Steroid hormones and growth factors affect lung cancer, and it is possible they act in concert to influence patient outcome.
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).
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.
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 receptor; progesterone receptor; aromatase; epidermal growth factor receptor; lung cancer
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.
non-small cell lung cancer (NSCLC); aromatase; CYP19; estrogen receptor (ER); epidermal growth factor receptor (EGFR); vascular endothelial growth factor (VEGF) receptor; anastrazole; exemestane; fulvestrant; vandetanib
Lung cancer is the leading cause of cancer-related deaths in both men and women worldwide. Despite advances in treatment, patients have few effective therapeutic options and survival rates remain low. Emerging evidence suggests that the hormones estrogen and progesterone play a key role in the progression of non-small-cell lung cancer (NSCLC). The aromatase enzyme, which is responsible for a key step in estrogen biosynthesis, elicits higher levels of estrogen in lung tumors as well as in metastases compared with nonmalignant tissues. Thus, aromatase may prove to be a key predictive biomarker for treatment of NSCLC. Epidemiologic and preclinical data show estrogens play a critical role in lung tumor development and progression. Two estrogen receptors, α and β, are expressed in normal and in cancerous lung epithelium, and estrogen promotes gene transcription that stimulates cell proliferation and inhibits cell death. Furthermore, expression of both forms of estrogen receptor, progesterone receptor and aromatase in NSCLC specimens has been correlated with worse clinical outcomes. Combination therapies that include estrogen receptor downregulators and aromatase inhibitors are currently being assessed in Phase I–II clinical trials among patients with advanced NSCLC. Results will help guide future lung cancer management decisions, with a goal of achieving more effective and less toxic treatments for patients.
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.
Progesterone; Estrogen; Steroid hormone receptor; Non-small cell lung cancer; VEGF; Progenitor cells; Cancer stem cells; Angiogenesis
A role for estrogen signaling in urothelial carcinoma of the bladder (UCB) is suggested to be associated with more advanced disease with worse outcomes in women. Estrogen receptor β (ERβ) is the predominant receptor in bladder tissues. We aimed to ascertain whether ERβ correlates with clinicopathological predictors of aggressive bladder cancer and worse survival outcomes. ERβ was measured by immunohistochemistry in malignant and adjacent benign bladder tissues in patients (N=72) with UCB who underwent radical cystectomy. ERβ expression was tested for statistical association with clinicopathological variables and patient survival. ERβ expression was determined in bladder cancer cell lines, and the effects of the selective estrogen modulator tamoxifen and the ERβ agonist diarylpropionitrile on cell growth were determined. The ERβ level was significantly higher in malignant vs. benign urothelium (P<0.001) and was strongly associated with aggressive tumor histology characterized by lymphovascular (P=0.008) and perineural (P=0.006) invasion, and clinical histories of pelvic irradiation (P=0.005), hydronephrosis (P=0.022) and no intravesical chemotherapy (P=0.038). All patients with a high (>70%) percentage of ERβ positivity in tissue with >3-month follow-up developed recurrent disease (P=0.009). Higher ERβ level was predictive of worse recurrence-free and overall survival following cystectomy, after adjustment for tumor stage, and remained significantly associated with recurrence-free survival in the multivariable analysis including tumor stage, nodal stage and lymphovascular invasion. Activation of ERβ in bladder cancer cell lines led to significant increases in proliferation, while pharmacological inhibition with tamoxifen blocked cell growth. Our study supports a role for ERβ in aggressive UCB. Pharmacological targeting of ERβ warrants further investigation as a therapeutic strategy in UCB.
epigenetic; hormonal; urothelial; transitional cell
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.
non-small cell lung cancer; estrogen receptor; 4,4’,4”-(4-propyl-[1H]-pyrazole-l,3,5-triyl)trisphenol; 2,3-bis(4-hydroxyphenyl)propionitrile; genistein
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.
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.
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.
Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.
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.
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.
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.
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.
Sub-study of trial P025 for advanced breast cancer.
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.
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.
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.
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.
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.
Aromatase, HER-2/neu, mammary tumorigenesis; Transgenic mice; Hormonal carcinogenesis
TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERα and ERβ) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β-hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERα-positive luminal MCF7 breast cancer cells. Low levels of ERα and ERβ mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17βHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17βHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17βHSD1, and 17βHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERα, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERβ, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.
Estrogen metabolism; Gene expression; MCF10A lineage; Breast cancer progression; Histone deacetylase inhibition
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.
non-small cell lung cancer; estrogen; aromatase; CYP19; promoter; promoter usage
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.
aromatase; estrogen; receptor; breast cancer; mammary; hyperplasia
Vascular endothelium expresses both the estrogen receptors (ERs) α and β, and ERα mediates development of early atherosclerosis in male mice. This process is thought to be testosterone-dependent. We hypothesized that male murine aortic endothelium produces robust levels of estradiol by aromatase conversion of testosterone, and that regulation of this process is mediated by the presence of ERs, primarily ERα. Aortic endothelium was isolated from ERα knockout (ERα -/-) and wild-type (ERα +/+) male mice and treated with testosterone or the 5α reduction product dihydrotestosterone (DHT), with or without the P450 aromatase inhibitor anastrazole, or a non-specific estrogen receptor antagonist. Aromatase gene expression and estradiol production were assayed. Treatment with testosterone, but not DHT, caused increased aromatase expression and estradiol production in ERα +/+ endothelium that was attenuated by disruption of ERα in the ERα -/- group. Anastrazole inhibition of aromatase reduced testosterone-induced aromatase expression and estradiol levels in both ERα -/- and ERα +/+ endothelium. Antagonism of both ERs decreased testosterone-induced aromatase expression in both wild-type and knockout groups. The effects of the receptor antagonist on estradiol production differed between the two groups, however, with a reduction in estradiol release from the ERα +/+ cells and complete abolition of estradiol release from the ERα -/- cells. Thus, estradiol production in vascular endothelium from male mice is robust, depends on the aromatic conversion of testosterone and requires functional ERα to achieve maximal levels of estradiol generation. Local vascular production of aromatase-mediated estradiol in response to circulating testosterone may affect ERα-dependent mechanisms to increase susceptibility to early atheroma formation in male mice. This pathway may have important therapeutic relevance for reducing the risk of atherosclerotic cardiovascular disease in human males.
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.
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.
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.
17β-estradiol; atrazine; estrogen receptor; GPR30; ovarian cancer cells
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.
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.
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).
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.
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.
aromatase inhibitor; HDAC; ERα; epigenetic silencing
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.
estrogen receptor β; immunohistochemistry; nevi; nevocytes; 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.
Breast Neoplasms; etiology; metabolism; pathology; Disease Progression; Estrogen Receptor alpha; deficiency; Estrogen Receptor beta; deficiency; Female; Humans; Neoplasms, Hormone-Dependent; etiology; metabolism; pathology
Estrogens play a salient role in the development and maintenance of both male and female nervous systems and behaviors. The plainfin midshipman (Porichthys notatus), a teleost fish, has two male reproductive morphs that follow alternative mating tactics and diverge in multiple somatic, hormonal and neural traits, including the central control of morph-specific vocal behaviors. After we identified duplicate estrogen receptors (ERβ1 and ERβ2) in midshipman, we developed antibodies to localize protein expression in the central vocal-acoustic networks and saccule, the auditory division of the inner ear. As in other teleost species, ERβ1 and ERβ2 were robustly expressed in the telencephalon and hypothalamus in vocal-acoustic and other brain regions shown previously to exhibit strong expression of ERα and aromatase (estrogen synthetase, CYP19) in midshipman. Like aromatase, ERβ1 label co-localized with glial fibrillary acidic protein (GFAP) in telencephalic radial glial cells. Quantitative PCR revealed similar patterns of transcript abundance across reproductive morphs for ERβ1, ERβ2, ERα and aromatase in the forebrain and saccule. In contrast, transcript abundance for ERs and aromatase varied significantly between morphs in and around the sexually polymorphic vocal motor nucleus (VMN). Together, the results suggest that VMN is the major estrogen target within the estrogen-sensitive hindbrain vocal network that directly determines the duration, frequency and amplitude of morph-specific vocalizations. Comparable regional differences in steroid receptor abundances likely regulate morph-specific behaviors in males and females of other species exhibiting alternative reproductive tactics.
duplicate estrogen receptors; auditory; vocal; midshipman fish; central pattern generator
To investigate the mechanisms by which breast cancer cells adapt and are able to grow during estrogen deprivation, human estrogen receptor-α (ERα)-positive breast cancer cells stably transfected with the aromatase gene (MCF-7Ca) were cultured in steroid-depleted medium for 6–8 months until they started proliferating. Compared with the parental MCF-7Ca cells, long-term estrogen-deprived UMB-1Ca cells exhibited increased aromatase activity (2000%), AIB1 expression (3500%) and ERα expression (100%). When MCF-7Ca cells were isolated from tumors of mice treated for 12 months with an aromatase inhibitor, letrozole, ERα was reduced (50%) whereas AIB1 levels were increased (>1000%), suggesting that the mechanism of estrogen deprivation might predetermine the signaling pathway utilized. To a lesser extent long-term estrogen-deprived MCF-7 cells (LTED) displayed an increase in AIB1, ERα and aromatase activity. Consistent with other findings, the growth of the LTED cells was inhibited by estradiol and antiestrogens, whereas the UMB-1Ca cells were slightly stimulated by estradiol and inhibited by antiestrogens and letrozole. In LTED cells treated with estradiol, levels of AIB1 and ERα (95%) were reduced. Interestingly, estradiol treatment caused no change in AIB1 and ERα expression in the UMB-1Ca cells which might explain the differential growth effect of the cells to estradiol. Together, these results demonstrate that estrogen deprivation results in the upregulation of the estrogen signaling pathway at the level of AIB1, ERα and aromatase, which might attenuate ER-mediated transcription representing one mechanism by which tumors adapt to proliferation in a low estrogenic environment.
aromatase; breast cancer; coactivators; estrogen receptor; long-term estrogen deprivation
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.
Adenoviridae; genetics; Breast Neoplasms; pathology; physiopathology; Cell Division; physiology; Cell Line; Cell Movement; physiology; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; physiology; Gene Expression Regulation; physiology; Gene Transfer Techniques; Genes, Reporter; physiology; Genetic Vectors; Humans; Neoplasm Invasiveness; pathology; physiopathology; Receptors, Estrogen; physiology