Anders Ström

Discovery of estrogen receptor α target genes and response elements in breast tumor cells

BackgroundEstrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes.ResultsOf around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs.ConclusionsOnly a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.

A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells

Transcriptional effects of estrogen result from its activation of two estrogen receptor (ER) isoforms; ERα that drives proliferation and ERβ that is antiproliferative. Expression of ERβ in xenograft tumors from the T47D breast cancer cell line reduces tumor growth and angiogenesis. If ERβ can halt tumor growth, its introduction into cancers may be a novel therapeutic approach to the treatment of estrogen-responsive cancers. To assess the complete impact of ERβ on transcription, we have made a full transcriptome analysis of ERα- and ERβ-mediated gene regulation in T47D cell line with Tet-Off regulated ERβ expression. Of the 35 000 genes and transcripts analysed, 4.1% (1434) were altered by ERα activation. Tet withdrawal and subsequent ERβ expression inhibited the ERα regulation of 998 genes and, in addition, altered expression of 152 non-ERα-regulated genes. ERα-induced and ERβ-repressed genes were involved in proliferation, steroid/xenobiotic metabolism and ion transport. The ERβ repressive effect was further confirmed by proliferation assays, where ERβ was shown to completely oppose the ERα–E2 induced proliferation. Additional analysis of ERβ with a mutated DNA-binding domain revealed that this mutant, at least for a quantity of genes, antagonizes ERα even more strongly than ERβ wt. From an examination of the genes regulated by ERα and ERβ, we suggest that introduction of ERβ may be an alternative therapeutic approach to the treatment of certain cancers.

Estrogen Receptor β Inhibits Angiogenesis and Growth of T47D Breast Cancer Xenografts

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERα and ERβ. ERα mediates the proliferative effect of estrogen in breast cancer cells, whereas ERβ seems to be antiproliferative. We engineered ERα-positive T47D breast cancer cells to express ERβ in a Tet-Off–regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERβ. The presence of ERβ resulted in a reduction in tumor growth. Comparison of the ERβ-expressing and non-ERβ–expressing tumors revealed that the expression of ERβ caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor β (PDGFβ). In cell culture, with the Tet-Off–regulated ERβ-expressing cells, expression of ERβ decreased expression of VEGF and PDGFβ mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFβ proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFβ promoter constructs revealed a repressive effect of ERβ at the promoter level of these genes. Taken together, these data show that introduction of ERβ into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis. (Cancer Res 2006; 66(23): 11207-13)

Tumor repressive functions of estrogen receptor beta in SW480 colon cancer cells.

Estrogen receptor beta (ERbeta) is the predominant ER in the colorectal epithelium. Compared with normal colon tissue, ERbeta expression is reduced in colorectal cancer. Our hypothesis is that ERbeta inhibits proliferation of colon cancer cells. Hence, the aim of this study has been to investigate the molecular function of ERbeta in colon cancer cells, focusing on cell cycle regulation. SW480 colon cancer cells have been lentivirus transduced with ERbeta expression construct with or without mutated DNA-binding domain or an empty control vector. Expression of ERbeta resulted in inhibition of proliferation and G(1) phase cell cycle arrest and this effect was dependent on a functional DNA-binding region. c-Myc is overexpressed in an overwhelming majority of colorectal tumors. By Western blot and real-time PCR, we found c-Myc to be down-regulated in the ERbeta-expressing cells. Furthermore, the c-Myc target gene p21((Waf1/Cip1)) was induced and Cdc25A was reduced by ERbeta at the transcriptional level. The second cdk2-inhibitor, p27(Kip1), was induced by ERbeta, but this regulation occurred at the posttranscriptional level, probably through ERbeta-mediated repression of the F-box protein p45(Skp2). Expression of the ERbeta-variant with mutated DNA binding domain resulted in completely different cell cycle gene regulation. We performed in vivo studies with SW480 cells +/- ERbeta transplanted into severe combined immunodeficient/beige mice; after three weeks of ERbeta-expression, a 70% reduction of tumor volume was seen. Our results show that ERbeta inhibits proliferation as well as colon cancer xenograft growth, probably as a consequence of ERbeta-mediated inhibition of cell-cycle pathways. Furthermore, this ERbeta-mediated cell cycle repression is dependent on functional ERE binding.

Dragon ERE Finder version 2: a tool for accurate detection and analysis of estrogen response elements in vertebrate genomes

We present a unique program for identification of estrogen response elements (EREs) in genomic DNA and related analyses. The detection algorithm was tested on several large datasets and makes one prediction in 13 300 nt while achieving a sensitivity of 83%. Users can further investigate selected regions around the identified ERE patterns for transcription factor binding sites based on the TRANSFAC database. It is also possible to search for candidate human genes with a match for the identified EREs and their flanking regions within EPD annotated promoters. Additionally, users can search among the extended promoter regions of approximately 11 000 human genes for those that have a high degree of similarity to the identified ERE patterns. Dragon ERE Finder version 2 is freely available for academic and non-profit users (http://sdmc.lit.org.sg/ERE-V2/index).

Inhibitory effects of estrogen receptor beta on specific hormone-responsive gene expression and association with disease outcome in primary breast cancer.

IntroductionThe impact of interactions between the two estrogen receptor (ER) subtypes, ERα and ERβ, on gene expression in breast cancer biology is not clear. The goal of this study was to examine transcriptomic alterations in cancer cells co-expressing both receptors and the association of gene expression signatures with disease outcome.MethodsTranscriptional effects of ERβ overexpression were determined in a stably transfected cell line derived from ERα-positive T-47D cells. Microarray analysis was carried out to identify differential gene expression in the cell line, and expression of key genes was validated by quantitative polymerase chain reaction. Microarray and clinical data from patient samples were then assessed to determine the in vivo relevance of the expression profiles observed in the cell line.ResultsA subset of 14 DNA replication and cell cycle-related genes was found to be specifically downregulated by ERβ. Expression profiles of four genes, CDC2, CDC6, CKS2, and DNA2L, were significantly inversely correlated with ERβ transcript levels in patient samples, consistent with in vitro observations. Kaplan-Meier analysis revealed better disease outcome for the patient group with an expression signature linked to higher ERβ expression as compared to the lower ERβ-expressing group for both disease-free survival (p = 0.00165) and disease-specific survival (p = 0.0268). These findings were further validated in an independent cohort.ConclusionOur findings revealed a transcriptionally regulated mechanism for the previously described growth inhibitory effects of ERβ in ERα-positive breast tumor cells and provide evidence for a functional and beneficial impact of ERβ in primary breast tumors.

Estrogen receptor beta in breast cancer—Diagnostic and therapeutic implications

More than 10 years have passed since the discovery of the second estrogen receptor, estrogen receptor beta (ERbeta). It is now evident that ERalpha is not the only ER in breast cancer cells; in fact, ERbeta is expressed in the majority of breast cancers although at lower levels than in the normal breast. In addition, ERbeta is expressed in breast cancer infiltrating lymphocytes, fibroblasts and endothelial cells, all known to influence tumor growth. By overexpressing or knocking-out ERbeta in breast cancer cell lines, several researchers have investigated its function with respect to proliferation and tumor growth. It appears that ERbeta is anti-proliferative, in many ways antagonising the function of ERalpha. Furthermore, phytoestrogens have a binding-preference for ERbeta and several epidemiological studies indicate a breast cancer preventing effect of this class of compounds. Tamoxifen is one of the standard, adjuvant treatments for ERalpha positive breast cancer, classically thought to mediate its effect through ERalpha. However, in several recent studies, ERbeta has been described as a potential marker for tamoxifen response. In summary, experimental, epidemiological as well as diagnostic studies point towards ERbeta as an important factor in breast cancer, opening up the possibility for novel ERbeta-selective therapies in the treatment of breast cancer.

Influence of Cellular ERα/ERβ Ratio on the ERα-Agonist Induced Proliferation of Human T47D Breast Cancer Cells

Breast cancer cells show overexpression of estrogen receptor (ER) α relative to ERβ compared to normal breast tissues. This observation has lead to the hypothesis that ERβ may modulate the proliferative effect of ERα. This study investigated how variable cellular expression ratios of the ERα and ERβ modulate the effects on cell proliferation induced by ERα or ERβ agonists, respectively. Using human osteosarcoma (U2OS) ERα or ERβ reporter cells, propyl-pyrazole-triol (PPT) was shown to be a selective ERα and diarylpropionitrile (DPN) a preferential ERβ modulator. The effects of these selective estrogen receptor modulators (SERMs) and of the model compound E2 on the proliferation of T47D human breast cancer cells with tetracycline-dependent expression of ERβ (T47D-ERβ) were characterized. E2-induced cell proliferation of cells in which ERβ expression was inhibited was similar to that of the T47D wild-type cells, whereas this E2-induced cell proliferation was no longer observed when ERβ expression in the T47D-ERβ cells was increased. In the T47D-ERβ cell line, DPN also appeared to be able to suppress cell proliferation when levels of ERβ expression were high. In the T47D-ERβ cell line, PPT was unable to suppress cell proliferation at all ratios of ERα/ERβ expression, reflecting its ability to activate only ERα and not ERβ. It is concluded that effects of estrogen-like compounds on cell proliferation are dependent on the actual ERα/ERβ expression levels in these cells or tissues and the potential of the estrogen agonists to activate ERα and/or ERβ.

Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERα/ERβ ratio

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.

Estrogen Receptors β1 and β2 Have Opposing Roles in Regulating Proliferation and Bone Metastasis Genes in the Prostate Cancer Cell Line PC3

The estrogen receptor (ER)β1 is successively lost during cancer progression, whereas its splice variant, ERβ2, is expressed in advanced prostate cancer. The latter form of cancer often metastasizes to bone, and we wanted to investigate whether the loss of ERβ1 and/or the expression of ERβ2 affect such signaling pathways in prostate cancer. Using PC3 and 22Rv1 prostate cancer cell lines that stably express ERβ1 or ERβ2, we found that the ERβ variants differentially regulate genes known to affect tumor behavior. We found that ERβ1 repressed the expression of the bone metastasis regulator Runx2 in PC3 cells. By contrast, RUNX2 expression was up-regulated at the mRNA level by ERβ2 in PC3 cells, whereas Slug was up-regulated by ERβ2 in both PC3 and 22Rv1 cells. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate carcinoma, was increased by ERβ2. In agreement with the increased Twist1 expression, we found increased expression of Dickkopf homolog 1; Dickkopf homolog 1 is a factor that has been shown to increase the RANK ligand/osteoprotegerin ratio and enhance osteoclastogenesis, indicating that the expression of ERβ2 can cause osteolytic cancer. Furthermore, we found that only ERβ1 inhibited proliferation, whereas ERβ2 increased proliferation. The expression of the proliferation markers Cyclin E, c-Myc, and p45(Skp2) was differentially affected by ERβ1 and ERβ2 expression. In addition, nuclear β-catenin protein and its mRNA levels were reduced by ERβ1 expression. In conclusion, we found that ERβ1 inhibited proliferation and factors known to be involved in bone metastasis, whereas ERβ2 increased proliferation and up-regulated factors involved in bone metastasis. Thus, in prostate cancer cells, ERβ2 has oncogenic abilities that are in strong contrast to the tumor-suppressing effects of ERβ1.