Supplementary Tables 1-3 from Gene Expression Preferentially Regulated by Tamoxifen in Breast Cancer Cells and Correlations with Clinical Outcome

Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC50 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (>= 0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 mu M compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, beta (beta-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by beta-CGRP.

Nuclear receptor estrogen receptor alpha (ER alpha) controls the expression of hundreds of genes responsible for target cell phenotypic properties, but the relative importance of direct versus tethering mechanisms of DNA binding has not been established. In this first report, we examine the genome-wide chromatin localization of an altered-specificity mutant ER with a DNA binding domain deficient in binding to estrogen response element (ERE)-containing DNA (DBDmut ER) versus wild-type ER alpha. Using high-throughput sequencing of ER chromatin immunoprecipitations (ChIP-Seq) and mRNA transcriptional profiling, we show that direct ERE binding is required for most of (75%) estrogen-dependent gene regulation and 90% of hormone-dependent recruitment of ER to genomic binding sites. De novo motif analysis of the chromatin binding regions in MDA-MB-231 human breast cancer cells defined unique transcription factor profiles responsible for genes regulated through tethering versus direct ERE binding, with Runx motifs enriched in ER-tethered sites. We confirmed a role for Runx1 in mediating ER alpha genomic recruitment and regulation of tethering genes. Our findings delineate the contributions of direct receptor ERE binding versus binding through response elements for other transcription factors in chromatin localization and ER-dependent gene regulation, paradigms likely to underlie the gene regulatory actions of other nuclear receptors as well.

OBJECTIVE: Selective estrogen receptor modulators (SERMs) demonstrate tissue specific estrogenic and antiestrogenic effects. SERMs improve bone density having little effect on endometrial and no effect on breast cell proliferation. Combining estrogen with SERM to form a TSEC has been demonstrated to provide an improved blend of tissue specific agonist and antagonist effects. TSECs alleviate vasomotor symptoms, although their action in breast and uterine cells is not completely understood. Proliferating cell nuclear antigen (PCNA) is one of the best characterized markers of proliferation. Here we evaluated the effects of raloxifene (RAL), tamoxifene (TAM), lasofoxifene (LAS) and bazedoxifene (BZA) alone and in combination with E2 on PCNA gene expression in uterine and breast cells. DESIGN: In vitro study in breast and uterine cancer cell lines. MATERIALS AND METHODS: Ishikawa and MCF-7 cells were treated for 24 h with 10nM E2, RAL, TAM, LAS, BZA, or P, either alone or with E2. RNA was extracted, reverse transcribed and RTPCR performed three times in triplicate. PCNA mRNA expression was normalized to β-actin. RESULTS: PCNA mRNA expression was increased in both cell lines treated with E2. In MCF7 cells BZA and LAS alone (p<0.01) but not TAM or RAL significantly decreased PCNA expression. The response to E2 was significantly attenuated by all TSECs (p<0.01) except for RAL+E2. In Ishikawa cells SERMs did not induce PCNA expression and all TSECs prevented estrogen induced PCNA expression. CONCLUSIONS: Estrogens induced proliferation of breast and uterine cells. Adding a SERM to estrogen, forming a TSEC, prevented the estrogen induced proliferation of both cell types. While TAM and RAL have shown clinical efficacy in prevention of breast cancer, these data suggest that other SERMs and TSECs have a similar or greater effect on suppressing breast cell proliferation without inducing uterine cell growth. TSECs are a promising menopausal therapy that will treat symptoms while demonstrating protective effects on breast and uterine cells.

Whereas estrogens exert their effects by binding to nuclear estrogen receptors (ERs) and directly altering target gene transcription, they can also initiate extranuclear signaling through activation of kinase cascades. We have investigated the impact of estrogen-mediated extranuclear-initiated pathways on global gene expression by using estrogen-dendrimer conjugates (EDCs), which because of their charge and size remain outside the nucleus and can only initiate extranuclear signaling. Genome-wide cDNA microarray analysis of MCF-7 breast cancer cells identified a subset of 17beta-estradiol (E2)-regulated genes ( approximately 25%) as EDC responsive. The EDC and E2-elicited increases in gene expression were due to increases in gene transcription, as observed in nuclear run-on assays and RNA polymerase II recruitment and phosphorylation. Treatment with antiestrogen or ERalpha knockdown using small interfering RNA abolished EDC-mediated gene stimulation, whereas GPR30 knockdown or treatment with a GPR30-selective ligand was without effect, indicating ER as the mediator of these gene regulations. Inhibitors of MAPK kinase and c-Src suppressed both E2 and EDC stimulated gene expression. Of note, in chromatin immunoprecipitation assays, EDC was unable to recruit ERalpha to estrogen-responsive regions of regulated genes, whereas ERalpha recruitment by E2 was very effective. These findings suggest that other transcription factors or kinases that are downstream effectors of EDC-initiated extranuclear signaling cascades are recruited to regulatory regions of EDC-responsive genes in order to elicit gene stimulation. This study thus highlights the importance of inputs from both nuclear and extranuclear ER signaling pathways in regulating patterns of gene expression in breast cancer cells.

Estrogen receptors alpha and beta (ERalpha and ERbeta) mediate the actions of estrogens in a variety of normal and cancer target cells. Estrogens differ in their preference for these ERs, and many phytoestrogens bind preferentially to ERbeta. To investigate how phytoestrogens such as genistein impact ER-regulated gene expression, we used adenoviral gene delivery of ERbeta coupled with ERalpha depletion with small interfering RNA to generate human breast cancer (MCF-7) cells expressing four complements of ERalpha and ERbeta. We examined the dose-dependent effects of genistein on genome-wide gene expression by DNA microarrays and monitored the recruitment of ERs and coregulators to responsive regions of estrogen-regulated genes. At a low (6 nm) concentration, genistein regulated gene expression much more effectively in cells coexpressing ERalpha and ERbeta than in cells expressing ERalpha alone, whereas at high concentration (300 nm), genistein induced transcriptome changes very similar to that of 17beta-estradiol. We demonstrate that ERbeta is preferentially activated by genistein and is recruited to estrogen-responsive genomic sites and that differential occupancy of ERalpha and ERbeta by genistein and 17beta-estradiol in turn influences the recruitment patterns of coregulators such as steroid receptor coactivator 3 (SRC3) and receptor-interacting protein 140 (RIP140). Our observations indicate that genistein is a potency-selective ligand for gene expression regulation by ERalpha and ERbeta and that the ability of ERalpha and ERbeta to serve as determinants of gene expression is greatly influenced by the nature of the ligand, by ligand dose, and by the differential abilities of ligand-ER complexes to recruit different coregulators at ER binding sites of hormone-regulated genes.

Estrogen receptors (cid:1) and (cid:2) (ER (cid:1) and ER (cid:2) ) mediate the actions of estrogens in a variety of normal and cancer target cells. Estrogens differ in their preference for these ERs, and many phytoestrogens bind preferentially to ER (cid:2) . To investigate how phytoestrogens such as genistein impact ER-reg-ulated gene expression, we used adenoviral gene delivery of ER (cid:2) coupled with ER (cid:1) depletion with small interfering RNA to generate human breast cancer (MCF-7) cells expressing four complements of ER (cid:1) and ER (cid:2) . We examined the dose-depen-dent effects of genistein on genome-wide gene expression by DNA microarrays and monitored the recruitment of ERs and coregulators to responsive regions of estrogen-regulated genes. At a low (6 n M ) concentration, genistein regulated gene expression much more effectively in cells coexpressing ER (cid:1) and ER (cid:2) than in cells expressing ER (cid:1) alone, whereas at high concentration (300 n M genistein induced transcriptome changes very similar to that of 17 (cid:2) -estradiol. We demonstrate that ER (cid:2) is preferentially activated by genistein and is recruited to estrogen-responsive genomic sites and that differential occupancy of ER (cid:1) and ER (cid:2) by genistein and 17 (cid:2) -estradiol in turn influences the recruitment patterns of coregulators such as steroid receptor coactivator 3 (SRC3) and receptor-interacting protein 140 (RIP140). Our ob-servations indicate that genistein is a potency-se-lective ligand for gene expression regulation by ER (cid:1) and ER (cid:2) and that the ability of ER (cid:1) and ER (cid:2) to serve as determinants of gene expression is greatly influenced by the nature of the ligand, by ligand dose, and by the differential abilities of li-gand-ER complexes to recruit different coregulators at ER binding sites of hormone-regulated genes. ( Molecular Endocrinology 22: 1032–1043, 2008)

Estrogens generally stimulate the proliferation of estrogen receptor (ER)-containing breast cancer cells, but they also suppress proliferation of some ER-positive breast tumors. Using a genome-wide analysis of gene expression in two ER-positive human breast cancer cell lines that differ in their proliferative response to estrogen, we sought to identify genes involved in estrogen-regulated cell proliferation. To this end, we compared the transcriptional profiles of MCF-7 and MDA-MB-231ER+ cells, which have directionally opposite 17beta-estradiol (E2)-dependent proliferation patterns, MCF-7 cells being stimulated and 231ER+ cells suppressed by E2. We identified a set of approximately 70 genes regulated by E2 in both cells, with most being regulated by hormone in an opposite fashion. Using a variety of bioinformatics approaches, we found the E2F binding site to be overrepresented in the potential regulatory regions of many cell cycle-related genes stimulated by estrogen in MCF-7 but inhibited by estrogen in 231ER+ cells. Biochemical analyses confirmed that E2F1 and E2F downstream target genes were increased in MCF-7 and decreased in 231ER+ cells upon estrogen treatment. Furthermore, RNA interference-mediated knockdown of E2F1 blocked estrogen regulation of E2F1 target genes and resulted in loss of estrogen regulation of proliferation. These results demonstrate that regulation by estrogen of E2F1, and subsequently its downstream target genes, is critical for hormone regulation of the proliferative program of these breast cancer cells, and that gene expression profiling combined with bioinformatic analyses of transcription factor binding site enrichment in regulated genes can identify key components associated with nuclear receptor hormonal regulation of important cellular functions.

AbstractThe beneficial effect of the selective estrogen receptor (ER) modulator tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like agonist effects. Here, we report that, in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of >60 genes, which are minimally regulated by estradiol (E2) or raloxifene in ERα-positive MCF-7 human breast cancer cells. This gene regulation by tamoxifen is mediated by ERα and reversed by E2 or ICI 182,780. Introduction of ERβ into MCF-7 cells reverses tamoxifen action on ∼75% of these genes. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, their expression level was examined in breast cancers of women who had received adjuvant therapy with tamoxifen. High expression of two of the tamoxifen-stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive cancers and hence seem to be markers of a poor prognosis. Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy. This may have a potential effect on the choice of tamoxifen versus aromatase inhibitors as adjuvant endocrine therapy. (Cancer Res 2006; 66(14): 7334-40)

Two subtypes of the estrogen receptor (ER), ERalpha and ERbeta, mediate the actions of estrogens, and although 70% of human breast cancers express ERbeta along with ERalpha, little is known about the possible comodulatory effects of these two ERs. To investigate this, we have used adenoviral gene delivery to produce human breast cancer (MCF-7) cells expressing different levels of ERbeta, along with their endogenous ERalpha, and have examined the effects of ERbeta and receptor occupancy, using ER subtype selective ligands, on genome-wide gene expression by microarray and pathway network analysis. ERbeta had diverse effects on gene expression, enhancing or counteracting ERalpha regulation for distinct subsets of estrogen target genes. Strikingly, ERbeta in the absence of estradiol (E2), elicited the stimulation or suppression of many genes that were normally only regulated by ERalpha with E2. In addition, ERbeta plus E2 elicited the expression of a unique group of genes that were not regulated by ERalpha plus E2 alone. The expression of genes in many functional categories were modulated by ERbeta, with the greatest numbers associated with transcription factors and signal transduction pathways. Regulation of multiple components in the TGFbeta and semaphorin pathways, and of genes controlling cell cycle progression and apoptosis, may contribute to the suppression of cell proliferation observed with ERbeta. Our observations suggest that the relative levels of ERbeta and ERalpha in breast cancers are likely to impact cell proliferation and the activities of diverse signaling pathways and their response to ER ligands and endocrine therapies.

Selective estrogen receptor modulators (SERMs) such as tamoxifen are effective in the treatment of many estrogen receptor-positive breast cancers and have also proven to be effective in the prevention of breast cancer in women at high risk for the disease. The comparative abilities of tamoxifen versus raloxifene in breast cancer prevention are currently being compared in the Study of Tamoxifen and Raloxifene trial. To better understand the actions of these compounds in breast cancer, we have examined their effects on the expression of ∼12,000 genes, using Affymetrix GeneChip microarrays, with quantitative PCR verification in many cases, categorizing their actions as agonist, antagonist, or partial agonist/antagonist. Analysis of gene stimulation and inhibition by the SERMs trans-hydroxytamoxifen (TOT) and raloxifene (Ral) or ICI 182,780 (ICI) and by estradiol (E2) in estrogen receptor-containing MCF-7 human breast cancer cells revealed that (a) TOT was the most E2-like of the three compounds, (b) all three compounds either partially or fully antagonized the action of E2 on most genes, with the order of antagonist activity being ICI > Ral > TOT, (c) TOT and Ral, but not ICI, displayed partial agonist/partial antagonist activity on a number of E2-regulated genes, (d) several stimulatory cell cycle-related genes were down-regulated exclusively by ICI, (e) the estrogen-like activity of Ral nearly always overlapped with that of TOT, indicating that Ral has little unique agonist activity different from that of TOT, and (f) some genes were specifically up-regulated by TOT but not Ral, ICI, or E2. Hence, gene expression profiling can discern fundamental differences among SERMs and provides insight into the distinct biologies of TOT, Ral, and ICI in breast cancer.

Estrogens exert many important effects in bone, a tissue that contains both estrogen receptors alpha and beta (ERalpha and ERbeta). To compare the actions of these receptors, we generated U2OS human osteosarcoma cells stably expressing ERalpha or ERbeta, at levels comparable with those in osteoblasts, and we characterized their response to 17beta-estradiol (E2) over time using Affymetrix GeneChip microarrays to determine the expression of approximately 12,000 genes, followed by quantitative PCR verification of the regulation of selected genes. Of the approximately 100 regulated genes we identified, some were stimulated by E2 equally through ERalpha and ERbeta, whereas others were selectively stimulated via ERalpha or ERbeta. The E2-regulated genes showed three distinct temporal patterns of expression over the 48-h time course studied. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ERbeta, in keeping with the selective E2 enhancement of the motility of ERbeta-containing cells. On genes regulated equally by E2 via ERalpha or ERbeta, the phytoestrogen genistein preferentially stimulated gene expression via ERbeta. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation.

Nuclear receptor (NR)-mediated transcription is driven by dynamic multiprotein coactivator complexes, the composition of which is thought to determine the biological activity of NRs at specific promoters. The extent to which NRs discriminate between a spectrum of potential binding partners is intuitively a function of the inherent affinities of these individual interactions. Using real time interaction analysis with BIAcore, we evaluated the affinities and kinetics of the interactions of full-length members of the SRC/p160 coactivator family with estrogen receptor a (ERalpha) and ERbeta bound to a variety of ligands. We substantiate that 17beta-estradiol enhances the affinity of ER-SRC/p160 interactions, whereas 4(OH)-tamoxifen, raloxifene, and ICI-182,780 inhibit these interactions. We show that a well defined, ER isoform-specific hierarchy governs the association of liganded ERs with full length SRC/p160 family members. Moreover, our data indicate that the interaction affinities of the full-length SRC/p160s with ERs are significantly higher then those of the NR interaction domains of the same coactivators, indicating that portions of coactivator molecules distinct from NR interaction domains might participate in receptor-coactivator complex formation. Finally, the interaction kinetics of SRC/p160s with ERs are consistent with a bipartite model, involving initial rapid formation of an unstable intermediate complex, and a subsequent slower reaction leading to its stabilization. We interpret our results as evidence that hierarchical coactivator interaction affinities are an important source of diversity in NR-mediated signaling and that the complexity of receptor-coactivator cross-talk might be best understood in the context of full-length molecules.

Estrogens are known to regulate the proliferation of breast cancer cells and to alter their cytoarchitectural and phenotypic properties, but the gene networks and pathways by which estrogenic hormones regulate these events are only partially understood. We used global gene expression profiling by Affymetrix GeneChip microarray analysis, with quantitative PCR verification in many cases, to identify patterns and time courses of genes that are either stimulated or inhibited by estradiol (E2) in estrogen receptor (ER)-positive MCF-7 human breast cancer cells. Of the >12,000 genes queried, over 400 showed a robust pattern of regulation, and, notably, the majority (70%) were down-regulated. We observed a general up-regulation of positive proliferation regulators, including survivin, multiple growth factors, genes involved in cell cycle progression, and regulatory factor-receptor loops, and the down-regulation of transcriptional repressors, such as Mad4 and JunB, and of antiproliferative and proapoptotic genes, including B cell translocation gene-1 and -2, cyclin G2, BCL-2 antagonist/killer 1, BCL 2-interacting killer, caspase 9, and TGFbeta family growth inhibitory factors. These together likely contribute to the stimulation of proliferation and the suppression of apoptosis by E2 in these cells. Of interest, E2 appeared to modulate its own activity through the enhanced expression of genes involved in prostaglandin E production and signaling, which could lead to an increase in aromatase expression and E2 production, as well as the decreased expression of several nuclear receptor coactivators that could impact ER activity. Our studies highlight the diverse gene networks and metabolic and cell regulatory pathways through which this hormone operates to achieve its widespread effects on breast cancer cells.

Analysis of estrogen receptor alpha and beta interplay with other transcription factors is critical to the understanding of how small molecules, the cognate ligands for these receptors, selectively regulate the mode and amplitude of gene transcription by affecting receptor activity. To better understand the molecular mechanisms of selective action of estrogen receptor ligands, we characterized estrogen receptor alpha and beta (ER) interaction with the p160 family of coactivators. We also investigated how these interactions are affected by binding of specific ligands. We show that ER or and beta utilize different LXXLL motifs for their interaction with p160 family members. We found that significant differences exist between the affinity of the nuclear receptor interacting domain (NRID) and interaction of separate LXXLL motifs with ERs. This result indicates that a single LXXLL motif is unlikely to be sufficient for interaction with receptors, and that regions other than LXXLL motifs also participate in ER-p160 complex formation. We found that ER alpha and beta have strong affinity preferences for particular coactivators. These results suggest that ER-mediated transcription is not driven by a random mixture of ER-coactivator complexes. We also show that some ER ligands are functionally specific. We describe a ligand that binds to both receptors, but enhances only ER beta interaction with SRC1 and SRC3 while exhibiting little effect on the ER alpha interaction with these proteins. Finally, we provide data that suggest how genistein may selectively recruit coactivators when liganded to ERs. It enhances the interaction of ERs with SRC1 and SRC3, but demonstrates a minimal effect on receptor interaction with DRIP205 and CBP.

The recent cloning of a second form of the estrogen receptor (ER-beta) has made it possible to map the distribution of ER-beta mRNA-containing perikarya in the rat hypothalamus. The present in situ hybridization histochemical studies have detected ER-beta mRNA in the medial preoptic area; the anterior periventricular, paraventricular, supraoptic, arcuate, medial tuberal and medial mammillary nuclei; the bed nucleus of the stria terminals, and zona incerta. As previously described for the classical ER (ER-alpha) mRNA, a dense accumulation of ER-beta mRNA-expressing perikarya is present in the medial preoptic area and bed nucleus of the stria terminalis. In contrast, ER-beta mRNA was also concentrated in the paraventricular and supraoptic nuclei, brain regions which contain little or no ER-alpha mRNA. Moreover, the arcuate and ventromedial nuclei, areas with abundant ER-alpha. contain only a weak level of ER-beta hybridization signal. The description of ER-beta mRNA-containing perikarya in the rat hypothalamus provides a foundation for further morphological and physiological studies aimed at elucidating the role of ER-beta in the hypothalamus.