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Molecular Endocrinology 12 (7): 973-985
Copyright © 1998 by The Endocrine Society

FRA-1 Expression Level Modulates Regulation of Activator Protein-1 Activity by Estradiol in Breast Cancer Cells

Alexandre Philips1,2, Catherine Teyssier1, Florence Galtier, Corinne Rivier-Covas, Jean-Marc Rey, Henri Rochefort and Dany Chalbos

Institut National de la Santé et de la Recherche Médicale Hormones and Cancer (U 148), and Université de Montpellier I 34090 Montpellier, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We compared the effect of estradiol on activator protein-1 (AP-1) activity in estrogen receptor positive (ER{alpha}+) and estrogen receptor negative (ER{alpha}-) human breast cancer cell lines transiently transfected with the AP-1-responsive reporter plasmid AP-1-TK-CAT and an ER{alpha} expression vector. While estradiol increased AP-1 activity in the ER{alpha}+ cell lines MCF7, ZR75.1, and T47D, it decreased (MDA-MB231 and BT20 cells) or had no significant effect (MDA-MB435 cells) on AP-1-mediated transcription in ER{alpha}- cells. Estradiol also inhibited AP-1 activity in ER{alpha}-MDA-MB231 cells stably transfected with ER{alpha} and in which ER{alpha} levels are close to those found in MCF7. Use of ER{alpha} mutant expression vectors demonstrated that the DNA-binding domain of ER{alpha} was needed for stimulation or inhibition of AP-1 activity by estradiol but suggested that ER{alpha} binding to estrogen-responsive elements was not required for these effects. Changes in regulation paralleled quantitative and qualitative changes in protein binding to AP-1 sites, as demonstrated by gel shift assay: protein binding was greater and DNA/protein complexes migrated faster for ER{alpha}- than for ER{alpha}+ cells. In fact, by Northern blot, a high level of Fra-1 mRNA was found in BT20 and MDA-MB231 cells as compared with ER{alpha}+ cells, and MDA-MB435 cells showed an intermediary level of expression. The differential expression of Fra-1 in MCF7 and MDA-MB231 cells was confirmed at the protein level by supershift experiments. In addition, overexpression of Fra-1 in MCF7 cells decreased the positive effect of estradiol while inhibition of Fra-1 expression in MDA-MB231 cells, by transient transfection of the Fra-1 antisense expression vector, abolished the negative effect of the hormone. In conclusion, we demonstrated that ER{alpha}- breast cancer cell lines differ from ER+ cells by a high level of AP-1 DNA-binding activity due, at least in part, to high Fra-1 constitutive expression. High Fra-1 concentration is crucial for the negative regulation of AP-1 activity by estradiol and thus may take part in estradiol-induced inhibition of cell proliferation in ER{alpha}- breast cancer cells transfected with ER{alpha} expression construct.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogens (1, 2), acting via nuclear steroid receptors, and growth factors such as epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), and transforming growth factor-{alpha}, which bind to tyrosine kinase-associated membrane receptors (3), are major mitogens of human breast cancers. The two regulatory pathways are interactive. Estrogens and growth factors have a synergic effect on the control of cell growth (4). In addition, antiestrogens, widely used in the treatment of breast cancer as inhibitors of estrogen-induced responses, also inhibit (in the absence of estrogens) the effect of growth factors on cell proliferation (5) and on regulation of specific genes (6, 7, 8).

Regulation of eukaryotic gene expression generally occurs at the transcription level due to the interaction of trans-acting regulatory proteins with sequence elements located in promoter regions of target genes. It is a complex phenomenon resulting from the intervention of multiple extracellular stimuli acting via different signal transduction pathways. One of these pathways involves steroid hormone receptors that act as ligand-activated transcription factors. These superfamily members are characterized by a highly conserved DNA-binding domain (DBD), which forms two zinc finger structures, and a less conserved COOH-terminal ligand-binding domain (LBD). Once activated, they induce transcription of target genes after binding to specific DNA sequences, called hormone-responsive elements, present in their promoter region (reviewed in Refs. 9, 10). Conversely, binding of growth factors to tyrosine kinase membrane receptors, through generation of second messengers activating a cascade of kinases, results in induction and/or activation of some transcription factors (11, 12). Among them, activator protein-1 (AP-1) consists of dimers of proteins encoded by fos and jun gene families, which have been widely implicated in differentiation, cell proliferation, and transformation (13). Jun (c-Jun, JunB, JunD) and Fos (c-Fos, Fra-1, Fra-2, and FosB) proteins share a conserved region containing the basic DBD and the leucine zipper dimerization motif. Jun proteins can form homodimers or heterodimers with proteins of the Fos family (14). These dimers regulate gene transcription through interactions with a specific DNA sequence, the 12-O tetra-decanoyl-phorbol-13 acetate (TPA) responsive element (TRE), also referred to as the AP-1 site (11, 14).

Transcriptional interferences between estrogens and growth factor pathways were recently observed. EGF and IGF-I were reported to increase estrogen responsive element (ERE)-mediated responses (for a review, see Ref. 15) by activating the estrogen receptor (ER{alpha}) by phosphorylation through the mitogen-activated protein kinase pathway (16, 17). We and others have previously shown that estradiol could modulate AP-1 activity (18, 19, 20, 21). In transient transfection experiments (19), estradiol increased basal AP-1-mediated transcription level in the ER{alpha} positive (ER{alpha}+) human breast cancer cell line MCF7. A positive effect of estrogens was also detected in the presence of IGF-I or EGF in conditions whereby the hormone did not modify the synthesis of c-Fos and c-Jun mRNA or after cotransfection with c-Fos and c-Jun expression vectors. Antiestrogens, which inhibited growth factor-induced cell proliferation, also inhibited growth factor-induced AP-1 activity (19); therefore, regulation of AP-1 activity by estradiol/ER{alpha} complexes was parallel to growth regulation.

While the role of estrogens in the promotion and development of breast cancer has been well documented for many years, the mechanism by which they stimulate the growth of hormone-responsive cancers is still poorly understood. Estrogens can directly increase proliferation of ER{alpha}+ epithelial cancer cells, as demonstrated by the use of established hormone-responsive human breast cancer cell lines (1, 2), and ER{alpha} is necessary for their mitogenic effect. However, the presence of ER{alpha} alone does not seem to be sufficient to promote estrogenic regulation of growth, as shown by studies on several types of ER{alpha}- cells (for a review, see Ref. 22) including breast cancer (23, 24, 25) and normal mammary cells (23). Surprisingly, when ER{alpha} was introduced by stable transfection in ER{alpha}- cells to restore estrogen regulation, estradiol inhibited cell proliferation, even when ER{alpha} was expressed at physiological levels. Estradiol treatment of stably ER{alpha}-transfected cells, which did not express endogenous ER{alpha}, was also shown to decrease the number and volume of lung metastases when injected in nude mice (25). The mechanisms involved in these opposite regulations are presently unknown. In transfected ER{alpha}- cells, estradiol efficiently induced transcription of an ERE-containing reporter construct and triggered the expression of some (but not all) endogenous estrogen-responsive genes (22, 23, 24).

Our hypothesis in the present study was that, in contrast to ERE-mediated responses, AP-1-responsive genes could be regulated differently by estradiol in ER{alpha}-transfected breast cancer cell lines expressing or not endogenous ER{alpha} and whose growth is respectively increased or decreased by the hormone. We therefore compared the effect of estradiol on AP-1 activity in both cell types. Using transient transfections, it was found that estradiol enhanced growth factor-induced AP-1-mediated transcription in all ER{alpha}+ breast cancer cell lines tested but inhibited AP-1 activity in ER{alpha}- cells cotransfected with an ER{alpha} expression vector. We further investigated the mechanism of this cell specificity. We presently report that ER{alpha}+ and ER{alpha}- cells express different concentrations of Fra-1, and that high Fra-1 expression level is responsible for the negative regulation of AP-1 activity by estradiol in ER{alpha}- breast cancer cell lines.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reverse Regulation of AP-1 Activity by Estradiol in ER{alpha}+ and ER{alpha}- Breast Cancer Cell Lines Transfected with ER{alpha}
We first compared estradiol regulation of transcriptional activity of AP-1 complexes (AP-1 activity) in MCF7 (ER{alpha}+) and MDA-MB231 (ER{alpha}-) human breast cancer cells. Cells were transfected with the reporter construct (AP-1)4-TK-CAT containing four AP-1 binding sites and the wild-type ER{alpha} expression vector HEGO. As shown in Fig. 1AGo, basal AP-1-mediated chloramphenicol acetyltransferase (CAT) activity was increased by estradiol in MCF7 cells but decreased by the hormone in MDA-MB231 cells. Experiments were also conducted in the presence of EGF or IGF-I, at concentrations that were maximally efficient for inducing c-Fos and c-Jun (4, 19) (Fig. 1AGo), or after cotransfection of the c-Jun expression vector (Fig. 1BGo). In MCF7 cells, 8 nM EGF and 5 nM IGF-I, respectively, increased AP-1-dependent transcription by 3.6- and 6.5-fold as compared with control cells. In MDA-MB231 cells, 8 nM EGF increased AP-1 activity by only 50%, 5 nM IGF-I had no significant effect (Fig. 1AGo), and both growth factors were not more efficient when they were tested in a wide range of concentrations between 0.1 nM and 1 µM (not shown). Induction of AP-1 activity by c-Jun overexpression was also much lower in MDA-MB231 cells that in MCF7 cells (1.5-fold and 10-fold induction as compared with control cells, respectively). Regardless of the experimental conditions, estradiol treatment enhanced AP-1 activity in MCF7 cells but inhibited AP-1 activity in MDA-MB231 cells. Similar effects of estradiol were obtained when the AP-1-responsive promoter was induced by cotransfection of c-Jun expression vector (Fig. 1BGo). In MDA-MB231 cells, estradiol inhibited AP1-mediated CAT activity in a dose-dependent manner (Fig. 1CGo). Doses of estradiol required to repress AP-1 activity in MDA-MB231 cells (half-maximal inhibition at about 50 pM, maximal inhibition at 0.1 nM) were the same as those previously reported to activate AP-1 activity in MCF7 cells (19). Modulation of AP-1 activity by estradiol in both cell lines was dependent on the integrity of AP-1 sites, since two mutations in the AP-1 binding site totally abolished the hormonal effect (Fig. 1DGo).



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  		Figure 1. Effect of Estrogen on AP-1 Activity in MCF7 and MDA-MB231 Breast Cancer Cell Lines Transfected with ER{alpha}

In all experiments, steroid-stripped MCF7 and MDA-MB231 cells were transfected for 16 h and then treated for 28 h with estradiol (E2, 1 nM; black bars) or vehicle (open bars), in combination with 8 nM EGF or 5 nM IGF-I when indicated. A, Cells were transfected with 100 ng of wild-type-ER{alpha} expression vector (HEGO, 40) and 1 µg of (AP-1)4-TK-CAT. B, Cells were transfected with HEGO (200 ng), (AP-1)4-TK-CAT (1 µg), and increasing concentrations (0, 50, 200, 500 ng) of pCI-c-Jun. C, MDA-MB231 cells, transfected with HEGO (200 ng) and 1 µg of (AP-1)4-TK-CAT reporter plasmid, were incubated with increasing concentrations of estradiol as indicated. D, Cells were transfected with HEGO (100 ng) and 1 µg mutated (AP-1)4-TK-CAT reporter plasmid, which does not allow binding of AP-1 factors. CAT activity was measured in whole cell extracts after normalization for ß-galactosidase activity, as described in the Materials and Methods. The results are the mean (±SD) of three independent experiments. They are expressed in arbitrary units; CAT activity measured in control cells was assigned a value of 1.

 
To determine whether estrogen stimulation of AP-1-dependent transcription was restricted to MCF7 cells or could be extended to other breast cancer cells, we then tested the effect of estradiol in several cell lines transfected with the reporter plasmid (AP-1)4-TK-CAT and the expression plasmid HEGO (Fig. 2Go). We have previously shown that, in MCF7 cells, estradiol, which induced c-Fos (4, 19), had no additive effect on the accumulation of c-Fos mRNA when it was induced by growth factors. The experiment was therefore conducted in the presence of 8 nM EGF to eliminate an effect of estradiol on c-Fos synthesis. As observed in MCF7 cells, estradiol increased AP-1-dependent transcription in two other wild-type ER{alpha}+ human breast cancer cell lines (T47D and ZR75.1). In contrast, and as in the MDA-MB231 cell line, AP-1 activity was decreased by estradiol in BT20 (~40% inhibition) and had no significant effect in MDA-MB435 cells. Comparable effects of estradiol were obtained in the absence of EGF (not shown).



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  		Figure 2. Effect of Estrogen on AP-1 Activity in ER{alpha}+ and ER{alpha}- Human Breast Cancer Cell Lines Transfected with ER{alpha}

All cell lines, expressing (ER+) or not (ER-) endogenous ER{alpha}, were grown in steroid-stripped medium and transfected with (AP-1)4-TK-CAT (1 µg) and HEGO (100 ng) as described in Materials and Methods and Fig. 1Go. Cells were then treated with 1 nM estradiol (black) or vehicle (open bars) in the presence of 8 nM EGF. The results are expressed as in Fig. 1Go. They are the mean (±SD) of three independent experiments. They are expressed in arbitrary units; CAT activity measured in control cells was assigned a value of 1.

 
ER{alpha} Concentration Is Not Responsible for the Reverse Regulation of AP-1 Activity
The reverse effect of estradiol/ER{alpha} complexes on AP-1 activity in MCF7 and MDA-MB231 cells could be the result of a different ER{alpha} expression level in the two cell lines. To eliminate this possibility, increasing amounts of ER{alpha} expression vector were transfected in cells together with the (AP-1)4-TK-CAT construct (Fig. 3AGo).



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  		Figure 3. Effect of ER{alpha} Concentration on Modulation of AP-1 Activity

A, Steroid-stripped MCF7 and MDA-MB231 cells were transfected with 1 µg (AP-1)4-TK-CAT and the indicated concentrations of the ER{alpha} expression vector HEGO for 16 h, as described in Fig. 1Go. They were then exposed for 28 h with 5 nMIGF-I (open bars) or 5 nM IGF-I plus 10 nM estradiol (black bars). The results shown are the mean (±SD) of triplicate from a single experiment representative of three separate assays. They are expressed in arbitrary units, CAT activity measured in cells not cotransfected with HEGO and cultivated in the absence of estradiol being assigned a value of 1. B, The steroid-stripped MDA-MB231 clones, HE5, HC1 (both stably transfected with the ER{alpha} expression vector pSG1/HEGO), and PB4 (stably transfected with the empty expression vector) were transiently transfected with (AP-1)4-TK-CAT (1 µg) and treated for 28 h with 10 nM estradiol (black bars) or vehicle (open bars) in the presence of 8 nM EGF. Results are expressed in arbitrary units; CAT activity measured in cells cultivated in the absence of estradiol was assigned a value of 1. They are the mean (±SD) of three independent experiments.

 
In the absence of added estradiol, transfection with the ER{alpha} expression vector enhanced IGF-I-induced AP-1 activity in MCF7 cells (2.7-fold induction for the higher HEGO concentration) and decreased AP-1 activity by approximately 25% in MDA-MB231 cells. Moreover, in MCF7 cells, the positive effect of estradiol, which was detected in the presence of endogenous ER{alpha} alone, was further increased after ER{alpha} overexpression. In contrast, in MDA-MB231 cells, estradiol inhibited AP-1-mediated CAT activity as soon as ER{alpha} expression vector was cotransfected. However, no regulation was observed in the absence of ER{alpha} expression, demonstrating that the receptor was necessary for the estradiol-induced decrease in AP-1 activity observed in these ER{alpha}- cells.

Regulation of AP-1 activity by estradiol was also analyzed in two MDA-MB231 clones (HC1 and HE5) stably transfected with HEGO, and expressing, respectively, 100 and 57 fmol of ER{alpha} per mg of cytosol protein, concentrations roughly equivalent to ER{alpha} concentration in MCF7 cells (25). As shown in Fig. 3BGo, estradiol decreased AP-1 activity in both sublines, while it had no effect in PB5 cells stably transfected with the empty expression vector. We therefore concluded that reverse regulation by estradiol in MCF7 and MDA-MB231 cells could not be explained by ER{alpha} expression levels and that introduction of ER{alpha} in wild-type ER{alpha}- cells was not sufficient to give them the characteristics of wild-type ER{alpha}+ cells with respect to the AP-1 response.

Induction of ERE-Mediated Responses Is Not Required for Modulation of AP-1 Activity by Estradiol
To determine whether induction of ERE-mediated responses was required for regulation of AP-1 activity, we tested the effect of estradiol in MCF7 and MDA-MB231 cells transfected with (AP-1)4-TK-CAT and expression vectors of ER{alpha} mutated in the DBD (Fig. 4Go).



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  		Figure 4. Effect of ER{alpha} Mutants on Modulation of AP-1 Activity by Estradiol

A, Steroid-stripped MCF7 cells were transfected with 1 µg (AP-1)4-TK-CAT alone or 1 µg (AP-1)4-TK-CAT and increasing concentrations (0.2, 0.4, and 0.8 µg) of expression vector coding for an ER{alpha} mutant or wild-type GR (HGO) as indicated. Mutants HE11 and HE91 are derived from HEO, which differs from the wild-type ER{alpha} by a Gly400/Val mutation in the LBD of the ER{alpha} protein (40 ). HE11 is deleted of the DBD ({Delta} 185–251) (41 ). HE91 harbors two point mutations (Glu203/Gly, Ala207/Val) in the DBD (26 ). Cells were then incubated for 28 h with 10 nM estradiol (black bars), 10 nM dexamethasone (gray bars) or vehicle (open bars) in the presence of 5 nM IGF-I. B, Steroid-stripped MDA-MB231 cells were transfected with 1 µg (AP-1)4-TK-CAT and 0.4 µg of the indicated expression vector. Cell treatments were as in panel A. Results are expressed in arbitrary units; CAT activity measured in cells transfected with (AP-1)4-TK-CAT alone and cultivated in the absence of estradiol was assigned a value of 1. The results shown are the mean (±SEM) of duplicate from a single experiment representative of three separate assays.

 
Increasing concentrations of ER{alpha} mutant expression vectors were transfected in MCF7 cells that express endogenous ER{alpha} (Fig. 4AGo). ER{alpha} mutants used in this study are derived from HEO, which differs from the wild-type ER{alpha} of MCF7 cells by a point mutation in the LBD of the protein, resulting in a lower affinity for estradiol (26). Overexpression of the HEO mutant increased the effect of estradiol on AP-1 activity; levels of induction by estradiol were higher than that obtained with the wild-type HEGO construct, and no significant effect was detected in the absence of hormonal treatment (compare Figs. 3Go and 4Go). In contrast to that observed with HEO, the HE11 mutant (27), lacking DBD, was inefficient in increasing the effect of estradiol on AP-1 activity. The results obtained with HE11 could be interpreted as a requirement for the ERE-mediated induction of an intermediary factor. Conversely, DBD could simply be required, for instance, as a structural domain necessary in protein-protein interactions. The HE91 mutant, which harbors two point mutations in the C-terminal side of the N-terminal zinc finger of ER{alpha}, was then used in an attempt to discriminate between these two possibilities. This mutant activates transcription from a reporter gene containing a glucocorticoid-responsive element, but not from a reporter gene containing a consensus ERE (28). Its introduction in MCF7 cells, increased the efficacy of estradiol in modulating AP-1 activity. In contrast, dexamethasone inhibited AP-1-dependent transcription in cells cotransfected with the wild-type glucocorticoid receptor (GR) expression vector HGO. Transfection of HEO in MDA-MB231 cells had no significant effect on AP-1 activity in the absence of added estradiol but increased its inhibition by estradiol more efficiently than HEGO transfection. In MDA-MB231cells, as in MCF7 cells, only HEO and HE91 allowed regulation of AP-1 activity, and the activated GR receptor had a negative effect (Fig. 4BGo).

These results therefore suggested that, in both cell lines, regulation of AP-1 activity by estradiol required the DBD of ER{alpha} but not the induction of ERE-controlled genes.

Differential Binding of trans-Acting Factors on AP-1 Sites in ER{alpha}+ and ER{alpha}- Cells
In an attempt to pinpoint the mechanisms involved in the reverse regulation of AP-1 activity in ER{alpha}+ and ER{alpha}- cells, we compared the ability of proteins from MCF7 and MDA-MB231 cells to bind the polyoma virus TRE motif in electrophoretic mobility shift assays.

When equal amounts of cellular proteins were analyzed, the intensity of the upper band was higher using MDA-MB231 cell extracts than with MCF7 cell extracts (Fig. 5AGo). In addition, the retarded complexes migrated differently for the two cell lines. This qualitative difference was highlighted after correction for specific binding when 3-fold more proteins were used for MCF7 cells than for MDA-MB231 cells (Fig. 5Go, B–D). In fact, DNA/proteins complexes migrated faster for MDA-MB231 cells than for MCF7 cells.



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  		Figure 5. In Vitro Binding Activity of MCF7 and MDA-MB231 Proteins to AP-1 Sequences

Double-stranded oligonucleotides, corresponding to the polyoma virus or collagenase TRE sequence, were 32P-labeled and incubated for 20 min with MCF7 and MDA-MB231 nuclear extracts, prepared as described in Materials and Methods, from cells incubated for 4 h with IGF-I (5 nM). Complexes were separated on a 5% nondenaturing acrylamide gel. The positions of specific retardations are indicated by filled arrows. A, MCF7 and MDA-MB231 nuclear extracts (10 µg proteins) were incubated with labeled polyoma virus TRE. B, Fifteen micrograms of MCF7 proteins and 5 µg of MDA-MB231 proteins were incubated with polyoma virus TRE. Ten- or 100-fold excess of nonradioactive collagenase TRE or vitellogenin ERE competitors were added with the labeled probe when indicated. C, Nuclear extracts (15 µg of MCF7 proteins and 5 µg of MDA-MB231 proteins) were incubated for 1 h at room temperature with 2 µg of polyclonal antibodies in the binding reaction mixture before addition of labeled polyoma virus TRE sequence. {alpha}Jun and {alpha}Fos antibodies, respectively, react with all the members of Jun and Fos families. Preimmune IgG (IgG, tracks 1 and 4) was used as control. The large arrow shows the position of complexes retarded by the presence of antibodies. D, Fifteen micrograms of MCF7 proteins and 5 µg of MDA-MB231 proteins were incubated with labeled collagenase TRE.

 
Differences in retardation actually reflected differences in protein binding to AP-1 sites. The upper band was efficiently blocked with an excess of unlabeled oligonucleotides containing the collagenase consensus TRE motif but not with oligonucleotides containing the vitellogenin ERE motif. As shown in Fig. 5CGo, it was supershifted after incubation with antibodies directed against all the members of Jun or Fos families. Anti-Fos antibodies almost completely supershifted the band, suggesting that AP-1 complexes were primarily constituted of heterodimers in cells treated for 4 h with the growth factor. However, anti-Jun antibodies only supershifted a small fraction of the DNA/protein complexes. This discrepancy is probably due to the weak efficiency of anti-Jun antibodies since, in the same experimental conditions, a larger fraction of the complexes was supershifted with antibodies specifically directed against the c-Jun protein (not shown). As shown in Fig. 5DGo, the same differences in migration were obtained when a labeled collagenase consensus TRE site was used in the assay.

We then investigated AP-1 DNA-binding activity in several ER{alpha}+ and ER{alpha}- breast cancer cell lines. Sp1 DNA-binding activity was tested in parallel as a control of the efficiency of nuclear protein extraction in the different cell lines. As shown in Fig. 6Go, where the same amounts of protein cell extracts from various cell lines were analyzed, higher protein binding to the polyoma virus TRE site and faster migration of DNA/protein complexes were detected for the three ER{alpha}- breast cancer cell lines tested (MDA-MB231, MDA-MB435, and BT20), as compared with the three ER{alpha}+ cell lines MCF7, T47D, and ZR75.1. In the two ER{alpha}+ MDA-MB231 clones (HE5 and HC1) the extent of retardation of TRE sequences was identical to that obtained in wild-type MDA-MB231 cells, demonstrating that ER{alpha} expression was not sufficient to explain the slower migration with ER{alpha}+ cells.



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  		Figure 6. In Vitro Binding Activity to AP-1 Sites of Breast Cancer Cell Proteins

Nuclear extracts (10 µg proteins) from ER{alpha}+ (T47D, ZR75.1, MCF7) and ER{alpha}- (MDA-MB231, MDA-MB435, BT20) cell lines were incubated with labeled double-stranded oligonucleotides containing either polyoma virus TRE sequence (top panels) or consensus sequence for Sp1 DNA-binding site (bottom panels), as described in Materials and Methods and Fig. 4Go. Complexes were separated on 5% nondenaturing acrylamide gels. Positions of specific (filled arrow) and nonspecific (open arrows) retardations are indicated.

 
Differential Expression of Fos Family Members in ER{alpha}+ and ER{alpha}- Breast Cancer Cell Lines
We then investigated whether the different results obtained with ER{alpha}+ and ER{alpha}- cells in gel retardation assays were due to a difference in the composition of AP-1 complexes in these cells.

We first compared by Northern blot experiments mRNA expression of Fos and Jun family members in MCF7 and MDA-MB231 cells cultivated for increasing periods of time in the presence of IGF-I (Fig. 7Go). In MCF7 cells, IGF-I induced c-Fos, c-Jun, and Fra-1 mRNAs. In contrast, in MDA-MB231 cells, mRNA levels were increased by TPA but not changed by IGF-I (Fig. 7AGo) or EGF (not shown) treatment. These results were in agreement with those obtained in transfection experiments where growth factors were only slightly efficient (EGF) or inefficient (IGF-I) in inducing AP-1 activity, contrary to what was observed in MCF7 cells (Fig. 1Go). Fra-2, FosB, JunB, and JunD mRNA expression were all at a very low level in both MCF7 and MDA-MB231 cells (not shown). The most obvious difference between the two cell lines was the high constitutive level of Fra-1 mRNA detected in MDA-MB231 cells. We therefore evaluated Fra-1 mRNA expression in other ER{alpha}- and ER{alpha}+ breast cancer cell lines (Fig. 7BGo). Fra-1 mRNA was, as in MDA-MB231 cells, highly expressed in ER{alpha}- BT20 cells. It was undetectable in T47D and ZR75.1 ER{alpha}+ cell lines under the same experimental conditions. An intermediary expression level was found in ER{alpha}- MDA-MB435 cells.



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  		Figure 7. Northern Blot Analysis of Members of AP-1 Complexes in Breast Cancer Cell Lines

A, MCF7 and MDA-MB231 cells were grown in steroid-stripped medium, as described in Materials and Methods. MCF7 cells were then incubated with IGF-I (5 nM) and MDA-MB231 cells with IGF-I (5 nM) or TPA (50 nM), for the indicated periods of time. Forty micrograms of total RNA were analyzed on a 1% agarose denaturing gel. After transfer onto nylon membrane, RNAs were hybridized to 32P-labeled probes. B, Total RNA was extracted from ER{alpha}+ (T47D, ZR75.1, MCF7) and ER{alpha}- (MDA-MB231, MDA-MB435, BT20) steroid-stripped cells and analyzed by Northern blot as described in panel A. The 36B4 probe, which corresponds to a constant RNA (48 ), was used as a loading control. The mRNA species lengths (kb) are indicated on the left. Two bands were detected with c-Jun (3.4 and 2.6 kb) and Fra-1 (3.3 and 1.7 kb) probes in agreement with the literature (43 50 ).

 
The differential expression of Fra-1 was then confirmed at the protein level by supershift experiments. Nuclear extracts from MCF7 and MDA-MB231 cells were preincubated with antibodies specific for each Fos protein before the addition of labeled oligonucleotides containing the polyoma virus TRE site (Fig. 8Go). Anti-FosB antibodies had no effect on DNA/protein complexes obtained from both cell lines. This absence of effect was not due to the inability of these antibodies to form a supershift complex in the assay since they were able to supershift in vitro translated FosB/c-Jun complexes bound to DNA (not shown). A slight supershift of DNA/protein complexes was observed with anti-c-Fos antibodies in MCF7 cells and with anti-Fra-2 antibodies in both cell lines. However, only antibodies directed against Fra-1 substantially supershifted complexes in MDA-MB231 cells. In contrast, they had no effect in MCF7 cells.



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  		Figure 8. Immunological Detection of Fos Proteins in Complexes Bound to AP-1 Sites in MCF7 and MDA-MB231 Cells

Extracts from steroid-withdrawn cells cultivated for 24 h with IGF-I (5 nM) were incubated for 1 h at room temperature with 2 µg of the indicated polyclonal antibodies in the binding reaction mixture before addition of labeled oligonucleotides containing polyoma virus TRE sequence. Two micrograms of preimmune IgG (IgG, tracks 1 and 4) were used as control. Positions of specific (filled arrow) and nonspecific (open arrow) retardations are indicated. The largest filled arrows mark antibody supershifts.

 
Fra-1 Concentration Modulates the Estradiol Effect on AP-1 Activity
Based on these results, we speculated that estradiol might decrease AP-1 activity when large quantities of Jun/Fra-1 complexes are present, as in ER{alpha}- cells. It would therefore be possible to reverse the estradiol effect by modulating the Fra-1 expression level.

To test this hypothesis, we first overexpressed Fra-1 in MCF7 cells. Cells were transfected with (AP-1)4-TK-CAT, HEGO, and increasing amounts of pCI-Fra-1 expression vector. Experiments were performed in cells cotransfected or not by pCI-c-Jun (Fig. 9AGo). In the absence of estradiol, Fra-1 overexpression increased the basal level of AP-1 activity but had no significant effect when c-Jun expression vector was cotransfected. In both cases, Fra-1 overexpression inhibited stimulation of AP-1-mediated transcription by estradiol. With the highest amount of pCI-Fra-1 expression vector and in the absence of c-Jun cotransfection, induction of CAT activity by estradiol decreased by 50%. In addition, the efficacy of estradiol in stimulating AP-1 activity, which was increased by c-Jun overexpression alone, was decreased by 75% when Fra-1 expression vector was cotransfected. To determine whether Fra-1 overexpression specifically inhibited the estradiol-induced AP-1 activity, the effect of increasing Fra-1 expression was tested in parallel in cells cotransfected by an ERE-containing reporter plasmid. As shown in Fig. 9Go, induction by estradiol of the ERE-ß-Globine-luciferase construct was not significantly altered by Fra-1 overexpression.



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  		Figure 9. Effect of Fra-1 Expression on Modulation of AP-1 Activity by Estradiol

A, Steroid-stripped MCF7 cells were transfected with HEGO (200 ng), increasing concentrations of pCI-Fra-1 (0, 0.4, 1.2 µg), and either 1 µg (AP-1)4-TK-CAT (left panel) or 1 µg ERE-ßGlobine-luciferase (right panel) reporter plasmids. They were cotransfected with 100 ng pCI-c-Jun expression plasmid when indicated. B, Steroid-stripped MDA-MB231 cells were transfected with HEGO (200 ng), increasing concentrations of pCI-Fra-1 antisense expression plasmid (0, 0.6, 1.2 µg), and either 1 µg (AP-1)4-TK-CAT (left panel) or 1 µg ERE-ßGlobine-Luciferase (right panel) reporter plasmids. Cells were then treated with or without 10 nM estradiol (E2) for 28 h, and CAT activity was evaluated as described in Fig. 1Go. Results are expressed in arbitrary units; CAT (left panels) or luciferase activity (right panels) measured in mock-transfected cells cultivated in the absence of estradiol was assigned a value of 1. They all represent the mean (±SD) of three independent experiments.

 
The crucial role of the Fra-1 concentration in controlling the sense of regulation of AP-1 activity by estrogens was confirmed in MDA-MB231 cells in which the Fra-1 expression level was decreased. As shown in Fig. 9BGo, transfection of increasing amounts of Fra-1 antisense expression vector lowered the basal AP-1 activity in a dose-dependent manner. The inhibitory effect of estradiol, which was 65% in control cells, was totally abolished with the highest concentration of antisense construct. By contrast Fra-1 antisense had only a slight effect on ERE-mediated transcription.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This study addressed the question of the cell specificity of interferences between AP-1 complexes and activated ER{alpha}. The estradiol-induced increase in AP-1 activity previously described in MCF7 cells was also observed in other human breast cancer cell lines expressing endogenous ER{alpha}. However, estradiol decreased this activity in ER{alpha}- cell lines, after transfection of an expression vector coding for ER{alpha}.

The difference in the regulation of AP-1 activity did not result from a difference of ER{alpha} expression in the two cell types. First, estradiol had an inhibitory effect in stably transfected MDA-MB231 cells that expressed ER{alpha} at a concentration comparable to that of wild-type MCF7 cells. Second, constitutive overexpression of ER{alpha} in MCF7 cells did not reverse the effect of estradiol but, conversely, enhanced the stimulation of AP-1 activity by the hormone.

Differences in the regulation of AP-1 activity reflected variations in protein binding to AP-1 sites. Protein binding was greater in ER{alpha}- than in ER{alpha}+ cells, in agreement with results of Dumont et al. (29) showing that AP-1 DNA-binding activity was increased in a MCF7 variant expressing reduced ER{alpha} amount. Retardation of TRE sequences was also qualitatively different in ER{alpha}+ and ER{alpha}- cells. ER{alpha}, which alone did not bind to AP-1 sites (Ref. 18 and our unpublished results), was however not responsible for the slower migration of protein/DNA complexes obtained from ER{alpha}+ cells. First, the same migration patterns were observed for wild-type MDA-MB231 cells and clones stably transfected with ER{alpha}. Second, migration was not modified by an excess of consensus vitellogenin ERE. Third, antibodies directed against ER{alpha} were unable to supershift the complexes, and, finally, the addition of baculovirus-produced mouse ER{alpha} did not modify the migration pattern obtained with MDA-MB231 cells (not shown).

The difference in migration thus most likely resulted from the composition of AP-1 complexes, which varied in both kinds of cells. In contrast to ER{alpha}+ cells, ER{alpha}- breast cancer cell lines expressed high constitutive AP-1 binding activity due, at least in part, to high Fra-1 levels. We do not know if high Fra-1 expression is related to the adverse prognosis of ER{alpha}- breast cancer cells, which are more aggressive and metastatic than the ER{alpha}+ forms. Fra-1 is significantly expressed in cycling cells, in contrast to c-Fos, and injection of neutralizing Fra-1 antibodies into Swiss 3T3 fibroblasts reduces DNA synthesis, especially in exponentially growing cells (30). It is devoid of the C-terminal transcriptional activation function (31), which is present in c-Fos and seems to be required for transformation, as measured by focus formation assay (31, 32), but not for tumor formation in nude mice, which was reported for Fos family members lacking this domain (32). In addition, although Fra-1 is, as c-Fos, capable of heterodimerization with Jun proteins and subsequently increases binding to the AP-1 site, it does not bind, unlike c-Fos, the TATA binding protein (TBP) (33). In contrast to c-Fos, its overexpression was reported to inhibit AP-1 activity controlled by c-Jun (34) that may explain the low efficacy of c-Jun overexpression in inducing AP-1-mediated transcription in MDA-MB231 cells in contrast to that observed in MCF7 cells (Fig. 1BGo). However, Fra-1 was also found to enhance the transcriptional activity of JunD (34) and increased the basal AP-1 activity in MCF7 cells (Fig. 9AGo), suggesting that expression of AP-1-controlled genes could differ in ER{alpha}+ and ER{alpha}- breast cancer cells.

Estradiol regulation of AP-1 activity was correlated with the Fra-1 expression level. Fra-1 expression was high in MDA-MB231 and BT20 cells, and AP-1 activity was inhibited by estradiol, whereas it was low in MCF7, ZR75.1, and T47D cells, and AP-1 activity was stimulated by the hormone in this case. In MDA-MB435 cells showing an intermediary level of expression, the estradiol effect was not significant. We therefore tried to reverse the hormonal effect by modulating the composition of AP-1 dimers. In MCF7 cells, increasing the Fra-1 concentration lowered the positive effect of estradiol. In addition, decreasing the endogenous level of Fra-1 in MDA-MB231 cells completely abolished the negative effect of the hormone. However, estradiol still induced AP-1 in MCF7 cells, and no positive effect was observed in MDA-MB231 cells. This could suggest that the Fra-1 level had only reached an intermediary expression level in transfected cells (Fra-1 expression in MDA-MB231 cells transfected with the Fra-1 antisense construct may, for instance, be lowered to a level comparable to that detected in wild-type MDA-MB435 cells). Conversely, Fra-1 expression level might be not sufficient to explain the opposite effect of estradiol, and regulation may implicate another factor. This factor may be the Fra-1 partner in AP-1 complexes whose expression (no differences were however detected in the expression level of Jun family mRNAs, not shown) or phosphorylation state may be different in ER{alpha}+ and ER{alpha}- cells. Alternatively, it may be a transcription intermediary factor such as CPB/p300 that has been implicated in mediating the transcriptional effect of both nuclear receptors and AP-1 (35). In any case, the Fra-1 concentration appeared crucial in the cell-specific effect of activated ER{alpha} on AP-1 activity: increasing its concentration lowered the positive effect, and a negative effect was only observed in cells when many Jun/Fra-1 complexes were present.

AP-1 complex content was previously reported to be important in the transcriptional interference between c-Jun and GR. Maroder et al. (36) showed that c-Jun overexpression increased dexamethasone stimulation of glucocorticoid-responsive element-dependent transcription in the CEM leukemic T cell line, whereas c-Fos overexpression had an inhibitory effect, suggesting an influence of the intracellular c-Fos level in this regulation. Promotor regions of proliferin (37, 38) and neurotensin/neuromedin N (NT/N) (39) genes, which can be bound by both GR and AP-1 complexes, were also described to be positively or negatively regulated by glucocorticoids, depending on the relative amount of c-Jun and Fos protein families. In both cases, glucocorticoids had a positive effect in the presence of c-Jun homodimers. However, c-Fos overexpression reversed the regulation of the proliferin gene (37), while it potentiated activation by the hormone of the NT/N gene (39). Conversely, overexpression of Fra-1 negatively regulated NT/N (39) but positively regulated proliferin (38) gene expression. In our study, using a single AP-1 site, glucocorticoids inhibited AP-1 activity in both MCF7 and MDA-MB231 cells, although they expressed different endogenous Fra-1 levels (Fig. 4Go).

The DBD was required for transcriptional interference between ER{alpha} and AP-1 complexes. However, ER{alpha} did not bind to AP-1 sites (Ref. 18 and our unpublished results) and the HE91 mutant (28), unable to activate transcription from an ERE-containing reporter gene, produced the same result as the wild-type receptor. These results therefore suggested that regulation of AP-1 activity resulted from protein-protein interactions. As for GR (39, 40), a physical in vitro interaction between ER{alpha} and c-Jun was recently reported by Webb et al. (21) and confirmed by us (C. Teyssier and D. Chalbos, unpublished data). This interaction is not sufficient to explain the cell-specific effect of estradiol (our results and Ref. 41). However, the presence of Fra-1 in AP-1 heterodimers might modify physical interactions between c-Jun and activated ER{alpha}.

Stable expression of ER{alpha} in ER{alpha}- cells results in estrogen-dependent inhibition of cell proliferation, whereas estradiol increases the growth of cells expressing endogenous ER{alpha}. As ERE-mediated transcription is increased by estrogens in both cell types (22), it was tempting to speculate that the opposite effect of estradiol on breast cancer cell proliferation, is the result of its reverse effect on some AP-1-controlled responses. Positive regulation of AP-1 activity, which was observed in all studied ER{alpha}+ breast cancer cells, may be necessary for stimulation of cell proliferation. Conversely, in ER{alpha}- breast cancer cells, high constitutive expression of Fra-1 leading to negative regulation (or no regulation for MDA-MB435) of AP-1 activity may explain the negative effect of the hormone on cell growth or at least the absence of a positive effect. However, ER{alpha}- MDA-MB453 breast cancer cells, in which estradiol was reported to increase AP-1 activity (21), might be an exception if activated ER{alpha} inhibits their proliferation. This could then suggest that negative regulation of breast cancer cell growth might also implicate alternative mechanism(s).

In conclusion, high constitutive expression of Fra-1 in ER{alpha}- breast cancer cells is responsible for high AP-1 DNA-binding activity. The Fra-1 concentration, which is low in ER{alpha}+ cells, is crucial in directing to a positive or negative regulation of AP-1 activity by estradiol and might play an important role on the opposite regulation of ER{alpha}- and ER{alpha}+ breast cancer cell proliferation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Human recombinant EGF and IGF-I were purchased from Euromedex (Souffelweyersheim, France) and Saxon Biochemicals (Hanover, Germany), respectively. 17ß-Estradiol was obtained from Hoechst-Marion-Roussel (Romainville, France) and prepared as 1000-fold concentrated stock solutions in ethanol. Anti-Jun antibodies (raised against amino acids 247–263 of mouse c-Jun), anti-Fos antibodies (amino acids 128–152 of human c-Fos), anti-c-Fos antibodies (amino acids 3–16 of human c-Fos), anti-Fra-1 antibodies (amino acids 3–22 of human Fra-1), anti-Fra-2 antibodies (amino acids 3–22 of human Fra-2), and anti-FosB antibodies (amino acids 102–117 of mouse FosB) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture
MCF7 cells were maintained in Ham’s F-12/DMEM (1;1), and all other cell lines were maintained in DMEM. All media were supplemented with 10% FCS and 50 µg/ml gentamycin. For transient transfection experiments, cells were stripped of endogenous steroids by successive passages in phenol red free medium containing 10% (2 days), and then 3% (3 days) dextran-coated charcoal (DCC)-stripped FCS (DCC-FCS) (19). They were then plated at about 80% confluence (106-2 x 106 cells per 35-mm diameter well) 24 h before transfection. For gel retardation assays and RNA extraction, cells, plated in T75 flasks (1:6 dilution), were stripped of endogenous steroids by successive passages in phenol red free medium containing 10% (2 days), 3% (3 days), and finally 1% DCC-FCS (2 days).

Plasmids
(AP-1)4-TK-CAT and (mutated AP-1)4-TK-CAT plasmids were derived from pG1–4XB and pG1–4XAB constructs, respectively (42). Four head-to-tail copies of the wild-type AP-1-responsive element of the polyoma virus enhancer were subcloned into the BamHI site of the eukaryotic expression vector pBLCAT 8PN (19). ERE-ßGlobine-Luciferase reporter plasmid was a gift of P. Balaguer (Montpellier, France). Expression vectors for GR (glucocorticoid receptor), ER{alpha}, and ER{alpha} mutants were kindly donated by P. Chambon (Strasbourg, France). PCI-Fra-1 and pCI-c-Jun were constructed into pCI vector (Promega, Madison, WI) by inserting whole cDNA sequences of human c-Fra-1 (43) and mouse c-Jun (44) under the control of the human cytomegalovirus immediate-early promoter. PCI-antisense Fra-1 construct harbored whole cDNA sequence of c-Fra-1 in antisense orientation.

Transient Transfection and CAT and Luciferase Assays
Twenty four hours after plating, the medium was changed and cells were transfected for 16 h using the calcium phosphate DNA coprecipitation method as previously described (19). When cells were transfected by an expression vector, the same amount of empty vector was transfected in control cells. Two micrograms of CMV-ß galactosidase expression plasmid were used for internal control of transfection efficiency, and pSPT18 DNA was added up to 5 µg total DNA per well. Cells were washed twice with phenol red free medium and treated, as indicated, for 24 h in phenol red free medium containing 1% DCC-FCS. CAT enzyme assays were performed in whole cell extracts after normalization for ß-galactosidase activity (45). Acetylated and nonacetylated forms of [14C]chloramphenicol were separated by TLC. Quantification was performed with a Fuji BAS1000 Bioimaging Analyzer (Raytest, Paris, France). For luciferase assay, cells were lysed for 15 min in the cell culture lysis reagent from Promega. Luciferase activity was measured, as described by Roux et al. (46), using an LKB luminometer (LKB Instruments, Rockville, MD) and normalized for ß-galactosidase activity (46).

Gel Retardation Assay
Cell extracts were prepared as described by Stein et al. (47), aliquoted (10 µg/µl), frozen on dry ice, and stored at -70 C. Protein concentrations were determined by a Bradford protein assay (Bio-Rad SA, Yvry Sur Seine, France). Gel retardation assays were performed using double-stranded oligonucleotides labeled by Klenow in the presence of [32P]dCTP. The sequences of the oligonucleotides were as follows. AP-1 binding sites: 5'-TCGACTGTGCTCAGTTAGTCACTTCC-3' and 5'-TCGAGGAAGTGACTAACTGAGCACAG-3' (polyoma virus TRE sequence) and 5'-CTAGCTGTCTGAGTCATGCA-3' and 5'-AGCTTGCATGACTCAGACAG-3' (collagenase TRE sequence). Consensus Sp1 DNA-binding site: 5'-TCGATCGGGGCGGGGCGA-3' and 5'-GCTCGCCCCGCCC-CGAT-3'. In a typical binding reaction mixture (20 µl final volume), 5–15 µg of proteins were incubated with 3 µg of poly(deoxyinosinic-deoxycytidylic)acid (Pharmacia Biotech, Saclay, France) in binding buffer [50 mM Tris-HCl (pH 8), 12.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol] for 20 min at room temperature. The 32P-labeled DNA (4 x 105 cpm, 0.5 pmol) was then added and the incubation continued for 20 min. The resulting DNA-protein complexes were resolved from the free probes by electrophoresis on a 5% nondenaturing polyacrylamide gel and visualized by autoradiography. In competition experiments, nonradioactive double-stranded competitor was added together with the labeled probe. For super-shift experiments, cell extracts were preincubated for 1 h, in binding buffer, with 2 µg antibodies before the addition of the labeled double-stranded oligonucleotide. The gels were dried and autoradiographed with intensifying screens at -80 C.

Northern Blotting
Total RNA (40 µg) was electrophoresed in 1% (wt/vol) agarose gel containing formaldehyde and transferred to Hybond-N+ membrane (Amersham Corp., Les Ulis, France). 36B4 (48), c-Jun (43), c-Fos (49), and Fra-1 (43) cDNA probes were 32P-labeled by multiprime DNA synthesis using an Amersham kit (SA, 109 cpm/µg). Hybridization in 50% formamide and wash conditions were performed as previously described (8). Filters were autoradiographed with intensifying screens at -80 C.


   ACKNOWLEDGMENTS
 
We are grateful to P. Chambon for providing ER{alpha} and GR expression vectors and 36B4 cDNA probe; B. Wasylyk for PB and PAB constructs; M. Piechaczyk for c-Jun and Fra-1 cDNA plasmids; and P. Balaguer for ERE-ßGlobine-Luciferase reporter construct. We also thank J.-Y. Cance for photographs and colleagues in the laboratory for critical reading of the manuscript.


   FOOTNOTES
 
Address requests for reprints to: Dany Chalbos, Unité 148 INSERM, 60 Rue de Navacelles, 34090 Montpellier, France. E-mail: chalbos{at}u148.montp.inserm.fr

This work was supported by the "Institut National de la Santé et de la recherche Médicale", the Université of Montpellier I, the "Association pour la Recherche sur le Cancer" (Grants 1250 and 1411), the "Ligue Nationale contre le Cancer," and the French "Ministère de la Recherche et de l’Enseignement Supérieur" (fellowship to C.T.).

1 The contributions of the two first authors of this manuscript should be considered equivalent. Back

2 Present address: UMR5535, IGGM, 1919 Route de Mende, 34293, Montpellier Cedex 5, France. Back

Received for publication October 17, 1997. Revision received February 20, 1998. Accepted for publication March 19, 1998.


   REFERENCES
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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