Estrogen-regulated Genes Research Articles

LIV‐1 regulates zinc uptake and E‐cadherin (CDH1) expression in MDA‐MB‐231 breast cancer cells but does not influence SNAI1

LIV‐1, the estrogen‐regulated gene encoding a member of the ZIP family of zinc transporters, has been implicated in both growth and metastasis of estrogen‐receptor (ER) positive and negative breast cancer cells. EGF treatment induces LIV‐1 protein expression in ER negative MDA‐MB‐231 cells by about 2‐fold whereas shRNA knock down reduces it 30%. Expression of E‐cadherin (CDH1) mRNA changed in parallel. Expression of SNAI1, thought to be a transcriptional repressor of CDH1, was also measured to further understand the linkage between LIV‐1 and CDH1. However, neither EGF nor LIV‐1 knockdown altered the expression of SNAI1 suggesting that this factor does not mediate influences of LIV‐1 on CDH1 in these cells. We also used 65Zn to characterize the role of LIV‐1 in zinc uptake. Knockdown of LIV‐1 reduced 65Zn uptake in parallel to the reduction in LIV‐1 expression, confirming its function as a zinc importer. Overall, LIV‐1 appears to play critical roles in both zinc homeostasis and the regulation of metastatic potential in MDA‐MB‐231 cells, though the mechanism of its linkage to CDH1 remains to be defined.

Estrogen-Regulated Prohibitin Is Required for Mouse Uterine Development and Adult Function

Estrogen signaling is pivotal for maintenance of female reproductive function in mammals. The physiological role of estrogen is mediated by estrogen receptors (ERs) and the steroid receptor coactivator family of transcriptional coregulators. Ablation of steroid receptor coactivator and ER coactivators in mice causes impaired female reproductive function. Recently we reported that prohibitin (PHB) can function as a corepressor for ERs in cultured cells. In this study, we demonstrate that PHB is an estrogen-regulated gene in vitro and in vivo, and its expression is induced by estrogen in the uterus, suggesting the existence of feedback regulatory loops. A conditional PHB knockout mouse model was generated by gene targeting to assess its in vivo function. Female mice with selective ablation of the PHB allele in the uterus were sterile, and their uteri were severely hypoplastic, indicating PHB is required for uterine development. Moreover, expression of ER and progesterone receptor target genes was selectively altered in response to hormone treatment. In summary, this study demonstrates that PHB is an estrogen-regulated gene and that PHB is essential for mouse uterine development and adult function and selectively required for estrogen-regulated gene expression.

Transcriptomic responses in Japanese medaka (Oryzias latipes) exposed to 17β-estradiol

The effects of 17 b -estradiol (E2) were evaluated using the medaka DNA microarray representing 36,398 genes. We first evaluated chronic effects on medaka exposed to E2 at different concentrations for 60 days posthatch. At ≥ 30 ng/L of E2 severe reproductive impairments such as sex reversal were observed. Larval medaka, Oryzias latipes , (within 24 hrs posthatch) were then exposed to E2 at various concentrations (3, 30, 100 ng/L) for up to 7 days. Microarray analyses of the E2-exposed larvae revealed that exposure to E2 up-regulated and down-regulated 339 and 105 genes, respectively. The up-regulated genes included ones involved in the p53 signaling pathway, apoptosis, and growth and development, in addition to well-known biomarkers such as vitellogenin and choriogenins. Down-regulated genes included heat shock proteins and estrogen receptors. Most of the up-regulated genes encoding the p53 signaling pathway, apoptosis, and growth and development exhibited a dose-dependent increase in gene expression, whereas the down-regulated genes in the heat shock protein category showed a dose-dependent decrease in gene expression. Time course experiments suggested that the E2 treatment attenuated the time-dependent changes in gene expressions of these genes. Among the genes related to oocyte maturation, estrogen-regulated genes such as choriogenins and vitellogenins were dramatically induced in response to E2 exposure, whereas other steroid-regulated genes such as zona pellucida-domain proteins did not change in gene expression by the E2 treatment. Results suggest that transcriptomic studies on larval medaka help elucidate the effects caused by endocrine disruptors on various biological pathways in vertebrate development.

Cocaine and amphetamine regulated transcript, type-2 vesicular glutamate transporter and calbindin co-expression define spiny stellate cells in layer IV of the granular cerebral cortex

Event Abstract Back to Event Cocaine and amphetamine regulated transcript, type-2 vesicular glutamate transporter and calbindin co-expression define spiny stellate cells in layer IV of the granular cerebral cortex C. S. Molnár1, M. Sárvári1, I. Kalló2, Z. Liposits1, 2 and E. Hrabovszky1* 1 Hungarian Academy of Sciences, Institute of Experimental Medicine, Hungary 2 Pázmány Péter Catholic University, Department of Neuroscience, Hungary Microarray experiments from our laboratory identified cocaine and amphetamine regulated transcript (CART) as an estrogen-regulated cortical gene. The present studies addressed the anatomical location and functional phenotype of cortical CART neurons using immunohistochemical (IHC) and in situ hybridization (ISH) strategies. IHC and ISH studies localized CART expressing neurons in layer IV throughout the granular cortex of the rat. CART neurons were found to be distinct from GABAergic interneurons expressing glutamic acid decarboxylase-65 mRNA. Out of the Ca-binding proteins calretinin, parvalbumin and calbindin, CART neurons only expressed low levels of calbindin. ISH studies established that the distribution of CART/calbindin neurons was identical with that of VGLUT2 mRNA expressing cells in layer IV. Morphological studies with IHC determined that CART/VGLUT2/calbindin neurons correspond to spiny stellate cells known to establish local efferent cortical connections. Future studies will use electrophysiological tools to address the effects of CART on cortical neuronal functioning and molecular approaches to study the regulation of cortical CART expression under various physiological and pathological conditions. Acknowledgements This work was supported by grants from the Hungarian Scientific Research Fund (OTKA K69127 T73002 and K83710), the Hungarian Health Research Council Fund (ETT 122/2009) and the European Community's Seventh Framework Programme (FP7/2007-2013; grant agreement n° 245009). Keywords: Molecular and cellular neurobiology, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Molecular and cellular neurobiology Citation: Molnár CS, Sárvári M, Kalló I, Liposits Z and Hrabovszky E (2011). Cocaine and amphetamine regulated transcript, type-2 vesicular glutamate transporter and calbindin co-expression define spiny stellate cells in layer IV of the granular cerebral cortex. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00001 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. E. Hrabovszky, Hungarian Academy of Sciences, Institute of Experimental Medicine, Budapest, Hungary, hrabovszky.erik@koki.mta.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers C. S Molnár M. Sárvári I. Kalló Z. Liposits E. Hrabovszky Google C. S Molnár M. Sárvári I. Kalló Z. Liposits E. Hrabovszky Google Scholar C. S Molnár M. Sárvári I. Kalló Z. Liposits E. Hrabovszky PubMed C. S Molnár M. Sárvári I. Kalló Z. Liposits E. Hrabovszky Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Persistent and non-persistent changes in gene expression result from long-term estrogen exposure of MCF-7 breast cancer cells

Life-long estrogen exposure is recognized as a major risk factor for the development of breast cancer. While the initial events in the regulation of gene expression by estrogen have been described in detail, far less is known of the role of estrogen in the long-term regulation of gene expression. In this study, we investigated the effects of long-term exposure of MCF-7 breast cancer cells to 1 nM 17β-estradiol on gene expression with the goal of distinguishing between gene expression that is continually reliant on estrogen receptor (ER) function as opposed to secondary and persistent effects that are downstream of ER. To assess the direct involvement of ER in the differential gene expression of long-term estrogen exposed (LTEE) cells in comparison with that of control cells, we exposed cultures to the selective estrogen receptor modulator raloxifene (RAL). cDNA microarray analysis showed that exposure to RAL inhibited expression of numerous characterized estrogen-regulated genes, including PGR, GREB1, and PDZK1. Genes that were increased in expression in LTEE cells yet were unaffected by RAL exposure included the aryl hydrocarbon receptor ( AHR) and numerous other genes that were not previously reported to be regulated by estrogen. Epigenetic regulation was evident for the AHR gene; AhR transcript levels remained elevated for several cell passages after the removal of estrogen. Signal transducer and activator of transcription 1 (STAT1); STAT1-regulated genes including ISG15, IFI27, and IFIT1; and MHC class I genes were also up-regulated in LTEE cells and were unaffected by RAL exposure. STAT1 is commonly overexpressed in breast and other cancers, and is associated with increased resistance to radiation and chemotherapy. This is the first study to relate estrogen exposure to increased STAT1 expression in breast cancer cells, an effect that may represent an additional role of estrogen in the pathogenesis of breast cancer.

Abstract P3-10-07: T5 Is a New Molecular Predictor of Distant Recurrence in Estrogen Receptor-Positive, HER2-Negative Breast Cancer

Abstract Background: Molecular tests predicting outcome of breast cancer patients may be useful for treatment decisions in addition to standard clinicopathologic features. Methods: Using human genome HG-U133A array and qRT-PCR datasets, we developed and validated a gene-expression signature predicting the likelihood of distant recurrence in postmenopausal, early-stage breast cancer patients with estrogen receptor-positive, HER2-negative tumors treated with adjuvant endocrine therapy. RNA levels assessed by qRT-PCR in formalin-fixed paraffin-embedded tumor specimens were used to calculate a risk score (T5) and to determine a risk group (low or high) for each patient. The prospectively defined T5 risk score was then validated independently in patients from two large randomized phase III trials. Distant recurrence-free survival and overall survival were analyzed with Cox models adjusted for clinicopathological factors. The primary endpoint was time to distant recurrence. Results: In a training set of 964 tumors, we identified a gene-expression signature consisting of three proliferation-related genes (BIRC5, UBE2C, DHCR7), five estrogen-regulated genes (RBBP8, IL6ST, AZGP1, MGP, STC2), and three reference genes (CALM2, OAZ1, RPL37A). For the validation, RNA analysis was possible in 1702 of 1725 (99%) tumors of both validation sets. Women were classified as having low risk (n=832; 49%) or high risk (n=870; 51%) by the T5 risk score. The T5 risk score provided prognostic information independent from clinicopathologic risk as estimated by Adjuvant!Online or Ki67 labeling index. Patients with a higher T5 risk score had a significantly shorter time to distant recurrence (adjusted hazard ratio, 1.24; 95% confidence interval [CI], 1.15 to 1.33; P<0.001) and overall survival (adjusted hazard ratio, 1.13; 95% CI, 1.06 to 1.19; P<0.001) compared to patients with a lower T5 risk score. The addition of the risk characterized by the T5 risk score to the clinicopathological risk resulted in 10-year distant recurrence-free survival rates of 95% in combined low risk patients and 82% in combined high risk patients (P<0.001). Conclusions: Using formalin-fixed paraffin-embedded tumor specimens, the multigene T5 risk score provides prognostic information independent of Adjuvant!Online or Ki67 labeling index. By combining the T5 risk score with clinicopathological risk, we were able to accurately identify breast cancer patients with low risk or high risk for distant recurrence. Using this new easy-to-use multigene tool in clinical practice will assist in optimizing adjuvant therapy by reducing both undertreatment and overtreatment and thus improves outcome and quality of life of patients with early-stage breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-07.

A Noncompetitive Small Molecule Inhibitor of Estrogen-regulated Gene Expression and Breast Cancer Cell Growth That Enhances Proteasome-dependent Degradation of Estrogen Receptor α

The mechanisms responsible for 17β-estradiol (E(2))-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor α (ERα) antagonists are not fully understood. We describe a new tool for dissecting ERα action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor α that does not compete with estrogen for binding to ERα. TPSF noncompetitively inhibits estrogen-dependent ERα-mediated gene expression with little inhibition of transcriptional activity by NF-κB or the androgen or glucocorticoid receptor. TPSF inhibits E(2)-ERα-mediated induction of the proteinase inhibitor 9 gene, which is activated by ERα binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ERα to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E(2)-ERα-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ERα-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ERαHA cells that overexpress ERα, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ERα protein levels in MCF-7 cells and several other cell lines without altering ERα mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ERα by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ERα. TPSF represents a novel class of ER inhibitor with significant clinical potential.

Estrogen Induces Distinct Patterns of MicroRNA Expression Within the Mouse Uterus

Control of estrogenic activity within the uterus is evident as unopposed estrogen action is associated with endometrial pathologies such as endometriosis and endometrial carcinoma. MicroRNAs (miRNAs) have emerged as important posttranscriptional regulators, which are postulated to fine-tune the actions of steroids in many systems including the uterus. The objective of the current study was to examine uterine expression of miRNAs in response to estrogen treatment within the mouse uterus using an ovariectomized, steroid-reconstituted mouse model. MicroRNA microarray analysis and subsequent quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) verification revealed that expression of mirn155, mirn429, and mirn451 was significantly increased by estrogen administration whereas mirn181b and mirn204 expression was significantly reduced. Pretreatment with the estrogen receptor (ER) antagonist ICI 182,780 confirmed that estrogen regulation was mediated via the classical ER pathway. This study demonstrates that estrogen regulates specific miRNAs within the murine uterus, which may participate in posttranscriptional regulation of estrogen-regulated genes.

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth.

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

Is It Time to ReSET the Standard for Estrogen Receptor Testing in Breast Cancer?

Is It Time to ReSET the Standard for Estrogen Receptor Testing in Breast Cancer?

Changes in expression of oestrogen regulated and proliferation genes with neoadjuvant treatment highlight heterogeneity of clinical resistance to the aromatase inhibitor, letrozole

IntroductionClinical resistance is a major factor limiting benefits to endocrine therapy. Causes of resistance may be diverse and the mechanism of resistance in individual breast cancers is usually unknown. The present study illustrates how changes in the expression of proliferation and oestrogen-regulated genes occurring during neoadjuvant treatment with the aromatase inhibitor, letrozole, may define distinctive tumour subgroups and suggest different mechanisms of resistance in clinically endocrine resistant breast cancers.MethodsPostmenopausal women with large primary oestrogen-receptor (ER)-rich breast cancers were treated neoadjuvantly with letrozole (2.5 mg daily) for three months. Clinical response was determined by ultrasound changes in tumour volume. Tumour ribonucleic acid (RNA) from biopsies taken before, after 14 days and after three months of treatment was hybridized on Affymetrix U133A chips. Changes in expression of KIAA0101, TFF3, SERPINA3, IRS-1 and TFF1 were taken as markers of oestrogen regulation and those in CDC2, CKS-2, Cyclin B1, Thymidine Synthetase and PCNA as markers of proliferation.ResultsFifteen tumours with < 50% volume reduction over three months of treatment were classified as being clinically non-responsive. Gene expression changes after 14 days of treatment with letrozole revealed different patterns of change in oestrogen regulated and proliferation genes in individual resistant tumours. Tumours could be separated into three different subgroups as follows: i) nine cases in which both proliferation and oestrogen signalling signatures were generally reduced on treatment (ii) four cases in which both signatures were generally unaffected or increased with treatment and (iii) two cases in which expression of the majority of oestrogen-regulated genes decreased whereas proliferation genes remained unchanged or increased. In 14 out of 15 tumours, RNA profiles were also available after three months of treatment. Patterns of change observed after 14 days were maintained or accentuated at three months in nine tumours but changes in patterns were apparent in the remaining five cancers.ConclusionsDifferent dynamic patterns of expression of oestrogen-regulated and proliferation genes were observed in tumours clinically resistant to neoadjuvant letrozole, thus illustrating heterogeneity of resistance and discriminating molecular sub-classes of resistant tumours. Molecular phenotyping might help to direct circumventing therapy suggesting the targeting of specific pathways in different tumour subtypes.

The liver receptor homolog-1 regulates estrogen receptor expression in breast cancer cells

Estrogen receptor-α (ER) is expressed in the great majority of breast cancers, and the inhibition of ER action is a key part of breast cancer treatment. The inhibition of ER action is achieved using anti-estrogens, primarily tamoxifen, and with aromatase inhibitors that inhibit estrogen biosynthesis, thereby preventing ER activation. However, resistance to these therapies is common. With the aim of identifying new molecular targets for breast cancer therapy, we have identified the liver receptor homolog-1 (LRH-1) as an estrogen-regulated gene. RNA interference and over-expression studies were used to investigate the role of the LRH-1 in regulating breast cancer growth and to identify the targets of an LRH-1 action. Promoter recruitment was determined using reporter gene and chromatin immunoprecipitation (ChIP) assays. We show that LRH-1 regulates breast cancer cell growth by regulating the ER expression. Reporter gene and in vitro DNA-binding assays identified an LRH-1-binding site in the ER gene promoter, and ChIP assays have demonstrated in vivo binding at this site. We also provide evidence for new LRH-1 variants in breast cancer cells arising from the use of alternative promoters. Previous studies have shown that LRH-1 functions in estrogen biosynthesis by regulating aromatase expression. Our findings extend this by highlighting LRH-1 as a key regulator of the estrogen response in breast cancer cells through the regulation of ER expression. Hence, inhibition of LRH-1 could provide a powerful new approach for the treatment of endocrine-resistant breast cancer.

In vitro and in vivo antitumor activity of melatonin receptor agonists

Melatonin has been shown to inhibit the proliferation of estrogen receptor α (ERα)-positive human breast cancer cells in vitro and suppress the growth of carcinogen-induced mammary tumors in rats. Melatonin's antiproliferative effect is mediated, at least in part, through the MT1 melatonin receptor and mechanisms involving modulation of the estrogen-signaling pathway. To develop melatonin analogs with greater therapeutic effects, we have examined the in vitro and in vivo antimitotic activity of two MT1/MT2 melatonin receptor agonists, S23219-1 and S23478-1. In our studies, both agonists are quite effective at suppressing the growth of MCF-7 human breast cancer cells. At a concentration of 10⁻⁶ m, S23219-1 and S23478-1 inhibited the growth of MCF-7 cells by 60% and 73%, respectively. However, S23478-1 is more effective than melatonin and S23219-1 at repressing the expression and transactivation of the ERα, and modulating the expression of pancreatic spasmolytic polypeptide (pS2), an estrogen-regulated gene. The melatonin agonist S23478-1 exhibited enhanced antitumor potency in the subsequent studies in our animal model. At a dosage of 25 mg/kg/day, S23478-1 is more efficacious than melatonin at inducing regression of the established N-nitroso-N-methyl-urea-induced rat mammary tumors. This dose of S23478-1 (25 mg/kg/day) generated a significant (P < 0.05) overall regression response of 52%. Furthermore, at this dosage, S23478-1 is more effective than melatonin at suppressing the estrogen-signaling pathway and promoting tumor cell apoptosis, significantly increasing the expression of the pro-apoptotic protein Bax, while decreasing the expression of ERα and the anti-apoptotic protein Bcl-2.

Estrogen regulation of X-box binding protein-1 and its role in estrogen induced growth of breast and endometrial cancer cells

BACKGROUND: X-box binding protein 1 (XBP1), a transcription factor involved in unfolded protein response, is also an estrogen-regulated gene and strongly correlates with estrogen receptor alpha (ERα) expression in breast cancers. We investigated the functional role of XBP1 in estrogen responsive breast and endometrial cancer cells as its functions are not fully understood. MATERIALS AND METHODS: ERα positive breast (MCF7) and endometrial (ECC1) cancer cells were used to study XBP1 gene regulation by 17-β-estradiol (E2) and to investigate the role of XBP1 in E2-mediated growth using short interfering RNA. Quantitative real-time PCR and Western blot were used to assess RNA and protein levels. Recruitment of ERα and other cofactors at the promoter and enhancer region of the XBP1 gene was investigated by chromatin immunoprecipitation. Estrogen responsive element (ERE)-mediated transcriptional activity was evaluated by a luciferase reporter assay. RESULTS: E2 induced the transcription of XBP1 in both MCF7 and ECC1 cells. E2-dependent recruitment of ERα, steroid receptor coactivator (SRC)-1 and SRC-3, and RNA polymerase II were observed at the promoter and/or enhancer region of the XBP1 gene. Depletion of XBP1 markedly inhibited the E2-induced growth in MCF7 and ECC1 cells. However, ERE-mediated transcription was not altered in XBP1-overexpressing or XBP1-depleted MCF7 cells. CONCLUSION: Our results confirm E2-induced transcription of XBP1 and demonstrate the crucial role of XBP1 in E2-induced growth of ERα positive breast and endometrial cancer cells without modulating the classical ERE-mediated transcription by ER. This knowledge creates new opportunities for therapeutic interventions.

Estrogen Regulation and Physiopathologic Significance of Alternative Promoters in Breast Cancer

Alternative promoters (AP) occur in >30% protein-coding genes and contribute to proteome diversity. However, large-scale analyses of AP regulation are lacking, and little is known about their potential physiopathologic significance. To better understand the transcriptomic effect of estrogens, which play a major role in breast cancer, we analyzed gene and AP regulation by estradiol in MCF7 cells using pan-genomic exon arrays. We thereby identified novel estrogen-regulated genes (ERG) and determined the regulation of AP-encoded transcripts in 150 regulated genes. In <30% cases, APs were regulated in a similar manner by estradiol, whereas in >70% cases, they were regulated differentially. The patterns of AP regulation correlated with the patterns of estrogen receptor alpha (ERalpha) and CCCTC-binding factor (CTCF) binding sites at regulated gene loci. Interestingly, among genes with differentially regulated (DR) APs, we identified cases where estradiol regulated APs in an opposite manner, sometimes without affecting global gene expression levels. This promoter switch was mediated by the DDX5/DDX17 family of ERalpha coregulators. Finally, genes with DR promoters were preferentially involved in specific processes (e.g., cell structure and motility, and cell cycle). We show, in particular, that isoforms encoded by the NET1 gene APs, which are inversely regulated by estradiol, play distinct roles in cell adhesion and cell cycle regulation and that their expression is differentially associated with prognosis in ER(+) breast cancer. Altogether, this study identifies the patterns of AP regulation in ERGs and shows the contribution of AP-encoded isoforms to the estradiol-regulated transcriptome as well as their physiopathologic significance in breast cancer.

Abstract 1235: The organic cation transporter SLC22A5 gene is regulated by the cooperative activity of the estrogen receptor and other transcription factors in breast cancer

Abstract The solute carrier family 22 member 5 (SLC22A5) gene encodes a polyspecific organic cation transporter that is associated with many diseases. However, the regulation of SLC22A5 and its relationship with breast cancer is not clearly defined. In a previous global gene expression analysis from our group, SLC22A5 was identified as an estrogen-regulated gene. In this study, we investigated the molecular mechanism by which estrogen receptor (ER) and other transcription factors regulate SLC22A5 gene expression in breast cancer. SLC22A5 is highly expressed in ER-positive breast cancer cell lines and ER-positive mammary tumor tissues compared to ER-negative cells and tissues. SLC22A5 expression is also correlated with the levels of ER expression in breast cancer cell lines and in mammary tumor tissue specimens. SLC22A5 mRNA expression is induced in MCF-7 breast cancer cells within half an hour after estrogen treatment and remains elevated for over 24 hours. Actinomycin D, but not cycloheximide, can inhibit this estrogen induction, indicating new protein synthesis is not needed for induction. These results suggest that estrogen directly activates SLC22A5 transcription. We next cloned the 6kb SLC22A5 promoter and luciferase experiments demonstrated that this portion of the SLC22A5 promoter is not responsive to estrogen treatment. However, we discovered that an intronic enhancer located approximately 9kb from the transcriptional start site confers estrogen responsiveness when inserted downstream of the SLC22A5 gene promoter-luciferase reporter construct. Several transcription factors binding sites, such as a cyclic adenosine monophosphate (cAMP) response element (CRE), an estrogen-response element (ERE), an activator protein-1 (AP-1) site and a half ERE, are juxtaposed in the middle of this intronic enhancer. Mutation or deletion of these elements abolishes the estrogen responsiveness. Chromatin-immunoprecipitation (ChIP) assay demonstrates the recruitment of the transcription factors onto the intronic enhancer region in an estrogen-dependent manner. We next investigated the effect of SLC22A5 knockdown on breast cancer cell growth. Results from these studies show that shRNA knockdown of SLC22A5 inhibits the proliferation of MCF-7 cells. This reduced proliferation is accompanied by decreased cyclin D1 levels. These results suggest that SLC22A5 is an estrogen-induced gene that is involved in regulating breast cell growth. Our results also suggest that this estrogen induction is mediated by a complex intronic enhancer. This estrogen-induced gene may serve as a novel breast cancer biomarker that may be targeted for improved treatment and prevention of breast cancer. 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 1235.

Abstract 605: Intermittent treatment of hormone-dependent breast cancer using exemestane: Translational research from lab bench to bedside

Abstract Although aromatase inhibitors (AIs) are useful in the treatment of hormone-responsive breast cancer, unfortunately, resistance to such therapy still develops. Based on our published preclinical results that ER, AREG and EGFR play important roles in the acquired resistance mechanism of exemestane (EXE), a steroidal AI, intermittent use of EXE was thought to delay the development of acquired resistance by preventing up-regulation of AREG and activation of EGFR-mediated pathways. This hypothesis was verified by preclinical cell culture and animal studies. Microarray analysis was performed using RNA isolated from week-14 (EXE-resistant) MCF7aro cells that were continuously treated with EXE, week-14 (EXE-responsive) cells that were intermittently treated with EXE (2 weeks on and 1 week off), and cells without EXE treatment. A selective number of estrogen-regulated genes, including AREG and SGK3 (serum/glucocorticoid regulated kinase family member 3), were differentially transcribed between the continuously treated samples and intermittently treated samples. Treatment of EXE resistant cells with siRNAs against AREG and SGK3 suppressed cell growth, supporting the roles of these two proteins in the development of resistance to EXE. Additional results from ChIP and gene expression analyses confirm that the expression of SGK3 can be up-regulated by E2 as well as EXE, and over-expression of SGK3 suppresses apoptosis and increases cell survival. Our animal experiments revealed that intermittent treatment of EXE (250 µg/day, 2 weeks on and 1 week off) was effective at suppressing androgen-induced MCF7aro tumor growth in nude mice, and that after 21 weeks of treatment, resistance resulted in continuous EXE treatment group, while the tumor volumes in the positive control group (androgen treated only) reached 10 cm3 at week 18. qPCR analysis indicated that SGK3 expression in tumors from continous EXE mice was two times that in tumors from the positive control mice, with lowest expression in tumors from the intermittent EXE group (30% of that of the positive control). An analysis on 11 pairs of malignant and adjacent normal tissue has revealed that SGK3 expression is significantly greater in breast tumor specimens than adjacent non-cancer specimens (P=0.006). Since results from these translational research studies provide an important mechanistic basis for intermittent EXE therapy, a clinical trial to evaluate the intermittent use of EXE on postmenopausal women with ER-positive metastatic breast cancer has been recently approved and initiated at the City of Hope NCI-designated Comprehensive Cancer Center. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 605.

The regulation of LIV‐1 mRNA in MDA‐MB‐231 human breast cancer cells and its association with E‐cadherin

LIV‐1 is an estrogen‐regulated gene encoding a member of the ZIP family of zinc transporters (SLC39A6). It is expressed in both estrogen‐receptor positive and negative breast cancer cells. In estrogen‐receptor positive MCF‐7 breast cancer cells, LIV‐1 expression is induced by both estrogen and insulin. LIV‐1 has also been observed to induce the expression of the cell adhesion protein E‐cadherin, thought to play a role in metastasis. In the present study, we investigated the regulation of LIV‐1 and its relationship to E‐cadherin in MDA‐MB‐231estrogen‐receptor negative breast cancer cells. Cells were treated with insulin and EGF for 24 h and the mRNA concentrations of LIV‐1 and E‐cadherin were analyzed by RT‐PCR. EGF treatment induced LIV‐1 mRNA by about 2‐fold whereas insulin increased expression only to a minor extent. The mRNA expression of E‐cadherin paralleled that of LIV‐1 with these treatments. MDA‐MB‐231 cells were also transfected with an shRNA construct designed to knock down LIV‐1 expression. This procedure reduced LIV‐1 mRNA concentrations by 30% and expression of E‐cadherin was reduced to a similar extent. This association between LIV‐1 and E‐cadherin expression in MDA‐MB‐231 cells suggests that LIV‐1 may be a regulator of E‐cadherin in both estrogen‐receptor positive and negative cells. The presence of LIV‐1 could serve as a prognostic marker for breast cancer patients with invasive tumors.

A CTCF-independent role for cohesin in tissue-specific transcription.

The cohesin protein complex holds sister chromatids in dividing cells together and is essential for chromosome segregation. Recently, cohesin has been implicated in mediating transcriptional insulation, via its interactions with CTCF. Here, we show in different cell types that cohesin functionally behaves as a tissue-specific transcriptional regulator, independent of CTCF binding. By performing matched genome-wide binding assays (ChIP-seq) in human breast cancer cells (MCF-7), we discovered thousands of genomic sites that share cohesin and estrogen receptor alpha (ER) yet lack CTCF binding. By use of human hepatocellular carcinoma cells (HepG2), we found that liver-specific transcription factors colocalize with cohesin independently of CTCF at liver-specific targets that are distinct from those found in breast cancer cells. Furthermore, estrogen-regulated genes are preferentially bound by both ER and cohesin, and functionally, the silencing of cohesin caused aberrant re-entry of breast cancer cells into cell cycle after hormone treatment. We combined chromosomal interaction data in MCF-7 cells with our cohesin binding data to show that cohesin is highly enriched at ER-bound regions that capture inter-chromosomal loop anchors. Together, our data show that cohesin cobinds across the genome with transcription factors independently of CTCF, plays a functional role in estrogen-regulated transcription, and may help to mediate tissue-specific transcriptional responses via long-range chromosomal interactions.

High expression of gabarapl1 is associated with a better outcome for patients with lymph node-positive breast cancer

Background:This study evaluates the relation of the early oestrogen-regulated gene gabarapl1 to cellular growth and its prognostic significance in breast adenocarcinoma.Methods:First, the relation between GABARAPL1 expression and MCF-7 growth rate was analysed. Thereafter, by performing macroarray and reverse transcriptase quantitative-polymerase chain reaction (RT–qPCR) experiments, gabarapl1 expression was quantified in several histological breast tumour types and in a retrospective cohort of 265 breast cancers.Results:GABARAPL1 overexpression inhibited MCF-7 growth rate and gabarapl1 expression was downregulated in breast tumours. Gabarapl1 mRNA levels were found to be significantly lower in tumours presenting a high histological grade, with a lymph node-positive (pN+) and oestrogen and/or progesterone receptor-negative status. In univariate analysis, high gabarapl1 levels were associated with a lower risk of metastasis in all patients (hazard ratio (HR) 4.96), as well as in pN+ patients (HR 14.96). In multivariate analysis, gabarapl1 expression remained significant in all patients (HR 3.63), as well as in pN+ patients (HR 5.65). In univariate or multivariate analysis, gabarapl1 expression did not disclose any difference in metastasis risk in lymph node-negative patients.Conclusions:Our data show for the first time that the level of gabarapl1 mRNA expression in breast tumours is a good indicator of the risk of recurrence, specifically in pN+ patients.