Estrogen Receptor Binding Sites Research Articles

Abstract 4759: HOXB7 functions as a co-activator of estrogen receptor in the development of tamoxifen resistance.

Abstract Background: Multiple factors including long term treatment with tamoxifen are involved in the development of selective estrogen receptor modulator (SERM)-resistance in ERα positive breast cancer. Increased expression of HER family members can also directly alter cellular responses to tamoxifen, but the mechanism underlying the increased expression of the HERs is not clear. In this report we show that HOXB7 is an upstream regulator of HER2 and ER expression. Results: Overexpression of HOXB7 results in upregulation of HER2 and ERα target genes, while knockdown of HOXB7 with siRNA in tamoxifen-resistant cell lines causes decrease of HER2 and ERα target genes expression and loss of tamoxifen resistance. To mediate upregulation of HER2 and ER-target genes occurring in tamoxifen resistance, the HOXB7-ERα complex recruits ERα coactivators and promotes HER2- and ER-target genes transcriptional activity through direct binding to the HER2 enhancer and regulatory regions of ER target genes. However, depletion of HOXB7 negatively affects the binding affinity of its cofactors to the HER2 enhancer element and ER-binding sites. Also, it is likely that miR196a controls HOXB7 expression; reducing miR196a levels in tamoxifen-resistant cells causes an increase of HOXB7 expression. Overexpression of miR196a sensitizes MCF-7 tamoxifen resistant cells to tamoxifen through downregulation of HER2 and ER-target genes while inhibition of miR196a by anti-miR196a inhibitors increases tamoxifen resistance through the upregulation of HER and ER target genes in MCF-7 cells. Further, miRNA 196a is regulated by c-MYC which undergoes stabilization through the EGFR-HER2 signaling pathway. Depletion of c-MYC by MYC shRNA enhances tamoxifen sensitivity by increasing the level of miR196a transcripts. Also, by c-MYC ChIP assay, we found that c-MYC inhibits miR196a expression through direct binding to its promoter region. Conclusions: Overexpression of HOXB7 is a key event in the initiation and maintenance of tamoxifen resistance. These studies suggest that HOXB7 acts as a key regulator orchestrating two major groups of target molecules, ERα and HER2, in the oncogene hierarchy. Blocking MYC expression or reexpression of miR196a in TAM-R cells may provide novel strategies for reversing SERM-resistance. Therefore, we have provided additional evidence that supports the thesis that functional antagonism of HOXB7 has the potential to circumvent tamoxifen resistance. Citation Format: Kideok Jin, Wei Wen Teo, Takahiro Yoshida, Sunju Park, Saraswati Sukumar. HOXB7 functions as a co-activator of estrogen receptor in the development of tamoxifen resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4759. doi:10.1158/1538-7445.AM2013-4759

Estrogenic Potency of Benzophenone UV Filters in Breast Cancer Cells: Proliferative and Transcriptional Activity Substantiated by Docking Analysis

The results from recent studies show that some benzophenones (BPs) and their hydroxylated metabolites can function as weak estrogens (E2) in the environment. However, little is known about the structure-activity relationship of these molecules. We have examined the effects of exposure to ten different BPs on the proliferation of estrogen receptor (ER)-positive breast cancer cells and on the transcriptional activity of E2-target genes. We analyzed two genes that are tightly linked with estrogen-mediated proliferation, the CXCL12 and amphiregulin genes and two classical estrogen-responsive genes, the pS2 and progesterone receptor. Significant differences in the BPs efficiency to induce cell proliferation and endogenous E2-target gene expressions were observed. Using ERE-, Sp1-, AP1- and C3-reporter genes that contain different ER-binding sites in their promoter, we also showed significant differences in the BPs efficiency in activation of the ER transactivation. Together, our analyzes showed that the most active molecule is 4-hydroxy-BP. Docking analysis of the interaction of BPs in the ligand-binding pocket of ERα suggests that the minimum structural requirement for the estrogenic activity of BPs is a hydroxyl (OH) group in the phenyl A-ring that allows interaction with Glu-353, Arg-394 or Phe-404, which enhances the stability between BPs and ERα. Our modeling also indicates a loss of interaction between the OH groups of the phenyl B-ring and His-524. In addition, the presence of some OH groups in the phenyl B-ring can create repulsion forces, which may constrain helix 12 in an unfavorable position, explaining the differential estrogenic effects of BPs. These results, together with our analysis of BPs for their potency in activation of cell proliferation and ER-mediated transcription, report an improved understanding of the mechanism and structure–activity relationship of BPs.

RNA sequencing of MCF-7 breast cancer cells identifies novel estrogen-responsive genes with functional estrogen receptor-binding sites in the vicinity of their transcription start sites.

Estrogen receptor α (ERα) is a key transcription factor in breast cancer, which plays an essential role in the pathophysiology of the disease by regulating the expression of various target genes. In the present study, we performed deep RNA sequencing (RNA-seq) as an unbiased high-throughput technique for comprehensive transcriptome analysis in ERα-positive human breast cancer MCF-7 cells, to facilitate the elucidation of ERα regulatory gene networks. From the 17,336 mapped RefSeq genes from the sequenced fragments of the cell samples treated with estrogen time dependently, substantial numbers of sequence reads were observed in 3,386 genes (>100 tags per million reads per sample at any of the six time points studied). ERα occupancy within and in the proximal regions of the genes (<10-kb upstream and downstream regions) was significantly enriched in the subgroup of the 3,386 genes compared to the whole 17,336 RefSeq genes. Of the 3,386 genes, we focused on 29 genes, which included ERα occupancy adjacent to their transcription start sites and whose expression was estrogen dependently altered by >3-fold. Knockdown studies using siRNAs specific to the 29 genes validated that prototypic ERα targets V-myc myelocytomatosis viral oncogene homolog and cyclin D1 promote both proliferation and migration of MCF-7 cells and further identified novel candidate ERα targets EIF3A and tumor protein D52-like 1, which will also facilitate the proliferation or migration of MCF-7 cells. Taken together, the present findings provide a valuable dataset that will elucidate ERα regulatory mechanisms in breast cancer biology, based on the integrative analysis of RNA-seq combined with the genome-wide information for ERα occupancy.

Abstract PD01-08: Differences in estrogen receptor signaling in non-malignant primary ER-positive breast epithelial cells and breast cancer.

Abstract Background: The estrogen receptor (ER) is expressed in ∼70% of sporadic breast cancer and activates genes driving cell proliferation and tumorigenesis. We have previously performed genome-wide analysis of ER binding sites in MCF-7 breast cancer cells, and identified distinct mechanisms of ER signaling. We have also previously used EpCAM and CD49f as markers to enrich for viable ER-positive (ER+) cells obtained from non malignant breast tissue. Here, we seek to elucidate differences in ER signaling between non-malignant and ER+ breast cancer cells. Methods: Primary breast epithelial cells were obtained from patients undergoing reduction mammoplasties and surgical excision of ER+ breast cancer. After dissociation of breast reductions into a single-cell suspension, ER+ mature luminal (ML; EpCAM+CD49f−) and luminal progenitor (LP; EpCAM+CD49f+) subpopulations were obtained by flow cytometry. Following estrogen stimulation, RNA was extracted for gene microarray analysis. ER chromatin immunoprecipitation and DNA sequencing (ChIP-seq) was performed. These results were compared to MCF-7 breast cancer cells. Results: Reduction mammoplasty and ER+ breast cancer tissues were analyzed, and compared to MCF-7 cells. Gene expression profiles were different between non-malignant tissue and ER+ breast cancer cells following estrogen stimulation, with a 2–3 fold higher number of ER regulated genes in ER+ breast cancer compared to ER+ non malignant cells, and few overlapping estrogen regulated genes. Genes that promotes cell cycling and cell proliferation were downregulated in non-malignant tissue, but were upregulated in breast cancer cells (P &amp;lt; 10–5). CYP1A1, a major estradiol metabolizing enzyme, was upregulated in normal cells but downregulated in ER+ breast cancer cells. Motif analysis of ER ChIP-seq data in normal and ER+ breast cancer tissues demonstrated an enrichment of ER motifs in the overlapping sites and an enrichment of FOXA1 motifs in ER+ breast cancer cells and TCF12 motifs in non-malignant ER+ epithelial cells. Conclusions: There are contrasting differences in ER signaling between normal mammary and breast cancer cells, with estrogen having anti-proliferative effects in normal luminal cells compared to pro-proliferative effects in breast cancer. ER ChIP-Seq has identified TCF12 as a major co-factor in non-malignant breast tissue whilst FOXA1 is a major co-factor in ER+ breast cancer. Our data provides evidence for key alterations in ER-signaling during tumorigenesis, and identifies potential mechanisms to target cancer specific ER signaling. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-08.

Estrogens Block p53 in Breast Cancer

In estrogen receptor–dependent breast cancer cells, estrogen signaling antagonizes the proapoptotic function of the tumor suppressor p53.

Estrogen receptor (ER) signaling in normal, BRCA (B) 1 and B2 mutation associated, and ER-positive breast cancer (BC) mammary cells.

576 Background: ER is expressed in ≈70% of sporadic BC and activates genes driving cell proliferation and tumorigenesis. B1 and B2 mutation associated BC have predominant triple negative and ER+ phenotypes respectively, yet hormonal manipulation with oophorectomy reduces BC risk in both B1 and B2 carriers. We have previously performed genome-wide analysis of ER binding sites in MCF-7 BC cells, and identified distinct mechanisms of ER signaling. Here, we seek to elucidate differences in ER signaling between non-malignant (normal, B1 and B2) and ER+ BC mammary cells. Methods: Breast tissue were obtained from patients undergoing reduction mammoplasties (normal), prophylactic mastectomies (B1 and B2 carriers) and surgical excision of ER+ BC. After dissociation into a single-cell suspension, ER+ EpCAM+CD49f- mature luminal (ML) and EpCAM+CD49f+ luminal progenitor (LP) subpopulations were obtained by flow cytometry. Following estrogen stimulation, RNA was extracted for gene microarray analysis. ER chromatin immunoprecipitation and DNA sequencing (ChIP-seq) was performed and DNA libraries prepared and sequenced. Results: Triplicates ofnormal, prophylactic B1 and B2, and ER+ BC tissues were analyzed.Gene ontology analysis indicates different gene expression signatures between non-malignant tissue and MCF-7 BC cells following estrogen stimulation. Genes that promotes cell cycling and proliferation were downregulated in non-malignant tissue, but were upregulated in BC cells (P &lt; 10-5). Hierarchical clustering of the gene microarray by mutation status revealed distinct LP gene signatures in normal, B1 and B2 breast tissue, whilst the ML signatures were largely indistinguishable between the three tissue types. Conclusions: There are contrasting differences in ER signaling between normal mammary and BC cells, with estrogen having anti-proliferative effects in normal luminal cells compared to pro-proliferative effects in BC. Our data suggest that alterations in ER signaling play a major role in breast tumorigenesis. Differences in the LP population between B1 and B2 breast tissue may account for their distinct BC phenotypes.

Abstract 803: HOXB7 functions as a co-activator of estrogen receptor in the development of tamoxifen resistance

Abstract Multiple factors including long term treatment with tamoxifen are involved in the development of selective estrogen receptor modulator (SERM)-resistance in ERα positive breast cancer. Increased expression of HER family members can also directly alter cellular responses to tamoxifen, but the mechanism underlying the increased expression of the HERs is not clear. In this report we show that HOXB7 interacts with ERα, and together they bind to the promoter or enhancer regions of ERα-regulated genes to enhance their transcription activity. We found a number of novel HOXB7 binding sites at promoter regions, intron regions and adjacent to ER-binding sites in ER-regulated genes through HOXB7 ChIP on chiP using an Affymetrix promoter array. Potentially, HOXB7 and ERα may co-regulate about 84% of ERα-target genes. HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen. Overexpression of HOXB7 results in upregulation of HER2 and ERα target genes while knockdown of HOXB7 with siRNA in tamoxifen-resistant cell lines causes decrease of HER2 and ERα target genes expression and loss of tamoxifen resistance. Our data shows that to mediate upregulation of HER2 and ER-target genes occurring in tamoxifen resistance, the HOXB7-ERα complex recruits ERα coactivators and promotes HER2- and ER-target genes transcriptional activity through direct binding to the HER2 enhancer and regulatory regions of ER target genes. However, depletion of HOXB7 negatively affects the binding affinity of its cofactors to the HER2 enhancer element and ER-binding sites. Also, we found that miR196a might control HOXB7 expression; reducing miR196a levels in tamoxifen-resistant cells causes an increase of HOXB7 expression. Further, miRNA 196a is regulated by c-MYC which undergoes stabilization through the EGFR-HER2 signaling pathway. These findings implicate overexpression of HOXB7 as a key event in the initiation and maintenance of tamoxifen resistance. These studies suggest that HOXB7 acts as a key regulator orchestrating two major groups of target molecules, ERα and HER2, in the oncogene hierarchy. Therefore, we have provided additional evidence that supports the thesis that functional antagonism of HOXB7 has the potential to circumvent tamoxifen resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 803. doi:1538-7445.AM2012-803

Association of double-positive FOXA1 and FOXP1 immunoreactivities with favorable prognosis of tamoxifen-treated breast cancer patients.

Breast cancer is primarily a hormone-dependent tumor that can be regulated by the status of the steroid hormones estrogen and progesterone. Forkhead box A1 (FOXA1) is a member of the forkhead box transcription factor family and functions as a pioneer factor of the estrogen receptor (ER) in breast cancer. In the present study, we demonstrate that FOXA1 mRNA was upregulated by estrogen and that estrogen receptor-α (ERα) recruitment to ER-binding sites in the vicinity of the FOXA1 gene was increased by estrogen in ERα-positive MCF-7 breast cancer cells. The estrogen-induced FOXA1 upregulation was repressed by 4-hydroxytamoxifen treatment. We also demonstrated that the proliferation and the migration of MCF-7 cells were decreased by FOXA1-specific small interfering RNA (siRNA; siFOXA1). Furthermore, siFOXA1 decreased the estrogen response element-driven transcription and the estrogen-dependent upregulation of ERα target genes in MCF-7 cells. Next, the immunohistochemical analyses of FOXA1 were performed using two groups of breast cancer specimens. The nuclear immunoreactivity of FOXA1 was detected in 80 (74%) of 108 human invasive breast cancers and was negatively correlated with tumor grade and positively correlated with hormone receptor status, including ERα and progesterone receptor, pathological tumor size, and immunoreactivity of FOXP1, another FOX family transcription factor. FOXA1 immunoreactivity was significantly elevated in the relapse-free breast cancer patients treated with tamoxifen. Notably, the double-positive immunoreactivities of FOXA1 and FOXP1 were significantly associated with a favorable prognosis for the relapse-free and overall survival of patients with tamoxifen-treated breast cancer, with lower P values compared with FOXA1 or FOXP1 immunoreactivity alone. These results suggest that FOXA1 plays an important role in the proliferation and migration of breast cancer cells by modulating estrogen signaling and that the double-positive immunoreactivities of FOXA1 and FOXP1 are associated with a favorable prognosis of tamoxifen-treated breast cancer.

Significant Association of Estrogen Receptor Binding Site Variation with Bipolar Disorder in Females

Major depression is nearly twice as prevalent in women compared to men. In bipolar disorder, depressive episodes have been reported to be more common amongst female patients. Furthermore, periods of depression often correlate with periods of hormonal fluctuations. A link between hormone signaling and these mood disorders has, therefore, been suggested to exist in many studies. Estrogen, one of the primary female sex hormones, mediates its effect mostly by binding to estrogen receptors (ERs). Nuclear ERs function as transcription factors and regulate gene transcription by binding to specific DNA sequences. A nucleotide change in the binding sequence might alter the binding efficiency, which could affect transcription levels of nearby genes. In order to investigate if variation in ER DNA-binding sequences may be involved in mood disorders, we conducted a genome-wide study of ER DNA-binding in patients diagnosed with major depression or bipolar disorder. Association studies were performed within each gender separately and the results were corrected for multiple testing by the Bonferroni method. In the female bipolar disorder material a significant association result was found for rs6023059 (corrected p-value = 0.023; odds ratio (OR) 0.681, 95% confidence interval (CI) 0.570–0.814), a single nucleotide polymorphism (SNP) placed downstream of the gene coding for transglutaminase 2 (TGM2). Thus, females with a specific genotype at this SNP may be more vulnerable to fluctuating estrogen levels, which may then act as a triggering factor for bipolar disorder.

Integrative genome-wide chromatin signature analysis using finite mixture models

Regulation of gene expression has been shown to involve not only the binding of transcription factor at target gene promoters but also the characterization of histone around which DNA is wrapped around. Some histone modification, for example di-methylated histone H3 at lysine 4 (H3K4me2), has been shown to bind to promoters and activate target genes. However, no clear pattern has been shown to predict human promoters. This paper proposed a novel quantitative approach to characterize patterns of promoter regions and predict novel and alternative promoters. We utilized high-throughput data generated using chromatin immunoprecipitation methods followed by massively parallel sequencing (ChIP-seq) technology on RNA Polymerase II (Pol-II) and H3K4me2. Common patterns of promoter regions are modeled using a mixture model involving double-exponential and uniform distributions. The fitted model obtained were then used to search for regions displaying similar patterns over the entire genome to find novel and alternative promoters. Regions with high correlations with the common patterns are identified as putative novel promoters. We used this proposed algorithm, RNA-seq data and several transcripts databases to find alternative promoters in MCF7 (normal breast cancer) cell line. We found 7,235 high-confidence regions that display the identified promoter patterns. Of these, 4,167 regions (58%) can be mapped to RefSeq regions. 2,444 regions are in a gene body or overlap with transcripts (non-coding RNAs, ESTs, and transcripts that are predicted by RNA-seq data). Some of these maybe potential alternative promoters. We also found 193 regions that map to enhancer regions (represented by androgen and estrogen receptor binding sites) and other regulatory regions such as CTCF (CCCTC binding factor) and CpG island. Around 5% (431 regions) of these correlated regions do not overlap with any transcripts or regulatory regions suggesting that these might be potential new promoters or markers for other annotation which are currently undiscovered.

Identification and Functional Annotation of Genome-Wide ER-Regulated Genes in Breast Cancer Based on ChIP-Seq Data

Estrogen receptor (ER) is a crucial molecule symbol of breast cancer. Molecular interactions between ER complexes and DNA regulate the expression of genes responsible for cancer cell phenotypes. However, the positions and mechanisms of the ER binding with downstream gene targets are far from being fully understood. ChIP-Seq is an important assay for the genome-wide study of protein-DNA interactions. In this paper, we explored the genome-wide chromatin localization of ER-DNA binding regions by analyzing ChIP-Seq data from MCF-7 breast cancer cell line. By integrating three peak detection algorithms and two datasets, we localized 933 ER binding sites, 92% among which were located far away from promoters, suggesting long-range control by ER. Moreover, 489 genes in the vicinity of ER binding sites were identified as estrogen response elements by comparison with expression data. In addition, 836 single nucleotide polymorphisms (SNPs) in or near 157 ER-regulated genes were found in the vicinity of ER binding sites. Furthermore, we annotated the function of the nearest-neighbor genes of these binding sites using Gene Ontology (GO), KEGG, and GeneGo pathway databases. The results revealed novel ER-regulated genes pathways for further experimental validation. ER was found to affect every developed stage of breast cancer by regulating genes related to the development, progression, and metastasis. This study provides a deeper understanding of the regulatory mechanisms of ER and its associated genes.

BMPR2 expression is suppressed by signaling through the estrogen receptor

BackgroundStudies in multiple organ systems have shown cross-talk between signaling through the bone morphogenetic protein receptor type 2 (BMPR2) and estrogen pathways. In humans, pulmonary arterial hypertension (PAH) has a female predominance, and is associated with decreased BMPR2 expression. The goal of this study was to determine if estrogens suppress BMPR2 expression.MethodsA variety of techniques were utilized across several model platforms to evaluate the relationship between estrogens and BMPR2 gene expression. We used quantitative RT-PCR, gel mobility shift, and luciferase activity assays in human samples, live mice, and cell culture.ResultsBMPR2 expression is reduced in lymphocytes from female patients compared with male patients, and in whole lungs from female mice compared with male mice. There is an evolutionarily conserved estrogen receptor binding site in the BMPR2 promoter, which binds estrogen receptor by gel-shift assay. Increased exogenous estrogen decreases BMPR2 expression in cell culture, particularly when induced to proliferate. Transfection of increasing quantities of estrogen receptor alpha correlates strongly with decreasing expression of BMPR2.ConclusionsBMPR2 gene expression is reduced in females compared to males in live humans and in mice, likely through direct estrogen receptor alpha binding to the BMPR2 promoter. This reduced BMPR2 expression may contribute to the increased prevalence of PAH in females.

Reprogramming clinical outcome

On binding to its target hormone, the oestrogen-receptor protein modulates the expression of many genes. Changes in the receptor's interaction with DNA have now been linked to clinical outcome in patients with breast cancer. See Letter p.389 Most breast tumours express the oestrogen receptor-α (ER), and this transcription factor is seen as an important drug target for the treatment of breast cancer. Jason Carroll and colleagues have mapped ER binding sites in solid primary cancers for the first time, and find distinct ER-binding profiles that are mediated in part by the FoxA1 DNA-binding protein and are associated with better or worse clinical outcome. As well as providing a potentially useful method of prognosis, this work may help to answer one of the major questions in breast cancer biology: how some ER+ tumours become drug resistant.

CBP Mediates NF-κB-Dependent Histone Acetylation and Estrogen Receptor Recruitment to an Estrogen Response Element in the BIRC3 Promoter

Estrogen receptor (ER) and NF-κB are transcription factors with profound effects on breast cancer cell proliferation and survival. While many studies demonstrate that ER and NF-κB can repress each other, we previously identified a gene signature that is synergistically upregulated by these two factors in more aggressive luminal B breast tumors. Herein, we examine a novel mechanism of cross talk between ER and NF-κB that results in the upregulation of the antiapoptotic gene BIRC3 (also known as cIAP2). We demonstrate that NF-κB, acting through two response elements, is required for ER recruitment to an adjacent estrogen response element (ERE) in the BIRC3 promoter. This effect is accompanied by a major increase in NF-κB-dependent histone acetylation around the ERE. Interestingly, CBP, a histone acetyltransferase previously implicated in repressive interactions between ER and NF-κB, plays a permissive role by promoting histone acetylation and ER recruitment, as well as enhanced expression of BIRC3. These findings suggest a new gene regulatory mechanism by which inflammation and NF-κB activation can influence ER recruitment to inherently inactive ER binding sites. This fine-tuning mechanism may explain how two factors that generally repress each other's activity may work together on certain genes to promote breast cancer cell survival and tumor progression.

Research resource: The estrogen receptor α cistrome defined by DamIP.

Gene expression is tightly regulated by transcription factors and cofactors that function by directly or indirectly interacting with DNA of the genome. Understanding how and where these proteins bind provides essential information to uncover genetic regulatory mechanisms. We have developed a new method to study DNA-protein interaction in vivo called DNA adenine methyltransferase (Dam)IP, which is based on fusing a protein of interest to a mutant form of Dam from Escherichia coli. We showed previously that DamIP can efficiently identify in vivo binding sites of Dam-tethered human estrogen receptor (hER)α. In current study, we present the cistrome of hERα determined by DamIP and high throughput sequencing (DamIP-seq). The DamIP-seq-defined hERα cistrome identifies many new binding regions and overlaps with those determined by chromatin immunoprecipitation (ChIP)-chip or ChIP-seq. Elements uniquely identified by DamIP-seq include a unique class of elements that show low, but persistent, hERα binding when reexamined by conventional ChIP. In contrast, DamIP-seq fails to detect some elements with very transient hERα binding. The methyl-adenine modifications introduced by Dam are stable and do not decrease over 12 d. In summary, the current study provides both an alternate view of the hERα cistrome to further understand the mechanism of hERα-mediated transcription and a new tool to explore other transcriptional factors and cofactors that is very different from conventional ChIP.

P4-01-06: Global Characterization of the SRC-1 Transcriptome Identifies Disintegrin C as an ER-Independent Mediator of Endocrine Resistant Breast Cancer.

Abstract Background: The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity leading to the development of a steroid independent tumour. The p160 steroid coactivataor protein SRC-1 drives this tumour adaptability. SRC-1 is central to the development of the endocrine resistant phenotype and is an independent predictor of disease free survival in tamoxifen-treated patients. Here, using discovery studies we identify Disintegrin C (DC), a multidomain transmembrane glycoprotein as a direct, ER-independent target of SRC-1. Methods: SRC-1-ChIP sequencing was performed in endocrine resistant (LY2) cells. Microarray experiments were performed on LY2 cells transfected with scrambled siRNA or SRC-1 siRNA, +/−4-OHT to assess SRC-1-dependent gene expression. Combining the ChIP-seq and expression array datasets, a total of 2,065 genes were significantly downregulated (p&amp;lt;0.05) following SRC-1 knockdown. DC was selected for validation as a potential novel metastatic oncogene. DC transcipt levels were measured following knockdown and over-expression of SRC-1 and ERα. Mouse xenograft and SRC-1 knockout models were used to examine DC expression in vivo. A TMA comprising 560 patients was stained for DC. Functional validation studies were performed using 3D culture and migration assays, with transient knockdown of DC and treatment with JC1, an inhibitory peptide against DC. Results: Treatment with 4-OHT significantly increased the number of ChIP-enriched intervals identified. The majority of peaks in the 4-OHT treated sample were close to the transcriptional start site, in the promoter, first exon or upstream of the promoter. As a defined ER coactivator, an overlap between ER binding sites and those identified for SRC-1 is expected. 43% of high confidence SRC-1 peaks in 4-OHT-treated LY2 cells contained an ERE binding motif. This raises the possibility that, in endocrine resistance, SRC-1 can drive transcription independent of ER. Treatment with 4-OHT resulted in substantial recruitment of SRC-1 to the DC promoter. In primary tumours there was a significant association between transcript levels of SRC-1 and DC. Cells derived from mammary tumours of the SRC-1-/−/PyMT mouse lacked DC, but there was high expression in the wild type PyMT mouse. Knockdown of ERα in LY2 cells did not alter DC expression. Knockdown of DC reduced cell migration and restored cell differentiation in resistant cells. In xenograft studies treatment with 4-OHT increased tumour volume in the resistant tumours and induced DC expression. Treatment with recombinant JC1 reduced cellular migration in endocrine resistant cells. Kaplan Meier estimates of disease free survival show that DC is a strong predictor of disease free survival in breast cancer (p&amp;lt;0.0001). SRC-1 and DC are significant independent predictors of disease recurrence (odds ratios 2.18 and 2.4). Conclusion: Our discovery studies have uncovered a steroid independent SRC-1 mediated network in endocrine resistant breast cancer. We identified a new SRC-1 target, DC. DC represents a rational new therapeutic target with a robust companion biomarker for treatment of endocrine resistant tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-06.

P3-10-02: Gene Expression Changes in Methylnitrosourea (MNU)-Induced ER+ Mammary Cancers Following Short-Term Treatment of Rats with the Aromatase Inhibitor Vorozole.

Abstract Aromatase inhibitors have proven to be highly effective in both therapy and prevention of ER+ breast cancer. Vorozole (R83842), a high affinity competitive inhibitor of aromatase (similar to letrozole and anastrozole) showed strong activity in early clinical trials against ER+ breast cancer. Furthermore, vorozole was highly effective in the prevention and therapy of ER+ cancers in the MNU-mammary cancer model (Lubet, et al., Carcinogenesis 19, 1345, 1998). In the present study, rats bearing mammary cancers induced by MNU were exposed to vorozole (1.25 mg/kg BW/day) for 5 days. Global gene expression analysis showed that 162 genes were down-regulated and 180 genes up-regulated in cancers treated with vorozole (p &amp;lt; 0.05 and fold change &amp;gt; 1.5). The genes modulated by vorozole were compared with two additional sets of data. First, thirty-two genes and a number of pathways exhibited significantly concordant changes with aromatase inhibitors both in the animal model and in at least three of four published human data sets. In particular, differentially expressed genes enriched in the cell cycle pathway that were related to chromosome condensation in prometaphase [including Aurora-A, Aurora-B, Bub1B, non-SMC condensin I complex, subunit H (BRRN1), Condensin, CAP-G, CAP-G/G2, CAP-H/H2, CAP-D2/D3, CAP-E, TOP2, Cyclin A, Cyclin B, CDK1, Histone H1 and inner centromere protein (INCENP)] were downregulated after treatment with the aromatase inhibitor. These results appear to be in agreement with the strong anti-proliferative effects of aromatase inhibitors in both animal and clinical studies. A second comparison was with an in vitro study in which estrogen was removed from MCF-7 cells in culture. Decreases in genes related to the E2F1 transcription factor were observed. In our study, 13 modulated genes exhibited E2F-1 binding sites in their promoter regions, and 7 genes contained both ER binding and E2F binding sites. We were able to confirm modulation of the cell cycle related and E2F-related genes in a large independent set of human samples treated with anastrozole. The results on RNA changes for Bub 1B, Cyclin A and CDK-1 were verified by employing IHC analysis. In summary, gene changes observed in the rat closely paralleled gene changes associated with aromatase treatment and estrogen withdrawal in humans. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-10-02.

P4-01-21: An Estrogen-Inducible Transcription Factor FOXP1 Promotes Estrogen-Dependent Cell Proliferation of Breast Cancer Cells and Is Associated with 5-Year Disease-Free Survival in Patients with Tamoxifen-Treated Breast Cancer.

Abstract Estrogen signaling pathways are involved in the growth and development of breast tumors through the activation of estrogen receptor a (ERα). ERα is expressed in most breast cancers and involves in estrogen-dependent proliferation by acting as transcription factors activating the expression of target genes. Various coregulators and transcription factors are associated with ERα-mediated transcriptional control of target genes. Therefore, a comprehensive understanding of estrogen signaling pathways in breast cancer is required for both treatment and diagnosis of the disease. Forkhead box P1 (FOXP1) is a member of the forkhead box transcription factor family and has been reported to be associated with various types of tumors. Here, we investigated the expression pattern of FOXP1 by immunohistochemistry in a series of 133 invasive breast cancers and compared it with clinicopathological factors. The expression of FOXP1 was detected in nuclei in 89 cases (67%) and correlated positively with tumor grade and hormone receptor status, including ERα and progesterone receptor (PgR), and negatively with pathological tumor size (pT). And in ERα-positive MCF-7 breast cancer cells, we demonstrated that FOXP1 mRNA was upregulated by estrogen and that ERα recruitment to ER binding sites within the FOXP1 gene region identified by ChIP-chip analysis was increased. We also demonstrated that proliferation of MCF-7 cells was increased by exogenously transfected FOXP1 and decreased by FOXP1-specific siRNA. Moreover, in MCF-7 cells, FOXP1 enhanced estrogen response element (ERE)-driven transcription. Finally, FOXP1 immunoreactivity was significantly more elevated in relapse-free breast cancer patients treated with tamoxifen than in relapse patients treated with it. Taken together, these results suggest that FOXP1 plays an important role in proliferation of breast cancer cells by modulating estrogen signaling and that FOXP1 immunoreactivity might be associated with the estrogen dependency of breast cancer clinically, which may predict favorable prognosis in the patients treated with tamoxifen. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-21.

Research Resource: Enhanced Genome-Wide Occupancy of Estrogen Receptor α by the Cochaperone p23 in Breast Cancer Cells

p23 is a chaperone with multiple heat shock protein 90 dependent and independent cellular functions, including stabilizing unliganded steroid receptors and modulating receptor-DNA dynamics. p23 protein is also up-regulated in several cancers, notably breast cancer. We previously demonstrated that higher expression of p23 in the estrogen-dependent breast cancer line MCF-7 (MCF-7+p23) selectively increased estrogen receptor (ER) target gene transcription and ER recruitment to regulatory elements, promoted cell invasion, and predicted a poor prognosis in breast cancer patients. To probe the impact of p23 on ER binding throughout the human genome, we compared ER occupancy in MCF-7+p23 cells relative to MCF-7-control cells by using chromatin immunoprecipitation followed by ultrahigh-throughput DNA sequencing in the absence and presence of 17β-estradiol (E2) treatment. We found that increased expression of p23 resulted in a 230% increase in the number of E2-induced ER-binding sites throughout the genome compared with control cells and also increased ER binding under basal conditions. Motif analysis indicated that ER binds to a similar DNA sequence regardless of p23 status. We also observed that ER tends to bind closer to genes that were induced, rather than repressed by either E2 treatment or p23 overexpression. Interestingly, we also found that the increased invasion of MCF-7+p23 cells was not only p23 dependent but also ER dependent. Thus, a small increase in the expression of p23 amplifies ER-binding genome wide and, in combination with ER, elicits an invasive phenotype. This makes p23 an attractive target for combating tumor cell metastasis in breast cancer patients.

FOXP1, an estrogen-inducible transcription factor, modulates cell proliferation in breast cancer cells and 5-year recurrence-free survival of patients with tamoxifen-treated breast cancer.

Breast cancer is primarily a hormone-dependent tumor that can be regulated by the status of steroid hormones, including estrogen and progesterone. Forkhead box P1 (FOXP1) is a member of the forkhead box transcription factor family and has been reported to be associated with various types of tumors. In the present study, we investigated the expression of FOXP1 in 133 human invasive breast cancers, obtained by core biopsy, by immunohistochemical analysis. Nuclear immunoreactivity of FOXP1 was detected in 89 cases (67%) and correlated positively with tumor grade and hormone receptor status, including estrogen receptor alpha (ERα) and progesterone receptor, and negatively with pathological tumor size. In ERα-positive MCF-7 breast cancer cells, we demonstrated that FOXP1 mRNA was upregulated by estrogen and increased ERα recruitment to ER binding sites identified by ChIP-on-chip analysis within the FOXP1 gene region. We also demonstrated that proliferation of MCF-7 cells was increased by exogenously transfected FOXP1 and decreased by FOXP1-specific siRNA. Furthermore, FOXP1 enhanced estrogen response element-driven transcription in MCF-7 cells. Finally, FOXP1 immunoreactivity was significantly elevated in relapse-free breast cancer patients treated with tamoxifen. These results suggest that FOXP1 plays an important role in proliferation of breast cancer cells by modulating estrogen signaling and that FOXP1 immunoreactivity could be associated with the estrogen dependency of clinical breast cancers, which may predict favorable prognosis in the patients treated with tamoxifen.