Estrogen receptor β represses Akt signaling in breast cancer cells via downregulation of HER2/HER3 and upregulation of PTEN: implications for tamoxifen sensitivity
© Lindberg et al.; licensee BioMed Central Ltd. 2011
Received: 9 November 2010
Accepted: 14 April 2011
Published: 14 April 2011
The inhibition of estrogen receptor (ER) α action with the ER antagonist tamoxifen is an established treatment in the majority of breast cancers. De novo or acquired resistance to this therapy is common. Expression of ERβ in breast tumors has been implicated as an indicator of tamoxifen sensitivity. The mechanisms behind this observation remain largely uncharacterized. In the present study, we investigated whether ERβ can modulate pathways implicated in endocrine resistance development.
T47-D and MCF-7 ERα-expressing breast cancer cells with tetracycline-regulated expression of ERβ were used as a model system. Expression levels and activity of known regulators of endocrine resistance were analyzed by performing quantitative polymerase chain reaction assays, Western blot analysis and immunostaining, and sensitivity to tamoxifen was investigated by using a cell proliferation kit.
Expression of ERβ in ERα-positive T47-D and MCF-7 human breast cancer cells resulted in a decrease in Akt signaling. The active form of an upstream regulator of Akt, proto-oncogene c-ErbB-2/receptor tyrosine kinase erbB-3 (HER2/HER3) receptor dimer, was also downregulated by ERβ. Furthermore, ERβ increased expression of the important inhibitor of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Importantly, ERβ expression increased the sensitivity of these breast cancer cells to tamoxifen.
Our results suggest a link between expression of ERβ and endocrine sensitivity by increasing PTEN levels and decreasing HER2/HER3 signaling, thereby reducing Akt signaling with subsequent effects on proliferation, survival and tamoxifen sensitivity of breast cancer cells. This study supports initiatives to further investigate whether ERβ presence in breast cancer samples is an indicator for endocrine response. Current therapies in ERα-positive breast cancers aim to impair ERα activity with antagonists or by removal of endogenous estrogens with aromatase inhibitors. Data from this study could be taken as indicative for also using ERβ as a target in selected groups of breast cancer.
Approximately two-thirds of breast cancers express estrogen receptors (ERs) and initially require estrogen to grow, and are therefore treated with ER antagonists, such as tamoxifen, or by depletion of endogenous estrogens with aromatase inhibitors [1, 2]. Two ERs, ERα and ERβ, have been identified . ERα plays an important role in the proliferation and progression of breast cancer, whereas a distinct function of ERβ in breast cancer initiation and development has not yet been clearly established. In in vitro settings, ERβ inhibits proliferation, migration and invasion of breast cancers cells [4–9] as well as the growth of breast tumor xenografts .
ERα is the marker of choice to decide endocrine treatment of breast cancer. However, in the case of tamoxifen treatment, despite the initial response to the therapy, one-third of patients will acquire resistance even though their ERα status may remain unchanged . ERβ has also been considered a marker of endocrine response. Lower expression of ERβ is found in tamoxifen-resistant tumors, and high levels of ERβ are sometimes associated with a better clinical outcome in ERα-expressing breast tumors . However, some studies have indicated that in high-grade, ERα-negative, node-positive breast tumors, ERβ presence appears to be a marker related to a more aggressive breast cancer .
Breast tumors overexpressing receptor tyrosine kinases (RTKs) are less likely to benefit from tamoxifen treatment [14–17]. Receptor tyrosine protein kinase erbB-3 (HER3) and proto-oncogene c-ErbB-2 (HER2) are members of the epidermal growth factor receptor (EGFR) family. HER3 lacks intrinsic kinase activity and relies on heterodimerization with other members of the EGFR family for transduction of signals. There is growing awareness of the importance of HER2/HER3 heterodimer formation in breast cancer progression, where coexpression of HER2 and HER3 has been shown to be a poor prognostic indicator associated with resistance to endocrine therapy and to HER tyrosine kinase inhibitors [18–22]. The majority of HER2-positive tumors are strongly positive for HER3 , which is also seen in mouse models of breast cancers, where high expression of HER2 is commonly associated with activated and overexpressed HER3 . Furthermore, inhibition of HER2 correlates with reduction in HER3 phosphorylation  and, correspondingly, inhibition of HER3 reduces phosphorylation of HER2 and abrogates HER2-mediated tamoxifen resistance .
Phosphatidylinositol 3-kinase (PI3K) promotes generation of phosphatidylinositol (3,4,5)-triphosphate (PIP3), which leads to phosphorylation and activation of the serine/threonine kinase Akt. The PI3K/Akt pathway plays important roles in regulating cell proliferation, growth, apoptosis and motility. Increased activity due to genetic changes is frequently seen in breast cancer, resulting in tumor progression, metastases and resistance to endocrine treatment [26–29]. Mutation of the PIK3CA gene, which encodes the p110α catalytic subunit of PI3K, leads to activation of Akt and is found in 18% to 40% of human breast cancers [30–32]. Stimulation of RTKs also activates Akt [33–35], and overexpression of HER2 is linked to elevated Akt activities [36–38]. In ERα-positive breast cancers treated with tamoxifen, detection of activated Akt at diagnosis has been shown to correlate to decreased overall survival .
Constitutive active Akt is also associated with loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression . PTEN is a tumor suppressor whose expression is often lost in breast cancers and associated with poor disease outcome [41–44]. PTEN antagonizes PI3K activity by dephosphorylating PIP3, resulting in lower levels of active Akt .
The goal of this study was to investigate whether ERβ1 (referred to hereinafter as ERβ) has any effect on the RTK/PI3K/Akt signaling pathway and thereby represents a regulator of tamoxifen sensitivity. We show that in ERα-positive breast cancer cells, expression of ERβ reduced Akt activation through downregulation of HER2/HER3 signaling and upregulation of PTEN and, importantly, increased sensitivity to tamoxifen. ERβ has sometimes been suggested as a predictor of endocrine response; however, the mechanisms underlying this response are still unknown. Here we suggest a link between expression of ERβ and endocrine sensitivity.
Materials and methods
T47-D cells with tetracycline-regulated expression of ERβ485 (T47-DERβ)  were routinely grown not expressing ERβ (-ERβ) in RPMI 1640 medium (Invitrogen, Paisley, United Kingdom) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 10 ng/mL doxycycline (Sigma, Stockholm, Sweden). For experiments, cells were grown for 24 to 168 hours prior to analysis in phenol red-free RPMI 1640 medium supplemented with 2% heat-inactivated FBS, 1% penicillin-streptomycin, 10 ng/mL -ERβ or 0.01 ng/mL doxycycline +ERβ in the presence of vehicle alone, ethanol and/or dimethyl sulfoxide (Sigma), or in 10 nM 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) (Tocris, Bristol, United Kingdom), 10 nM 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) (Tocris), 10 nM 17β-estradiol (E2) (Sigma), 10 nM 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY) (Tocris), 100 nM ICI 182, 789 (ICI) (Tocris) or 100 to 1,000 nM 4-hydroxy-tamoxifen (4-OH-T) (Sigma). MCF-7 breast cancer cells with tetracycline-regulated expression of ERβ485 were treated in a similar manner. T47-DPBI cells (Mock) were used as controls.
Quantitative real-time polymerase chain reaction assays
Cells were grown in six-well tissue culture plates for 24 to 96 hours and lysed in TRIzol reagent (Invitrogen), then RNA was extracted and cDNA was synthesized as described previously . Quantitative real-time polymerase reaction assays (qRT-PCR) were performed with SYBR Green PCR Master Mix in an ABI PRISM 7500 (Applied Biosystems, Foster City, USA). The following primers were used: 18S forward 5'-CCTGCGGCTTAATTTGACTCA-3', reverse 5'-AGCTATCAATCTGTCAATCCTGTCC-3'; ERβ forward 5'-ACTTGCTGAACGCCGTGACC-3', reverse 5'-CAGATGTTCCATGCCCTTGTT-3'; HER2 forward 5'-AAAGGCCCAAGACTCTCTCC-3', reverse 5'-CAAGTACTCGGGGTTCTCCA-3'; HER3 forward 5'-GTCATGAGGGCGAACGAC-3', reverse 5'-AGAGTCCCAGGACACACTGC-3'; and PTEN forward 5'-GGGGAAGTAAGGACCAGAGAC-3', reverse 5'-TCCAGATGATTCTTTAACAGGTAGC-3'. Quantification was carried out following the supplier's protocols using the standard curve method.
Cells grown on plates were washed with ice-cold phosphate-buffered saline (PBS), transferred to Eppendorf tubes and pelleted by centrifugation. Cell pellets were freeze-thawed and resuspended with PBS-TDS buffer (PBS with 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid and phosphatase inhibitors (Sigma)), incubated for 30 minutes on ice and centrifuged at 11,000 rpm for 10 minutes at 4°C. Supernatants were collected for further analysis. Protein quantification was carried out using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, USA).
Western blot analysis
Forty micrograms of total cellular protein were separated using 7.5% SDS-polyacrylamide gel electrophoresis and electrotransferred onto a nitrocellulose membrane (Hybond-C; AmershamBuckinghamshire, United Kingdom). After blocking in 5% milk protein (wt/vol) in PBS, 0.1% Tween 20 (vol/vol) membranes were sequentially incubated with primary and secondary antibodies. The following antibodies were used: anti-ERβ (14021; Abcam, Cambridge, United Kingdom), GTX110607 (GeneTex, Irvine, USA), anti-phospho-HER3 tyr1289 (21D3; Cell Signaling Technology, Danvers, USA), anti-phospho-Akt pathway sampler kit (9916; Cell Signaling Technology), anti-phospho-HER2 antibody sampler kit (9923; Cell Signaling Technology), anti-PTEN (26H9; Cell Signaling Technology); anti-α-tubulin (11H10; Cell Signaling Technology), anti-EGFR (E12020; Transduction Laboratories, Franklin Lakes, USA), anti-HER3 (sc-285; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-actin (Sigma). The secondary antibodies were horseradish peroxidase-conjugated (Sigma). Visualization was carried out using the ECL Plus kit (Amersham) or the SuperSignal West Pico kit (Pierce Biotechnology). At least three independent experiments were carried out.
Cells were cultured on sterilized glass coverslips in high- or low-doxycycline conditions for 4 days as described above. The cells were fixed by ice-cold methanol and ice-cold acetone for 10 minutes and 1 minute, respectively. Blocking of nonspecific binding was done with BlockAce (Dainippon Pharmaceutical, Osaka, Japan) for 1 h at room temperature. The samples were then incubated overnight at 4°C with the following antibodies at the indicated dilutions in 10% BlockAce in PBS: anti-HER2, 1:150 (29D8; Cell Signaling Technology), and anti-PTEN, 1:100 (26H9; Cell Signaling Technology). After washes with PBS, samples were incubated with corresponding Alexa Fluor 568-conjugated secondary antibody 1:500 (Invitrogen) and Hoechst 33342 5 μg/mL (Molecular Probes, Eugen, USA) in PBS for 1 hour at room temperature. Samples were mounted with VECTASHIELD (Vector Laboratories, Burlingame, USA) after washes with PBS. Negative controls were incubated without primary antibody. To compare staining intensity between different samples, pictures were obtained with fixed exposure time. Staining was repeated three times to confirm consistent results.
Pictures of fluorescence staining were captured with a Zeiss Axioplan 2 microscope using Zeiss Plan-Apochromat 63×/1.40 oil lens (Carl Zeiss, Oberkochen, Germany). Images were acquired with a Zeiss AxioCam MRm camera under the same settings. Captured images were processed using the AxioVision Rel 4.6 program and edited using Adobe PhotoShop C54 software (Adobe, San Jose, USA), and the same adjustments were applied to all images.
T47-DERβ and MCF-7ERβ cells were cultured for 3 days in high (-ERβ) or low (+ERβ) doxycycline concentrations in the absence or presence of vehicle, E2 or WAY. On the third day, cells were replated on 96-well plates and allowed to adhere for 24 hours. Thereafter increasing concentrations of 4-OH-T were added. Growth medium was changed every other day. Cell viability was measured after 0, 5 and 7 days of incubation with 4-OH-T using a colorimetric assay (WST-1; Roche, Basel, Switzerland) following the manufacturer's suggestions. Measurement of absorbance was done using a SpectraMax 250 microplate reader (Molecular Devices, Sunnyvale, USA) against a background control as blank.
Differences between more than two groups were compared by one-way analysis of variance and Tukey's multiple posttest using GraphPad software (GraphPad, San Diego, USA).
Results and discussion
AKT signaling is repressed by ERβ
Since addition of the ERβ ligand DPN exerted no stable, repeatable additional effect to that already observed following ERβ expression (Figure 1 and 1 data not shown), we investigated whether ER antagonists would prevent ERβ-induced decrease of Akt phosphorylation. For this purpose, ICI 182, 780 (ICI), a selective ER downregulator, and the selective estrogen modulator 4-OH-T were used. As expected, ICI induced complete downregulation of ERα (Figure 1D). ERβ protein levels were partially downregulated by ICI, whereas 4-OH-T had no significant effect on either ERα or ERβ protein levels (Figure 1E). Furthermore, ERα protein levels were reduced in cells expressing ERβ (Figures 1D and 1E). This latter finding was consistently observed in all inducible systems that we tested. Treatment with ICI or 4-OH-T did not inhibit the ERβ-induced decrease of pAkt levels. However, in ICI- or 4-OH-T-treated cells, the ERβ-induced decrease of pAkt levels was less than that in cells not exposed to ICI or 4-OH-T, suggesting a weak antagonistic action of ICI and 4-OH-T. In summary, in two different ERα-expressing human breast cancer cell lines, ERβ expression clearly reduced activation of the Akt signaling pathway.
ERβ regulation of HER2 and HER3 expression
Neither ICI nor 4-OH-T prevented ERβ-induced downregulation of HER3 protein levels (Figure 3B). qRT-PCR analysis showed that ICI and 4-OH-T both increased overall HER3 mRNA levels, which could be indicative of ERα, similarly to ERβ, having a repressive effect on HER3 mRNA expression. However, the ERα-selective ligand PPT had no effect on HER3 protein expression. Further studies are needed to explain this difference. ICI, but not 4-OH-T, clearly did not inhibit ERβ-induced downregulation of HER3 mRNA. The ICI-induced increase and ERβ-induced downregulation of HER3 mRNA levels in ICI-treated cells correlated well with HER3 protein levels. This was not obvious in 4-OH-T treated cells, where a difference was seen at the protein level but not at the mRNA level.
ERβ downregulates heregulin-induced activation of HER2/HER3 dimer and Akt
Heregulin-β1 (HRG-β1), a member of the EGFR family, is a ligand for HER3. As HER3 has no intracellular tyrosine kinase domain, it partners with other members of the EGFR family to initiate intracellular signaling. The preferred dimerization partner is HER2, which has tyrosine kinase activity. In the intracellular domain of HER3, there are six tyrosines that, upon phosphorylation by HER2, will serve as docking sites for the p85 adaptor subunit of PI3K. Thus, HRG-β1 activation of the HER2/HER3 dimer results in strong activation of the PI3K/Akt signaling pathway.
Exposure of T47-DERβ cells to HRG-β1 for 30 minutes also dramatically increased levels of pAkt (Figure 4A). At this time point, ERβ expression did not decrease levels of phosphorylated Akt. However, a time study of HRG-β1-stimulated cells showed that from 2 hours onward, ERβ presence decreased levels of phosphorylated Akt (Figure 4B). One possible explanation for this could be that in the acute phase after HRG-β1 addition, there was a massive activation of Akt due to the already mutated PIK3CA in T47-DERβ cells, an activation that ERβ could not inhibit. However, ERβ could decrease levels of phosphorylated Akt after its peak activity, when the activity was still clearly above that in unstimulated cells (Figure 4A).
Exposure of cells to DPN, E2 or WAY (another ERβ selective agonist) did not influence levels of HRG-β1-induced phosphorylated HER2, HER3 and Akt (Figure 4A andAdditional file 3). To further investigate the Akt pathway in the context of endocrine sensitivity and ERβ expression, in addition to HRG-β1 treatment, cells were further treated with ICI or 4-OH-T (Figure 4A). ICI and 4-OH-T exposure both increased levels of phosphorylated HER2 and HER3 in the absence or presence of ERβ. An effect that may be related to increased total HER2 levels in cells treated with ICI or 4-OH-T (Figures 3A and 4A). However, levels of phosphorylated HER2 and pHER3 were clearly lower when ERβ was present.
PTEN levels increase following ERβ expression
Expression of ERβ sensitizes breast cancer cells to tamoxifen
In several experiments, the addition of agonist or antagonist did not increase or decrease the effect of ERβ expression on its own. This could indicate that in these breast tumor cells, ERβ is activated in a ligand-independent manner; for example, it is phosphorylated in the AF-1 domain and then is also less inhibited by antagonists that have a focus on ligand-binding and the AF-2 domain . Further studies are needed to clarify this hypothesis with mapping of phosphorylated sites of ERβ in these cells. Interestingly, a recent report  shows that ERβ phosphorylated at serine 105 is associated with a good prognosis in breast cancer. A future challenge is to develop ligands that, in this setting, that is, ERβ-expressing breast cancers with increased kinase activity, could activate or increase the inhibitory effect of ERβ on Akt signaling.
Our results suggest a link between expression of ERβ and endocrine sensitivity by increasing PTEN levels and decreasing HER2/HER3 signaling, thereby reducing Akt signaling with subsequent effects on proliferation, survival and tamoxifen sensitivity of breast cancer cells. This study supports initiatives to further investigate whether ERβ presence in breast cancer samples is an indicator for endocrine response. Current therapies in ERα-positive breast cancers aim to impair ERα activity with antagonists or by removal of endogenous estrogens with aromatase inhibitors. Data from this study could be taken as indicative for using ERβ as a target in selected groups of breast cancer.
epidermal growth factor receptor
receptor tyrosine kinase erbB-3
ICI 182: 789
polymerase chain reaction
phosphatase and tensin homologue deleted on chromosome 10
quantitative real-time polymerase chain reaction
receptor tyrosine kinase
We thank Dr Anders Ström for providing us with T47-D-inducible and MCF-7 ERβ-inducible cells. This study was supported by grants from the Swedish Cancer Fund and from the Welch Foundation. Luisa Helguero is supported by the Portuguese Science and Technology Foundation projects Ciencia 2008 and PTDC/SAU-ONC/112671/2009. The funding agencies had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.
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