Background
Breast cancer is the most frequent cause of mortality from cancer in women. Therapy of ERα-positive tumors using anti-estrogens, like Tamoxifen and aromatase inhibitors achieves an overall survival of about 82% after eight years [
1]. Triple-negative breast cancers (TNBCs) that do not express ERα and progesterone receptors and do not overexpress Her-2neu gene product are not susceptible to endocrine therapy. Mortality of patients with TNBC is double as high as for carriers of ERα-positive tumors [
1]. For this reason, there is an urgent need for development of innovative, targeted therapies for this group of patients.
In the last years a number of new therapeutic approaches were tested with limited success. Treatment with platinum compounds resulting in a response rate of 30% could be increased to 49% by the combination with Cetuximab, an antibody to the epidermal growth factor receptor [
2]. The DNA-repair enzyme, poly-ADP-ribose polymerase (PARP), was also found to be a promising target in TNBC [
3,
4].
For many years, it was assumed that an estrogen receptor resides at the plasma membrane. G-protein coupled receptor 30 (GPR30) was identified to be responsible for most rapid signaling events of 17β-estradiol [
5,
6]. Before identification of GPR30 as third kind of estrogen receptors, other authors supposed that rapid estrogen signaling is initiated by a divergent membrane bound ERα [
7]. GPR30 expression is prevalent in TNBC and associated with a higher recurrence rate [
8].
In early experiments, almost before function of GPR30 was described, a rapid increase of cAMP was observed after stimulation of MCF-7 breast cancer cells with 17β-estradiol [
9]. Only some years later it was discovered, that binding of 17β-estradiol to GPR30 increases adenylate cyclase activity and MAP-kinase Erk 1 [
6,
9,
10]. Increased cAMP leads to phosphorylation of CREB that subsequently binds to cAMP-response elements (CRE) on promoters of mitogenic genes [
11]. Activation of MAP-kinase finally leads to enhanced proliferation of breast cancer cells. The signaling via βγ-subunits in TNBC has been briefly described [
12]. In addition to 17β-estradiol, selective estrogen receptor modulator Tamoxifen and complete ERα antagonist Fulvestrant bind to GPR30 and activate certain signaling pathways in breast cancer cells, thus leading to stimulation of proliferation [
5]. According to these observations GPR30 has been proposed to be an excellent new therapeutic target for the treatment of TNBC [
13].
Recently, we reported that in TNBC cell lines an increase of proliferation by17β-estradiol was dependent on GPR30, as it was completely prevented by knock-down of GPR30 expression using specific siRNA [
12].
Dennis et al. developed G15, a specific inhibitor of GPR30 signaling [
14]. In addition, estriol has been shown to be a potent inhibitor of GPR30 [
15]. In this report we have analyzed the efficacy of estriol as inhibitors of GPR30 on growth inhibition of TNBC cells. Similar attempts using G15 in TNBC were less successful than with estriol and are additionally presented as supplementary material.
Methods
Cell lines, chemicals and cell culture
TNBC cell lines HCC1806, HCC70 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC, Manassas, Virginia, USA). In order to guarantee the identity of the cell lines over the years, cells were expanded after purchase and aliquots were stored in liquid nitrogen. Every half year a new frozen stock was opened and expanded to carry out the experiments. Cells were cultured in MEM (Biochrom, Berlin, Germany) supplemented with 2 mM glutamine, 6 ng/ml insulin, 10 ng/ml transferrin, penicillin (50 U/ml), streptomycin (50 μg/ml) from Gibco (Paisley, UK), and 5% fetal bovine serum (Biochrom, Berlin).
17β-estradiol (E2), estriol, insulin, and transferrin were from Sigma-Aldrich (Deisendorf, Germany). G15 was purchased from R & D systems (Wiesbaden, Germany). Primers for PCR and biotin-labeled oligonucleotide probes for electrophoretic mobility shift assay were produced by MWG-eurofins (Ebersberg, Germany).
Proliferation assays
Proliferation assays for 17β-estradiol in the absence and in the presence of estriol were performed in phenol red-free medium supplemented with charcoal depleted serum (CD-FCS) as previously described [
16]. CD-FCS was prepared according to the procedure described by Stanley et al. [
17].
Proliferation assays were performed at least three times in quadruplicates with different passages. Means and standard deviations of the optical density (OD) of the replicates were calculated.
Treatment of cells
For stimulation of TNBC cells to analyze signal transduction of GPR30, 4×106 cells were plated in culture medium into 25 cm2-culture flasks. After attachment, cells were serum starved for 24 hours to synchronize the 17β-estradiol-starved cells in G0-phase. Serum starved cells were treated for 30 minutes either with 10-4 M estriol or solvent (0.1% ethanol) and subsequently stimulated with 10-8M 17β-estradiol in 0.1% ethanol for 10 min or 20 minutes. Cells were harvested and cell pellets lysed in 100 μl Cell lytic M (Sigma, Deisendorf, Germany), supplemented with protease-inhibitor (Sigma, Deisendorf, Germany) and phosphatase-inhibitor (Sigma, Deisendorf, Germany).
Western blots
Lysates of cells were cleared at 15000 g for 5 minutes and the protein concentration in the supernatant was determined using the method of Bradford. 50 μg of each sample were separated in a 7.5% polyacrylamid gel, blotted on PVDF-membrane and sequentially detected with rabbit-anti-human primary antibodies. GPR30 expression was detected with anti-GPR30 (sc-48524) from Santa Cruz (Dallas, TX), anti-phospho-Src (2113), anti-Src (2109), anti-S133phospho-CREB (9198), were all purchased from Cell Signaling and anti-CREB (04-767) from Millipore. Antibody to phospho Tyr1173EGF-receptor (324864) was from Calbiochem (Darmstadt, Germany), anti-EGF-receptor antibody (2235) from Epitomics (Hamburg, Germany) and anti-actin from Sigma Chemicals (Deisendorf, Germany). All primary antibodies were used diluted 1:2000 in TBST.
After washing in TBST blots were incubated with a 1:20.000 dilution of horseradish peroxidase conjugated goat-anti-rabbit antibody (ECL, GE-Healthcare, Freiburg, Germany). After washing, blots were incubated with the chemoluminescence reagent Femto (Thermo-Scientific, Rockford, IL) and scanned on a Licor C-digit chemoluminescence detector (Licor, Lincoln, NE). Densitometric evaluations of the protein bands were performed using the analysis tool of the Image Studio Digits Vers.4 delivered with the Licor C-Digit chemoluminescence detector (Licor, Lincoln, NE) and were normalized to actin.
Analysis of GPR30 signaling
GPR30 signal transduction and gene expression of c-fos, cyclin D1 and aromatase were analyzed as previously described [
12].
In detail, from TNBC cells, pretreated with estriol or not and subsequently stimulated for 30 minutes with 10-8M 17β-estradiol, RNA was purified using the RNeasy-kit (Qiagen, Hilden, Germany).
200 ng of each RNA were transcribed using 400 u Superscript reverse transcriptase (Invitrogen, Karlsruhe, Germany) in the presence of 0.5 μM oligo-dT primer for 60 min at 37°C. 5 μl of the resulting cDNA were amplified with 1 u Taq polymerase (Peqlab, Erlangen, Germany) in the presence of 200 μM dNTPs and 200 nM of the appropriate primers described in [
12].
Optimal PCR conditions for each gene were ascertained, guaranteeing that generation of the PCR-products was in the exponential phase. Therefore cDNA of c-fos was amplified by 32 cycles, cyclin D1 by 35 cycles, and aromatase by 28 cycles. As reference the RNA of the ribosomal protein L7 was amplified by 20 cycles.
PCR-products were separated in a 2% agarose gel (Type IV, special high EEO, Sigma Chemicals, Steinheim, Germany) in 0.5x TBE buffer at 100 V for 30 min. Gels were stained in ethidium bromide (2 μg/ml) for 30 min and photographed on a transiluminator using a CDS camera (TD20, Kodak, Rochester).
Electrophoretic mobility shift assay
The effects of estriol on the downstream signaling of GPR30 were analyzed using an electrophoretic mobility shift assay (EMSA) as previously described [
18] using oligonucleotides representing the promoter sequence from -14 to +11containing a CRE (bold) labeled at the 3’end by Biotin.
Cyclin D1-sense: AACAACAGTAACGTCACACGGACTA.
Cyclin D1-antisense: TTGTTGTCATTGCAGTGTGCCTGA.
Statistical analysis
The data were tested for significant differences by one-way analysis of variance followed by Student–Newman–Keuls’ test for comparison of individual groups, after a Bartlett test had shown that variances were homogenous.
Discussion
TNBC is characterized by the lack of expression of estrogen receptor α (ERα) and progesterone receptors and no overexpression of Her-2. This makes these tumors refractory for standard antiestrogen therapy with Tamoxifen and for antibody therapy using Trastuzumab. GPR30, a membrane-bound receptor for estrogens responsible for fast non-genomic effects of 17β-estradiol, might be a promising target in TNBC. Immunohistochemical staining of sections of a small cohort of TNBCs for GPR30 revealed that most of these tumors strongly expressed GPR30. GPR30 expression correlated with a higher recurrence rate of TNBC [
8]. Experimentally, it has been shown that 17β-estradiol also induces proliferation in immortalized breast epithelial cell line MCF10A in a GPR30-dependent manner as well as does the GPR30-specific agonist G1 [
23]. Only recently, we have used GPR30 specific siRNA to knock-down GPR30 expression in TNBC making the treated cells refractory to growth stimulation by 17β-estradiol [
12]. There are meanwhile a number of clinical investigations discussing an involvement of GPR30 in malignant transformation of breast cancer cells [
24‐
26]. In addition, GPR30 was found to play a growth promotion role in ovarian cancer [
27]. All these observations strongly support our assumption that stimulation of GPR30 by circulating 17β-estradiol contributes to the malignant behavior of TNBC. GPR30 was also expressed in the TNBC cell lines used in the present study, except MDA-MB-231 that were used as negative control (Figure
2B and D). Stimulation of TNBC cell lines with the ligands of GPR30, 17β-estradiol and 4-Hydroxy-tamoxifen leads to activation of src-kinase and EGF-receptor phosphorylation. Recently we reported that knock-down of GPR30 expression using siRNA completely prevented activation of these kinases and abrogated the stimulation of cell proliferation by 17β-estradiol in TNBC cell lines [
12]. Therefore, a search for potent pharmacological inhibitor of GPR30 might prove valuable for the treatment of TNBC.
G15, a substance from a compound library, was reported to be selective for GPR30 and inhibited signal transduction of GPR30 [
14]. The naturally occurring estrogen metabolite estriol has also been shown to be a GPR30 antagonist in estrogen receptor-negative breast cancer cell line SKBr3 [
15]. SKBr3 is not a representative of TNBC cell lines, because this cell line overexpresses Her-2. But as we show in the present report, estriol is a potent GPR30 antagonist in a number of TNBC cell lines. Treatment of TNBC cell lines with estriol led to a significant reduction in cell number to 16% of control at 8×10
-5 M estriol, the highest concentration achievable in aqueous solution. The inhibitory effect of estriol on cell proliferation correlated with the amount of GPR30 expressed in the various TNBC cell lines.
Phosphorylation of src-kinase and transactivation of EGF-receptor by 17β-estradiol were almost as successfully prevented as was observed after knock-down of GPR30 using siRNA [
12]. In addition to the inhibition of EGF-receptor activation, estriol also proved to be effective against the Gα-dependent signaling of GPR30. Phosphorylation of the cAMP-responsive element binding protein (CREB) by PKA was already prominent in the absence of17β-estradiol implying that in addition PKA is activated by other signaling pathways, too. ATF1, a relative to CREB, is phosphorylated almost to the same extent as CREB after stimulation of TNBC cells with 17β-estradiol. This phosphorylation is even more clearly prevented by the pretreatment with 10
-4 M estriol.
Electrophoretic shift assays (EMSAs) were performed with a labeled oligonucleotide representing the CRE in the promoter of the cyclin D1 promoter at position -14 to +11 from the transcription start site. In TNBC cells treated with 10-8 M 17β-estradiol an oligonucleotide band shifted by about 40 kD increased in intensity. In cells pretreated with 10-4 M estriol the amount of shifted oligonucleotide was lower than in cells treated with 17β-estradiol alone. Again, this finding provides evidence, that induction of cyclin D1 expression by 17β-estradiol is GPR30 dependent and prevented by estriol treatment. EMSAs showed an additional band at about 150 kD that was detectable in all samples independent of the various treatments of the TNBC cells. The origin of this band is not yet clear. In supershift experiments using an antibody against phosphorylated CREB this band disappeared from the blot because it was shifted to much higher molecular weight. By this supershift experiment this unknown additional band in EMSAs is at least identified to contain CREB. This band probably arises from CREB complexed with other proteins bound to the oligonucleotide of the cyclin D1 promoter.
Using an oligonucleotide with CRE found further upstream at position –517 to –493 we did not observe a binding of CREB to this oligonucleotide at any treatment condition (data not shown).RT-PCR examinations of mRNA from estriol/estradiol treated cells for cyclin D1 expression confirmed the results of the CREB binding to a cAMP-responsive element of the cyclin D1 promoter, as observed in EMSA tests (Figure
7). All the above mentioned results clearly indicate the involvement of GPR30 in the growth regulation of TNBC by 17β-estradiol.
In addition, we analyzed the GPR30 dependent regulation aromatase expression in TNBC cells. Treatment with 17β-estradiol induced aromatase expression in TNBC cell lines and estriol prevented this induction completely (Figure
5). The induction of aromatase expression by 17β-estradiol is in accordance with a report of Lin et al. [
20] who observed a novel signaling paradigm in endometrial cancer cells initiated by estrogenic activation of GPR30. They showed that PI3K and MAPK signal transduction cascades activated by GPR30 converge on nuclear hormone receptor SF-1 that modulates transcription of the aromatase gene [
20]. This novel GPR30/SF-1 pathway increases local concentrations of estrogen, and mediates autocrine proliferative effect on cells expressing GPR30. We hypothesize, that in TNBC cells, an autocrine circuit of 17β-estradiol exists that upregulates its own synthesis via stimulation of GPR30. In detail, 17β-estradiol stimulates aromatase expression via GPR30 and the additionally synthesized 17β-estradiol further stimulates GPR30 exponentially leading to growth stimulation of the TNBC cells. Estriol is capable to disrupt this vicious circle by inhibition of GPR30 as we were able to show that induction of aromatase expression by 17β-estradiol is suppressed by treatment with 10
-4 M estriol. As final consequence of inhibition of GPR30 activity growth stimulation of TNBC cells by 17β-estradiol is clearly prevented by estriol.
High plasma levels of estriol are detected during pregnancy and women who were multiparous have a more than one-third lower risk of breast cancer [
28]. The reduced risk for basal-like breast cancer, the major form of TNBC, observed in women with increasing number of breastfed children might be an additional indication for a protective effect of estriol for TNBC [
29]. But it should be considered, that serum-levels at the third trimester of pregnancy were estimated to be at 4×10
-8 M [
30] and this is a 1000 times lower concentration than we applied in the proliferation assay of TNBC cell lines.
Due to its limited solubility in culture medium the effects of estriol could not be further increased. The synthetic GPR30 antagonist G15 was even less soluble in culture medium (maximal solubility 10
-5 M) and therefore less effective in inhibiting EGF-receptor transactivation (data not shown) and less effective in prevention of proliferation (see Additional file
2).
Other GPR30 antagonists are presently under investigation in several laboratories. In 2011, Dennis et al. described another GPR30 antagonist (G36) having a slightly higher affinity to GPR30 than G15 but no information on the aqueous solubility of this compound was given by the authors [
14]. Lappano et al. [
31] introduced MIBE, an inhibitor, that acts on both GPR30 and estrogen receptor α [
31]. Further a tricarbonyl-Re/Tc(I) chelate with GPR30 antagonistic properties has been described [
32]. All these compounds are worth testing their efficiency on TNBC.
While measuring the effects of estriol on GPR30 in TNBC cell lines we were aware that besides ERα and GPR30 (GPER) there is a third receptor for estrogens, ERβ, in many different cell types. In addition, it is frequently described, that ERβ is an opponent of ERα, and from our results we cannot certainly reason that the inhibitory action of estriol in TNBC is solely exerted by GPR30. But Western blots of proteins from our tested cell lines revealed a very low expression of ERβ in these cell lines (see Additional file
3: Figure S3).
Up to now, no targeted therapy for TNBC was shown to be successful. Promising candidates, like PARP inhibitors, taking advantage of a disturbed DNA-repair due to frequent BRCA1-mutations present in TNBC [
3,
4] and the use of the EGF-R antibody Cetuximab for therapy targeting the overexpression of the EGF-receptor in TNBC were of limited success [
2‐
4].
Our observation, that growth of TNBC is stimulated by 17β-estradiol via GPR30 indicates that reduction of 17β-estradiol in TNBC patients by application of aromatase inhibitors might remain a therapeutic option in triple-negative breast cancer. The efficacy of estriol in inhibiting proliferation of TNBC cells should be further evaluated in vivo.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RG and CG together developed the conception of the project. RG carried out all experiments, performed data analysis and drafted the manuscript. CG participated in the design of the study and the statistical analysis and he supervised the drafting of the manuscript. GE critically revised the manuscript and approved the final version. All authors read and approved the final manuscript.