Pure Antiestrogen ICI Research Articles

Evidence that Adiponectin Fuels LKB1/ERα Interaction in Breast Cancer Cells

BACKGROUNDObesity is characterized by a chronic and excessive growth of adipose tissue and it is increasingly recognized as an oncogenic factor. Due to its endocrine function, adipose tissue secretes several bioactive molecules known as adipocytokines. Among these, adiponectin plays a key role in the pathogenesis of different obesity‐associated diseases, including malignancies, such as breast cancer.The main pathway through which adiponectin exerts its effects is LKB1/AMPK/mTOR signaling. It has been reported that LKB1 plays an essential role in mammary carcinogenesis, regulating apoptosis, proliferation, angiogenesis, energy metabolism and cell polarity. Moreover, low levels of LKB1 have been associated with a more aggressive tumor phenotype and correlate with a poor prognosis. In contrast, LKB1 overexpression induces inhibition of tumor growth, angiogenesis and metastases. In the recent years, our research group has identified ERα as an important regulator of adiponectin action in breast cancer, clarifying that the role of this cytokine seems to be dependent on cell phenotypes.Here, we provided novel insights about the effects of adiponectin on the functional interaction between ERα and LKB1 in ERα‐positive (MCF‐7 and ZR75) and negative (MDA‐MB‐231) breast cancer cells.METHODSRNA levels were assessed by RT‐PCR. Protein expressions were evaluated by immunoblotting in the presence or absence of ERα siRNA and antiestrogen ICI 182, 780. To study promoter activity, transient transfection assays were done. The recruitment of ERα on LKB1 promoter was evaluated by ChIP and DAPA assays. Immunofluorescence assays were performed to evaluate LKB1 localization and function.RESULTSWe demonstrated that adiponectin increased mRNA and protein levels of LKB1 in ERα‐positive breast cancer cells MCF‐7 and ZR75, whereas no significant changes were observed in ERα‐negative MDA‐MB‐231 cells. This up‐regulatory effect was due to the direct action of adiponectin on the promoter region of LKB1 gene. For instance, in MCF‐7 cells, transiently transfected with plasmids containing the full length or a series of 5′ deleted segments of human LKB1 promoter, we identified a sequence (−1626 to −1598) containing Estrogen Responsive Elements (ERE). ChIP and DAPA assays evidenced an increased association of ERα on ERE‐containing sequence proving the recruitment of ERα on LKB1 promoter in adiponectin‐treated cells. Noteworthy, in the presence of ERα siRNA and the pure antiestrogen ICI 182, 780 the upregulation of LKB1 expression was no longer noticeable. It is extremely intriguing to remark how the increasing intracellular pool of LKB1 upon adiponectin was mostly sequestered in the nuclear compartment as coactivator of ERα, which in turn uncoupled it from AMPK interaction, generally leading to the inhibition of cellular energy expenditure.CONCLUSIONSOur results demonstrated how ERα may impact LKB1/AMPK functional interaction upon adiponectin exposure and in such way may negatively interfere in switching cellular energy balance.Support or Funding InformationAssociazione Italiana Ricerca sul Cancro (AIRC) Grants IG‐18602; Progetti di Ricerca di Interesse Nazionale‐Ministero Istruzione Università e Ricerca Grant 2015B7M39T_001This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fatty acid synthase regulates estrogen receptor-\u03b1 signaling in breast cancer cells

Fatty acid synthase (FASN), the key enzyme for endogenous synthesis of fatty acids, is overexpressed and hyperactivated in a biologically aggressive subset of sex steroid-related tumors, including breast carcinomas. Using pharmacological and genetic approaches, we assessed the molecular relationship between FASN signaling and estrogen receptor alpha (ERα) signaling in breast cancer. The small compound C75, a synthetic slow-binding inhibitor of FASN activity, induced a dramatic augmentation of estradiol (E2)-stimulated, ERα-driven transcription. FASN and ERα were both necessary for the synergistic activation of ERα transcriptional activity that occurred following co-exposure to C75 and E2: first, knockdown of FASN expression using RNAi (RNA interference) drastically lowered (>100 fold) the amount of E2 required for optimal activation of ERα-mediated transcriptional activity; second, FASN blockade synergistically increased E2-stimulated ERα-mediated transcriptional activity in ERα-negative breast cancer cells stably transfected with ERα, but not in ERα-negative parental cells. Non-genomic, E2-regulated cross-talk between the ERα and MAPK pathways participated in these phenomena. Thus, treatment with the pure antiestrogen ICI 182 780 or the potent and specific inhibitor of MEK/ERK, U0126, was sufficient to abolish the synergistic nature of the interaction between FASN blockade and E2-stimulated ERα transactivation. FASN inhibition suppressed E2-stimulated breast cancer cell proliferation and anchorage-independent colony formation while promoting the reduction of ERα protein. FASN blockade resulted in the increased expression and nuclear accumulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, two critical mediators of the therapeutic effects of antiestrogen in breast cancer, while inactivating AKT, a key mediator of E2-promoted anchorage-independent growth. The ability of FASN to regulate E2/ERα signaling may represent a promising strategy for anticancer treatment involving a new generation of FASN inhibitors.

The function of membrane-associated molecules in acquired resistance to antiestrogens in breast cancer

Long-term clinical adjuvant antihormone therapy for breast cancer has significantly improved survival of estrogen receptor (ER)-positive breast cancer patients, but acquired resistance to antiestrogens is a major challenge in clinic. The evolution of acquired resistance to selective estrogen receptor modulators (SERMs) is unique because the growth of resistant tumors is dependent on SERMs. Thus, it appears that acquired resistance to SERMs is initially able to utilize either estrogen (E 2 ) or a SERM as the growth stimulus in the ER-positive SERM-resistant breast tumors. However, no mechanism has been established to explain this paradox. Our newly established cell model MCF-7: PF, for the first time, replicates Phase I acquired resistance to SERMs in vitro . The cells are stimulated to grow robustly with E 2 and SERMs through the ER which is confirmed by the evidence that pure antiestrogen ICI 182,780 (ICI) completely blocks the stimulation induced by E 2 or SERMs. In contrast to E 2 that activates classical ER-target genes, SERMs continue to function as effective antiestrogens to inhibit classical ER-target genes, even at the time of growth stimulation. A significant alteration of ER function observed in SERM-resistant cells is the enhancement of the non-genomic pathway of ER and the activation of multiple membrane function-associated molecules including focal adhesion molecules and adapter proteins to further increase phosphorylation of insulin-like growth factor-1 receptor (IGF-1R). Inhibition of membrane-associated signaling, IGF-1R and focal adhesion kinase (FAK), completely abolishes 4-OHT-stimulated cell growth. Overall, the constant nuclear pressure causes broad activation of membrane-associated signaling to aid breast cancer cell survival during the selection process required for acquired SERM resistance. The targeting of these membrane function-associated pathways and seeking new unanticipated combination therapies may have further clinical potential to decipher and treat endocrine-resistant breast cancer.

Elucidation of Underlying Mechanisms by Which Millettia macrophylla Benth Induces Its Estrogenic Activity.

Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its secondary metabolites to ERα and ERβ. In vivo, the cotreatment of extracts and ICI 182,780 significantly abrogated the biological responses induced by the extracts alone. Taken together, these results indicate that the active principles of M. macrophylla induce their beneficial effects on menopausal symptoms by activating the ERs.

Neonatal exposure to zearalenone induces long term modulation of ABC transporter expression in testis

Mycotoxin zearalenone (ZEN) is a cereal contaminant produced by various species of Fusarium fungi. When interacting with estrogen receptors, ZEN leads to animal fertility disturbances and other reproductive pathologies. Few data are available on the effects of perinatal exposure to ZEN, particularly in the blood–testis barrier. The aim of this study was to assess the impact of ZEN in adult rats exposed neonatally. We focused on the expression and cellular localization of major ABC transporters expressed in adult rat testis, comparing ZEN effects with those of Estradiol Benzoate (EB) neonatal exposure. Dose-dependent and long term modulations of mRNA and protein levels of Abcb1, Abcc1, Abcg2, Abcc4 and Abcc5 were observed, along with Abcc4 protein cellular delocalization. ZEN exposure of SerW3 Sertoli cells showed modulation of Abcb1, Abcc4 and Abcc5. Comparison with EB exposure showed similar modulation profiles for Abcg2 but differential modulations for Abcb1, Abcc1, Abcc4 and Abcc5 in vivo, and a similar profile for Abcb1 modulation by ZEN and EB, but differential modulation for Abcc4 and Abcc5 in vitro. ZEN and EB effects were inhibited by in vitro addition of the pure anti-estrogen ICI 182.780, suggesting the at least partial implication of ZEN estrogenic activity in these modulations. These results suggested that ZEN neonatal exposure could affect the exposure of testis to ABC transporter substrates, and negatively influence spermatogenesis and male fertility.

AKT-aro and HER2-aro, models for de novo resistance to aromatase inhibitors; molecular characterization and inhibitor response studies

Aromatase inhibitors (AI) are currently the first line therapy for estrogen receptor (ER)-positive postmenopausal women. De novo AI resistance is when a patient intrinsically does not respond to an AI therapy as well as other targeted endocrine therapy. To characterize this type of resistance and to examine potential therapies for treatment, we have generated two cell models for de novo resistance. These models derive from MCF-7 cells that stably overexpress aromatase and Akt (AKT-aro) or HER2 (HER2-aro). Evaluation of these cell lines revealed that the activities of aromatase and ER were inhibited by AI and ICI 187280 (ICI) treatment, respectively; however, cell growth was resistant to therapy. Proliferation in the presence of the pure anti-estrogen ICI, indicates that these cells do not require ER for cell growth and distinguishes these cells from the acquired AI resistant cells. We further determined that the HSP90 inhibitor 17-DMAG suppressed the growth of the AI-resistant cell lines studied. Our analysis revealed 17-DMAG-mediated decreased expression of growth promoting signaling proteins. It was found that de novo AI resistant AKT-aro and HER2-aro cells could not be resensitized to letrozole or ICI by treatment with 17-DMAG. In summary, we have generated two cell lines which display the characteristics of de novo AI resistance. Together, these data indicate the possibility that HSP90 inhibitors may be a viable therapy for endocrine therapy resistance although additional clinical evaluation is needed.

The terminal substituents of 7α, 6-hexanyl derivatives of estradiol determine their selective estrogen receptor modulator versus agonist activities

Pure antiestrogens were clinically developed as alternative therapies for estrogen receptor (ER) positive breast cancers. Unlike the selective estrogen receptor modulators (SERMs), these antiestrogens are devoid of tissue-specific ER agonist activity. Many of these compounds are steroidal in nature, containing an estradiol (E2) structural core with long alkyl side chains at the C-7α position. Two novel 7α-substituted E2 derivatives were evaluated that retain high binding affinity for ER. Compared to known pure antiestrogens, these compounds, referred to as compound 13 (C13) and C14, contain shorter 7α alkyl side chains and differ only in their terminal substituent: a hydroxyl moiety versus a benzyloxy group, respectively. Herein we assessed the effects of these compounds on ER transcriptional activity and report that despite their similar overall structure, C13 and C14 produce distinct cell type-specific responses. Of note, C13 functions as a mixed agonist/antagonist in Hela cells, inducing only weak ER transcriptional activity while preventing coactivator recruitment and stabilizing ER expression. However, this compound effectively stimulates ER activity in MCF-7 cells, does not increase ER levels and promotes cell proliferation on par with E2. Conversely, C14 stimulates transcriptional activity in both cell types and enhances ER–coactivator interactions. The activities of both compounds were inhibited by the pure antiestrogen ICI 182,780. Taken together, these results reveal that C13 is a SERM while C14 is an ER agonist, and indicate that the terminal modification of the C-7α hexanyl side chain of these estradiol derivatives is an important determinant of the biocharacter of these ER ligands.

Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon

Excessive Cl − secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl − secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl − secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl − secretion in rat colonic mucosal sheets was studied by current–voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K + channel and apical Cl − channel activity, respectively. 17β-Estradiol dose-dependently inhibited secretory responses to both toxins with IC 50 values of approximately 1 nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17β-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17β-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCδ activation. In nystatin-permeabilized tissues, apical Cl − currents were unaffected by 17β-estradiol treatment while basolateral K + current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17β-estradiol inhibits enterotoxin-induced Cl − secretion via a PKCδ-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17β-estradiol inhibition of Cl − secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.

Abstract 2294: The nuclear association among ERα – PR – AIB1 is modulated by progestins and antiprogestins in experimental breast cancer

Abstract The nuclear receptor coactivator AIB1 (SRC-3) is intimately associated with hormone action in estrogen receptor (ER)-positive breast cancer, and its overexpression has been associated with early resistance to endocrine therapy. In two models of ER+, progesterone receptor (PR)+ breast cancer, human T47D cells growing in vitro and C4-HD and C4-HI mouse mammary carcinomas maintained by syngeneic transplantations in BALB/c mice, we have shown that progestins induce a nuclear physical association between ERα and PR isoform A (PRA). We hypothesize that this interaction is necessary to induce progestin-induced cell proliferation. While both the pure antiestrogen ICI 182780 (ICI) and the antiprogestin RU486 inhibit tumor growth, only ICI blocked the ERα-PRA nuclear interaction induced by medroxyprogesterone acetate (MPA). RU486 showed an agonist-like effect stimulating the association between both receptors. The aim of this study was to investigate the interaction of ERα with PR isoform B (PRB) in both models and to evaluate the role of AIB1 in progestin-induced proliferation and in RU486-induced inhibition of cell proliferation. MPA (10nM) treatment induced an early increase in the nuclear co-localization between pSer118 ERα and pSer162 PRB in epithelial cells from the mouse model (p<0.001), and between total ERα with pSer162 PRB in T47D cells (p<0.001), as shown by confocal microscopy. By co-immunoprecipitation assays we corroborated that both PR isoforms interact with ERα in the cell nuclei. We also found that MPA increased the nuclear association of pSer294 PR and ERα with AIB1 in the murine epithelial tumor cells (p<0.001). These results were corroborated by co-immunoprecipitation in nuclear extracts from untreated or MPA-treated T47D cells. RU486 (10nM) induced similar ERα-PR interactions as MPA, but no association between PR and AIB1 was observed in RU486-treated cells. Our data support the hypothesis that a nuclear complex of ERα, both PR isoforms, and AIB1 mediates the proliferative effects of MPA. In addition, the results suggest that the lack of association of ERα-PR complexes with AIB1 may explain the antagonist effect of RU486 in these cells, supporting the hypothesis that the inhibitory effects of RU486 are related to genomic events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2294. doi:10.1158/1538-7445.AM2011-2294

Research Resource: Estrogen-Driven Prolactin-Mediated Gene-Expression Networks in Hormone-Induced Prostatic Intraepithelial Neoplasia

Cotreatment with testosterone (T) and 17β-estradiol (E2) is an established regimen for inducing of prostatic intraepithelial neoplasia (PIN) and prostate cancer in rodent models. We previously used the pure antiestrogen ICI 182,780 (ICI) and bromocriptine, a dopamine receptor agonist, to inhibit PIN induction and systemic hyperprolactinemia in Noble rats and found that the carcinogenic action of T+E2 is mediated directly by the effects of E2 on the prostate and/or indirectly via E2-induced hyperprolactinemia. In this study, we delineate the specific action(s) of E2 and prolactin (PRL) in early prostate carcinogenesis by an integrated approach combining global transcription profiling, gene ontology, and gene-network mapping. We identified 2504 differentially expressed genes in the T+E2-treated lateral prostate. The changes in expression of a subset of 1990 genes (∼80%) were blocked upon cotreatment with ICI and bromocriptine, respectively, whereas those of 262 genes (∼10%) were blocked only by treatment with ICI, suggesting that E2-induced pituitary PRL is the primary mediator of the prostatic transcriptional response to the altered hormone milieu. Bioinformatics analyses identified hormone-responsive gene networks involved in immune responses, stromal tissue remodeling, and the ERK pathway. In particular, our data suggest that IL-1β may mediate, at least in part, hormone-induced changes in gene expression during PIN formation. Together, these data highlight the importance of pituitary PRL in estrogen-induced prostate tumorigenesis. The identification of both E2- and pituitary PRL-responsive genes provides a comprehensive resource for future investigations of the complex mechanisms by which changes in the endocrine milieu contribute to prostate carcinogenesis in vivo.

Abstract 4610: Nuclear interaction between estrogen and progesterone receptors in experimental breast cancer is blocked by antiestrogens but not by RU 486

Abstract The expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) in breast cancer is used as a prognostic factor and identifies a group of tumors which may respond to an endocrine therapy aimed to block estrogen synthesis or ER activation. There is however, compelling evidence that indicates that PR is also involved in breast cancer growth. We have developed an experimental model of breast cancer in BALB/c female mice in which mammary carcinomas are induced by the administration of medroxyprogesterone acetate (MPA). The hormone-dependent (HD) tumor, C4-HD, grows in vivo only in the presence of MPA, and expresses ERα and both PR isoforms (PRA and PRB). PR are essential for tumor growth in this model, since antisense oligonucleotides of PR and different antiprogestins inhibit their growth. Interestingly, 17β-estradiol (E2) and the pure antiestrogen ICI 182780 (ICI) also induce tumor regression. It is controversial how progesterone (Pg) induces non genomic effects. Although it has been demonstrated that Pg binding to cytosolic PRB activates c-Src through an interaction with ERα, others have reported that this interaction is not necessary for c-Src activation in human T47D breast cancer cells. In our Laboratory, we have shown a nuclear physical association between ERα and PR in C4-HD tumors, and we postulate that this interaction is necessary to induce progestin stimulated cell proliferation. The aim of this study was to further investigate this nuclear interaction in C4-HD and in T47D epithelial cells. We found, by confocal immunofluorescence, that MPA (10nM) treatment induced an early increase in the nuclear co-localization between ERα and PRA in both tumor models (p<0.001). These results were corroborated by co-immunoprecipitation assays showing that both PR isoforms interact with ERα in the cell nuclei. We also found nuclear interaction between both pSer118 ERα and pSer294 PR after 15 minutes of MPA incubation in C4-HD cells (p<0.001), indicating that this complex could be transcriptionally active. E2 (1nM) and ICI (1μM) treatment inhibited this interaction whereas the antiprogestin RU 486 (RU, 10nM), which also inhibits cell proliferation as E2 and ICI, behaved as a PR agonist supporting previous data of our lab that indicates that RU activates MAPK as MPA, and that the inhibitory effects induced by RU are due to genomic events. Our data support the hypothesis that ERα is involved in MPA-induced cell proliferation; we postulate however that the nuclear interaction between ERα and PR participates in the progestin induced stimulatory effects in these breast cancer models. In addition, the results shown herein reinforce our previous findings in which we showed early agonistic effects of RU mediating non genomic events, despite inducing tumor regression. These results highlight the importance of targeting both ERα and PR in breast cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4610.

Bisphenol A impacts cardiomyocyte differentiation in vitro by modulating cardiac protein expression

introduction: Bisphenol A (BPA) is an environmental toxin commonly found in plastics and is able to mimic the actions of endogenous steroid hormones. BPA binds and activates intracellular estrogen receptors (eRα and eRβ) and estrogen related receptor γ (eRRγ), all of which are present in cardiomyocytes. however, it is unclear how BPA impacts the heart. We hypothesized that BPA modulates the expression of proteins regulating cardiac structure, energy and calcium homeostasis during cardiomyocyte differentiation in vitro. Methods: We differentiated h9C2 cells into cardiomyocytes in hormone-replete (Rm) or hormone-depleted (hD) media. We co-treated the cells with graded amounts of Bpa and pure anti-estrogen ICI 182,780, which blocks eRα and eRβ activity. Immunoblotting measured the expression of the structural protein β-myosin heavy chain (βmhC), calcium homeostasis protein sarcoendoplasmic reticulum calcium aTpase (seRCa2a), and the cardiac energy-producing protein creatine kinase (CK). results: expression of these proteins was hormone-dependent during cardiomyocyte differentiation, with expression highest in Rm media after 72 or 96 hours of differentiation. adding 10-8 m BPA to hD media increased cardiac structural (βmhC), energy (CK), and calcium homeostasis (seRCa2a) protein expression. Conversely, 10-7 m BPA added to Rm media decreased protein expression. Co-treatment with ICI 182,780 reduced Bpa-mediated induction of seRCa2a and CK expression in hD media. discussion: BPA modulates cardiac structure, calcium and energy homeostasis protein expression during cardiomyocyte differention in vitro. moreover, the data suggest that BPA mediates these changes in protein expression through activation of cardiomyocyte eRα, eRβ, or eRRγ.

Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of testicular steroidogenesis: a possible estrogenic effect of di(n-butyl) phthalate

Although di(n-butyl) phthalate (DBP), a suspected endocrine disruptor, induces testicular atrophy in prepubertal male rats, whether it exerts estrogenic activity in vivo remains a matter of debate. In the present study, we explored the estrogenic potency of DBP using 3-week-old male rats, and then examined the relationship between estrogen-induced spermatogenic cell apoptosis and testicular steroidogenesis. Daily exposure to DBP for 7 days caused testicular atrophy due to loss of spermatogenic cells, whereas testicular steroidogenesis was almost the same with the control values. A single exposure of DBP decreased testicular steroidogenesis in addition to decreasing the level of serum LH at 3 h after DBP treatment, with an extremely high incidence of apoptotic spermatogenic cells at 6 h after administration. To elucidate the estrogenic activity of DBP, we carried out an inhibition study using pure antiestrogen ICI 182,780 (ICI) in a model of spermatogenic cell apoptosis induced by DBP or estradial-3-benzoate (EB). Although both the DBP- and EB-treated groups showed a significant increase in spermatogenic cell apoptosis, ICI pretreatment significantly decreased the number of apoptotic spermatogenic cells in these two groups. In contrast, testicular steroidogenesis and serum FSH were significantly reduced in all the treated groups, even in the DBP+ICI and EB+ICI groups. Taken together, these findings led us to conclude that estrogenic compounds such as DBP and EB induce spermatogenic cell apoptosis in prepubertal rats, probably by activating estrogen receptors in testis, and that reduction in testicular steroidogenic function induced by estrogenic compounds is not associated with spermatogenic cell apoptosis.

Gene expression profiling in Ishikawa cells: A fingerprint for estrogen active compounds

Several anthropogenous and naturally occurring substances, referred to as estrogen active compounds (EACs), are able to interfere with hormone and in particular estrogen receptor signaling. EACs can either cause adverse health effects in humans and wildlife populations or have beneficial effects on estrogen-dependent diseases. The aim of this study was to examine global gene expression profiles in estrogen receptor (ER)-proficient Ishikawa plus and ER-deficient Ishikawa minus endometrial cancer cells treated with selected well-known EACs (Diethylstilbestrol, Genistein, Zearalenone, Resveratrol, Bisphenol A and o,p′-DDT). We also investigated the effect of the pure antiestrogen ICI 182,780 (ICI) on the expression patterns caused by these compounds. Transcript levels were quantified 24 h after compound treatment using Illumina BeadChip Arrays. We identified 87 genes with similar expression changes in response to all EAC treatments in Ishikawa plus. ICI lowered the magnitude or reversed the expression of these genes, indicating ER dependent regulation. Apart from estrogenic gene regulation, Bisphenol A, o,p′-DDT, Zearalenone, Genistein and Resveratrol displayed similarities to ICI in their expression patterns, suggesting mixed estrogenic/antiestrogenic properties. In particular, the predominant antiestrogenic expression response of Resveratrol could be clearly distinguished from the other test compounds, indicating a distinct mechanism of action. Divergent gene expression patterns of the phytoestrogens, as well as weaker estrogenic gene expression regulation determined for the anthropogenous chemicals Bisphenol A and o,p′-DDT, warrants a careful assessment of potential detrimental and/or beneficial effects of EACs. The characteristic expression fingerprints and the identified subset of putative marker genes can be used for screening chemicals with an unknown mode of action and for predicting their potential to exert endocrine disrupting effects.

495 POSTER Enhanced expression of Annexin IV in clear cell carcinoma of the ovary and its association with chemo-resistance

The selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT) is a full agonist at the transforming growth factor (TGF) α gene in ER negative breast cancer cells stably transfected with ERα cDNA (Levenson et al., Br. J. Cancer 77 (1998) 1812–1819). E2 and 4-OHT increase TGFα mRNA and protein in a concentration dependent manner. The responses to E2 and 4-OHT are blocked by the pure antiestrogen ICI 182,780, which does not induce TGFα. Transfected MDA-MB-231 cells contain functional ERα but no ERβ function was detected. Neo transfected cells that did not express ERα or cells stably transfected with the DNA binding domain mutant C202R/E203V which prevents gene activation did not induce TGFα mRNA after either E2 or 4-OHT treatment. An examination of the time course for either 10 nM E2 or 1 μM 4-OHT for MDA-MB-231 cells stably transfected with cDNA for ERα showed increases in TGFα mRNA within 2 or 3 h respectively. Cells pretreated with cycloheximide (1 μg/ml) showed induced TGFα mRNA in response to E2 or 4-OHT but TGFα mRNA induction was blocked by actinomycin D (1 μg/ml). We conclude that both E2 and 4-OHT induce TGFα by direct interaction of ERα with DNA and that ERβ is not involved in the estrogen-like response to 4-OHT in the MDA-MB-231 cells.

In vivo profiling of estrogen receptor/specificity protein-dependent transactivation.

17beta-Estradiol (E2) activates the estrogen receptor (ER) through multiple genomic and nongenomic pathways in various tissues/organs. ERalpha/specificity protein-dependent activation of E2-responsive genes containing GC-rich promoters has been identified in breast and other cancer cell lines, and in this study, we describe transgenic animals overexpressing a transgene containing three tandem GC-rich sites linked to a minimal TATA or thymidine kinase promoter and a luciferase gene. Several mouse lines expressing the transgenes were characterized and, in line 15, E2 induced a 9-fold increase in luciferase activity in the female mouse uterus, and the synthetic estrogens bisphenol A and nonylphenol also induced uterine luciferase activity. The pure antiestrogen ICI 182,780 induced luciferase activity in the mouse uterus, and similar results were observed for ICI 182,780 in breast cancer cells transfected with this construct. Differences in the ER agonist and antagonist activities of E2, nonylphenol, bisphenol A, and ICI 182,780 were investigated in the male testis and penis and the male and female stomach in line 15 transgenic mice. All of these tissues were hormone responsive; however, the patterns of induced or repressed luciferase activity were ligand structure, tissue, and sex dependent. These results demonstrate for the first time hormonal activation or repression of a GC-rich promoter in vivo, and the results suggest that the ERalpha/specificity protein pathway may contribute to E2-dependent induction and repression of genes.

Estrogen Receptor-α Hinge-Region Lysines 302 and 303 Regulate Receptor Degradation by the Proteasome

Cellular levels of estrogen receptor-alpha (ERalpha) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ERalpha and the protein degradation machinery facilitate the down-regulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ERalpha, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We used ERalpha-negative breast cancer C4-12 cells to generate cells stably expressing wild-type (wt)ERalpha or ERalpha with lysine-to-alanine substitutions at K302 and K303 (ERalpha-AA). In the unliganded state, ERalpha-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared with wtERalpha, due to its elevated association with the ubiquitin ligase carboxy terminus of Hsc70-interacting protein (CHIP) and the proteasome-associated cochaperone Bag1. Treatment of C4-12 cells with either 17beta-estradiol (E2) or the pure antiestrogen ICI 182,780 (ICI) induced rapid degradation of wtERalpha via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ERalpha-AA was less efficiently degraded. Furthermore, ERalpha-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor down-regulation. The reduced stability of ERalpha-AA in the unliganded state and the increased stability of ERalpha-AA in the liganded state were concordant with reporter gene assays demonstrating that ERalpha-AA has lower basal activity but higher E2 inducibility than wtERalpha. These data provide the first evidence that K302/303 protect ERalpha from basal degradation and are necessary for efficient E2- and ICI-induced turnover in breast cancer cells.

Peroxisome proliferator-activated receptor-gamma agonist, troglitazone has anticancer effect on breast cancer cells

Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat as they do not respond to hormone therapy. In an attempt to determine if expressing the human estrogen receptor in an ectopic manner could restore the hormone responsiveness of these cells, we have expressed the human ER in ER-negative MDA-MB 231 breast cancer cells using a recombinant adenovirus gene delivery system that allows high level expression of ER in essentially all cells. In these cells, the ER was correctly translated, had a wild type hormone binding affinity (Kd=0.6 nM), bound well to estrogen response element-containing DNA, and showed an activation pattern of estrogen response element-reporter gene activity by estrogen and antiestrogens very similar to that observed in MCF-7 breast cancer cells containing endogenous ER (stimulation by estrogen, no stimulation by the antiestrogens trans-hydroxytamoxifen or ICI 164 384, and blockade of estradiol stimulation by trans-hydroxytamoxifen or ICI 164 384). Intriguingly, estradiol stimulation of these cells was also able to induce expression of pS2, an estrogen regulated gene considered to be a favorable prognostic marker for endocrine therapy in ER-positive breast cancer cells. Expression of the ER had no effect by itself on the proliferation rate of MDA-MB 231 cells. However, treatment of the ER-containing cells with estradiol or with the pure antiestrogen ICI 164 384 suppressed proliferation of the cells while the antiestrogen trans-hydroxytamoxifen had little effect on proliferation; and cotreatment with trans-hydroxytamoxifen reversed the estradiol- or ICI 164 384-evoked suppression of proliferation. To understand the mechanism underlying the inhibition of proliferation by estradiol, we examined the expression of several growth related endogenous genes. c-Myc protooncogene expression was strongly inhibited by treatment with estradiol as was expression of BRCA1 and BRCA2 genes, which is in agreement with their mitogenic-dependent expression, while expression of β-actin, a housekeeping gene, was not affected by hormone treatment. Together, these data suggest that reexpressing the human ER in breast cancer cells that no longer express this protein renders them sensitive to hormone treatment. The ability of the antiestrogen ICI 164 384 to suppress the proliferation of ER-negative breast cancer cells that reexpress ER might be useful ultimately as an endocrine gene therapy approach for controlling the growth of ER-negative breast cancer cells. The application of recombinant adenoviruses expressing the human ER presents interesting features which might be used as a basis for designing more powerful and effective treatments for ER-negative breast cancers.

The Role of Amphiregulin in Exemestane-Resistant Breast Cancer Cells: Evidence of an Autocrine Loop

Exemestane-resistant breast cancer cell lines (i.e., ExeR), derived from MCF-7 cells expressing a high level of aromatase (MCF-7aro), were generated in our laboratory. The epidermal growth factor (EGF)-like protein amphiregulin (AREG) was highly expressed in ExeR cells based on cDNA microarray analysis. The high levels of AREG mRNA in ExeR cell lines were confirmed by real-time reverse transcription-PCR. The high levels of AREG protein in ExeR cell lysates and culture media were confirmed by Western blot analysis and ELISA, respectively. Furthermore, our Western blot analysis showed that whereas no AREG was detected in the DMSO control, overnight treatment of parental MCF-7aro cells with 1 micromol/L exemestane strongly induced the expression of AREG. This induction was totally blocked by 100 nmol/L of pure antiestrogen ICI 182,780, implying estrogen receptor (ER) dependence of exemestane-induced AREG expression. MCF-7aro cells were not able to proliferate in hormone-free medium, but were able to proliferate in conditioned medium from ExeR cells, similar to the treatment of recombinant human AREG. Small interference RNA targeting AREG inhibited ExeR proliferation, confirming that AREG is truly functioning as a growth factor of ExeR cells. The specific inhibitors to ER (ICI 182,780), EGF receptor (EGFR; AG1478), and mitogen-activated protein kinase (MAPK; U0126) all showed dose-dependent suppression of the proliferation of ExeR cells, indicating the involvement of the ER, EGFR, and MAPK pathways. Based on these findings, we propose a possible mechanism that underlies exemestane resistance: exemestane induces AREG in an ER-dependent manner. AREG then activates the EGFR pathway and leads to the activation of the MAPK pathway that drives cell proliferation.

17β-Estradiol inhibits osteoprotegerin production by the estrogen receptor-α-positive human breast cancer cell line MCF-7

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-α, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p<0.0001 by ANOVA). The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17β-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.