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Molecular Endocrinology 20 (5): 996-1008
Copyright © 2006 by The Endocrine Society

Activation Function-1 Domain of Estrogen Receptor Regulates the Agonistic and Antagonistic Actions of Tamoxifen

Selina Glaros, Natasha Atanaskova, Changqing Zhao, Debra F. Skafar and Kaladhar B. Reddy

Departments of Pathology (S.G., N.A., K.B.R.) and Physiology (C.Z., D.F.S.), Wayne State University School of Medicine, The Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201

Address all correspondence and requests for reprints to: Kaladhar B. Reddy, Department of Pathology, Wayne State University, 540 East Canfield Avenue, Detroit, Michigan 48201. E-mail: kreddy{at}med.wayne.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ER{alpha}-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ER{alpha}, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ER{alpha}, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ER{alpha} regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE EFFECTS OF estrogens are mediated primarily via estrogen receptor {alpha} and ß (ER{alpha} and ERß), which are members of the nuclear hormone receptor superfamily (1). 17ß-Estradiol (E2) binding to its receptor induces the ligand-binding domain to undergo a characteristic conformational change, whereupon the receptor dimerizes, binds to DNA, and subsequently stimulates gene expression (2). ER{alpha} is stimulated by two distinct activation regions, activation function-1 (AF-1) and AF-2 (3, 4, 5). AF-1 is located in the N-terminal A/B domain and is constitutively activated or exerts ligand-independent transcriptional activity (6, 7, 8). AF-2 is located in the C-terminal ligand-binding domain and exerts ligand-dependent transcriptional activity (9). AF-1 and AF-2 activate transcription independently or synergistically and act in a promoter-specific and cell-specific manner (10). ER is also activated by ligand-independent mechanisms that involve cross talk from peptide and growth factor signal transduction pathways. Both ligand-dependent and ligand-independent activation of ER are modulated by receptor phosphorylation (11, 12). The major phosphorylation sites of ER residues in the N-terminal domain are serine 104, 106, 118, and 167. Mutations of serine 104, 106, and 118 to alanine result in a general decrease of ER transcriptional activity (12, 13). Phosphorylation of serine 167 was shown to be important in DNA binding by the receptor (14). Numerous signaling pathways regulate ER phosphorylation (6, 13, 15, 16). Interactions with coregulatory proteins are important mechanisms mediating E2 and selective ER modulator action.

Protein kinase B (PKB)/AKT family is composed of three closely related isoforms, AKT-1 (PKB{alpha}), AKT-2 (PKBß), and AKT-3 (PKB{gamma}), which are expressed at mRNA levels in all normal tissues (17). Elevated AKT-1 kinase activity (16, 18) and AKT-2 kinase activity have been reported in breast carcinomas (18, 19). Expression of AKT-3 has been shown to be up-regulated in ER-negative breast cancer tumors, suggesting a role for AKT-3 in this aggressive phenotype of breast cancer (20, 21). Overexpression of AKT is associated with tamoxifen resistance in breast cell lines (22, 23) and human breast tumors (24, 25). The AKT phosphorylation site (RXRXXS/T, where X is any residue) is present in ER{alpha} but not in ERß. This suggests the possibility that AKT-induced changes in ER signaling are mediated through ER{alpha}. However, the mechanisms by which activated AKT regulates tamoxifen sensitivity or resistance are not clear at present.

Approximately 70% of all breast cancers are dependent for their growth on estrogen and a functional estrogen receptor {alpha} (ER{alpha}). Hence, ER-positive breast cancer is usually treated through hormone reduction using aromatase inhibitors (26, 27) or antiestrogens such as tamoxifen (28). The most commonly used antiestrogen is tamoxifen, and it is beneficial in pre- and postmenopausal women whose tumors are ER positive; the optimal treatment period is 5 yr (29). However, most patients undergoing long-term treatment of breast cancer with tamoxifen eventually experience recurrence of tumor growth. One of the reasons for this treatment failure is the acquisition by the tumor of the ability to respond to tamoxifen as a stimulatory rather than an inhibitory ligand (30, 31). It is widely documented that the inappropriate activation of growth factor-signaling cascades can promote antiestrogen failure in breast cancer cells. This phenomenon has been described for the overexpression of multiple growth factors and their receptors, including epidermal growth factor and TGF{alpha} acting through the epidermal growth factor receptor (EGFR) (32, 33). Furthermore, heregulins are known to act through HER-3 and HER-4 (34, 35), and IGF-I and -II act through the type I IGF receptor (IGF-IR) (36, 37). It is also known that HER-2 contributes to antiestrogen failure either directly when overexpressed (38, 39, 40) or indirectly through heterodimerization with other ErbB receptor family members (33). However, the molecular mechanisms by which overexpression of growth factors and their receptors lead to antiestrogen resistance have not been adequately established.

To elucidate the signaling pathways involved in regulating tamoxifen resistance in ErbB-overexpressing cells, we expressed EGFR, myristoylated AKT-2 (myr-AKT-2), constitutively activated MEK-1, or both myr-AKT and activated-MEK in ER-positive MCF-7 human breast tumor cells. Our results show that overexpression of EGFR or the activation of AKT in MCF-7 cells leads to ligand-independent phosphorylation of Ser167 in the AF-1 domain of ER{alpha}; these phosphorylation events lead to enhanced ER{alpha}-amplified in breast cancer 1 (AIB1) interactions and tamoxifen resistance. However, inhibition of AKT by short inhibitory RNA (siRNA) in EGFR- or MEK/AKT-expressing cells reverses tamoxifen resistance. Simultaneous inhibition of AKT and MAPK by siRNA restores 70–80% of the tamoxifen sensitivity in these cells. Our findings provide insight into the molecular mechanism by which ligand-independent activation of ER{alpha} leads to tamoxifen resistance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ErbB Kinase Activity Is Required for Ligand-Independent Activity of ER{alpha} and Tamoxifen Resistance in Vitro
Multiple lines of experimental evidence suggest that overexpression of ErbB receptors confers antiestrogen resistance to breast tumor cells (41). However, the mechanism by which overexpression of ErbB receptors leads to tamoxifen resistance is not adequately established. To determine the molecular mechanism by which ErbB receptor overexpression leads to tamoxifen resistance, we developed MCF-7 cells that stably overexpress EGFR (EGFR/MCF-7 cells) (6, 42) or activated MEK (MEK/MCF-7 cells) (6). To stimulate signaling in MCF-7 and EGFR/MCF-7 cells, all experiments were done in 5% charcoal-stripped serum unless otherwise stated. Our data show that MEK/MCF-7 cells have higher MAPK activity without significant alteration of AKT activity. However, in EGFR/MCF-7 cells, both MAPK and AKT activation were higher compared with the control cells (Fig. 1AGo). To confirm that activated AKT and/or MAPK can cross talk and phosphorylate ER{alpha} (a nuclear receptor) at different sites, we performed indirect immunofluorescence using antibodies directed specifically against phospho-Ser118 and phospho-Ser167 on ER. Our data in Fig. 1BGo indicate that activated MAPK (ERK 1 and 2) can phosphorylate the ER at Ser118 (green), but it does not have a significant effect on Ser167 phosphorylation (red). On the other hand, in EGFR/MCF-7 cells, phosphorylation of ER{alpha} was observed at Ser118 (green) by MAPK and Ser167 (red) by AKT (Fig. 1BGo). Activation of AKT and MAPK was also seen in HER-2-overexpressing MCF-7 cells (HER-2/MCF-7 cells) (data not shown). In MCF-7 cells no phosphorylation was detected (Fig. 1BGo). Our data also show that ER is predominantly located in the nucleus as seen by green or red fluorescence in the 4',6-diamidino-2-phenylindole (DAPI)-stained blue nucleus. Nucleus is stained blue by DAPI in all the cells. The specificity of phospho-Ser118 and phospho-Ser167 ER antibodies was further confirmed by Western blot analysis (Fig. 1CGo).


Figure 1
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  		Fig. 1. Overexpression of EGFR Leads to MAPK and AKT Activation, and Phosphorylation of Ser118 and Ser167 in AF-1 Domain of ER

Cells were grown in DMEM with serum to near confluence. The seeding medium was removed, cells were washed twice with PBS, and the medium was replaced with phenol red-free DMEM with 5% charcoal stripped serum. phospho-MAPK or phospho-AKT levels were evaluated 48 h later by Western immunoblot analysis. A, Western blot analysis shows predominant phosphorylation of MAPK in MEK/MCF-7 cells, phosphorylation of both MAPK and AKT in EGFR/MCF-7 cells, and no significant changes in the phosphorylation of either MAPK or AKT in MCF-7 control cells. These results are representative of three independent experiments. The blot was stripped and reprobed with GAPDH antibody to assess equal loading of proteins. B, Immunofluorescence data show that MEK/MCF-7 cells have elevated ER phosphorylation at Ser118 (green) and EGFR/MCF-7 cells have elevated levels of ER phosphorylation at both Ser118 (green) and Ser167 ER (red) compared with control cells. The nucleus of the cells was stained blue by DAPI in all the immunofluorescence experiments. C, Western blot analysis shows predominant phosphorylation of Ser 118 by MAPK in MEK/MCF-7 cells and phosphorylation of both Ser118 by MAPK and Ser 167 by AKT in EGFR/MCF-7 cells. These results are representative of three independent experiments. The blot was stripped and reprobed with GAPDH antibody to assess equal loading of proteins. p-ER, phospho-ER; p-MAPK, phospho-MAPK; p-AKT, phospho-AKT.

 
To further determine whether AKT and MAPK-induced phosphorylation of the AF-1 domain in ER enhances transcriptional activity of ER and its response to antiestrogens such as tamoxifen and ICI 182,780 in ER{alpha}-positive MCF-7 cells, we transiently transfected the cells with estrogen-response element (ERE)-tkLuc, containing a Vitellogenin A2-derived ERE as described previously (6) or the corresponding empty vector, tkLuc (with no ERE) along with the internal control plasmid pRL, to correct for transfection efficiency. Cotransfection of constitutively active MEK, which activates MAPK, resulted in approximately 7-fold increase in reporter activity compared with control. E2 further enhanced ERE-tkLuc activity to approximately 14-fold. Tamoxifen blocked E2- and MEK-induced reporter activity (Fig. 2AGo). Transfection of the myr-AKT-2 expression vector resulted in approximately 7-fold increase in reporter activity compared with control, and E2 enhanced reporter activity by about 9-fold. However, tamoxifen was unable to block AKT-induced ERE-reporter activity (Fig. 2BGo). ICI 182,780, a pure antiestrogen, blocked both MEK- and AKT-induced transcriptional activity. One reason may be that ICI 182,780 prevents activation of AF-1 and AF-2, whereas tamoxifen blocks only AF-2 activation (43). These data suggest that expression of either activated MAPK or AKT can induce increase in the transcriptional activity of ER{alpha}. However, amoxifen is unable to block AKT-induced transcriptional activation in vitro.


Figure 2
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  		Fig. 2. AKT and MAPK Increase Ligand-Independent Activity of ER{alpha}

MCF-7 cells were transiently transfected with activated MEK (panel A) or myr-AKT-2 (panel B) along with EREtkLuc (the ERE-containing reporter plasmid) or tkLuc (the corresponding empty vector with no ERE) and cotransfected with pRL (an internal reporter plasmid to control for transfection efficiency). Then cells were treated with E2 (10–8), tamoxifen (10–6), ICI 182,780 (10–7), or ethanol vehicle for 48 h and then harvested. Cell extracts were prepared and analyzed for luciferase activity, as described in Materials and Methods. The magnitude of activation obtained from tkLuc-transfected cell extracts (as determined after normalization to pRL activity) was used to calculate reporter activity. Transactivation is reported as the fold induction relative to the basal level of luciferase activity in cells transfected with the empty tkLuc reporter vector, which is arbitrarily set at 1.0-fold. Cotransfection of activated MEK or myr-AKT led to increases in estrogen-independent activity and further increases on estrogen-stimulation. Tamoxifen significantly inhibited MEK-induced transcriptional activity. However, in myr-AKT-transfected cells tamoxifen was unable to block AKT-induced transcriptional activation. ICI-182,780 blocked both MEK and AKT-induced transcriptional activity. The data shown here are from three separate experiments, represented as the means ± SE. Tam, Tamoxifen; ICI, ICI 182,780.

 
To determine the physiological significance of MAPK- and AKT-induced phosphorylation of ER{alpha} and its effects on the antiestrogen tamoxifen in vitro, we treated the MCF-7, MEK/MCF-7, EGFR/MCF-7, and HER-2/MCF-7 cells with increasing concentration of tamoxifen (4-OHT, 10–10 to 10–6 M) and determine its effects on cell proliferation. The proliferation of MCF-7 and MEK/MCF-7 cells was inhibited 60–70% by 10–6 M tamoxifen. In contrast, only 20–30% inhibition was observed in EGFR- and HER-2-overexpressing cells (Fig. 3AGo). These data suggest that EGFR- or HER-2-overexpressing cells are less sensitive or resistant to tamoxifen treatment in vitro.


Figure 3
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  		Fig. 3. Overexpression of EGFR Leads to Tamoxifen Resistance Both in Vitro and in Vivo

A, Effect of MEK, EGFR, or HER-2 overexpression on tamoxifen resistance in vitro. MCF-7 (control cells), MEK/MCF-7, EGFR/MCF-7, and HER-2/MCF-7 cells were grown as described in Fig. 1Go. These cells are treated with increasing concentrations of tamoxifen. Cells were counted on d 5. MCF-7 and MEK/MCF-7 cells were inhibited by 50–70% in the presence of tamoxifen at 10–6 M compared with control cells. However, EGFR or HER-2 overexpression cells were inhibited by 20–30% compared with control cells. These results are representative of three independent experiments. B, EGFR overexpression leads to tamoxifen resistance in vivo. EGFR/MCF-7 cells formed large tumors compared with MEK/MCF-7 cells in the presence of slow-release E2 pellet in ovariectomized SCID mice. In EGFR/MCF-7 cells, tamoxifen was unable to inhibit tumor growth significantly (P = 0.23). However, in MEK/MCF-7 cells, tamoxifen significantly inhibited tumor growth (P < 0.05) (panel B). TAM, Tamoxifen.

 
To further determine whether EGFR overexpression leads to tamoxifen resistance in vivo, we injected MEK/MCF-7 and EGFR/MCF-7 cells subcutaneously on either side of an ovariectomized SCID mouse in the presence or absence of 17ß-estradiol (0.72 mg/pellet, 90-d slow-release pellet). Once xenografts had reached a volume of 2200 ± 128 (mean ± SE), tumor-bearing mice were randomly allocated to no treatment or treatment with tamoxifen. In MEK/MCF-7 tumors, E2-stimulated tumor growth (6137 ± 210, mean + SE) and tamoxifen significantly blocked E2-induced tumor growth (2983 ± 180, mean ± SE) (P < 0.05). However, in EGFR/MCF-7 cells, estrogen significantly enhanced tumor growth (8476 ± 380, mean ± SE) whereas tamoxifen was unable to inhibit the tumor growth significantly (7646 ± 460, mean ± SE) (P = 0.23) (Fig. 3BGo). The above data suggest that overexpression of EGFR leads to tamoxifen resistance both in vitro and in vivo.

AKT-Induced Phosphorylation of Ser167 in AF-1 Domain of ER{alpha} Plays a Major Role in Tamoxifen Resistance
Activation of ErbB receptors leads to activation of multiple downstream signaling molecules such AKT, MAPK, signal transducer and activator of transcription, Cyclin D, etc. (44, 45). This could lead to activation of ER{alpha} through other signaling molecules in addition to AKT and MAPK. To determine precisely whether AKT- and/or MAPK-induced activation of Ser167 and Ser118 in ER plays a major role in tamoxifen resistance, we generated a series of stable clones in MCF-7 cells that express myristoylated AKT (myr-Akt) alone, or express both myr-AKT and activated MEK. These clones were confirmed by Western immunoblot analysis (Fig. 4AGo). The various clones identified by Western blot analysis were further tested to determine whether activated AKT and/or MAPK can cross talk and phosphorylate ER{alpha} by immunofluorescence using specific antibodies directed against phospho-Ser118 and phospho-Ser167 on ER{alpha}. The data in Fig. 4BGo show that MAPK phosphorylates Ser118 (green) in MEK/MCF-7 cells and that AKT phosphorylates Ser167 (red) in AKT/MCF-7 cells. However, in MEK/AKT/MCF-7 cells, both Ser118 (by MAPK) and Ser167 (by AKT) are phosphorylated. We further determined the effect of estrogen and tamoxifen on cell proliferation in MCF-7 cell lines expressing activated MEK alone, AKT alone, or both MEK and AKT. MCF-7 cells transfected with empty vector were used as control cells. The proliferation of MCF-7 control cells was inhibited by 65–70% in the presence of E2 plus tamoxifen compared with E2 alone. However, in AKT/MCF-7 cells, E2 plus tamoxifen inhibited cell proliferation by 25%, in MEK/MCF-7 cells by 50%, and in MEK-AKT/MCF-7 cells by only 8% (Fig. 4CGo). These data suggest that activated AKT or AKT-MAPK pathways play a major role in the regulation of antiestrogen resistance to tamoxifen in ER-positive MCF-7 breast tumor cells.


Figure 4
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  		Fig. 4. Expression of Activated AKT Leads to Tamoxifen Resistance

A, Western blot analysis show enhanced phosphorylation of both MAPK and AKT in MEK/AKT-overexpressing MCF-7 clones (MEK-AKT/MCF-7 cells) compared with control MCF-7 cells, and AKT/MCF-7 clones show enhanced phosphorylation of AKT. The blot was stripped and reprobed with GAPDH antibody to assess equal loading of proteins. B, We further confirmed that activated MEK specifically phosphorylates ER{alpha} at Ser118 (green), myr-AKT at Ser167 (red) and MEK/AKT at Ser 118 (green) and Ser167 (red) by immunohistochemical staining. C, Tamoxifen was able to inhibit E2-mediated cell proliferation by approximately 50–70% in MCF-7 and MEK/MCF-7 cells, and by about 25% in AKT/MCF-7 cells. In contrast, tamoxifen’s ability to inhibit cell proliferation was only about 8% in MEK-AKT/MCF-7 cells. The data shown here are from three separate experiments, represented as the means ± SE. pMAPK, phospho-MAPK; p-AKT, phospho-AKT; p-ER, phospho-ER; C, control; TAM, tamoxifen.

 
It has been proposed that E2 signaling is mediated by recruiting transcriptional coactivators to the ER (46). AIB1, also called steroid receptor coactivator 3, receptor-associated coactivator 3, acetyltransferase, and p300/cAMP response element-binding protein binding protein interacting protein, is an ER coactivator that was previously shown to play an important role in hormone-mediated breast cancer progression (45, 47). Tamoxifen inhibits coactivator recruitment and promotes corepressor association with ER to inhibit estrogen-dependent gene transcription in tamoxifen-sensitive breast cancer cells (48). To evaluate the effect of cross talk on ER-coactivator interactions, we determined the interaction of ER-AIB1 by coimmunoprecipitation and Western blot analysis in MCF-7, MEK/MCF-7, AKT/MCF-7, and EGFR/MCF-7 cells in the presence or absence of E2 and/or Tam. In the MCF-7 and MEK/MCF-7 cells, ER-AIB1 interactions increased in the presence of E2 compared with control and decreased in the presence of E2 plus tamoxifen (Fig. 5Go, A and B); these cells are sensitive to tamoxifen (Fig. 2AGo). However, in AKT/MCF-7 and EGFR/MCF-7 cells, both E2 and E2 plus tamoxifen increased ER-AIB1 interactions (Fig. 5Go, C and D), and these cells are resistant to tamoxifen.


Figure 5
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  		Fig. 5. AKT Activation Enhances Interaction between ER and AIB1 in the Presence of Tamoxifen

The coimmunoprecipitation studies in tamoxifen-sensitive MCF-7 and MEK/MCF-7 cells show that E2 enhanced ER{alpha}-AIB1 interactions, and tamoxifen reduced the ER{alpha}-AIB1 interactions (A and B). However, in tamoxifen-resistant EGFR/MCF-7 and AKT/MCF-7 cells both E2 and tamoxifen enhanced the ER{alpha}-AIB1 interactions (C and D). These experiments were performed on at least on three independent occasions. C, Control; TAM, tamoxifen.

 
To further examine whether serine-167 is important for ER{alpha} transcriptional activity and to determine whether it alters ER-AIB1 interaction, we transiently transfected human wild-type ER or ER{alpha} mutant(s) (S167E or S167A) into HeLa cells. Studies have shown that well-characterized ER-negative HeLa cells exhibited 70–80% transfection efficiency (data not shown). Substitution of serine with glutamic acid at Ser-167 of ER mimics ER phosphorylation at 167, whereas substitution with alanine eliminates it. HeLa cells were transiently transfected with human ER{alpha} mutant(s) for 48 h after which the transcriptional activity and ER-AIB1 interaction were determined by coimmunoprecipitation and Western blot analysis as described preciously (6). Transfection with the phosphorylation mimic ER{alpha} mutant (S167E) resulted in approximately 2-fold increase in ERE-reporter activity compared with control, and E2 further enhanced ERE-tkLuc activity to approximately 3.5-fold. However, tamoxifen was unable to block S167E mutant activity (data not shown). To directly investigate whether ER phosphorylation at Ser167 enhances ER-AIB1 interactions in the presence of tamoxifen, coimmunoprecipitation and Western blot analysis were performed. In cells transfected with the S167E ER mutant, ER-AIB1 interactions increased in the presence of E2 and tamoxifen (Fig. 6BGo). In contrast, in cells transfected with either wild-type ER or S167A ER mutant, ER-AIB1 interactions increased in the presence of E2 compared with control and were reduced in the presence of E2 plus tamoxifen (Fig. 6Go, A and C). These results suggest that phosphorylation of ER{alpha} at Ser-167 causes the receptor to recruit coactivators such as AIB1 in the presence of tamoxifen to enhance estrogen-dependent gene transcription; this could enhance the agonist actions of tamoxifen in ER-positive cells, leading to a tamoxifen-resistant phenotype.


Figure 6
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  		Fig. 6. ER{alpha} Mutant (S167E) Increases ER{alpha}-AIB1 Interaction in the Presence of Tamoxifen

ER-negative HeLa cells were transfected with wild-type ER (A), S167E (B), or S167A ER{alpha} mutant plasmid (C). After 48 h, coimmunoprecipitation experiments were done using anti-AIB1 antibody and separated by SDS-PAGE. After transfer, the membrane was probed with anti-ER{alpha} antibody and then stripped and reprobed with anti-AIB1 antibody. In S167E-expressing cells E2 and tamoxifen enhanced ER{alpha}-AIB1 interaction. In S167A or wild-type ER-transfected cells, E2 enhanced ER{alpha}-AIB1 interactions and tamoxifen reduced them. These experiments were performed on at least three independent occasions. C, Control; TAM, tamoxifen.

 
Inhibition of AKT Sensitizes the Resistant Cells to Tamoxifen
To further validate the significance of AKT and MAPK in the regulation of the agonistic and antagonistic effects of tamoxifen in MCF-7 cells, we examined the effects of inhibiting AKT and /or MAPK by siRNA in EGFR/MCF-7 and in MEK-AKT/MCF-7 cells. We transfected AKT or MEK siRNA to block AKT and/or MAPK activition in EGFR/MCF-7 cells. Our data show that MEK siRNA significantly blocked phosphorylated MAPK levels, and the inhibition persisted up to 120 h. However, MEK siRNA had no effect on total MAPK levels (data not shown). Random siRNA, which was used as a control, had no effect on phosphorylated MAPK levels, as shown by immunoblot analysis (Fig. 7AGo). Similarly, AKT siRNA significantly blocked phosphorylated AKT levels, and the inhibition persisted up to 120 h. By contrast, random siRNA had no effect on phosphorylated AKT levels (Fig. 7BGo). These cells were further tested to determine whether inhibition of AKT and/or MAPK by siRNA blocks AKT- or MAPK-induced ER{alpha} phosphorylation by immunofluorescence assay using specific antibodies directed against phospho-Ser118 or phospho-Ser167 on ER. Our data show that inhibition of MEK by siRNA specifically blocks Ser118 phosphorylation in AF-1 domain of ER{alpha}; random siRNA had no effect (Fig. 7CGo, top panel). Similarly, inhibition of AKT by siRNA specifically blocked Ser167 phosphorylation; random siRNA had no effect (Fig. 7CGo, bottom panel). We further evaluated the effect of tamoxifen (10–6 M) in the presence of MEK siRNA and/or AKT siRNA on cell proliferation. EGFR/MCF-7 cell proliferation was inhibited by 15–20% in the presence of E2 plus tamoxifen compared with E2 treatment (Fig. 7DGo). However, when cells are treated with MEK siRNA plus E2 and tamoxifen, inhibition increased to 20–25% compared with the E2 group. When cells are treated with AKT siRNA plus E2 and tamoxifen, inhibition increased to 50–55%. Finally in the presence of AKT and MEK siRNA plus E2 and tamoxifen, inhibition increased to 70–75% (Fig. 7DGo). To further confirm whether MEK and AKT play a major role in antiestrogen resistance, we blocked AKT and/or MEK with siRNA in MEK-AKT/MCF-7 cells. The data obtained in MEK-AKT/MCF-7 cells (Fig. 8Go, A–D) are comparable to the data observed in EGFR/MCF-7 cells (Fig. 7Go). Cells grown under these conditions appear to remain viable, as indicated by their morphological appearance, by lack of floating cells and by the ability of the cells to exclude trypan blue (>90% viable). These data suggest that AKT or AKT and MAPK phosphorylation of ER{alpha} plays a major role in tamoxifen resistance, and inhibiting AKT or AKT and MAPK significantly restores tamoxifen sensitivity in these cells.


Figure 7
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  		Fig. 7. Inhibition of AKT in EGFR/MCF-7 Cells Reverses Tamoxifen Resistance

In EGFR/MCF-7 cells MEK siRNA and AKT-2 siRNA significantly blocks phospho-MAPK (panel A) and phosphor-AKT (panel B) levels up to 120 h. However, random siRNA had no effect up to 120 h. Immunofluorescence shows that inhibition of phosphorylated MAPK levels by MEK siRNA specifically blocked ER phosphorylation at Ser118 and AKT siRNA blocked ER phosphorylation at Ser167 in EGFR/MCF-7 cells (panel C). Random siRNA had no effect on the phosphorylation of Ser118 or Ser167. Panel D shows EGFR/MCF-7 cell proliferation data in the presence of AKT and/or AKT siRNA and tamoxifen. E2 + tamoxifen blocked about 20% of E2-induced growth. However, with the combination of MEK siRNA + tamoxifen, resistance is reversed by approximately 20–25%; with AKT siRNA + tamoxifen, resistance is reversed by approximately 50–55%. In the presence of both MEK siRNA + AKT siRNA + tamoxifen, resistance is reversed by about 70–75%. Cell viability in these studies is more than 90% as shown by the ability of cells to exclude trypan blue. The data shown here are from three separate experiments, represented as the means ± SE. pMAPK, phospho-MAPK; p-ER, phospho-ER; p-AKT, phospho-AKT; TAM, tamoxifen.

 

Figure 8
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  		Fig. 8. Inhibition of AKT in MEK-AKT/MCF-7 Cells Reverses Tamoxifen Resistance

In MEK-AKT/MCF-7 cells MEK siRNA and AKT-2 siRNA significantly blocks phosphorylated MAPK (panel A) and AKT (panel B) levels up to 120 h. However, random siRNA had no effect even after 120 h. Immunofluorescence shows that inhibition of phosphorylated MAPK levels by MEK siRNA specifically blocked ER phosphorylation at Ser118, and AKT siRNA blocked ER phosphorylation at Ser167 in MEK-AKT/MCF-7 cells (panel C). Random siRNA had no effect on Ser phosphorylation. Panel D shows MEK-AKT/MCF-7 cell proliferation data in the presence of AKT and/or MEK siRNA and tamoxifen. E2 + tamoxifen blocked approximately 10% of E2-induced growth. However, the combination of MEK siRNA + tamoxifen reversed resistance by about 30–35%; the combination of AKT siRNA + tamoxifen reversed resistance by about 70–75%, and in the presence of both MEK siRNA + AKT siRNA + tamoxifen, the tamoxifen resistance is reversed by approximately 80–85%. Cell viability in these studies is more than 90%, as shown by the ability of cells to exclude trypan blue. The data shown here are from three separate experiments, represented as the means ± SE. p-MAPK, phospho-MAPK; p-ER, phospho-ER; TAM, tamoxifen.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The development of acquired resistance to antihormonal agents in breast cancer is a major therapeutic problem. In this report we examined the mechanism(s) underlying the ligand-independent activation of ER{alpha} by growth factor signaling and its effect on the agonist and antagonist actions of tamoxifen. Our data using EGFR/MCF-7 and AKT/MCF-7 cells show that activated AKT can phosphorylate Ser 167 in the AF-1 domain of ER{alpha} (Fig. 4BGo). This enhances the interaction between ER{alpha} and AIB1 in the presence of both estrogen and tamoxifen (Fig. 5Go, C and D), leading to tamoxifen resistance (Fig. 4CGo). We have shown that in MEK/MCF-7 cells, activated MAPK phosphorylates Ser 118 in the AF-1 domain of ER{alpha} (Fig. 4BGo), and this enhances the interaction between ER{alpha} and AIB1 in the presence of estrogen, whereas tamoxifen reduces ER{alpha}-AIB1 interaction (Fig. 5BGo), and these cells maintain sensitivity to tamoxifen (Fig. 4CGo). The data obtained with S167E and S167A ER{alpha} mutants and AKT siRNA suggest that, in a Tam-resistant MCF-7 system, AKT is the dominant pathway responsible for antiestrogen resistance. Taken together the above data suggest that different serine phosphorylation sites in AF-1 domain of ER{alpha} significantly influence the agonistic and antagonistic action of tamoxifen.

It was previously shown that the AF-1 region of ER{alpha} contains phosphorylation sites for a number of kinases including MAPK, AKT, and cyclin A/cdk2 (13, 15, 49). Some of these sites are conserved between ER{alpha} and ERß (50). However, the AKT phosphorylation site is present in ER{alpha} (50) but not in ERß, suggesting the possibility that AKT-induced agonistic effects of tamoxifen are mediated through ER{alpha}. The precise details of how the EGFR/HER-2 kinase pathway modulates ER function remain to be elucidated. We currently favor a model in which ER{alpha} is a substrate of ErbB-induced AKT. The strongest evidence in favor of this model is that AKT-induced phosphorylation of Ser-167 in ER{alpha} leads to enhanced ER{alpha}-AIB1 interaction and tamoxifen resistance both in EGFR/MCF-7 and AKT/MCF-7 cells. Our experimental data with AKT and MEK siRNA showed that reduction of AKT alone was sufficient to reverse tamoxifen resistance by 50–70% in EGFR/MCF-7 and MEK-AKT/MCF-7 tamoxifen-resistant cells. However, inhibition of both AKT and MAPK further enhanced sensitivity to tamoxifen by 70–80% (Figs. 7Go and 8Go). In addition, other members of the ER signaling pathway may be substrates of MAPK and AKT-mediated signaling. There is ample precedent for phosphorylation of steroid hormone receptor coactivators and corepressors by growth factor-mediated signaling (46, 51). To date, MAPK has been strongly implicated in modulating coregulator function, but there is no knowledge about such regulation through the AKT pathway in breast cancer cells. Further experiments are required to determine whether AKT signaling can affect ER function through phosphorylation of coregulators.

EGFR/HER-2 are overexpressed in a variety of human tumors, including breast cancers, and are associated with a poor prognosis (52) and resistance to chemo- and endocrine therapy (33, 40, 53). In vitro studies have implicated EGFR in acquiring resistance to antiestrogen therapy (32, 54). Similarly, EGFR signaling has been linked to the progression of androgen-responsive prostate cancer to androgen-independent/hormone-refractory tumors (55). Unfortunately, most of the drugs, especially against the EGFR/HER-2 pathway, have failed to elicit a significant response in many solid tumors including breast cancer (41). One of the reasons for the limited success of drugs targeted against EGFR/HER-2 to reverse or prevent the antiestrogen resistance may be due to constitutive activation of downstream signaling molecules such as phosphatidylinositol 3-kinase/AKT-, Ras/MAPK-, or protein kinase C-signaling pathways that support growth in the presence of tamoxifen (23, 56). However, a recent study suggests that breast and prostate cancer cells can acquire resistance to gefitinib (ZD1839/Iressa an EGFR inhibitor) by increasing other signaling pathways such as IGF-1 receptor pathway (57). Because AKT is one of the major downstream signaling molecules for ErbB and IGF-mediated signaling, inhibiting AKT could prevent endocrine therapy resistance.

We have previously shown that MAPK-induced Ser118 phosphorylation lowers the estrogen requirement for optimal tumor growth, yet these tumors are still sensitive to antiestrogens such as tamoxifen and ICI 182,780 (6). Consistent with our data, clinical studies also suggest that Ser118 phosphorylation of ER{alpha} is associated with better disease outcome in women treated with tamoxifen (58, 59). In contrast, clinical data also show that the presence of activated AKT in breast cancer predicts a worse outcome among endocrine-treated patients (24, 25). Taken together, our data show that different serine phosphorylation sites in AF-1 domain of human ER{alpha} significantly influence the agonist and antagonist actions of tamoxifen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Lines and Reagents
DMEM, phenol red-free DMEM, recombinant human insulin, and fetal bovine serum were from Life Technologies (Gaithersburg, MD). Charcoal-treated fetal bovine serum was from Cocalico Biologicals (Cocalico, PA). The E2 and 4-hydroxytamoxifen were from Sigma Chemical Co. (St. Louis, MO). The E2 pellets (0.72-mg pellet, 90-d release) and tamoxifen (5 mg pellet, 90-d release) were obtained from Innovative Research of America (Sarasota, FL). Phospho-MAPK antibody was from New England Biolabs (Beverly, MA). phospho-Akt (Ser 473) rabbit polyclonal antibody was from Cell Signaling Technology (Beverly, MA). ER antibody was from NeoMarkers (Freemont, CA). AIB1 antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit polyclonal was obtained from Trevigen (Gaithersburg, MD). Human breast cancer MCF-7 cells were purchased from American Type Culture Collection (Manassa, VA). The cells were grown in DMEM supplemented with 10 µg/ml insulin, 5% fetal calf serum, and antibiotics (penicillin/streptomycin).

Transfection and Establishment of Stable Cell Lines
Constitutively activated MAPK kinase (MEK) cDNA was kindly provided by Dr. Ahn (University of Colorado, Boulder, CO). In brief, constitutively activated MEK was constructed by combining a deletion of a helix encompassing residues 32–51 with the substitution of glutamic or aspartic acid for Ser218 and Ser222 (60). For establishment of clonal cell lines, cells were grown to approximately 60–70% confluency in complete medium, harvested by trypsinizing, and suspending in complete medium at 1 x 105 cells/ml. After mixing 0.4 ml of cells with DNA (1 µg of pMCL hemagglutinin-tagged MEK/cytomegalovirus promoter) and electroporating in a Bio-Rad Gene Pulsar (Bio-Rad Laboratories, Hercules, CA) at 950 µF and 0.22 kV/cm (t = 20–30 msec), cells were allowed to stand at room temperature for 10 min before the addition of 5 ml of complete medium and incubation for 48 h. Cells were then split and grown in the presence of hygromycin to isolate at least 20 clonal hygromycin-resistant cell lines. Immunoblot analysis and kinase assay were done to select the constitutively activated MEK expressing (MEK/MCF-7 cell) and control MCF-7 cell lines.

MCF-7 cells were stably transfected with full-length EGFR cDNA (pRCMV3 vector obtained from Dr. Gordan Gill, University of California, San Diego, CA), myr-AKT-2 by electroporation using a Bio-Rad gene pulsar at 950 µF and 0.22 kV/cm (t = 12–14 msec). Stable transfectants were selected in the presence of 250 µg/ml G418 (Life Technologies) for 2–3 wk. Individual antibiotic-resistant colonies were isolated and screened for the expression of the corresponding protein by immunoblot analysis using anti-EGFR antibody (clone-528) or AKT antibody. All cell lines were routinely tested for mycoplasma contamination and found to be negative.

ER-Dependent Reporter Transcriptional Activity
MCF-7 cell lines were propagated in DMEM containing 5% dextran-coated charcoal (DCC)-stripped fetal bovine serum for 3 d before the onset of experiments. For experiments, the cells were seeded into 24-well plates at a density of 1 x 105 cells per well and allowed to reach approximately 60% confluence. Using Superfect (QIAGEN, Chatsworth, CA) according to the manufacturer’s instructions, the cells were then transfected with 1.0 µg/well of either the estrogen-responsive reporter, ERE-tkLuc, containing a Vitellogenin A2-derived ERE as described elsewhere (61, 62) or the corresponding empty vector, tkLuc (with no ERE), along with 0.1 µg/well of the internal control plasmid pRL, used to correct for transfection efficiency. After 4 h, the transfected cells were washed and then incubated in fresh DMEM containing 5% DCC, supplemented with E2 (10–8 M), ICI 182,780 (10–7 M), 4-hydroxytamoxifen (10–6 M), and/or 0.1% ethanol vehicle alone for 48 h. The cells were harvested and the cell extracts were assayed for luciferase activities using the dual-luciferase reporter system (Promega Corp., Madison, WI) according to the protocol specified by the manufacturer. The magnitude of activation in tkLuc-transfected cells was determined after normalization to the activity of pRL and then taken as 1.0-fold. This value was used to calculate the relative (fold) change in transcriptional activities of ERE-tkLuc-transfected cells, after normalization to pRL activity. All data have been normalized as the ratio of raw light units to pRL units corrected for pRL activity and are shown as the means ± SE from three separate experiments (performed in triplicate).

The Immunofluorescence Analysis
Cell were plated on coverslips in a 24-well dish and grown in 5% DCC in DMEM phenol red-free medium for 48 h and then treated as indicated in the figure legend. The cells were washed with PBS and were fixed with 100% methanol for 5 min. The fixed cells were blocked using 5% BSA/PBS solution for 30 min and incubated overnight with primary ER-Ser118 antibody diluted (1:50) and /or primary Ser167 antibody diluted (1:50) in Tris buffer saline [50 mM Tris-HCl (pH 7.4), 150 mM NaCl] (Cell Signaling Technology). Cells were incubated with Alexa 488 goat antimouse or antirabbit antibody and DAPI (Molecular Probes, Eugene, OR) for 1 h (1:100). The coverslips were mounted on slides with Slow Fade reagent (Molecular Probes), and images were captured using fluorescence microscopy.

Western Immunoblot Analysis
Western blotting was performed as described previously (63) using a standard protocol. Crude protein extracts were obtained by lysing 5 x 106 cells in a buffer [50 mM Tris-HCl (pH 7.6), 1% Nonidet P-40, 2 mM EDTA, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM sodium ortho-vanadate, 2 mM EGTA, 4 mM sodium p-nitro phenyl phosphate, 100 mM sodium fluoride] supplemented with protease inhibitors [leupeptin (0.5%), aprotinin (0.5%), and phenylmethylsulfonyl fluoride (0.02%)]. Samples containing 50 µg of total protein were electrophoresed on 7.5 or 10% sodium dodecyl sulfate-polyacrylamide gels and transferred on to nitrocellulose membrane by electroblotting. To determine an ER-AIB1 association, 1 mg of cell lysate was precipitated with the AIB1 antibody and protein G-Sepharose (Sigma, St. Louis, MO) followed by immunoblot analysis for ER{alpha} and/or coactivators. Membranes were probed with antibodies as indicated, followed by horseradish peroxidase-conjugated mouse or rabbit secondary antibodies and enhanced chemiluminescence detection (Amersham Biosciences Corp, Piscataway, NJ). Intensities of the bands were quantified using Unscan-it software (Silk Scientific, Inc., Orem, UT) and normalized to the corresponding GAPDH levels.

RNA Interference Assay
Cells were plated in 24-well tissue culture plates (Corning Laboratories, Houston, TX), at a density of 1 x 104 cells per well, in DMEM containing 5% fetal bovine serum. After 24 h, the seeding medium was removed, cells were washed twice with PBS, and the medium was replaced with phenol red-free DMEM with 5% charcoal-stripped serum. To suppress AKT-2 and/ or MEK-1 expression, cells were transfected with 0.8 mg/well of Smart-pool siRNA duplexes (AKT-2 and/or MEK-1) from Upstate Biotechnology, Inc. (Lake Placid, NY) using Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Cells were harvested and extracts prepared for immunoblot analysis at 0, 48, 72, and 120 h.

Tumorigenesis in Scid-Beige Mouse
MEK/MCF-7 cells (1 x 106 cells per mouse) or EGFR/MCF-7 cells were suspended in matrigel and injected sc on either side of the mouse in Scid-beige ovariectomized mice (Taconic Farms, Germantown, NY). The estrogen-treated mice received sc implantation of E2 pellets (E2-0.72 mg/biodegradable carrier-binder pellet) at the time of cell inoculation. Pellets containing the antiestrogen Tamoxifen (5 mg/biodegradable carrier-binder pellet) were implanted after 30 d. All pellets were 90-d biodegradable slow-release pellets obtained from Innovative Research of America (Sarasota, FL). Five animals were included in each group in two sets of independent experiments. Tumor growth was monitored by caliper measurements twice weekly by measuring the length (a) and width (b) of tumor and volumes calculated as (a x b2)/2.


   ACKNOWLEDGMENTS
 
We thank Dr. N. Ahn (University of Colorado, Boulder, CO) for providing activated MEK cDNA and Dr. J. Q. Cheng (University of South Florida, Tampa, FL) for providing AKT cDNA.


   FOOTNOTES
 
This work was supported in part by National Institutes of Health Grant RO1 CA 83964-01 (to K.B.R) and the Department of Pathology, Wayne State University.

The authors have nothing to declare.

First Published Online February 2, 2006

Abbreviations: AF-2, Activation function 2; AIB1, amplified in breast cancer 1; DAPI, 4',6-diamidino-2-phenylindole; DCC, dextran-coated charcoal; E2, 17 ß-estradiol; EGFR, epidermal growth factor receptor; ER, estrogen receptor; ERE, estrogen response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PKB/Akt, protein kinase B; MEK, MAPK kinase; myr-Akt, myristoylated AKT; siRNA, short inhibitory RNA.

Received for publication July 12, 2005. Accepted for publication January 25, 2006.


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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα
Coregulators:   AIB1
Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen



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