- Email: [email protected]
Estrogen Receptor β as a Pharmaceutical Target
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Special Issue[5_TD$IF]: Precision Medicine
Review
Estrogen Receptor b as a Pharmaceutical Target Margaret Warner,1 Bo Huang,1 and Jan-Ake Gustafsson1,2,* A major issue in clinical endocrinology[6_TD$IF] today is how to use hormones to achieve the health benefits that they clearly can provide but avoid the negative side effects, that is, how to develop more precise medicines. This problem of how to use hormones is pervasive in clinical endocrinology. It is true for estrogen, progesterone, androgen, vitamin D, and thyroid hormone, and the problem is amplified in the case of new ligands for the more recently discovered nuclear receptors. Selective targeting of hormone receptor subtypes is one attractive way to harness the beneficial effects of hormones while reducing unwanted side effects. Here, we focus on estrogen receptor (ER)b, which has promise as a selective target in hormone replacement therapy, and in breast and prostate cancers. Nuclear Receptors and Their Ligands Although the receptors for five major steroid hormones have been identified, we know that there are more nuclear receptors than those that mediate the actions of identified hormones [1,2] but we do not know whether any of the ligands will turn out to be steroids that are at present classified as inactive metabolites of known steroid hormones or, indeed, whether such metabolites are modulators of signaling of the known steroid receptors. Ligands for nuclear receptors are not all steroids but include cholesterol metabolites, fatty acids, and metabolites of vitamin A [3]. The major classical reproductive, thyroid, and adrenal hormones are part of the hypothalamic–pituitary–gonadal/adrenal/thyroid axes. However, there are endogenous ligands for nuclear receptors that do not fall into this category. In recent years, an oxysterol, 27-hydroxy cholesterol, has been identified as an agonist of ER/ [4] and an antagonist of ERb [5]. The significance of this novel estrogen is under investigation but it reinforces the point that there is a lot to be learnt even about receptors that have been targeted in health and disease for decades. The fact that these two ERs have varying distributions in the body (Figure 1) and display differences in their pharmacology (Figure 2) opens the door for more precise modulation of estrogen signaling, as discussed below. Oxysterols: Oxysterols are endogenous ligands for the nuclear receptor liver X receptor (LXR) [6,7]. There is no clear evidence that LXRs are part of an endocrine system (no secretory gland, although several target organs have been identified). Instead, they may be part of a paracrine or autocrine system. In addition to their recognized functions in cholesterol homeostasis and the immune system [8], LXRs are expressed in some specific parts of the brain and appear to have a modulatory function in neurons [8–11]. Both LXR/ and b are expressed in the choroid plexus where they control the cerebral spinal fluid [12]. LXRb is expressed in vasopressin neurons and affects water balance in the body [13] and in microglia where it regulates neuroinflammation [14]. The powerful anti-inflammatory actions of LXR suggest that ligands for these receptors might be good alternatives for glucocorticoids in treatment of inflammation but to date no selective LXR agonists are available for clinical testing. There is crosstalk between LXRs and
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Trends Targeting of nuclear receptor subtypes represents a way to differentiate between positive and negative health benefits of hormone therapy. ER/ and ERb have different distributions and functions in the body. Selective targeting of ERb may be preferable to estrogen as hormone therapy in menopausal women. ERb agonists may be beneficial in prostate cancer by keeping tumors to a low grade. A subset of triple-negative breast cancers is positive for ERb, suggesting it might be a target in this difficult-to-treat disease.
1 Center for Nuclear Receptors and Cell Signaling, Department of Cell Biology and Biochemistry, University of Houston, Texas, USA 2 Center for Innovative Medicine, Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Sweden
*Correspondence[7_TD$IF]: [email protected] (J.-A. Gustafsson).
http://dx.doi.org/10.1016/j.tips.2016.10.006 © 2016 Elsevier Ltd. All rights reserved.
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Central nervous system (ER-α, ER-β)
Lungs (ER-β) Bone (ER-α, ER-β) Cardiovascular system (ER-α, ER-β) Liver (ER-α)
Gastrointestinal tract (ER-β)
Bladder (ER-β) Prostate (ER-β) Testes/Ovary (ER-α, ER-β)
Figure 1. ER/ and ERb Have Different Distributions throughout the Body. ERb has less of a role in reproductive processes, suggesting it may be a therapeutic target in other areas. [2_TD$IF]Abbreviation: ER, estrogen receptor.
Figure 2. ERb Has a Modulatory Role on Both ER and AR Signaling. ERb
ERβ – Proliferaon + + Differenaon + + Apoptosis –
ERα AR
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can inhibit androgen- and estrogenmediated proliferation. ER/ and AR both mediate differentiation, therefore, complete androgen or estrogen ablation is probably not a good idea in treatment of androgen- or estrogen-dependent cancers. ERb ligands will enhance ERb signaling and modulate ER/ and AR signaling without the need for complete ablation of the hormones. Abbreviations: AR, androgen receptor; ER, estrogen receptor.
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ERs because they share a common signaling coactivator RAP[8_TD$IF]250 (nuclear receptor-activating protein 250[9_TD$IF]) [15,16]. Thus, as knowledge expands, so too does our awareness of the complexity of estrogen signaling.
Hormone Replacement after the Menopause The problems with estrogen replacement after menopause are many, and at present, there is no consensus on the safety of estrogen replacement after the menopause. Perhaps, this predicament is most surprising because clinicians have been using estrogen replacement for protection of bone [17] and improvement of mood and cognition [18,19] for many years and have been using an ER blocker to treat breast cancer for decades [20,21]. Estrogen replacement has now fallen into disfavor because of a reported increased risk of breast cancer associated with its use [17,21]. In some ways, the debate surrounding how and when to administer estrogen typifies the issues surrounding therapeutic exploitation of hormones. The question is what is the desirable level of estrogen in a postmenopausal woman and how should estrogen be delivered to produce this level. Can the dose and target be refined so as to favor the beneficial properties? It is well recognized that estrogen has two types of functions. One function is in female reproduction (growth of the mammary gland and endometrium and ovulation), which requires high levels of estrogen such as those achieved at mid-cycle. The other effects of estrogen have to do with immune suppression (including regulation of osteoclasts and bone resorption), maintenance of healthy mucous membranes, regulation of epithelial proliferation in the gastrointestinal tract and, in men, the epithelium of the prostate. So, the key question is what level of estrogen is optimal when estrogen is no longer needed for its role in reproduction.
ERb This question regarding estrogen replacement should have been reformulated in 1996 when the second ER was discovered [22]. ERb has little role in the proliferation of the mammary gland or endometrium and is not expressed in the pituitary, so ligands for this receptor will not cause castration effects in men. Although questions have been raised about its physiological functions [23], ERb does have a major role in the immune system, cardiovascular system, nervous system, and in the prostate [24]. One of the more interesting facts that we have learnt over the years about ERb is that it appears to be a tumor suppressor gene [25] and its expression is lost in early stages of ductal breast (ductal in situ) [26] and prostate (Gleason grade 3+3) [27] cancers. Another important observation is that ERb-selective agonists can increase expression of ERb in cells where its expression has been downregulated [28]. This raises the hope that, in the clinic, ERb agonists can be used to increase ERb expression in early stages of cancer and prevent proliferation and cancer progression. So, instead of asking how much estrogen is needed to keep ER/ active without being too active, we have a much simpler question: how to specifically activate ERb? Twenty years after its discovery, pharmaceutical companies and academic chemists have produced good ERb-selective agonists. These appear to be safe drugs [29]. They do not cause growth of the breast or uterus but they do inhibit epithelial proliferation in the prostate without any effect on the pituitary [30]. In the treatment of perimenopausal symptoms, estradiol is effective, but prolonged use of estradiol is associated with an increased risk of breast cancer [31]. The questions that have to be re-addressed are: why are there vascular problems in the perimenopausal period; why does estradiol help; and are there pharmacological interventions that may pose fewer adverse risks than estradiol?
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Although there has been a focus on the benefits of estradiol and the role of estrogen in proliferation of the breast, two of the hormones whose level are increased when estradiol levels fall are follicle-stimulating hormone (FSH) and activin. Both hormones have effects on the vascular system and bone, and in addition, activin is associated with tumorigenesis [32–37]. Activins are members of the transforming growth factor (TGF)b superfamily. The inhibitory wing of the TGFb system is composed of inhibin and its receptor TGFB3R and follistatin; all of which are ERb regulated. Loss of inhibin signaling is the most likely reason for the development of pituitary and granulosa cell tumors in ERb / mice [38], similar to tumors in inhibin / mice [39]. Thus, some of the vascular effects that occur at menopause could be caused, indirectly, by loss of estrogen and, more directly, by increase in FSH and activin. If this is the case, the target in treating the symptoms of menopause should be activin signaling and one way to do this is to increase inhibin signaling. Because of its effects on TGFb signaling, ERb agonists should be beneficial pharmaceuticals in the treatment of vascular disturbances (hot flushes and night sweats). Genistein, a phytoestrogen, has been tested in postmenopausal women and found to improve some of the cardiovascular symptoms of the menopause [40,41] but so far, phytoestrogens are not dispensed clinically and their use remains at the level of the health food stores. The problem is that the available natural phytoestrogens do not have a good selectivity for ERb and at high doses will activate ER/. Because there are so many of these remedies being marketed, the risk is that women will be consuming too much of these phytoestrogens and there will be activation of ER/. It is often argued that the menopause is not a disease and, therefore, women should not be treated with pharmaceuticals for menopausal symptoms. However, cognitive decline and breast cancer are serious diseases, and it is easy to justify use of pharmaceuticals to prevent or delay onset of these diseases if the pharmaceuticals are safe and do not increase the risk of other diseases. Whether or not ERb agonists may be used to reduce the risk of development of breast cancer and cognitive decline will have to be tested in clinical trials. ERb in Breast Cancer There is a wealth of data on the expression of ERb in breast cancer but no consensus about its role in the etiology and progression of this group of diseases. One of the reasons for lack of consensus in observations in different laboratories is the use of antibodies with different specificities. In our own work, we initially used an antibody directed against the N terminus of ERb and we found expression of ERb in breast cancer [42]. This antibody was specific and did not recognize ER/. However, it recognized all of the splice variants of ERb [43,44]. It is important to be able to distinguish between ERb1, the ligand-activated, antiproliferative form of ERb, and the splice variants, which are all different from ERb1 in the ligand-binding domain. These variants do not bind to estrogens. In addition, unlike ERb1, ERb splice variants increase proliferation rather than suppress it and can oppose ERb1 actions [45,46]. With the use of specific antibodies targeting the C terminus of ERb, we found that ERb1 is lost early in ductal breast cancer [26]. However, ERb1 is expressed in lobular breast cancers and in a small fraction (20%) of triplenegative breast cancers (Figure 3). It is of particular interest to us that lobular cancer is considered as a disease of loss of anoikis and not of proliferation [47,48] and that these cancers express high levels of nuclear phosphatase and tensin homolog (PTEN) [26]. PTEN is a known tumor suppressor gene that, in addition to regulating the AKT pathway at the cell membrane, has antiproliferative actions in the cell nucleus [49,50]. If ERb facilitates the entry of PTEN into the nucleus, this might be one of the mechanisms through which it exerts its antiproliferative effects. The significance of ERb1 expression in triple-negative breast cancer is not yet clear. Expression of ERb1 should repress proliferation even in the absence of ligand and can stimulate proliferation in the presence of tamoxifen [51]. High expression of ERb in the face of a proliferating cancer may mean that the ERb signaling pathway is not functional. Some possible culprits that could cause
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(A)
(B)
(C)
(D)
(E)
(F)
Figure 3. Representative Staining of ERb in TNBC (Scale bar = 50 mm). [3_TD$IF](A–D) show positive staining of ERb. In [4_TD$IF](E and F) TNBC are samples that do not express ERb. Twenty percent of TNBC samples examined express strong nuclear ERb staining. Abbreviations: ER, estrogen receptor; TNBC, triple-negative breast cancer.
defective signaling are: (i) the presence of ERb inhibitors within the cancers; (ii) increased expression of co-repressors; and (iii) decreased expression of coactivators. One candidate for inhibition of ERb function is high levels of 27-hydroxycholesterol, an endogenous ER/ agonist and ERb antagonist [52,53]. Whatever the reason, the presence of ERb in a subgroup of triple-negative breast cancers is of potential clinical interest because of the lack of targets for treatment in this disease. The aim with this group of patients would be to activate ERb and determine whether there are any positive effects. The easiest way to do this is to identify PDX[10_TD$IF] (patient derived xenograft) expressing ERb and examine the effects of a selective ERb agonist on growth of these xenografts in mice. ERb in Prostate Cancer (PCa) Localized PCa of low Gleason grades (well differentiated) is not fatal but metastatic castrationresistant PCa (CRPCa) is. It is still thought that PCa develops progressively from prostatic
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(A)
(B)
20 µm
Figure 4. Ducts of Ventral Prostate Are Composed of Single Layers of Columnar or Cuboidal Epithelial Cells. (A). In ERb knockout mouse ventral prostate the epithelium is multi-layered (B). [2_TD$IF]Abbreviation: ER, estrogen receptor.
intraepithelial neoplasia to cancers of low and subsequently higher Gleason grades and because androgen ablation eventually leads to CRPCa [54–56], therefore, it is reasonable to consider a strategy that prevents progression of PCa by intervening in early stages of development of the disease and to avoid androgen ablation. ERb agonists prevent proliferation of prostatic epithelium [57]. If early-grade cancer does not proliferate, there will be no clonal expansion of cells that harbor mutations and less chance of them developing into more aggressive cancer. Estradiol itself has been used to treat PCa but the doses used were too high and via ER/, and it caused chemical castration and cardiovascular disease. Androgen ablation, whether medical or surgical, is the first choice in treatment of metastatic PCa. Although there has been development of better and more potent androgen receptor antagonists, resistance to these drugs continues to develop and the result is CRPCa. Alternative approaches to androgen ablation need to be considered to prevent development of CRPCa. If prostate cancer can be kept at a low Gleason Grade, we will solve the problem of death and morbidity from this disease. Prostate cancer, like breast cancer, is a hormone-driven disease. Breast cancer results from the biological nature of the epithelium, which constantly proliferates throughout life. Unlike the breast, which undergoes continuous cycles of proliferation and involution throughout reproductive life, in the adult prostate, proliferating cells are rare except under conditions when there is androgen-stimulated growth (Figure 4). In such an indolent organ, carcinogenesis would be expected to be a lengthy process and for this reason, it has been postulated that PCa is initiated early in life. One alternative suggestion is that the prostate does undergo periods of proliferation throughout life. One possible reason for prostate growth is acute increase in levels of estrogen, which will shut off gonadotropin release from the pituitary and thus stop the production of testosterone from the testis. Androgens are the precursors of estrogen, thus, lack of testosterone eventually leads to a fall in estrogen production and the testes regain synthesis of testosterone. There are increases in estrogen levels in men during inflammatory processes and in visceral obesity [58]. Unlike the aromatase promoter in the gonads, which is regulated by cAMP and gonadotropins, in adipose tissue, aromatase expression is regulated by cytokines and tumor necrosis factor / [59]. During alternating cycles of estrogen and androgen levels, reduction in androgen levels causes atrophy of the prostate, and restoration of androgens causes regrowth of the prostatic epithelium. Thus, inflammation and obesity could predispose to increased risks of clonal expansion of cells harboring oncogenic mutations. Such a mechanism might explain the relationship between visceral obesity and PCa [60]. At present, it is not clear what is the biological advantage of why inflammation turns on aromatase but it may be part of the process of terminating the inflammatory response. If this
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is the case, an ERb-selective ligand should be more effective than estradiol in repressing nuclear factor- kB signaling without suppressing the pituitary. Clearly, more information is needed if we are going to avoid the recent mistakes made with ligands for another nuclear receptor, peroxisome proliferator-activated receptor g. The major lesson learnt from the past is that if we are going to use nuclear receptors as targets, we have to understand the biology and endocrinology of the systems in which they participate. It is of great value to remember the old saying that you can never only change one parameter in endocrinology.
Outstanding Questions Can ERb agonists keep prostate cancer at low Gleason grades? Is there a subpopulation of triple-negative breast cancer that can respond to ERb agonists? Will LXR agonists replace glucocorticoids as anti-inflammatory agents?
Concluding Remarks The concept of hormone receptors in precision medicine typically refers to cancer, but as we have seen here, selective targeting of receptor subtypes may have benefits in cancer and beyond. From its profile of actions, ERb agonists should be useful in hormone replacement therapy, early stages of PCa, that population (20%) of triple-negative breast cancer that expresses ERb, ERb-positive lobular breast cancer [26], neuroinflammatory diseases [46], and some forms of hypertension [61]. Non-endocrinologists express some skepticism about the idea that a single nuclear receptor could have such a wide array of functions (see Outstanding Questions), but endocrinologists are fully aware that individual nuclear receptors like the glucocorticoid receptor, the thyroid hormone receptor, and ER/ each has effects on multiple organ systems. For clinical endocrinologists, a receptor that has anti-inflammatory and antiproliferative functions (and is expressed in the epithelium of the gastrointestinal tract, prostate, mammary gland, as well as in B lymphocytes and vascular smooth muscle) should represent an interesting target for treatment of several endocrine disorders. Recently, there have been several publications on compounds selectively targeting ERb (e.g. [62]), and more of this research is to be expected in the next few years.
Can ERb agonists be used to prevent cognitive decline and vascular disturbances after menopause?
[1_TD$IF]Disclaimer [12_TD$IF]Statement
The authors report no conflict of interest[13_TD$IF]. Acknowledgments This study was supported by grants from the Swedish Cancer Society, CIMED, the Cancer Prevention and Research Institute of Texas (Grant RP110444-P1), and the Robert A. Welch Foundation (E-0004).
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Special Issue[5_TD$IF]: Precision Medicine
Review
Estrogen Receptor b as a Pharmaceutical Target Margaret Warner,1 Bo Huang,1 and Jan-Ake Gustafsson1,2,* A major issue in clinical endocrinology[6_TD$IF] today is how to use hormones to achieve the health benefits that they clearly can provide but avoid the negative side effects, that is, how to develop more precise medicines. This problem of how to use hormones is pervasive in clinical endocrinology. It is true for estrogen, progesterone, androgen, vitamin D, and thyroid hormone, and the problem is amplified in the case of new ligands for the more recently discovered nuclear receptors. Selective targeting of hormone receptor subtypes is one attractive way to harness the beneficial effects of hormones while reducing unwanted side effects. Here, we focus on estrogen receptor (ER)b, which has promise as a selective target in hormone replacement therapy, and in breast and prostate cancers. Nuclear Receptors and Their Ligands Although the receptors for five major steroid hormones have been identified, we know that there are more nuclear receptors than those that mediate the actions of identified hormones [1,2] but we do not know whether any of the ligands will turn out to be steroids that are at present classified as inactive metabolites of known steroid hormones or, indeed, whether such metabolites are modulators of signaling of the known steroid receptors. Ligands for nuclear receptors are not all steroids but include cholesterol metabolites, fatty acids, and metabolites of vitamin A [3]. The major classical reproductive, thyroid, and adrenal hormones are part of the hypothalamic–pituitary–gonadal/adrenal/thyroid axes. However, there are endogenous ligands for nuclear receptors that do not fall into this category. In recent years, an oxysterol, 27-hydroxy cholesterol, has been identified as an agonist of ER/ [4] and an antagonist of ERb [5]. The significance of this novel estrogen is under investigation but it reinforces the point that there is a lot to be learnt even about receptors that have been targeted in health and disease for decades. The fact that these two ERs have varying distributions in the body (Figure 1) and display differences in their pharmacology (Figure 2) opens the door for more precise modulation of estrogen signaling, as discussed below. Oxysterols: Oxysterols are endogenous ligands for the nuclear receptor liver X receptor (LXR) [6,7]. There is no clear evidence that LXRs are part of an endocrine system (no secretory gland, although several target organs have been identified). Instead, they may be part of a paracrine or autocrine system. In addition to their recognized functions in cholesterol homeostasis and the immune system [8], LXRs are expressed in some specific parts of the brain and appear to have a modulatory function in neurons [8–11]. Both LXR/ and b are expressed in the choroid plexus where they control the cerebral spinal fluid [12]. LXRb is expressed in vasopressin neurons and affects water balance in the body [13] and in microglia where it regulates neuroinflammation [14]. The powerful anti-inflammatory actions of LXR suggest that ligands for these receptors might be good alternatives for glucocorticoids in treatment of inflammation but to date no selective LXR agonists are available for clinical testing. There is crosstalk between LXRs and
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Trends Targeting of nuclear receptor subtypes represents a way to differentiate between positive and negative health benefits of hormone therapy. ER/ and ERb have different distributions and functions in the body. Selective targeting of ERb may be preferable to estrogen as hormone therapy in menopausal women. ERb agonists may be beneficial in prostate cancer by keeping tumors to a low grade. A subset of triple-negative breast cancers is positive for ERb, suggesting it might be a target in this difficult-to-treat disease.
1 Center for Nuclear Receptors and Cell Signaling, Department of Cell Biology and Biochemistry, University of Houston, Texas, USA 2 Center for Innovative Medicine, Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Sweden
*Correspondence[7_TD$IF]: [email protected] (J.-A. Gustafsson).
http://dx.doi.org/10.1016/j.tips.2016.10.006 © 2016 Elsevier Ltd. All rights reserved.
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Central nervous system (ER-α, ER-β)
Lungs (ER-β) Bone (ER-α, ER-β) Cardiovascular system (ER-α, ER-β) Liver (ER-α)
Gastrointestinal tract (ER-β)
Bladder (ER-β) Prostate (ER-β) Testes/Ovary (ER-α, ER-β)
Figure 1. ER/ and ERb Have Different Distributions throughout the Body. ERb has less of a role in reproductive processes, suggesting it may be a therapeutic target in other areas. [2_TD$IF]Abbreviation: ER, estrogen receptor.
Figure 2. ERb Has a Modulatory Role on Both ER and AR Signaling. ERb
ERβ – Proliferaon + + Differenaon + + Apoptosis –
ERα AR
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can inhibit androgen- and estrogenmediated proliferation. ER/ and AR both mediate differentiation, therefore, complete androgen or estrogen ablation is probably not a good idea in treatment of androgen- or estrogen-dependent cancers. ERb ligands will enhance ERb signaling and modulate ER/ and AR signaling without the need for complete ablation of the hormones. Abbreviations: AR, androgen receptor; ER, estrogen receptor.
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ERs because they share a common signaling coactivator RAP[8_TD$IF]250 (nuclear receptor-activating protein 250[9_TD$IF]) [15,16]. Thus, as knowledge expands, so too does our awareness of the complexity of estrogen signaling.
Hormone Replacement after the Menopause The problems with estrogen replacement after menopause are many, and at present, there is no consensus on the safety of estrogen replacement after the menopause. Perhaps, this predicament is most surprising because clinicians have been using estrogen replacement for protection of bone [17] and improvement of mood and cognition [18,19] for many years and have been using an ER blocker to treat breast cancer for decades [20,21]. Estrogen replacement has now fallen into disfavor because of a reported increased risk of breast cancer associated with its use [17,21]. In some ways, the debate surrounding how and when to administer estrogen typifies the issues surrounding therapeutic exploitation of hormones. The question is what is the desirable level of estrogen in a postmenopausal woman and how should estrogen be delivered to produce this level. Can the dose and target be refined so as to favor the beneficial properties? It is well recognized that estrogen has two types of functions. One function is in female reproduction (growth of the mammary gland and endometrium and ovulation), which requires high levels of estrogen such as those achieved at mid-cycle. The other effects of estrogen have to do with immune suppression (including regulation of osteoclasts and bone resorption), maintenance of healthy mucous membranes, regulation of epithelial proliferation in the gastrointestinal tract and, in men, the epithelium of the prostate. So, the key question is what level of estrogen is optimal when estrogen is no longer needed for its role in reproduction.
ERb This question regarding estrogen replacement should have been reformulated in 1996 when the second ER was discovered [22]. ERb has little role in the proliferation of the mammary gland or endometrium and is not expressed in the pituitary, so ligands for this receptor will not cause castration effects in men. Although questions have been raised about its physiological functions [23], ERb does have a major role in the immune system, cardiovascular system, nervous system, and in the prostate [24]. One of the more interesting facts that we have learnt over the years about ERb is that it appears to be a tumor suppressor gene [25] and its expression is lost in early stages of ductal breast (ductal in situ) [26] and prostate (Gleason grade 3+3) [27] cancers. Another important observation is that ERb-selective agonists can increase expression of ERb in cells where its expression has been downregulated [28]. This raises the hope that, in the clinic, ERb agonists can be used to increase ERb expression in early stages of cancer and prevent proliferation and cancer progression. So, instead of asking how much estrogen is needed to keep ER/ active without being too active, we have a much simpler question: how to specifically activate ERb? Twenty years after its discovery, pharmaceutical companies and academic chemists have produced good ERb-selective agonists. These appear to be safe drugs [29]. They do not cause growth of the breast or uterus but they do inhibit epithelial proliferation in the prostate without any effect on the pituitary [30]. In the treatment of perimenopausal symptoms, estradiol is effective, but prolonged use of estradiol is associated with an increased risk of breast cancer [31]. The questions that have to be re-addressed are: why are there vascular problems in the perimenopausal period; why does estradiol help; and are there pharmacological interventions that may pose fewer adverse risks than estradiol?
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Although there has been a focus on the benefits of estradiol and the role of estrogen in proliferation of the breast, two of the hormones whose level are increased when estradiol levels fall are follicle-stimulating hormone (FSH) and activin. Both hormones have effects on the vascular system and bone, and in addition, activin is associated with tumorigenesis [32–37]. Activins are members of the transforming growth factor (TGF)b superfamily. The inhibitory wing of the TGFb system is composed of inhibin and its receptor TGFB3R and follistatin; all of which are ERb regulated. Loss of inhibin signaling is the most likely reason for the development of pituitary and granulosa cell tumors in ERb / mice [38], similar to tumors in inhibin / mice [39]. Thus, some of the vascular effects that occur at menopause could be caused, indirectly, by loss of estrogen and, more directly, by increase in FSH and activin. If this is the case, the target in treating the symptoms of menopause should be activin signaling and one way to do this is to increase inhibin signaling. Because of its effects on TGFb signaling, ERb agonists should be beneficial pharmaceuticals in the treatment of vascular disturbances (hot flushes and night sweats). Genistein, a phytoestrogen, has been tested in postmenopausal women and found to improve some of the cardiovascular symptoms of the menopause [40,41] but so far, phytoestrogens are not dispensed clinically and their use remains at the level of the health food stores. The problem is that the available natural phytoestrogens do not have a good selectivity for ERb and at high doses will activate ER/. Because there are so many of these remedies being marketed, the risk is that women will be consuming too much of these phytoestrogens and there will be activation of ER/. It is often argued that the menopause is not a disease and, therefore, women should not be treated with pharmaceuticals for menopausal symptoms. However, cognitive decline and breast cancer are serious diseases, and it is easy to justify use of pharmaceuticals to prevent or delay onset of these diseases if the pharmaceuticals are safe and do not increase the risk of other diseases. Whether or not ERb agonists may be used to reduce the risk of development of breast cancer and cognitive decline will have to be tested in clinical trials. ERb in Breast Cancer There is a wealth of data on the expression of ERb in breast cancer but no consensus about its role in the etiology and progression of this group of diseases. One of the reasons for lack of consensus in observations in different laboratories is the use of antibodies with different specificities. In our own work, we initially used an antibody directed against the N terminus of ERb and we found expression of ERb in breast cancer [42]. This antibody was specific and did not recognize ER/. However, it recognized all of the splice variants of ERb [43,44]. It is important to be able to distinguish between ERb1, the ligand-activated, antiproliferative form of ERb, and the splice variants, which are all different from ERb1 in the ligand-binding domain. These variants do not bind to estrogens. In addition, unlike ERb1, ERb splice variants increase proliferation rather than suppress it and can oppose ERb1 actions [45,46]. With the use of specific antibodies targeting the C terminus of ERb, we found that ERb1 is lost early in ductal breast cancer [26]. However, ERb1 is expressed in lobular breast cancers and in a small fraction (20%) of triplenegative breast cancers (Figure 3). It is of particular interest to us that lobular cancer is considered as a disease of loss of anoikis and not of proliferation [47,48] and that these cancers express high levels of nuclear phosphatase and tensin homolog (PTEN) [26]. PTEN is a known tumor suppressor gene that, in addition to regulating the AKT pathway at the cell membrane, has antiproliferative actions in the cell nucleus [49,50]. If ERb facilitates the entry of PTEN into the nucleus, this might be one of the mechanisms through which it exerts its antiproliferative effects. The significance of ERb1 expression in triple-negative breast cancer is not yet clear. Expression of ERb1 should repress proliferation even in the absence of ligand and can stimulate proliferation in the presence of tamoxifen [51]. High expression of ERb in the face of a proliferating cancer may mean that the ERb signaling pathway is not functional. Some possible culprits that could cause
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(A)
(B)
(C)
(D)
(E)
(F)
Figure 3. Representative Staining of ERb in TNBC (Scale bar = 50 mm). [3_TD$IF](A–D) show positive staining of ERb. In [4_TD$IF](E and F) TNBC are samples that do not express ERb. Twenty percent of TNBC samples examined express strong nuclear ERb staining. Abbreviations: ER, estrogen receptor; TNBC, triple-negative breast cancer.
defective signaling are: (i) the presence of ERb inhibitors within the cancers; (ii) increased expression of co-repressors; and (iii) decreased expression of coactivators. One candidate for inhibition of ERb function is high levels of 27-hydroxycholesterol, an endogenous ER/ agonist and ERb antagonist [52,53]. Whatever the reason, the presence of ERb in a subgroup of triple-negative breast cancers is of potential clinical interest because of the lack of targets for treatment in this disease. The aim with this group of patients would be to activate ERb and determine whether there are any positive effects. The easiest way to do this is to identify PDX[10_TD$IF] (patient derived xenograft) expressing ERb and examine the effects of a selective ERb agonist on growth of these xenografts in mice. ERb in Prostate Cancer (PCa) Localized PCa of low Gleason grades (well differentiated) is not fatal but metastatic castrationresistant PCa (CRPCa) is. It is still thought that PCa develops progressively from prostatic
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(A)
(B)
20 µm
Figure 4. Ducts of Ventral Prostate Are Composed of Single Layers of Columnar or Cuboidal Epithelial Cells. (A). In ERb knockout mouse ventral prostate the epithelium is multi-layered (B). [2_TD$IF]Abbreviation: ER, estrogen receptor.
intraepithelial neoplasia to cancers of low and subsequently higher Gleason grades and because androgen ablation eventually leads to CRPCa [54–56], therefore, it is reasonable to consider a strategy that prevents progression of PCa by intervening in early stages of development of the disease and to avoid androgen ablation. ERb agonists prevent proliferation of prostatic epithelium [57]. If early-grade cancer does not proliferate, there will be no clonal expansion of cells that harbor mutations and less chance of them developing into more aggressive cancer. Estradiol itself has been used to treat PCa but the doses used were too high and via ER/, and it caused chemical castration and cardiovascular disease. Androgen ablation, whether medical or surgical, is the first choice in treatment of metastatic PCa. Although there has been development of better and more potent androgen receptor antagonists, resistance to these drugs continues to develop and the result is CRPCa. Alternative approaches to androgen ablation need to be considered to prevent development of CRPCa. If prostate cancer can be kept at a low Gleason Grade, we will solve the problem of death and morbidity from this disease. Prostate cancer, like breast cancer, is a hormone-driven disease. Breast cancer results from the biological nature of the epithelium, which constantly proliferates throughout life. Unlike the breast, which undergoes continuous cycles of proliferation and involution throughout reproductive life, in the adult prostate, proliferating cells are rare except under conditions when there is androgen-stimulated growth (Figure 4). In such an indolent organ, carcinogenesis would be expected to be a lengthy process and for this reason, it has been postulated that PCa is initiated early in life. One alternative suggestion is that the prostate does undergo periods of proliferation throughout life. One possible reason for prostate growth is acute increase in levels of estrogen, which will shut off gonadotropin release from the pituitary and thus stop the production of testosterone from the testis. Androgens are the precursors of estrogen, thus, lack of testosterone eventually leads to a fall in estrogen production and the testes regain synthesis of testosterone. There are increases in estrogen levels in men during inflammatory processes and in visceral obesity [58]. Unlike the aromatase promoter in the gonads, which is regulated by cAMP and gonadotropins, in adipose tissue, aromatase expression is regulated by cytokines and tumor necrosis factor / [59]. During alternating cycles of estrogen and androgen levels, reduction in androgen levels causes atrophy of the prostate, and restoration of androgens causes regrowth of the prostatic epithelium. Thus, inflammation and obesity could predispose to increased risks of clonal expansion of cells harboring oncogenic mutations. Such a mechanism might explain the relationship between visceral obesity and PCa [60]. At present, it is not clear what is the biological advantage of why inflammation turns on aromatase but it may be part of the process of terminating the inflammatory response. If this
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is the case, an ERb-selective ligand should be more effective than estradiol in repressing nuclear factor- kB signaling without suppressing the pituitary. Clearly, more information is needed if we are going to avoid the recent mistakes made with ligands for another nuclear receptor, peroxisome proliferator-activated receptor g. The major lesson learnt from the past is that if we are going to use nuclear receptors as targets, we have to understand the biology and endocrinology of the systems in which they participate. It is of great value to remember the old saying that you can never only change one parameter in endocrinology.
Outstanding Questions Can ERb agonists keep prostate cancer at low Gleason grades? Is there a subpopulation of triple-negative breast cancer that can respond to ERb agonists? Will LXR agonists replace glucocorticoids as anti-inflammatory agents?
Concluding Remarks The concept of hormone receptors in precision medicine typically refers to cancer, but as we have seen here, selective targeting of receptor subtypes may have benefits in cancer and beyond. From its profile of actions, ERb agonists should be useful in hormone replacement therapy, early stages of PCa, that population (20%) of triple-negative breast cancer that expresses ERb, ERb-positive lobular breast cancer [26], neuroinflammatory diseases [46], and some forms of hypertension [61]. Non-endocrinologists express some skepticism about the idea that a single nuclear receptor could have such a wide array of functions (see Outstanding Questions), but endocrinologists are fully aware that individual nuclear receptors like the glucocorticoid receptor, the thyroid hormone receptor, and ER/ each has effects on multiple organ systems. For clinical endocrinologists, a receptor that has anti-inflammatory and antiproliferative functions (and is expressed in the epithelium of the gastrointestinal tract, prostate, mammary gland, as well as in B lymphocytes and vascular smooth muscle) should represent an interesting target for treatment of several endocrine disorders. Recently, there have been several publications on compounds selectively targeting ERb (e.g. [62]), and more of this research is to be expected in the next few years.
Can ERb agonists be used to prevent cognitive decline and vascular disturbances after menopause?
[1_TD$IF]Disclaimer [12_TD$IF]Statement
The authors report no conflict of interest[13_TD$IF]. Acknowledgments This study was supported by grants from the Swedish Cancer Society, CIMED, the Cancer Prevention and Research Institute of Texas (Grant RP110444-P1), and the Robert A. Welch Foundation (E-0004).
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