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.

Two subtypes of the estrogen receptor (ER), ER␣ and ER␤, mediate the actions of estrogens, and although 70% of human breast cancers express ER␤ along with ER␣, 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 ER␤, along with their endogenous ER␣, and have examined the effects of ER␤ and receptor occupancy, using ER subtype selective ligands, on genome-wide gene expression by microarray and pathway network analysis. ER␤ had diverse effects on gene expression, enhancing or counteracting ER␣ regulation for distinct subsets of estrogen target genes. Strikingly, ER␤ in the absence of estradiol (E2), elicited the stimulation or suppression of many genes that reproductive physiology and development, and in the functioning of numerous nonreproductive tissues as well. Estrogen hormones also influence the growth of various cancers (1), including breast and endometrial cancers. Although estrogens were originally thought to signal through only one form of estrogen receptor (ER), the complexity of estrogen physiology was compounded when a second form of the ER (termed ER␤, ESR2) was cloned (2, 3). Since then, much effort has gone into investigating the specific roles of the two receptor subtypes in diverse estrogen target tissues (4 -7).Although ER␤ is normally coexpressed with ER␣ in many tissue types, and the majority of human breast cancers express ER␤ along with ER␣ (8 -10), it is not fully known how the presence of both receptors and their relative levels control cellular responses to estrogen. These two transcription factors have a similar domain structure and very similar DNA binding domains, but have substantial differences in their ligand binding domains and especially in their N-terminal activation function regions (1, 11). Examination of their separate activities in osteosarcoma cells indicated distinct as well as overlapping gene regulatory activities (12, 13). Because these receptors are able to heterodimerize when present in the same cell (14), their joint actions and possible comodulatory effects on gene regulation are issues of importance.Although it is well documented that ER␣-positive breast cancer cells show enhanced proliferation in response to estrogen (15, 16), the manner in which ER␤ impacts estrogen mitogenicity and the changes in gene expression that underlie these effects are less clear, although several reports support the role of ER␤ as a negative regulator of ER␣ (17)(18)(19).To better understand the role of ER␤ in influencing estrogen action, we have used adenoviral gene delivery of ER␤ and gene expression microarray analyses to investigate gene regulatory effects of ER␤ in breast cancer cells expressing ER␣, as well as to distill the information into gene networks and pathways responsible for controlling estrogen activities. Our results indicate that ER␤ can modulate ER␣ gene expression in both an enhancing and a suppressing fashion, and...

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.

Estrogen is of great importance in the regulation of uterine function. The aim of this study was to examine the individual physiological roles of each of the two receptors for estradiol, estrogen receptor (ER) alpha and ERbeta, and their potential comodulatory effects on gene expression and uterine growth using recently developed ER subtype-selective agonist ligands. The use of ER subtype-selective ligands provides an alternative, complementary approach to the use of receptor knockout mice. Administration of the ERalpha-selective ligand propyl pyrazole triol (PPT) to immature mice resulted in a significant increase in uterine weight, as well as bromodeoxyuridine incorporation and proliferating cell nuclear antigen expression in luminal epithelial cells. PPT also increased complement component 3, lactoferrin, and glucose-6-phosphate dehydrogenase (G6PDH), and decreased androgen receptor (AR) and progesterone receptor (PR) mRNA levels in uterine tissue, as did estradiol (E(2)). However, when compared with E(2), PPT was less effective in stimulating uterine weight, complement component 3, and G6PDH expression but was as effective as E(2) in regulating lactoferrin, AR, and PR expression. In contrast to the action of the ERalpha agonist PPT, the ERbeta agonist diarylpropionitrile (DPN) did not increase uterine weight or luminal epithelial cell proliferation at a dose that reduced G6PDH and elicited a decrease in PR and AR mRNA and protein expression. Interestingly, DPN reduced the uterine weight stimulation by PPT, and enhanced the effect of PPT in decreasing uterine PR and AR mRNA. These findings with ER subtype-selective ligands indicate that ERalpha is the major regulator of estrogen function in the uterus, but that ERbeta does exert effects on some uterine markers of estrogen action. In addition, ERbeta can modulate ERalpha activity in a response-specific and dose-dependent manner.

Approximately 75% of breast tumors express the estrogen receptor (ER), and women with these tumors will receive endocrine therapy. Unfortunately, up to 50% of these patients will fail ER-targeted therapies due to either de novo or acquired resistance. ER-positive tumors can be classified based on gene expression profiles into Luminal A- and Luminal B-intrinsic subtypes, with distinctly different responses to endocrine therapy and overall patient outcome. However, the underlying biology causing this tumor heterogeneity has yet to become clear. This review will explore the role of inflammation as a risk factor in breast cancer as well as a player in the development of more aggressive, therapy-resistant ER-positive breast cancers. First, breast cancer risk factors, such as obesity and mammary gland involution after pregnancy, which can foster an inflammatory microenvironment within the breast, will be described. Second, inflammatory components of the tumor microenvironment, including tumor-associated macrophages and proinflammatory cytokines, which can act on nearby breast cancer cells and modulate tumor phenotype, will be explored. Finally, activation of the nuclear factor κB (NF-κB) pathway and its cross talk with ER in the regulation of key genes in the promotion of more aggressive breast cancers will be reviewed. From these multiple lines of evidence, we propose that inflammation may promote more aggressive ER-positive tumors and that combination therapy targeting both inflammation and estrogen production or actions could benefit a significant portion of women whose ER-positive breast tumors fail to respond to endocrine therapy.