Estrogen Receptor β in Prostate Cancer

Estrogens, via the interaction with their receptors, play important roles in the control of cellular growth and differentiation. Specifically, estrogens regulate the growth and development of the mammary gland in embryogenesis as well as in pre- and postpubertal periods, and of ovarian follicles during the reproductive cycle. 1 Importantly, estrogens are known to play a role in the development of the male reproductive system as well. 2 The effects of estrogens on prostate epithelium are still primarily unknown although estrogens have been used in the treatment of prostate cancer because of their growth-inhibitory effects. However, either because of toxicity and/or because of poor response rates, treatment of prostate cancer with estrogen has been discontinued.

Estrogenic activity is mediated by the physical interaction between the estrogen receptors (ERs) and the hormone with subsequent activation of the receptors. These belong to a superfamily of nuclear receptors that are ligand-dependent transactivators and include receptors for retinoic acid, vitamin D, steroid, and thyroid hormones as well as orphan receptors for which ligands are yet to be found. 3

Although it was initially thought that estrogens mediate their action through a single receptor, the estrogen receptor α (ERα), a second ER has been identified relatively recently from a rat prostate library and termed ERβ. 4 Whereas these two receptors share structural similarities (47% identity) and some functional properties, it is clear that individual characteristics allow them to have distinct biological functions.

Human ERs (hERs) have six functional regions that vary in their degree of conservation. The least conserved region among species, within the receptor superfamily and between ERα and ERβ, is the hypervariable amino-terminal, important for transactivation. In contrast, the DNA-binding domain is highly conserved (96% identity). Interestingly, there is 59% identity at the amino-acid level between ERα and ERβ in the ligand-binding domain. Although both receptors bind the natural ligand 17β estradiol with about equal affinity, 5 phytoestrogens and selective estrogen receptor modulators (SERMs) can bind ERα and ERβ selectively. 6 However, the putative diverse biological function of the two receptors should not only be ascribed to different ligands but, more importantly, to their different tissue distribution. When inactive, ERs exist as monomers bound to heat shock proteins. 7 On activation, they can form homodimers (ERα/ERα; ERβ/ERβ) or heterodimers (ERα/ERβ). If, for instance, ERα and ERβ are co-expressed in a given tissue or tumor, the formation of a heterodimer will likely yield a different transcriptional profile from that obtained if homodimers are generated in the presence of natural ligands or SERMs.

Although androgens are required for the normal development and function of the male reproductive system, the role of estrogens is still unclear. ERα, ERβ, and ERα/β knock-out mice have been generated to study the biological effects in different tissues including the male reproductive system. 2,8-10 The main findings in these knock-out strains are the requirement of ERα for ovulation and that of ERβ for granulosa cell proliferation in the female, whereas male infertility was found in the ERα but not in the ERβ knock-outs (βERKO). As far as the prostate is concerned, a recent study 10 reports no abnormalities in epithelial proliferation in either ERβ or double ERα and ERβ knock-outs (ERαβKO) despite previous reports suggesting such abnormalities. 9 The absence of prostatic lesions in the ERβ knock-outs does not contradict experimental evidence pointing to a role for ERβ as an inhibitor of prostatic growth, as suggested by Leav and colleagues in this issue of The American Journal of Pathology 11 and by the same group of investigators in another recent report. 12 In fact, a case in point is that of the cell-cycle inhibitor p27. p27 has been found to be associated with aggressive behavior in a wide variety of tumors. 13 Its targeted disruption in mice results merely in pituitary tumors. 14-16 However, when these mice are challenged by carcinogens or radiation 17 or are crossed with mouse strains with known susceptibility to prostate cancer such as the PTEN heterozygous-deficient mice, 18 tumors develop at high rates. Studies of this sort will prove useful in both βERKO and ERαβKO genetically engineered strains to determine the putative anti-proliferative function of ERβ in the prostate.

The discovery of ER β has thus lead to a re-evaluation of the biological functions of estrogens as well as of estrogen antagonists in prostate cancer. It has recently been shown that SERMs function as estrogens in some tissues whereas in others have anti-estrogenic effects. 6 This is thought to result from the induction of distinct conformations in the two ERs, allowing the recruitment of different co-regulators. These, in turn, are thought to determine the transcriptional profile induced by ligands. In fact, opposite transcriptional events occur when the two receptors are exposed to tamoxifen in vitro. 19 Thus, by defining the molecular mechanisms of ER signaling in prostate cancer cells it should be theoretically possible to design new SERMs with significant anti-tumoral activity.

Before antibodies became available, localization of the ER subtypes was accomplished either by steroid autoradiography, 20,21 in situ hybridization, 22 or, more recently, by reverse transcriptase-polymerase chain reaction. 12,23 Therefore, in most of the studies ERβ expression in both cell lines and tissues has been investigated at the RNA level. Interestingly, only ERβ, and not ERα transcripts were previously detected in rodent prostate epithelial cells. ERβ mRNA appeared to be expressed in both basal and luminal cells. 22 ERα transcripts were also found to be re-expressed after treatment with de-methylation agents in human prostate cell lines suggesting transcriptional regulation of this receptor. 12 Again, at the RNA level, the prostate carcinoma cell lines LNCaP and DU-145 were previously shown to express exclusively ERβ whereas PC-3 cells expressed transcripts from both receptors. 12 In addition, ERβ transcripts were found to be decreased in both localized and hormone refractory prostate cancers relative to normal prostate tissue when these were measured by quantitative reverse transcriptase-polymerase chain reaction, suggesting that loss of ERβ correlated with disease progression. 23

To date, several antibodies have been generated to the various regions of ERβ and used in Western blot analysis, immunoprecipitation, and mobility shift assays. 7 Some of these antibodies directed at rat or mouse ERβ 10,24-27 or human ERβ 28,29 have also been used in immunohistochemical studies. In this issue, Leav and colleagues 11 describe for the first time the localization of the ERβ protein in a spectrum of human prostate specimens, ranging from normal, to dysplastic (prostate intraepithelial neoplasia), to invasive and metastatic cancers. To accomplish this, they generated a polyclonal antibody raised against a peptide from the C-terminal region of the receptor. The peptide was chosen on the basis of having no homology to the corresponding region of ERα. As far as normal tissue is concerned, ERβ was found to be expressed in basal cells of normal prostatic acini as well as in stromal cells. Of note, ERβ was not expressed in secretory cells. Importantly, the only previous study in which ERβ was detected in the human prostate using an antibody raised against the N-terminus region of the receptor, also localized ERβ in basal acinar cells and in stromal cells. 29

As expected, ERα was expressed only in stromal cell nuclei. Ho and colleagues 12 have also recently shown that primary, nontransformed, human epithelial prostate cultures express ERβ but not ERα mRNA. In this article, 11 they confirm ERβ protein expression in prostate basal cells in culture (PrEC; Clonetics, Inc. San Diego, CA).

Interestingly, Leav and colleagues 11 show that high-grade dysplasia does not express ERβ. Antiproliferative effects of tamoxifen and the anti-estrogen ICI-182,780 were previously shown to be reversed by ERβ antisense in prostate cells. 12 Furthermore, increased expression of ERβ was found to be associated with estrogen-mediated protection from Apc-associated tumor formation in min mice. 30 Finally, ERβ protein down-regulation is associated with mitogenic activity in premalignant lesions of the breast. 31 Taken together, these findings raise the attractive possibility that lack of expression of ERβ may contribute to the initial phases of epithelial tumorigenesis. In terms of the early phases of prostate intraepithelial dysplasia, Leav and colleagues 11 found expression of ERβ in low-grade prostate intraepithelial neoplasia lesions but lack of expression in cells displaying high-grade dysplasia. Thus, ERβ may be expressed in the very early phases of dysplasia (low grade), perhaps to counteract proliferative stimuli but lack of ERβ expression occurs, and may in fact be required, in the lesions with high-grade cytological atypia, known to be associated with invasive carcinomas.

The pattern of expression in the invasive cancers examined was, as expected, more complex. Using the ERβ-specific antibody they generated, Leav and colleagues 11 showed that the majority of moderately differentiated tumors expressed ERβ although a difference was observed when ERβ-negative clear cell carcinomas of the transition zone were compared to ERβ-positive tumors arising in the peripheral zone. ERβ was also widely expressed in androgen-independent, metastatic tumors. Although obviously difficult to compare, the immunohistochemical findings seem to contradict the mRNA data previously reported in prostate cancer. 23 However, little is known about posttranscriptional regulation of ERβ and it may be possible that the ERβ protein is more stable in androgen-independent, metastatic tumors despite reduced transcriptional activity for this gene. The finding of increased ERβ in both moderately differentiated cancers and aggressive metastatic tumors is also contradictory. In essence, is ERβ the good guy or the bad guy? Is its down-regulation associated with tumorigenesis and/or tumor progression or is the expression of ERβ in prostate tumors of advanced stage transducing signals that favor tumor aggressiveness? Obviously, more detailed studies in human prostate cancer, complete of biochemical correlates, need to be performed to determine the precise role of ERβ in these tumors.

The finding of positivity of tumor cells for ERβ in metastatic, androgen-independent lesions, however, could be of potential therapeutic interest, provided the receptor is functional in this setting. To this end, it is interesting to note that many genes expressed by the basal cells, which do not depend on androgens for survival, such as bcl-2, 32-34 Her-2-neu, 35 and prostate stem cell antigen 36 get re-expressed in advanced, androgen-independent cancers. Intriguingly, ERβ seems to follow the same path. Except for the keratin profile and the almost universal expression of the androgen receptor in these tumors, metastatic, androgen-independent prostate tumors thus seem to display a basaloid phenotype. This should lend itself to targeted therapeutic manipulations of this late-stage, fatal type of prostate cancer, as Leav and colleagues 11 suggest.

In summary, the different biological activities of ERα and ERβ, may be ascribed to a variety of factors that will need to be carefully studied in human prostate carcinomas now that tools such as laser capture microdissection, quantitative real-time polymerase chain reaction, and immunohistochemistry are available. These factors include recruitment of different co-activators and co-repressors to modify transcription and homodimeration versus heterodimerization of these receptors. Because heterodimerization can only occur in cells that co-express these receptors, assessment of expression of both ERs as well as that of co-regulators, in normal and tumor tissues, becomes of paramount importance. Leav and colleagues 11 have begun to address this issue in normal and neoplastic human prostate using a novel, anti-hERβ antibody. They have shown that ERβ is expressed in basal cells in normal prostatic acini, it is not expressed in high-grade dysplasia, is expressed in the majority of moderately differentiated, invasive cancers whereas it is lost with increasing Gleason score. Interestingly, ERβ was somewhat surprisingly expressed in androgen-independent metastatic tumors. The data provided generate more questions than they answer. In addition, one must bear in mind that the number of cases assessed is small and that the antibody has not been validated by other groups. Therefore, additional immunohistochemical studies to assess ERβ expression in prostate cancer are necessary.

In conclusion, ERβ may play a significant role in prostate cell differentiation and proliferation and may modulate both the initial phases of prostate carcinogenesis and androgen-independent tumor growth. Whether ERβ enhances or suppresses prostate cancer development and/or progression remains to be established.