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mediates gene expression changes and growth response in ovarian cancer cells exposed to estrogen
Cancer Research UK Centre, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, UK
(Requests for offprints should be addressed to S Langdon; Email: simon.langdon{at}cancer.org.uk)
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Abstract |
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(ER). Consistent with this, these gene expression changes were blocked by the anti-estrogen tamoxifen. The use of ER- and ERß-specific ligands allowed molecular dissection of the E2 response and showed that ER activation was responsible for the observed changes in gene expression, whereas ERß played no significant role. Inhibition of de novo protein synthesis by cycloheximide was used to distinguish between primary and secondary target genes regulated by E2. Actinomycin D was used to show that changes in gene expression levels induced by E2 were a result of changes in transcription and not due to changes in mRNA stability. The results presented here demonstrate that estrogen-driven growth of epithelial ovarian carcinoma is mediated by activation of ER-mediated, and not ERß-mediated, transcription.
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Introduction |
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Estrogens are major regulators of growth and differentiation in normal ovaries and in the development and progression of ovarian carcinoma, but the mechanisms of action remain unclear. The biological effects of estrogens are mediated by two forms of estrogen receptor (ER) that are encoded by separate genes, ER and ERß (Greene et al. 1985, Green et al. 1986, Ponglikitmongkol et al. 1988, Mosselman et al. 1996, Ogawa et al. 1998). The ER, a member of the nuclear receptor super-family, is an estrogen ligand-activated transcription factor that binds to an estrogen-responsive element (ERE) in the promoter region of target genes, regulating their transcription (Evans 1988, Green & Chambon 1988). There has been much speculation about the relative roles of ER and ERß in this disease, as ERß levels are high in the normal ovary and predominate over ER, while the converse is true in ovarian cancers (Brandenberger et al. 1997, Enmark et al. 1997, Kuiper et al. 1997, Pujol et al. 1998, Rutherford et al. 2000, Bardin et al. 2004, Lindgren et al. 2004). A recent study has suggested that ERß loss is an important event in the development of ovarian cancer (Bardin et al. 2004). In this report, we focus on the role of ER, the isoform that is retained in ovarian cancers.
The significance of estrogen in the etiology of ovarian carcinoma has been emphasized by the fact that anti-estrogenic intervention will inhibit the growth of ovarian carcinoma in vitro and in vivo (Langdon et al. 1990, 1994a), and recently it has been reported that estrogen replacement therapy induces ovarian cancer (Lacey et al. 2002). Clinical trials with the aromatase inhibitor letrozole, which acts by depleting levels of estrogen available to the ER, have shown clinical benefit in a sub-group of ovarian cancer patients (Bowman et al. 2002, Papadimitriou et al. 2004) as have trials using tamoxifen (Hatch et al. 1991, Ahlgren et al. 1993). These studies further support the view that certain ER-positive ovarian cancers show growth dependency on estrogen. As approximately two-thirds of all ovarian tumors express ER at the time of diagnosis (Slotman & Rao 1988), identifying the downstream targets of estrogen will reveal putative functional pathways regulated by activated ER, and provide a greater understanding of the roles estrogens play in ovarian cancer. Estrogen regulation of protein expression has been well documented in breast cancer models but to date little is known about estrogen-regulated gene expression in ovarian cancer. Genes which have been shown to be regulated, thus far, include c-myc (Chien et al. 1994, Hua et al. 1995), progesterone receptor (Nash et al. 1989, Langdon et al. 1994b), cathepsin D (CTSD) (Galtier-Dereure et al. 1992, Rowlands et al. 1993), fibulin-1 (Clinton et al. 1996, Moll et al. 2002) and insulin-like growth factor binding proteins (Krywicki et al. 1993).
Here we have used the BD Atlas Human Cancer Expression Array, containing 1200 gene probes, to monitor changes in gene expression in response to the naturally occurring estrogen 17-beta estradiol (E2), in an ER-positive, estrogen-responsive ovarian cancer cell line. The results reveal that E2 directly regulates the transcription of a plethora of genes involved in many aspects of cellular function such as DNA repair, extracellular matrix, apoptosis and signal transduc-tion. Using a range of different cell lines and ER- and ERß-specific ligands, we have been able to show that changes in gene expression are related to ER status and hormone responsiveness.
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Materials and methods |
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The ovarian cancer cell lines, PEO1, PEO1CDDP, PEO4, PEO6, PEO14 and PEO16 were established within this unit (Langdon et al. 1988); SKOV-3, CaOV3 and MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA); OVCAR-3, OVCAR-4 and OVCAR-5 were obtained from Dr T C Hamilton (Fox Chase Institute, Philadelphia, PA, USA); 41M, 59M, OAW42, A2780, ZR-75-1 and MDA-MB-231 cells were obtained from the European Collection of Cell Cultures (ECACC; Porton Down, Wilts, UK). All cell lines were routinely cultured at 37°C, 90% humidity and 5% CO2 in RPMI 1640 (Life Technologies, Paisley, Strathclyde, UK) containing 10% heat-inactivated FCS, 100 µg/ml streptomycin and 100 IU/ml penicillin. Where speci-fied, cells were treated with 25 µg/ml cycloheximide (Sigma, UK) to block protein synthesis, 5 µg/ml actinomycin D (Sigma, UK) to block transcription or 0.3 µM tricostatin A (Calbiochem, La Jolla, CA, USA) to inhibit histone deacetylase activity. An ER agonist (propyl pyrazole triol (PPT)) and an ERß-specific agonist (diarylpropionitrile (DPN)) were obtained from Tocris, Bristol, Avonmouth, UK and were used at the concentrations shown.
Western blotting
Cell lines were grown to 70% confluence in 92-cm tissue culture dishes in the presence of RPMI 1640 containing 10% FCS. The medium was removed and replaced with RPMI 1640 without phenol red but containing 5% double charcoal-stripped FCS (DCS-FCS). After a further 48–72 h, estradiol (1 nM) was added to cells. To harvest, cells were washed twice in ice-cold PBS, and lyzed in 50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5 mM EDTA, 2 mM Na3VO4, 10 mM NaOMb, 150 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mM dithiothreitol, and spun for 20 min at 16 000 g at 4°C. The protein content of the resulting supernatant was determined by the Bradford assay (Bio-Rad Laboratories, UK). Total protein (5–30 µg) was separated on 10% SDS-acrylamide gel and transferred onto a nitrocellulose membrane. Membranes were blocked in Tris-buffered saline (pH 7.4) containing 0.1% Tween (TBST) and 10% milk. Blots were incubated in antibodies for 1 h at room temperature. The following antibodies were used: anti-ER (1 : 200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-ERß (1 : 500; Upstate Biotechnology, Lake Placid, NY, USA), and anti-fibronectin, (1 : 200; Santa Cruz Biotechnology). Immunoreactive bands were detected by enhanced chemiluminescence (Santa Cruz Biotechnology).
Growth assays
Exponentially growing cells were harvested by trypsinization and plated in 24-well plates at a density of 5 x 104 cells/well in RPMI 1640 containing 10% FCS. After 24 h to allow for attachment, the medium was removed and replaced with RPMI 1640 without phenol red and 5% DCS-FCS; E2 and tamoxifen were added to cells, and this was designated day 0. Cells were harvested from wells on day 5 and counted on a cell counter (Coulter Electronics Ltd, Luton, UK). Changes in growth were calculated relative to controls incubated in DCS-FCS-containing media.
Oligonucleotide array
Differential gene expression was performed using the Atlas Cancer 1.2 K cDNA Expression Array (BD, Palo Alto, CA, USA), according to the manufacturer’s protocol.
cDNA synthesis
Total cellular RNA was extracted from cells in log phase growth using TRI reagent (Sigma, UK). RNA from each 75-cm2 flask was treated with 20 units DNase 1 (Roche, East Sussex, UK), in 50 µl, to remove genomic DNA contamination. Forty nanograms RNA were used per RT-PCR using a one-step RT-PCR kit (Qiagen) with product-specific primers. To detect low abundance products, RNA was first reverse transcribed with a first-strand cDNA synthesis kit (Roche) using the random primers provided. One microgram RNA yielded 10 µl cDNA, of which 1 µl was used for each subsequent PCR with each primer pair.
Real-time quantitative PCR
Primers were chosen with the assistance of the computer application Primer 3.0 http://focker.wi.mit.edu/primer3/. We performed BLASTN searches against dbEST and non redundant sequence databases of GenBank to confirm the total gene specificity of the nucleotide sequences chosen as primers. Single product amplification was checked for each primer pair by analysis of product melt curves (ROTORGENE software Cambridge, UK).
All the PCRs were performed using a Corbett Research Rotorgene 2000 instrument. The thermal cycling conditions comprised an initial Taq heat-activation step at 95°C for 10 min and 45 cycles of 95°C for 15 s, 55°C for 30 s and 72°C for 45s. Annealing temperature was dropped to 50°C for amplification of ChIP DNA. Specific PCR ampli-fication products were detected by the fluorescent double-stranded DNA binding dye SYBR Green. Primer sequences used are available upon request. Experiments were performed at least three times with at least triplicates for each data point.
ERE searching
Human and mouse promoter sequences of E2-regulated genes were obtained from EntrezGene (NCBI; www.ncbi.nlm.nih.gov), Promoser (Boston University) and UCSC genome browser; http://genome.ucsc.edu. A region 4 kb upstream and 1 kb downstream of the transcriptional start site (TSS) was selected and scanned for potential ERE using MatInspector (Genomatix; www.genomatix.de). The level of conservation of a 19 bp sequence, containing the predicted ERE, was measured by using ClustalW (EBI; www.ebi.ac.uk) to align human and mouse orthologous promoters.
Luciferase reporter assay
ER transcriptional activity in PEO1 cells was measured using the Dual Luciferase Reporter Assay (Promega, UK) according to the manufacturer’s protocol. The cells were co-transfected with luciferase reporter construct driven by either the consensus ERE from Xenopus vitellogenin A2 gene (GGTCA nnn TGACC) (Vit-ERE-tk-luc) (Klein-Hitpass et al. 1986) or the region –1908 bp to +136 bp, relative to the transcriptional start site, from the rat fibronectin promoter (PGL2F1900) (Lee et al. 2000), and pRL-tk (renilla luciferase) to correct for transfection efficiency. Cells, starved of estrogen for 48 h in phenol red-free medium with 5% charcoal-stripped serum, were transfected using lipofectin reagent (Invitrogen, UK) according to the manufacturer’s recommendations. Cells were treated with 1 nM 17ß-estradiol 24 h after transfection and luciferase activity was measured in lyzed cells 24 h later using a microplate luminometer (Berthold, Bad Wildbad, Germany).
Chromatin immunoprecipitation
Cells were grown to 95% confluence on 10-cm dishes in phenol red-free RPMI 1640 supplemented with 5% double charcoal-stripped FCS for at least 48 h. Following the addition of 17ß-estradiol for various times, cells were washed twice with PBS and cross-linked with 1% formaldehyde at room temperature for 10 min followed by 5 min incubation with 0.125 M glycine. Cells were then rinsed with ice-cold PBS and collected into PBS. Cells were washed sequentially with 1 ml ice-cold PBS, buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5) and buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5), then resuspended in 0.3 ml lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1), 1 x protease inhibitor cocktail (Roche) and sonicated three times for 10 s to give fragments in the range 0.8 to 2.5 kb. For each immuno precipitate, 0.1 ml lysate was diluted in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1) followed by immunoclearing with 2 µg sonicated salmon sperm DNA and 20 µl protein A-sepharose for 1 h at 4°C. Immunoprecipitation was performed overnight at 4°C with specific antibodies to either ER (Santa Cruz HC20) or acetylated histone H4 (Upstate #06-866). After immunoprecipitation, 20 µl protein A-Sepharose and 2 µg salmon sperm DNA were added and the incubation was continued for another hour. Precipitates were washed sequentially for 15 min each in TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Precipitates were then washed three times with TE buffer (50 mM Tris-HCl, pH 8.1; 1 mM EDTA) and extracted twice with 1% SDS, 0.1 M NaHCO3. Eluates were pooled and heated at 65°C for at least 6 h to reverse the formaldehyde cross-linking. DNA fragments were purified with a QIAquick Spin Kit (Qiagen, UK). For real-time PCR, 5 µl from 50 µl elution were used. IP was normalized to input DNA.
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Results |
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To enable us to relate E2-directed transcription to ER status and hormone responsiveness in ovarian cancer, we first assessed the expression of ER and ERß in 16 ovarian cancer cell lines. Western blotting with antibodies directed against either ER or ERß revealed that ER protein is expressed in PEO1, PEO1CDDP, PEO4, PEO6 and SKOV3 cells at levels similar to that observed in the ER-positive breast cell lines ZR-75-1 and MCF-7 (Fig. 1
). ERß was expressed to varying degrees in all cell lines tested.
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Figure 2 shows E2-induced changes in cell number relative to a control sample not exposed to E2. Both ER-positive PEO1 and PEO4 cell lines were growth stimulated by E2 implying the presence of functional ER. SKOV-3 cells, despite expressing ER, did not show an increase in growth, relative to control, after treatment with E2. PEO14 cells, negative for ER, also displayed a lack of growth-responsiveness to E2. A further 9 ER-negative, ERß-positive ovarian cell lines (PEO16, 41M, 59M, OVCAR-3, OVCAR-4, OVCAR-5, A2780, CAOV3 and OAW42) were tested for growth sensitivity to E2 but did not respond despite expressing ERß. These data suggest that expression of ER is necessary for E2-induced growth and ERß alone does not play a significant role in the growth response.
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(PPT) (Stauffer et al. 2000) or ERß (DPN) (Meyers et al. 2001) (Fig. 2B
). While PPT stimulated proliferation in a similar manner to E2, DPN failed to induce a change in proliferation until concentrations reached 1 µM. At this high concentration, DPN has been shown to cross-activate ER (Meyers et al. 2001). These results again indicate that growth response to estrogen in these cell lines is mediated by ER and not ERß. 17ß-Estradiol-mediated changes in gene expression
To identify genes regulated by E2 in the PEO1 cell line, RNAs from untreated and E2-treated cells were compared. RNAs from untreated cells and cells exposed to 10–10 M E2 for 24 h were isolated, labeled and hybridized to BD Atlas Cancer 1.2 K cDNA microarrays and analyzed by phosphorimaging (Fig. 3A). BD Atlas-Image software was used to generate a color schematic normalized comparison of the two arrays to demonstrate the extent of gene expression changes in PEO1 cells upon E2 stimulation (Fig. 3B). Figure 3C shows semi-quantitative similarities and differences between gene expression profiles of the two RNA populations. Each point in the scatterplot represents the expression values of a gene in two separate treatments, one value on the X-axis (untreated control) and the other one on the Y-axis (E2-treated). The expression levels of the majority of genes were unaffected (lined up on the identity line); however, 5 genes were up-regulated and 23 genes were down-regulated by more than threefold in E2-treated cells compared with untreated control cells (Table 1). Interestingly, genes up-regulated by E2, such as FOSL1, CTSD and TFAP4, are known to play a role in promoting cell proliferation. Of particular note, however, was the number of down-regulated genes, especially those involved in maintenance of the cytoskeleton, suggesting a role for E2 signaling in invasion and metastasis.
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17ß-Estradiol-mediated changes correlate with ER expression and growth responsiveness
The expression of a randomly selected subset of genes, identified as E2 regulated in the PEO1 cell line, was assessed in the growth-responsive cell line PEO4 and the non-growth-responsive SKOV3 and PEO14 cell lines. Figure 4 shows that E2 also induced a change in expression of these genes in PEO4 cells, whereas PEO14 and SKOV3 cells were non-responsive. This implies that the expression of functional ER is necessary to mediate E2 regulation of these genes.
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shows that OHT effectively blocked the ability of E2 to mediate gene expression changes in the PEO1 and PEO4 cell lines.
Assaying gene expression levels in PEO1 and PEO4 cells treated with ER and ERß agonists demonstrated that activation of ER alone is sufficient to cause the effects observed with E2 treatment for all of the 8 genes tested (Fig. 5). DPN was unable to induce gene expression changes even at concentrations up to 1 µ M (data not shown). This suggests that E2-mediated gene expression changes are mediated by activation of ER and not the consequence of activation of ERß.
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To establish whether changes in transcript levels of E2-modulated genes were due to a change in transcription rate or a change in mRNA stability, the rate of decay of each transcript, in actinomycin D-treated (to block further RNA synthesis) PEO1 cells pre-incubated with 1 nM E2 for 48 h, was measured and compared with an untreated control. RT-PCR was then used to measure the levels of target mRNA at various time points after actinomycin D treatment. Figure 6A demonstrates that up-regulation of expression of FOSL1 and TRAP1 genes was not a result of E2-mediated stabilization of mRNA. Interestingly, down-regulated mRNA transcripts IGFBP3, BENE, FN1, PLAU and TGFBI were stabilized in the presence of E2 (estradiol-reduced expression levels of LCN2 were too low to accurately measure decay rate). These results are consistent with the observed changes in mRNA expression being due to E2-regulated transcription rate changes and not due to effects on mRNA stability.
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It is likely that some of these genes may be regulated because of secondary effects induced by the primary targets of ER. To identify primary targets, we analyzed the effect of E2 in the absence and presence of cycloheximide (CHX). The inhibition of protein synthesis by CHX prevents most of the secondary gene regulation that is not transactivated directly by ER. ER protein levels remained relatively stable in the presence of CHX throughout the experiment. PEO1 cells were pretreated with CHX for 10 min prior to E2 stimulation for a further 24 h. Six out of the subset of eight gene products tested were shown to be direct targets of E2: E2 induced a change in mRNA expression level in the presence of CHX (Fig. 6B). Preventing de novo protein synthesis greatly reduced the effects of E2 on the expression of PLAU and TRAP1 suggesting that these genes are, at least partly, regulated by transcriptional targets of ER.
Estrogen response elements in regulated genes
The promoters of some genes in this study have previously been shown to have estrogen-responsive elements, which bind estrogen receptor (e.g. CTSD). To investigate whether the novel estrogen-regulated genes identified in this study contain potential binding sites for ER, we performed a computer-assisted analysis of the 5°-flanking sequence of each gene. Using MatInspector professional software (www.genomatrix.gsf.de), we examined whether ER consensus sites were contained within the promoter region or the first intron of the 28 estradiol-responsive genes. To improve specificity, we used threshold values of 1.0 for core similarity and 0.85 for matrix similarity. Altogether 27 of the 28 genes investigated demonstrated at least a half-palindromic sequence GGTCA within 4kb of the transcription start site (TSS) (Table 1). Based on the hypothesis that functionally important non-coding genomic sequences will be conserved during evolution, murine orthologous counterparts for each promoter were retrieved from the UCSC genome browser and sequences aligned using ClustalW. Comparative geno-mic analysis showed that 10 out of 25 promoters tested contained a conserved ERE sequence (sequence similarity score across 19 bp sequence > 0.6, or complete conservation of half-site ERE). The genes CASP4 and KRT4 do not have mouse orthologs.
E2-mediated down-regulation of FN1 in ovarian cancer cells involves recruitment of ER and histone deacetylase activity to the FN1 promoter
Of particular interest was the number of down-regulated genes identified as ER targets. FN1, although previously identified as being up-regulated in response to estradiol in cardiac fibroblasts (Mercier et al. 2002) and during mouse mammary gland development (Woodward et al. 2001), was one gene that was strikingly down-regulated in ER-positive ovarian cancer cells. Figure 7A shows the location of a highly conserved putative estrogen response element in human, mouse and rat genomes. In accordance with the mRNA changes observed in response to estradiol, protein levels of FN1 are also greatly reduced in the presence of E2. Interestingly, full serum, not double charcoal stripped, possesses enough hormonal activity to reduce expression of FN1 protein. Only stripped FCS allows full de-repression of FN1 expression. Induction of E2-responsive progesterone receptor protein is also shown together with expression of beta-actin as a loading control. To assess whether E2 causes transcriptional repression at the FN1 pro-moter, we transfected ovarian PEO1 cells with a luciferase reporter construct of the FN1 promoter region –1908 to + 136 and measured luciferase activity after treatment with E2 and compared the results with that of vit-ERE-luc, which is known to be induced by E2. Results show that, as expected, E2 induced expression of luciferase through the vit ERE in ovarian cancer cells. However, the activity of the FN1 promoter was greatly reduced by the addition of E2. These results are in agreement with the idea that E2 causes down-regulation of FN1 through an ERE in the promoter region. Down-regulation of the FN1 promoter is shown in Fig. 7D to be sensitive to the histone deacetylase inhibitor trichostatin A (TSA) as FN1 promoter driven luciferase activity and endogenous FN1 mRNA and protein levels are restored when TSA is added to E2-treated cells. This suggests that down-regulation of FN1 occurs through recruitment of histone deacetylase activity to the promoter region. To demonstrate that E2 recruits ER to the promoter of FN1, chromatin immuno-precipitation was used. Figure 7E shows that upon E2 stimulation, ER is recruited to the endogenous FN1 promoter with a concurrent decrease in the level of acetylated histone H4. Together, these results present a suggestion that E2 treatment causes recruitment of a transcriptional repression complex of ER and histone deacetylases to the FN1 promoter causing a reduction in FN1 mRNA and protein levels.
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mediates both the growth response and gene expression changes in ovarian cancer cells exposed to E2. Ovarian cancer cell lines expressing only ERß are growth unresponsive to E2. Expression levels of ERß are high in the normal ovary and, in a number of studies, this isoform predominates over ER not only in the normal ovary but also in benign ovarian tumors (Brandenberger et al. 1997, Enmark et al. 1997, Kuiper et al. 1997, Pujol et al. 1998). ERß appears protective, as adenoviral delivery of this gene into ovarian cancer cells has been shown to inhibit cell proliferation and motility and enhance apoptosis (Bardin et al. 2004). Progression to ovarian cancer leads to a change in the ratio of ER to ERß, with levels of ER being generally higher than ERß (Brandenberger et al. 1997, Enmark et al. 1997, Kuiper et al. 1997, Pujol et al. 1998, Rutherford et al. 2000, Bardin et al. 2004, Lindgren et al. 2004), further suggesting that either ERß confers a protective effect and/or ER promotes growth and invasion. This is further supported by a study comparing metastatic ovarian cancer with primary ovarian cancers wherein only ER was found in metastatic disease with ERß being no longer present (Rutherford et al. 2000). These data are all consistent with the idea that different ER isoforms are performing distinct roles in the ovary – ER function being associated with malignant growth and progression, whilst ERß plays a protective role.
We are not aware of any ER-negative ovarian cancer cell lines that respond to estrogen and these data are in line with previous reports that the presence of this receptor at moderate to high levels is essential for a growth function. A recent clinical trial, investigating the effectiveness of the aromatase inhibitor letrozole in ovarian cancer treatment, showed a clear association between response and higher levels of expression of ER (Bowman et al. 2002). However, in that trial, as in the current study with SKOV-3, it is apparent that not all ER-positive cells respond to E2. This may be due to disabling mutations of the ER, or the absence of critical cofactors (co-activators), or the presence of repressors, which will markedly influence the transcriptional (and growth) response (Shang et al. 2000). Alternatively, other growth regulatory pathways, such as the erbB2 receptor signaling pathways, may be up-regulated, essentially bypassing estrogen’s control (Dowsett 2001). This may explain the lack of E2 response in the SKOV-3 cell line, as it markedly overexpresses erbB2 (Hua et al. 1995).
Since the functionality of ER will be exhibited as changes in transcription of regulated genes, gene changes were sought by use of microarray technology after E2 exposure. A limited number of genes have already been identified as being estrogen regulated in ovarian cancer. These include genes linked to proliferation (c-myc (Chien et al. 1994, Hua et al. 1995) and insulin-like growth factor binding proteins (IGFBPs) (Krywicki et al. 1993)), differentiation (progesterone receptor (Nash et al. 1989, Langdon et al. 1994b)) and to invasion (CTSD (Galtier-Dereure et al. 1992, Rowlands et al. 1993) and fibulin-1 (Clinton et al. 1996, Moll et al. 2002)). Two of these genes, namely IGFPB3 and CTSD, were present on the microarray and shown to be modulated by E2. In the present study we identified regulators of the cell cycle (CCNB1), apoptosis (TNFSF7, TRAP1, UBL1 and CASP4), transcription (FOSL1, TFAP4, EIF2B1) and signaling (NOTCH4, IGFPB3, BENE, LCN2, GRSF1). Many of these modulated genes are also linked to either the cytoskeleton and extracellular matrix (CTSD, CDH6, CYR61, KRTs 4,7 and 13, VIM, TGFBI, DES, AKAP12, TRAM1, MMPs 11 and 17, PLAU) and hence could be involved in tumor spread and metastasis. Other than CTSD and IGFBP3, we are only aware that CCNB1 (Zoubine et al. 1999), CYR61 (Tsai et al. 2002), FN1 (Woodward et al. 2001), PLAU (Levenson et al. 1998) and LCN2 (Seth et al. 2002) have been reported as being modulated by E2. To confirm that these changes were mediated via ER, reversals by the ER competitor OHT were demonstrated. Similarly, these gene expression changes were not seen in the ER-negative PEO14 cell line. Use of the ER and ERß specific agonists indicated that the E2-modulated changes in expression could be reproduced by the ER agonist (PPT) while the ERß specific agonist (DPN) had no effect. As for the growth changes, these data support the importance of ER over ER in this disease. A recent report exploring tissue-specific responsiveness to estrogen concluded that ER mediated estrogen regulation even in tissues possessing high levels of ERß expression (Jelinsky et al. 2003) and our results in malignant tissue are in line with this.
Of the 8 genes studied as being either direct or indirect targets of E2, 6 demonstrated equivalent expression changes in the presence of CHX, indicating no requirement for new protein synthesis, thereby implicating a direct action of E2 on these target genes. For TRAP1 and PLAU, there was some inhibition of the expression changes in the presence of CHX suggesting that expression of these genes is at least partially regulated by downstream effectors involving new protein synthesis.
The presence of an estrogen receptor binding site was sought in the promoter regions of the target genes identified, and all but one of these genes possessed at least a half-palindromic sequence GGTCA in the 4 kb region prior to the transcriptional start site, with most within a 2 kb region. Previous reports have shown that such incomplete motifs might transduce ER signaling (Tora et al. 1988). Indeed, very few estrogen-regulated genes contain the consensus ERE palindromic sequence (GGTCAnnnTGACC) and there is abundant evidence that half-ERE sites mediate many of estrogen’s actions through co-operation with other binding sites e.g. Sp1 (reviewed in Klinge 2001, O’Lone et al. 2004). The use of actinomycin D suggested that the observed changes in mRNA expression were due to E2-regulated transcription rate changes and not due to effects on mRNA stability, as the up-regulated genes (FOSL1 and TRAP1) were not stabilized in the presence of both E2 and actinomycin D and the down-regulated genes showed higher rather than lower transcript levels in the presence of both E2 and actinomycin D.
Several genes, identified as being E2-regulated in ovarian cancer cells, have previously been shown to be E2 targets in other tissue types. However, the effects of E2 observed in previous studies opposed the effects observed in this study. For example, Cyr61, identified here as a down-regulated E2 target, has previously been shown to be induced by E2 in the mammary adenocarcinoma cell line MCF-7 (Sampath et al. 2001). PLAU, also down-regulated by E2 in this study, has been shown to be up-regulated by E2 during fracture healing (Hatano et al. 2004), and is also upregulated in response to E2 in several breast cell lines (Seth et al. 2002). FN1, which was down-regulated in our study yet up-regulated in other cell types (Woodward et al. 2001, Mercier et al. 2002), was investigated further. We have shown that in ovarian cancer cells the promoter region of the FN1 gene is responsive to E2, and that as well as recruiting ER to the promoter, a TSA-sensitive histone deacetylase activity is also recruited. Reduction in acetylation of histone H4 is known to be associated with transcriptional repression, providing a plausible mechanism for E2-mediated down-regulation of this gene.
In conclusion, these results strongly support a role for ER but not ERß in the growth regulation of ovarian cancer and are consistent with the observed enhanced ratio of ER over ERß observed in ovarian cancers. We have identified a number of novel estrogen-regulated genes and have shown that some previously identified targets of E2 respond differently to E2 in ovarian cancer cells. These targets will be further explored as potential mediators of estrogen’s action in this particular disease.
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Acknowledgements |
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References |
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