obstructive voiding of 2. Agents that bind to both ERs with equal affinity, but only BPH, confirmatory clinical evidence is still lacking. activate transcription with ERb, for example, MF101, nyasol and liquiritigenin 3. Agents which have both characteristics of greater binding and Pro-apoptotic effects transcriptional activity with ERb, for example, diarylpropionitrile. ERb also induces apoptosis in the prostate in addition to having anti-proliferative effect,25 and mechanism of ERb action is mediated by tumor necrosis factora (TNFa) and is androgen ERb-MODULATION IN BPH: HOW DOES IT WORK? independent.54 In our study3 we found that BP sensitizes BPH Anti-proliferative response stromal cells to caspase-8-mediated apoptosis though extrinsic The activation of ERb leads to an anti-proliferative response that is pathway by significantly upregulating the mRNA levels of TNF not influenced by alterations in systemic androgen levels or family death receptors and its ligands (Fas/FasL). Human BPH

& 2013 Macmillan Publishers Limited Prostate Cancer and Prostatic Disease (2013), 226 – 232 Selective estrogen receptor modulators M Garg et al 228 36 explants exposed to SERMs at 5.0 mM concentration for 7 days were as demonstrated in studies with knockout mice models. These studied, which revealed the apoptosis of mainly the stromal cells findings suggest that ERb-specific agonists have sufficient in BPH tissue, which was maximum in case of hydroxytamoxifen potential to be used as new agents for the pharmacological and minimum in case of ormeloxifene. Ki-67 labeling was notably intervention and management of prostatic hyperplasia. reduced in SERM-treated BPH explants as compared with control. Our in vivo studies in rat3 indicated that irrespective of the Ex vivo data by Glienke et al.55 clearly demonstrate that the molecular structure and mechanism of action, the SERMs administration of hydroxytamoxifen at concentrations from 10 to universally and significantly reduce prostate weight. This 20 mM induces apoptosis in human prostate stromal cells. The response was better in combination with a 5a-reductase induction of apoptosis was more effective with hydroxytamoxifen inhibitor, finasteride. We found that the ventral prostate of adult than with tamoxifen though clinical studies could not confirm the mature rats receiving tamoxifen, BP and ormeloxifene each at effects of tamoxifen treatment on BPH. 1.0 mg kg À 1 dose for 21 days regressed significantly by 37%, 32%, ERb agonists induce apoptosis in different cells of estrogen- 36% respectively. deficient aromatase knockout mice, that is, luminal, prostatic Histological investigations on human BPH tissue3 revealed that stromal and basal epithelial cells.54 ERb agonists fail to respond in finasteride, when used alone, effectively reduced acinar diameter the TNFa knockout mice, demonstrating TNFa-induced apoptosis but cause marginal decrease in epithelial cell height, while SERMs in the stromal cells. In addition to the direct effect of ERb agonists caused extreme thinning of epithelium along with dilation of acini. on the stromal nodules of BPH, they also disrupt stromal–epithelial This was accompanied with complete disappearance of epithelial interactions, which are important for proliferation and differentia- invaginations, a feature most prominently seen in BPH tissue. tion of prostatic epithelial cell.56 By targeting the stromal However, when both finasteride and SERM were used in component, the aberrant cycle of paracrine signaling breaks, combination, a substantial reduction in both epithelial cell and thus ERb agonists may affect both stroma and epithelial cells, height and acinar diameter was evident. Thus, SERMs can explaining their potential therapeutic use. The pathway of potentiate the action of 5-a reductase inhibitors for a better apoptosis induced by ERb agonist is different from that of response. castration. While ERb agonists induce apoptosis via activation Yang et al.39 studied the effect of raloxifene on prostatic stromal of caspase-8, castration does so by using caspase-957 and as hyperplasia in rats. Quantitative analysis of rat prostate indicated opposed to apoptosis induced by castration, ERb agonists cause that in the tamoxifen and raloxifene groups, the number of acini apoptosis in both the androgen-dependent or -independent cells was decreased, and the appearance of epithelial cells changed (Figure 1 and Table 1). from columnar to flat or low columnar type. In addition, the smooth muscle cell layer that surrounds the acini became thinner. Further improvement had not been seen with combination of ERb MODULATION: VALIDATION IN ANIMAL BPH MODEL AND finasteride and raloxifene compared with that of raloxifene alone. HUMAN CELL LINES Yang showed that both the proliferation of prostatic stromal and Recently, responses of ERb-selective ligands have been demon- epithelial cells could be inhibited by SERMs. The immuno- strated in various animal studies. Norman58 proved in rodent histochemistry staining results showed that raloxifene was found prostate models that involution of the ventral prostate occurs with to be a more potent agent that caused reversal of smooth muscle the use of ERb-selective ligands. Also with the administration of an cell thickness. Herein, authors provide the strong evidence that ERb-specific agonist, prostatic epithelial hyperplasia ablates raloxifene may have a beneficial role in the rat prostate whereas an ERa-specific agonist did not produce any such effect hyperplasia.

Figure 1. Proposed mechanisms involved in beneﬁcial effect of selective estrogen receptor modulator (SERMs) in BPH. EGFR, epidermal growth factor receptor; IGF-1, insulin-like growth factor-1; TNF-a, tumor necrosis factor a;TGFb, tumor growth factor.

Prostate Cancer and Prostatic Disease (2013), 226 – 232 & 2013 Macmillan Publishers Limited Selective estrogen receptor modulators M Garg et al 229 Table 1. In vitro experimental studies showing effects of SERMs in human BPH cell lines

Study Estrogen/SERMs Response

Climent K M et al.12 Estradiol Increased proliferation of BPH-derived prostate stromal cells in culture Kumar et al.2 4-Hydroxytamoxifen Reduced stromal cell viability. BP sensitizes BPH stromal cells to caspase-8- BP mediated apoptosis Tamoxifen Ormeloxifene. Rui-Yang et al.38 Raloxifene Antagonized the estrogen-stimulated proliferation in prostate stromal cells Tamoxifen Finasteride Yatkin E et al.49 Fispemifene Anti-inﬂammatory action by decreasing the number and density of inﬂamed acini Wolfgang Glienke et al.54 4-Hydroxytamoxifen Induces apoptosis in human prostate stromal cells Abbreviations: BP, benzopyran; SERM, selective estrogen receptor modulator.

Table 2. Animal and human studies showing response of SERMs in prostate diseases

Study SERMs/b-selective ligands Response

Norman ERb-selective ligands Involution of the ventral prostate in rodent prostate models et al.58 Kumar et al.2 Tamoxifen Significantly reduce prostate weight by 37%, 32%, 36%, respectively BP Ormeloxifene Yang Rui Tamoxifen and raloxifene groups No. of acini decreased. Epithelial cells changed from columnar to flat or low columnar et al.38 type McPherson ERb agonist 8b-VE2 Apoptosis in different prostatic cells in ArKO mice independent of androgens et al.53 Corrada et al.76 Tamoxifen Tamoxifen resulted in decreased prostate volume in seven beagles with spontaneous BPH Gonzalez Tamoxifen Randomized trial study in dogs BPH with 28.5% decrease in prostate volume et al.77 Steiner et al.78 Toremifene in the doses of 60 mg Significantly increased bone mineral density in 46 men of prostate cancer receiving a per day GnRH agonist Price et al.79 Toremifene In randomized study of 514 men with high-grade PIN, toremifene decreased incidence of prostate cancer by 1 year Abbreviations: ER, estrogen receptor; GnRH, gonadotropin-releasing hormone; PIN, prostatic intraepithelial neoplasia; SERM, selective estrogen receptor modulator.

Gene expression profiling along with studies in bERKO mice Ho and Habib64 in their recent study discussed that the further enhanced our knowledge about molecular basis of estrogens effect the prostate through various mechanisms, such ERb-mediated effects within cells and their importance in as aromatase expression and apoptosis, and via prostaglandin E2 pathophysiology of BPH (Table 2). and SERMs can influence these intraprostatic estrogen levels and Different theories emphasizing the causative mechanism of BPH are potential therapeutic targets for the treatment of BPH. pathogenesis have been described in literature. In dihydrotestos- With these studies, there is ample evidence that SERMs may be terone hypothesis, it is postulated that castration in men before of benefit for the treatment or prevention of BPH, and it is high puberty lead to failure of BPH to develop.59 Embryonic reawakening time to explore these agents through clinical prospective trials to theory60 enumerates reawakening of the embryonic induction ascertain their efficacy in treatment of prostatic hyperplasia, potential of prostatic stroma through involvement of various subject to satisfactory pharmacokinetic and toxicity testing.65,66 mechanisms including growth factors such as transforming growth factor.61 Last, in stem cell theory, BPH develops through stem cells number increase or in clonal expansion of transit or LIMITATIONS AND APPROACHES FOR IDENTIFYING ERb- amplifying cells.62 SELECTIVE LIGANDS As discussed, SERMs have beneficial effects on BPH by The ERb through regulating different physiological functions has a modifying the growth factors. More recently Hu et al.63 in their potential of becoming therapeutic target for various pathological experimental work cultured stem/progenitor cells from normal states. However, the clinical application of SERMs for the human prostate using a prostasphere assay and showed that treatment of BPH may not be as simple as it appears to be, as these cells express high levels of ER including ERa,ERb and GPR30 there are several other aspects that can complicate this issue, but not androgen receptors. These stem cells had a proliferative including the variability of ERa and ERb expression in prostate response on exposure to 1 nM estradiol-17b and are specific diseases. Because of the structural characteristics and similar targets for estrogen action in the prostate. On stimulatory ligand-binding cavity of ERa and ERb, it has been difficult to response of these receptors, augmented signals alter the normal identify ERb ligands with high affinity, potency and selectivity.67 cell proliferative and apoptotic pathways in a number of estrogen- Thus, it is challenging to develop ERb-selective ligands for target tissues including prostate. This molecular aspect highlights therapeutic use and this is one of the reason for the hinderance the potential for SERMs in targeting diseased stem cells by of use of these agents, although their beneficial effects are known modifying these signals for the treatment of prostate diseases. for long time. Furthermore, the effect of expression or function of

& 2013 Macmillan Publishers Limited Prostate Cancer and Prostatic Disease (2013), 226 – 232 Selective estrogen receptor modulators M Garg et al 230 ER splice variants and mutations on prostate growth has not been vertebral fractures in men with prostate cancer on androgen- fully understood.68,69 ERs may exert both ligand-dependant deprivation therapy compared with the placebo group. Although and -independent activities through genomic and non-genomic thromboembolic events were more frequent, there was a pathways.70,71Although these factors may be more crucial in significant improvement in bone mineral density and serum lipid prostate cancer as compared with BPH, these are the certain profile in toremifene group.86 Although in an another phase III aspects in estrogen signaling and transcription that require further randomized double-blind study of 3 years, in which 1590 patients investigation and research. with high-grade prostatic intraepithelial neoplasia were randomized Nevertheless, since the discovery of ERb in 1996, development to receive 20 mg toremifene or placebo, the toremifene was not of newer more selective ERb ligands has been a field of active found to decrease the incidence of prostate cancer compared with research, and great efforts are going on in this field.58,72,73 With that of placebo.87 These preliminary studies indicate that SERMs are the advancement in techniques, several different mechanisms well tolerated in men, and have been shown to even ameliorate the have been used to identify and create the ERb-selective ligands; side effects of androgen ablation therapy in men with prostate for example, Neubauer et al.74 developed a ERb agonist, which is a cancer.86,88 inhibitor of prostate growth but has no action on the immune Because of the continuous increment in our understanding of system, while a ERb agonist, which has immune suppressant behavior of ER in a tissue-selective manner and with the property without effecting the prostate, was described by Elloso developments of newer more specific SERMs and other b-slective et al.75 Structural core of known ERb-selective ligands are modified ligands, in future, these drugs may become integral part of to new compounds with greater selectivity and/or potency, such treatment regime of BPH therapy. as isoflavone core structure of genistein.76 By modifications of the phenolic hydroxyl groups or nitrile groups of ER77 or by using cells with stably integrated estrogen-responsive element genes, new CONCLUSION better molecules may be developed. Also it is now known With the discovery of ERb-specific SERMs, our knowledge about that ligand binding does not always correlate directly with the basic pathophysiology and underlying cellular signaling transcriptional activity, and ER ligands may be receptor subtype mechanism has been potentiated and this has opened up new selective in either concentration-dependent manner or at the pathways in pharmacological management of BPH. There are transcriptional level. With these aspects in mind, future various preclinical studies in literature that support the rationale development of SERMs or b-selective ligands may be designed 78 for evaluation of clinical application of SERM alone or in with dimer selectivity as well as tissue and subtype selectivity. combination with existing androgen blockade for the manage- Additional obstacle in BPH study is the lack of ideal ment of BPH. ERb appears to mediate beneficial effects by experimental model for BPH. Some animals such as dogs, mouse favorably altering the growth factor milieu, and exerting anti- and rodents have prostate that is hormone responsive and thus proliferative, anti-inflammatory and pro-apoptotic effects. suitable in BPH research, although their prostate anatomy varies significantly from that of humans.79 As such, bladder outlet obstruction resulting from BPH has been insufficiently described in these models. But with the genetic manipulations and CONFLICT OF INTEREST advancements in molecular altered pathways, some of these The authors declare no conflict of interest. problems have been tackled and transgenic animal models have been developed and utilized.80 Nicholson et al.81 recently described a model of BPH and bladder outlet obstruction REFERENCES induced in mice with testosterone (T) and 17b-estradiol (E(2)) 1 Tang J, Yang J. Etiopathogenesis of benign prostatic hyperplasia. 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