Related Articles

Purpose

Steroid hormones and growth factors affect lung cancer, and it is possible they act in concert to influence patient outcome.

Experimental Design

Primary lung tumors and normal lung tissue were analyzed for expression and localization of estrogen receptor α and β–1 ERα and ERβ), aromatase, progesterone receptor (PR), and epidermal growth factor receptor (EGFR).

Results

Tumors expressed higher levels of ERβ compared to matched normal lung, while the reverse was true of PR. High cytoplasmic ERβ expression was identified as an independent negative prognostic predictor of overall survival (OS) (HR=1.67), and low total PR was identified as an independent negative predictor of time to progression (TTP) (HR=1.59). After adjusting for stage, age, sex and smoking, combined high cytoplasmic ERβ and low total PR showed enhanced effects on OS (HR=2.64) and on TTP (HR=6.02). Further effects on OS were observed when EGFR expression was included (HR=5.32). Patients with low cytoplasmic ERβ, low aromatase, low EGFR and high total PR had shorter OS than patients with the opposite pattern (HR= 6.60). Contribution of these markers to survival showed no significant sex differences in a multivariable model. ERα was elevated in tumors but was not predictive of survival, and appears to represent a variant ERα protein that is only recognized by a C-terminal antibody.

Conclusions

Hormonal and EGFR pathways together may contribute to lung cancer prognosis. Lung tumors with high ERβ–1 /low PR may define patients with aggressive biology. A validation study is necessary to fully assess the predictive value of these markers.

Estrogen signaling is critical in the progression of tumors that bear estrogen receptors. In most patients with breast cancer, inhibitors that block interactions of estrogen with its receptors or suppress the production of endogenous estrogens are important interventions in the clinic. Recent evidence now suggests that estrogen also contributes to the pathogenesis of non–small cell lung cancer (NSCLC). We used a human lung cancer xenograph model system to analyze the effect of aromatase or estradiol on tumor growth. We further examined the level of protein expression of aromatase in 422 patients with NSCLC using a high-density tissue microarray. Results were confirmed and validated on an independent patient cohort (n = 337). Lower levels of aromatase predicted a greater chance of survival in women 65 years and older. Within this population, the prognostic value of aromatase was greatest in earlier stage lung cancer (stage I/II). In addition, for women with no history of smoking, lower aromatase levels were a strong predictor of survival. Our findings implicate aromatase as an early-stage predictor of survival in some women with NSCLC. We predict that women whose lung cancers have higher levels of aromatase might be good candidates for targeted treatment with aromatase inhibitors.

Lung cancer has become increasingly common in women, and gender differences in the physiology and pathogenesis of the disease have suggested a role for estrogens. In the lung recent data have shown local production of estrogens from androgens via the action of aromatase enzyme and higher levels of estrogen in tumor tissue as compared with surrounding normal lung tissue. High levels of aromatase expression are also maintained in metastases as compared with primary tumors. Consistent with these findings, clinical studies suggest that aromatase expression may be a useful predictive biomarker for prognosis in the management of non-small cell lung cancer (NSCLC), the most common form of lung malignancy. Low levels of aromatase associate with a higher probability of long-term survival in older women with early stage NSCLC. Treatment of lung NSCLC xenografts in vivo with an aromatase inhibitor (exemestane) alone or combined with standard cisplatin chemotherapy elicits a significant reduction in tumor progression as compared to paired controls. Further, lung cancer progression is also governed by complex interactions between estrogen and growth factor signaling pathways to stimulate the growth of NSCLC as well as tumor-associated angiogenesis. We find that combination therapy with the multitargeted growth factor receptor inhibitor vandetanib and the estrogen receptor antagonist fulvestrant inhibit tumor growth more effectively than either treatment administered alone. Thus, incorporation of antiestrogen treatment strategies in standard antitumor therapies for NSCLC may contribute to improved patient outcome, an approach that deserves to be tested in clinical trials.

Lung cancer is the most common cause of cancer mortality in male and female patients in the US. Although it is clear that tobacco smoking is a major cause of lung cancer, about half of all women with lung cancer worldwide are never-smokers. Despite a declining smoking population, the incidence of non-small cell lung cancer (NSCLC), the predominant form of lung cancer, has reached epidemic proportions particularly in women. Emerging data suggest that factors other than tobacco, namely endogenous and exogenous female sex hormones, have a role in stimulating NSCLC progression. Aromatase, a key enzyme for estrogen biosynthesis, is expressed in NSCLC. Clinical data show that women with high levels of tumor aromatase (and high intratumoral estrogen) have worse survival than those with low aromatase. The present and previous studies also reveal significant expression and activity of estrogen receptors (ERα, ERβ) in both extranuclear and nuclear sites in most NSCLC. We now report further on the expression of progesterone receptor (PR) transcripts and protein in NSCLC. PR transcripts were significantly lower in cancerous as compared to non-malignant tissue. Using immunohistochemistry, expression of PR was observed in the nucleus and/or extranuclear compartments in the majority of human tumor specimens examined. Combinations of estrogen and progestins administered in vitro cooperate in promoting tumor secretion of vascular endothelial growth factor and, consequently, support tumor-associated angiogenesis. Further, dual treatment with estradiol and progestin increased the numbers of putative tumor stem/progenitor cells. Thus, ER- and/or PR-targeted therapies may offer new approaches to manage NSCLC.

In non-small cell lung cancer (NSCLC) cells, 17β-estradiol increases transcription, activates MAPK, and stimulates proliferation. We hypothesize that estrogen receptor β (ERβ) mediates these responses because it, but not ERα, is detected in our NSCLC cell lines. To test this, we determined the effects of the ERβ-selective agonists genistein (GEN) and 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) and the ERα-selective agonist 4,4’,4”-(4-propyl-[1H]-pyrazole-l,3,5-triyl)trisphenol (PPT) in 201T cells. The cells were transfected with either an ERα or an ERβ expression vector and an estrogen response element (ERE)-tk-luciferase reporter construct. PPT increased luciferase activity in cells expressing ERα but not ERβ GEN and DPN selectively increased luciferase activity in ERβ-transfected cells at concentrations ≤ 10 nM. Fulvestrant blocked the GEN and DPN-mediated increases, indicating that transcription was ER-dependent. GEN but not PPT mediated a significant 1.5-fold increase in reporter activity upon transfection with ERE-tk-luciferase alone, demonstrating that endogenous ERβ activates transcription. PPT and DPN increased MAPK phosphorylation (2.5-fold and 3.7-fold, respectively). However, only DPN stimulated 201T growth in vitro (p = 0.008) and in vivo (p = 0.05). We conclude that ERβ mediates genomic and non-genomic responses to estrogen in 201T cells and that activation of both pathways may be necessary for increased proliferation of these cells.

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E2) are distinct from those in non-E2-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E2 proliferative H1793 and non-E2-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E2 or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

Estrogens play essential roles in the progression of mammary and prostatic diseases. The transcriptional effects of estrogens are transduced by two estrogen receptors, ERα and ERβ, which elicit opposing roles in regulating proliferation: ERα is proliferative while ERβ is anti-proliferative. Exogenous expression of ERβ in ERα-positive cancer cell lines inhibits cell proliferation in response to estrogen and reduces xenografted tumor growth in vivo, suggesting that ERβ might oppose ERα's proliferative effects via formation of ERα/β heterodimers. Despite biochemical and cellular evidence of ERα/β heterodimer formation in cells co-expressing both receptors, the biological roles of the ERα/β heterodimer remain to be elucidated. Here we report the identification of two phytoestrogens that selectively activate ERα/β heterodimers at specific concentrations using a cell-based, two-step high throughput small molecule screen for ER transcriptional activity and ER dimer selectivity. Using ERα/β heterodimer-selective ligands at defined concentrations, we demonstrate that ERα/β heterodimers are growth inhibitory in breast and prostate cells which co-express the two ER isoforms. Furthermore, using Automated Quantitative Analysis (AQUA) to examine nuclear expression of ERα and ERβ in human breast tissue microarrays, we demonstrate that ERα and ERβ are co-expressed in the same cells in breast tumors. The co-expression of ERα and ERβ in the same cells supports the possibility of ERα/β heterodimer formation at physio- and pathological conditions, further suggesting that targeting ERα/β heterodimers might be a novel therapeutic approach to the treatment of cancers which co-express ERα and ERβ.

Background

New, third-generation aromatase inhibitors (AIs) have proven comparable or superior to the anti-estrogen tamoxifen for treatment of estrogen receptor (ER) and/or progesterone receptor (PR) positive breast cancer. AIs suppress total body and intratumoral estrogen levels. It is unclear whether in situ carcinoma cell aromatization is the primary source of estrogen production for tumor growth and whether the aromatase expression is predictive of response to endocrine therapy. Due to methodological difficulties in the determination of the aromatase protein, COX-2, an enzyme involved in the synthesis of aromatase, has been suggested as a surrogate marker for aromatase expression.

Methods

Primary tumor material was retrospectively collected from 88 patients who participated in a randomized clinical trial comparing the AI letrozole to the anti-estrogen tamoxifen for first-line treatment of advanced breast cancer. Semi-quantitative immunohistochemical (IHC) analysis was performed for ER, PR, COX-2 and aromatase using Tissue Microarrays (TMAs). Aromatase was also analyzed using whole sections (WS). Kappa analysis was applied to compare association of protein expression levels. Univariate Wilcoxon analysis and the Cox-analysis were performed to evaluate time to progression (TTP) in relation to marker expression.

Results

Aromatase expression was associated with ER, but not with PR or COX-2 expression in carcinoma cells. Measurements of aromatase in WS were not comparable to results from TMAs. Expression of COX-2 and aromatase did not predict response to endocrine therapy. Aromatase in combination with high PR expression may select letrozole treated patients with a longer TTP.

Conclusion

TMAs are not suitable for IHC analysis of in situ aromatase expression and we did not find COX-2 expression in carcinoma cells to be a surrogate marker for aromatase. In situ aromatase expression in tumor cells is associated with ER expression and may thus point towards good prognosis. Aromatase expression in cancer cells is not predictive of response to endocrine therapy, indicating that in situ estrogen synthesis may not be the major source of intratumoral estrogen. However, aromatase expression in combination with high PR expression may select letrozole treated patients with longer TTP.

Trial registration

Sub-study of trial P025 for advanced breast cancer.

Background

Recent evidence suggests that estrogen signaling may be involved in the pathogenesis of non-small cell lung cancer (NSCLC). Aromatase is an enzyme complex that catalyses the final step in estrogen synthesis and is present in several tissues, including the lung. In the current study we investigated the activity of the aromatase inhibitor exemestane in human NSCLC cell lines H23 and A549.

Results

Aromatase expression was detected in both cell lines. H23 cells showed lower protein and mRNA levels of aromatase, compared to A549 cells. Exemestane decreased cell proliferation and increased apoptosis in both cell lines, 48 h after its application, with A549 exhibiting higher sensitivity than H23 cells. Aromatase protein and mRNA levels were not affected by exemestane in A549 cells, whereas an increase in both protein and mRNA levels was observed in H23 cells, 48 h after exemestane application. Moreover, an increase in cAMP levels was found in both cell lines, 15 min after the administration of exemestane. In addition, we studied the effect of exemestane on epidermal growth factor receptor (EGFR) localization and activation. Exemestane increased EGFR activation 15 min after its application in H23 cells. Furthermore, we demonstrated a translocation of EGFR from cell membrane, 24 h after the addition of exemestane in H23 cells. No changes in EGFR activation or localization were observed in A549 cells.

Conclusion

Our findings suggest an antiproliferative effect of exemestane on NSCLC cell lines. Exemestane may be more effective in cells with higher aromatase levels. Further studies are needed to assess the activity of exemestane in NSCLC.

In humans, aromatase (CYP19) gene expression is regulated via alternative promoters. Activation of each promoter gives rise to a CYP19 mRNA species with a unique 5′-untranslated region. Inhibition of aromatase has been reported to downregulate lung tumor growth. The genetic basis for CYP19 gene expression and aromatase activity in lung cancer remains poorly understood. We analyzed tissues from 15 patients with non-small cell lung cancer (NSCLC) to evaluate CYP19 promoter usage and promoter-specific aromatase mRNA levels in NSCLC tumor tissues and adjacent non-malignant tissues. CYP19 promoter usage was determined by multiplex RT-PCR and aromatase mRNA levels were measured with real-time RT-PCR. In non-malignant tissues, aromatase mRNA was primarily derived from activation of CYP19 promoter I.4. Although promoter I.4 usage was also dominant in tumor tissues, I.4 activation was significantly lower compared with adjacent non-malignant tissues. Activity of promoters I.3, I.1 and I.7 was significantly higher in tumor tissues compared with non-malignant tissues. In 4 of 15 cases of non-small cell lung cancer, switching from CYP19 promoter I.4 to the alternative promoters II, I.1 or I.7 was observed. In 9 cases, there were significantly higher levels of aromatase mRNA in lung tumor tissues compared with adjacent non-malignant tissues. These findings suggest aberrant activation of alternative CYP19 promoters that may lead to upregulation of local aromatase expression in some cases of NSCLC. Further studies are needed to examine the impact of alternative CYP19 promoter usage on local estrogen levels and lung tumor growth.

A majority of breast cancers are hormone-responsive, and require estrogen for growth, and respond to hormonal therapy that blocks estrogen receptor action. Breast tumors with low levels of or completely lacking estrogen receptor fail to respond to antiestrogen therapy yet require estrogen for tumor initiation. To address the importance of local estrogen in oncogene-mediated breast tumorigenesis, we have crossed MMTV-aromatase with MMTV-HER2/neu and examined the incidence of breast cancer in double transgenic mice in comparison with parental strains. Double transgenic mice show normal mammary development and express both transgenes at similar levels to that of parental strains. Tumor incidence in double transgenic mice (<5%) decreased compared to HER2/neu mice (> 65%). In addition to a significant decrease in tumorigenesis, these mice expressed ERα as well as high levels of ERβ along with decreased levels of cyclin D1 and phosphorylated pRb among other changes. Furthermore, experiments using THC (ERα- agonist and ERβ-antagonist) clearly demonstrate the critical role of ERβ in HER2/neu-mediated tumorigenesis. These studies provide the first genetic evidence that estrogen receptor, mainly ERβ than ERα and its dependent changes play an important role in regulating mammary tumorigenesis. These findings provide further evidence for development and testing of novel therapeutic approaches based on selective regulation of estrogen receptors (ERα and β) - dependent actions for the treatment and prevention of breast cancers.

Factors associated with increased estrogen synthesis increase breast cancer risk. Increased aromatase and estrogen receptor α (ERα) in both normal epithelium and ductal carcinoma in situ lesions are found in conjunction with breast cancer, leading to the idea that altered estrogen signaling pathways predispose the mammary gland to cancer development. Here, we developed a transgenic mouse that conditionally expresses aromatase in the mammary gland, and used it along with a deregulated ERα expression model to investigate the molecular pathways involved in the development of mammary gland preneoplasia and carcinoma. Both increased ERα and aromatase expression led to the development of preneoplasia, but increased preneoplasia, in addition to carcinoma, was found in aromatase over-expressing mice. Increased prevalence of mammary pathological changes in mice expressing aromatase correlated with increased Cyclin E and Cyclin-dependent kinase 2 expression. Gain of both ERα and aromatase increased expression of ERα and progesterone receptor, but aromatase produced a higher increase than ERα, accompanied by higher levels of downstream target genes cyclin D1, c-Myc and RANKL. In summary, while gain of both ERα and aromatase activate abnormal growth pathways in the mammary gland, aromatase induced a wider range of abnormalities that was associated with a higher prevalence of mammary preneoplasia and cancer progression.

Objectives

Although renal angiomyolipoma (AML) occurs more commonly in females than males, the origin of this difference in incidence by sex is unknown. Therefore, we investigated the expression of the androgen receptor (AR), estrogen receptor subtypes alpha (ERα) and beta (ERβ), progesterone receptor, and the enzyme aromatase in renal AML.

Methods

We evaluated specimens from 110 patients who had undergone resection of a renal AML, including 90 women and 20 men. Immunohistochemistry was performed using monoclonal antibodies on paraffin-embedded tissue sections. Expression was correlated with patient demographics and tumor pathologic features.

Results

ERβ was expressed in 100% (106 of 106) of the AML specimens evaluated. Of the 104 specimens that could be assessed for the AR, 82 (79%) demonstrated staining. Of 110 lesions, 31 (28%), 42 (38%), and 11 (10%) expressed ERα, progesterone receptor, and aromatase, respectively. The level of ERβ expression was not associated with patient age (P = 0.92), sex (P = 0.82), a diagnosis of tuberous sclerosis (P = 0.56), or histologic subtype of AML (P = 0.94). A trend was found toward increased AR expression in men (P = 0.069) and younger patients (P = 0.052), and ERα was expressed in the AML specimens from 5 (71%) of 7 patients with tuberous sclerosis compared with 26 (25%) of 103 without tuberous sclerosis (P = 0.018). Both AR and ERα expression were more common in the triphasic subtype of AML than in the lipomatous tumors (P = 0.046 for both).

Conclusions

The results of our study have shown that ERβ expression is ubiquitous in renal AML, and the AR is found in most tumors. ERα and progesterone receptor were expressed in approximately one third of cases. These data suggest a potential role for hormones in the pathogenesis and management of renal AML.

Background

Atrazine, one of the most common pesticide contaminants, has been shown to up-regulate aromatase activity in certain estrogen-sensitive tumors without binding or activating the estrogen receptor (ER). Recent investigations have demonstrated that the orphan G-protein–coupled receptor 30 (GPR30), which is structurally unrelated to the ER, mediates rapid actions of 17β-estradiol and environmental estrogens.

Objectives

Given the ability of atrazine to exert estrogen-like activity in cancer cells, we evaluated the potential of atrazine to signal through GPR30 in stimulating biological responses in cancer cells.

Methods and results

Atrazine did not transactivate the endogenous ERα in different cancer cell contexts or chimeric proteins encoding the ERα and ERβ hormone-binding domain in gene reporter assays. Moreover, atrazine neither regulated the expression of ERα nor stimulated aromatase activity. Interestingly, atrazine induced extracellular signal-regulated kinase (ERK) phosphorylation and the expression of estrogen target genes. Using specific signaling inhibitors and gene silencing, we demonstrated that atrazine stimulated the proliferation of ovarian cancer cells through the GPR30–epidermal growth factor receptor transduction pathway and the involvement of ERα.

Conclusions

Our results indicate a novel mechanism through which atrazine may exert relevant biological effects in cancer cells. On the basis of the present data, atrazine should be included among the environmental contaminants potentially able to signal via GPR30 in eliciting estrogenic action.

The initiation of breast cancer is associated with increased expression of tumor-promoting estrogen receptor α (ERα) protein and decreased expression of tumor-suppressive ERβ protein. However, the mechanism underlying this process is unknown. Here we show that PES1 (also known as Pescadillo), an estrogen-inducible protein that is overexpressed in breast cancer, can regulate the balance between ERα and ERβ. We found that PES1 modulated many estrogen-responsive genes by enhancing the transcriptional activity of ERα while inhibiting transcriptional activity of ERβ. Consistent with this regulation of ERα and ERβ transcriptional activity, PES1 increased the stability of the ERα protein and decreased that of ERβ through the ubiquitin-proteasome pathway, mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). Moreover, PES1 transformed normal human mammary epithelial cells and was required for estrogen-induced breast tumor growth in nude mice. Further analysis of clinical samples showed that expression of PES1 correlated positively with ERα expression and negatively with ERβ expression and predicted good clinical outcome in breast cancer. Our data demonstrate that PES1 contributes to breast tumor growth through regulating the balance between ERα and ERβ and may be a better target for the development of drugs that selectively regulate ERα and ERβ activities.

Approximately 25% of breast cancers do not express the estrogen receptor (ERα) and consequently do not respond to endocrine therapy. In these tumors, ERα repression is often due to epigenetic modifications such as methylation and histone deacetylation. For this reason, we investigated the ability of the histone deacetylase inhibitor entinostat (ENT) to trigger re-expression of ERα and aromatase in breast cancer cells, with the notion that this treatment would restore sensitivity to the aromatase inhibitor letrozole. ENT treatment of tumor cells increased expression of ERα and aromatase along with the enzymatic activity of aromatase, in a dose-dependent manner both in vitro and in vivo. Notably, ERα and aromatase upregulation resulted in sensitization of breast cancer cells to estrogen and letrozole. Tumor growth rate was significantly lower in tumor xenografts following treatment with ENT alone and in combination with letrozole compared to control tumors (p >0.001). ENT plus letrozole also prevented lung colonization and growth of tumor cells with a significant reduction (p>0.03) in both visible and microscopic foci. Our results demonstrate that ENT treatment can be used to restore the letrozole responsiveness of ER-negative tumors. More generally, they provide a strong rationale for immediate clinical evaluation of combinations of histone deacetylase and aromatase inhibitors to treat ER-negative and endocrine-resistant breast cancers.

Estrogen levels increase during pregnancy and clinical evidence has long suggested that melanocytes are estrogen-responsive. We hypothesized that nevi from pregnant patients would exhibit increased expression of estrogen receptor β (ERβ) and thus enhanced potential to respond to altered estrogen levels. Normal, dysplastic and congenital nevi (n = 212) were collected from pregnant and non-pregnant women ranging from 18 to 45 years of age. Immunohistochemical staining was performed on these nevi using antibodies specifically directed against estrogen receptor α (ERα) and ERβ. ERα was not observed in any lesions; thus, ERβ was the predominant estrogen receptor in melanocytic cells from all types of nevi. Enhanced positivity for ERβ in normal nevi during pregnancy was noted, compared with non-pregnant controls including nevocytes residing in both the epidermal and dermal micro-environments (P = 0.005 and P = 0.001 respectively). Nevi with increasingly melanocytic atypia showed increased ERβ in nevocytes nested within the epidermis. No additional increase in ERβ in atypical nevi was observed during pregnancy. For normal and congenital nevi, regardless of pregnancy status, dermally associated nevocytes tended to have greater ERβ immunoreactivity. Significant decreases in ERβ immunoreactivity were observed in congenital nevi from pregnant women compared with normal and dysplastic nevi from pregnant women. Our data suggest that nevi possess the capacity to be estrogen-responsive. Factors such as pregnancy and degree of atypia are associated with enhanced ERβ with the exception of congenital nevi where the melanocytes were unique in their response to pregnancy.

The characterization of estrogen receptor beta (ERβ) brought new insight into the mechanisms underlying estrogen signaling. Estrogen induction of cell proliferation is a crucial step in carcinogenesis of gynecologic target tissues and the mitogenic effects of estrogen in these tissues (e.g. breast, endometrium and ovary) are well documented both in vitro and in vivo. There is also an emerging body of evidence that colon and prostate cancer growth is influenced by estrogens. In all of these tissues, most studies have shown decreased ERβ expression in cancer as compared to benign tumors or normal tissues, whereas ERα expression persists. The loss of ERβ expression in cancer cells could reflect tumor cell dedifferentiation but may also represent a critical stage in estrogen-dependent tumor progression. Modulation of the expression of ERα target genes by ERβ, or ERβ specific gene induction could indicate that ERβ has a differential effect on proliferation as compared to ERα. ERβ may exert a protective effect and thus constitute a new target for hormone therapy, e.g. via ligand specific activation. The potential distinct roles of ERα and ERβ expression in carcinogenesis, as suggested by experimental and clinical data, are discussed in this review.

Recent studies indicate that the expression of ERβ in breast cancer is lower than in normal breast, suggesting that ERβ could play an important role in carcinogenesis. To investigate this hypothesis, we engineered estrogen-receptor negative MDA-MB-231 breast cancer cells to reintroduce either ERα or ERβ protein with an adenoviral vector. In these cells, ERβ (as ERα) expression was monitored using RT-PCR and Western blot. ERβ protein was localized in the nucleus (immunocytochemistry) and able to transactivate estrogen-responsive reporter constructs in the presence of estradiol. ERβ and ERα induced the expression of several endogenous genes such as pS2, TGFα or the cyclin kinase inhibitor p21, but in contrast to ERα, ERβ was unable to regulate c-myc proto-oncogene expression. The pure antiestrogen ICI 164, 384 completely blocked ERα and ERβ estrogen-induced activities. ERβ inhibited MDA-MB-231 cell proliferation in a ligand-independent manner, whereas ERα inhibition of proliferation is hormone-dependent. Moreover, ERβ and ERα, decreased cell motility and invasion. Our data bring the first evidence that ERβ is an important modulator of proliferation and invasion of breast cancer cells and support the hypothesis that the loss of ERβ expression could be one of the events leading to the development of breast cancer.

We reported previously that both subtypes of estrogen receptors, ERα and ERβ, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ERα and ERβ mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells obtained from six individuals with three immortalized urothelial (E6, E7, and UROtsa) and two bladder cancer cell lines (HTB-9 and T24). We found that all these cells express similar levels of ERβ, but immortalized and cancer cells express much higher amounts of ERα than primary cells. Higher levels of ERα mRNA were also observed in the biopsies of bladder transitional cell carcinoma compared with sample from the same bladder unaffected by tumor. Using the ERα-selective agonist PPT, the ERβ-selective agonist DPN, and specific small interfering RNA against ERα or ERβ, we found that ERβ predominantly mediates estrogen-induced G1/S transition and cell proliferation in the primary urothelial cells. By contrast, ERα predominantly mediates estrogen-induced G1/S transition and cell proliferation in bladder cancer cell lines. Furthermore, we found that 17β-estradiol (E2) rapidly induces phosphorylation of extracellular signal-regulated kinases, but U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, does not affect E2-induced urothelial cell proliferation. E2 up-regulated cyclin D1 and cyclin E expression in both the primary and bladder cancer cells, and the cancer cells have higher cyclin D1 and cyclin E expression during G0/G1 phases. Our data suggest that estrogen exerts its effects through different ER subtypes in urothelial cells. Increased expression of ERα may contribute to early induction of cyclin D1 and cyclin E during the cell cycle in bladder cancer cells.

Background

Estrogen receptors alpha (ERα) and beta (ERβ) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERβ being able to modulate the effects of ERα on gene transcription and cell proliferation. ERβ is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERβ in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology.

Results

Expression of full-length ERβ in hormone-responsive, ERα-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERβ and 6024 ERα binding sites in estrogen-stimulated cells, comprising sites occupied by either ERβ, ERα or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb. These putative primary ERβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERβ binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions.

Conclusions

Results indicate that the vast majority of the genomic targets of ERβ can bind also ERα, suggesting that the overall action of ERβ on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.

Breast cancer cells show overexpression of estrogen receptor (ER) α relative to ERβ compared to normal breast tissues. This observation has lead to the hypothesis that ERβ may modulate the proliferative effect of ERα. This study investigated how variable cellular expression ratios of the ERα and ERβ modulate the effects on cell proliferation induced by ERα or ERβ agonists, respectively. Using human osteosarcoma (U2OS) ERα or ERβ reporter cells, propyl-pyrazole-triol (PPT) was shown to be a selective ERα and diarylpropionitrile (DPN) a preferential ERβ modulator. The effects of these selective estrogen receptor modulators (SERMs) and of the model compound E2 on the proliferation of T47D human breast cancer cells with tetracycline-dependent expression of ERβ (T47D-ERβ) were characterized. E2-induced cell proliferation of cells in which ERβ expression was inhibited was similar to that of the T47D wild-type cells, whereas this E2-induced cell proliferation was no longer observed when ERβ expression in the T47D-ERβ cells was increased. In the T47D-ERβ cell line, DPN also appeared to be able to suppress cell proliferation when levels of ERβ expression were high. In the T47D-ERβ cell line, PPT was unable to suppress cell proliferation at all ratios of ERα/ERβ expression, reflecting its ability to activate only ERα and not ERβ. It is concluded that effects of estrogen-like compounds on cell proliferation are dependent on the actual ERα/ERβ expression levels in these cells or tissues and the potential of the estrogen agonists to activate ERα and/or ERβ.

The role of estrogens in the increased risk of lung adenocarcinoma in women remains uncertain. We reported that lung adenocarcinoma cell lines from female, but not male, patients with non–small cell lung cancer respond proliferatively and transcriptionally to estradiol (E2), despite equal protein expression of estrogen receptors (ER) α and β. To test the hypothesis that nuclear localization of ERα corresponds to genomic E2 activity in lung adenocarcinoma cells from females, cell fractionation, immunoblot, and confocal immunohistochemical microscopy were performed. We report for the first time that E2 increases phospho-serine-118-ERα (P-ser118-ERα) and cyclin D1 (CCND1) nuclear colocalization in H1793, but not A549 lung adenocarcinoma cells, derived from a female and male patient, respectively. ERβ was primarily in the cytoplasm and mitochondria, independent of E2 treatment, and showed no difference between H1793 and A549 cells. E2 induced higher transcription of endogenous ERα-regulated CCND1 in H1793 than in A549 cells. Likewise, higher rapid, non-genomic E2-induced extracellular signal–regulated kinase 1/2 activation was detected in H1793 compared with A549 cells, linking extracellular signal–regulated kinase activation to increased P-ser118-ERα. Furthermore, E2 increased cyclin D1 and P-ser118-ERα nuclear localization in H1793, but not A549 cells. Together, our results indicate that nuclear localization of P-ser118-ERα provides one explanation for sex-dependent differences in E2-genomic responses in lung adenocarcinoma cell lines.

The estrogen receptor α (ERα)-mediated pathway plays a critical role in breast cancer development and progression. KiSS1 was previously described as a metastasis suppressor gene in certain carcinomas. However, the role of KiSS1/GPR54 in breast cancer remains controversial. Whether the function of the KiSS1/GPR54 system depends on estrogen signaling in the breast cancer cell remains to be determined. This study aimed to determine the expression profiles of the KiSS1/GPR54, ERα, ERβ, aromatase and cyclin D1 genes in human breast cancer tissues, and to identify a possible link between the expression levels of the studied genes and the selected clinical and pathological features. The study subjects comprised 59 females treated surgically for primary breast cancer. Total RNA was extracted from frozen breast cancer tissues, and expression levels were examined to determine any correlations. We observed strong positive correlations between the expression levels of the studied genes. The expression of ERα correlated positively with progesterone receptors (PRs), and in these tumors we also observed positive correlations between KiSS1, GPR54 and cyclin D1 mRNAs and the ERα protein. ER-positive breast tumors exhibited higher KiSS1 and GPR54 levels than the ER-negative tumors. The expression levels of the ERα and GPR54 transcripts were higher in the moderately differentiated tumors (G2) compared to the poorly differentiated high-grade (G3) cancers. We also found that HER-2/neu status in breast cancer is negatively associated with GPR54 mRNA expression. Decreasing GPR54 mRNA expression levels in HER-2/neu (+) tumors may be associated with the deregulation of the classical estrogen-mediated signaling pathway in breast tumors, and therefore, with promotion of tumor invasiveness. Our findings indicate that genes involved in the KiSS1/GPR54 system, as well as in the estrogen signaling pathway, may be utilizable molecular factors in pathogenesis studies of breast cancer.

Estrogenic effects are mediated through two estrogen receptor (ER) subtypes, ERα and ERβ. Estrogens are the most commonly prescribed drugs to treat menopausal conditions, but by non-selectively triggering both ERα and ERβ pathways in different tissues they can cause serious adverse effects. The different sizes of the binding pockets and sequences of their activation function domains indicate that ERα and ERβ should have different specificities for ligands and biological responses that can be exploited for designing safer and more selective estrogens. ERα and ERβ regulate different genes by binding to different regulatory elements and recruiting different transcription and chromatin remodeling factors that are expressed in a cell-specific manner. ERα- and ERβ-selective agonists have been identified that demonstrate that the two ERs produce distinct biological effects. ERα and ERβ agonists are promising new approach for treating specific conditions associated with menopause.