Estrogen Receptor Expression and Function in Long- Term Estrogen ...

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 10 Printed in U.S.A.

Estrogen Receptor Expression and Function in LongTerm Estrogen-Deprived Human Breast Cancer Cells* MEEI-HUEY JENG, MARGARET A. SHUPNIK, TIMOTHY P. BENDER, ERIC H. WESTIN, DEBDUTTA BANDYOPADHYAY, RAKESH KUMAR, SHIGERU MASAMURA†, AND RICHARD J. SANTEN Department of Internal Medicine (M.-H.J., M.A.S., R.J.S.), Department of Microbiology (T.P.B.), University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; Department of Medicine (E.H.W.), The Robert C. Byrd Health Science Center, West Virginia University, Morgantown, West Virginia 26506; Laboratory of Cell Growth Regulation, University of Texas M.D. Anderson Cancer Center (D.B., R.K.), Houston, Texas 77030; and Department of Medicine, Pennsylvania State University (S.M.), School of Medicine, Hershey, Pennsylvania 17033 ABSTRACT Hormone-dependent breast cancer responds to primary therapies that block estrogen production or action, but tumor regrowth often occurs 12–18 months later. Additional hormonal treatments that further reduce estrogen synthesis or more effectively block its action cause additional remissions, but the mechanisms responsible for these secondary responses are not well understood. As a working hypothesis, we postulated that primary hormonal therapy induces adaptive changes, resulting in enhanced estrogen receptor (ER) expression and target gene activation and, further, that secondary treatment modalities interfere with these receptor-mediated transcriptional pathways. To test this hypothesis, we used an MCF-7 breast cancer model system involving deprivation of estradiol in culture for a prolonged period. These long-term estradiol-deprived (LTED) cells adapt by acquiring the ability to regrow in the absence of added estradiol. The experimental paradigm involved the comparison of wild-type cells with LTED cells. As endpoints, we directly assessed ER expression at the messenger RNA-, protein-, and ligand-binding levels and ER functionality by quantitating reporter gene activation and expression of endogenous estrogen target gene messenger RNA, as well as ER

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OMEN with hormone-dependent breast cancer respond to estrogen deprivation with tumor regression. Initial treatment strategies include blockade of estrogen action with antiestrogens and reduction of estrogen secretion by medical or surgical oophorectomy. Initial tumor regressions average 12–18 months but are often followed by relapse and disease recurrence. Secondary therapies are designed to further lower estrogen levels with aromatase inhibitors or to block estrogen action more completely with pure antiestrogens (1). These interventions frequently induce further remissions averaging an additional 12–18 months. Though Received February 10, 1998. Address all correspondence and requests for reprints to: Meei-Huey Jeng, Department of Internal Medicine, Division of Hematology/Oncology, University of Virginia Health Sciences Center, Box 513, Charlottesville, Virginia 22908. E-mail: [email protected]. * This work was supported by Grants NIH-RO-1– 65622– 04 (to R.J.S.), HD-25719 (to M.A.S.), GM-55985 and CA-44579 (to T.P.B.), and CA65746 (to R.K.). † Current address: Department of Surgery, Ichikawa General Hospital, Tokyo Dental College, 5–11-13 Sugano, Ichikawa-city, Chiba, 2720824 Japan.

coactivator levels. Our data demonstrated an adaptive increase in ER expression and in basal ER functionality, as assessed by read-out of three different transfected reporters in LTED, as opposed to wild-type MCF-7 cells. Increased reporter gene read-out was dramatically inhibited by the pure antiestrogen ICI 182,780. As verification that endogenous (as well as transfected) estrogen target genes had enhanced transcription, we found that the basal levels of c-myb and c-myc message were substantially increased in LTED cells and could be inhibited by antiestrogen. Interestingly, the levels of c-myb and c-myc message in the LTED cells seemed to be increased out of proportion to the degree of ER reporter gene activation and were similar to those in wild-type cells maximally stimulated with estradiol. In addition, not all estrogen-responsive genes were activated, because transforming growth factor-a message level was not increased in LTED cells. Up-regulation of the steroid receptor coactivator SRC-1 did not seem to mediate the process of enhanced ER-induced transcription. Considering these observations together, we suggest that long-term estradiol deprivation causes adaptive processes that not only involve up-regulation of the ER but also influence the specificity and magnitude of activation of estrogen-responsive genes. (Endocrinology 139: 4164 – 4174, 1998)

these clinical observations are well documented, the molecular and tumor biologic mechanisms that explain these secondary responses are not well understood and are the subject of the present report. Several investigators have used in vitro systems to model the transitional events that occur after initial exposure to hormonal manipulations. These models use wild-type MCF-7, ZR-75–1, or T47D (2–7) breast cancer cells, which require the presence of estradiol for growth, as do hormonedependent breast tumors, in both pre- and postmenopausal women. To mimic the clinical effects of primary hormonal therapy, cultured cells are deprived of estrogen, for a long term, by growing them in media treated specifically to remove substantial amounts of estrogen. After a period of proliferative quiescence lasting 1–3 months, the return of proliferation mimics the relapses observed 12–18 months after primary hormonal therapy in patients. In the cellular model, as in patients, tumor cell regrowth often can be secondarily inhibited by pure antiestrogens. These in vitro model systems have provided a means to gain insight into the mechanisms for relapse and secondary

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INCREASED ER AFTER ESTROGEN DEPRIVATION

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TABLE 1. Comparison of ER and PgR levels of MCF-7 wild-type and LTED cellsa Basal ER level Wild-type

[3H]E2-bound ER 65 fmol/1 3 106 cells Ref. 10 Immunoreactive ER 100% Ref. 5 [3H]E2-bound ER 3.5 pmol/mg DNA Ref. 6 [3H]E2-bound ER 1.8 3 104 sites/cell Ref. 3

a b

Basal PgR level

LTED . 4 months

Wild-type

LTED . 4 months

[3H]E2-bound ER 204 fmol/1 3 106 cells Immunoreactive ER 315% [3H]E2-bound ER 6.5 pmol/mg DNA [3H]E2-bound ER 9.5 3 104 sites/cell

[3H]R5020-bound PgR 8 pmol/1 3 106 cells [3H]R5020-bound PgR 0.3 pmol/mg DNA [3H]R5020-bound PgR b 0.6 3 104 sites/cell

Fold induction of PgR by 10210 Wild-type

M

E2

LTED . 4 months

[3H]R5020-bound PgR 8-fold increase 13-fold increase 5 pmol/1 3 106 cells 3 [ H]R5020-bound PgR 23-fold increase 51-fold increase 0.1 pmol/mg DNA [3H]R5020-bound PgR b15-fold increase b6-fold increase b 1.1 3 104 sites/cell

Data were taken directly from published literatures, as cited. These data were not previously reported.

responses in patients. Long-term estradiol deprivation (LTED) results in a 2- to 4-fold increase in estrogen receptor (ER) binding sites and protein (Table 1) (2, 3, 5, 8 –12) but minimal or no rise in progesterone receptor (PgR) (2, 3, 5, 6, 8 –12). Although the ER is capable of activation by exogenous estrogen and of stimulating PgR synthesis in LTED cells, proliferation is not increased further by addition of estrogen. It has been proposed that the adaptation and regrowth process (2, 3, 5, 6, 8 –12) involves increased sensitivity to, or secretion of, growth factors, rather than enhanced ERmediated transcription. Thus antiestrogens, which inhibit the growth of these cells, are thought to block growth factor actions as a mechanism for inhibiting cellular proliferation. Our prior studies and those of others suggest an alternative hypothesis, where ER-mediated transcription is indeed functionally involved in the regrowth process (6). This concept is based on the observation that exogenous estrogen can rescue the inhibitory effects of antiestrogens on cell growth. Because this evidence is indirect, the present study sought to examine ER expression and ER-mediated transcription directly in our LTED cell model. Additionally, we examined whether ER target genes, which are involved in cellular proliferation [i.e. c-myb, c-myc, and transforming growth factor-a (TGFa)], rather than in differentiated function (i.e. PgR), are stimulated. Our results demonstrated an increase in ER expression and transcriptional activation, as reflected by exogenously transfected reporter activity. Interestingly, however, we observed differential expression of endogenous estradiol responsive genes with a substantial increase in c-myb and c-myc but minimal or no increase in TGFa or PgR. These results provide direct evidence that estradiol deprivation induces adaptive mechanisms that involve ER expression and transcription. They also suggest a level of regulation that allows a selective increase in transcription of certain estrogen-responsive genes involved in cellular proliferation but not of the PgR gene, which generally mediates differentiated cellular function. Materials and Methods Tissue culture Wild-type human breast cancer MCF-7 cells were maintained in improved MEM [zinc option Richter’s modification (IMEM) containing 10% FBS and 10 mm HEPES]. LTED cells were established and fully characterized as reported previously (6). Briefly, wild-type MCF-7 cells were deprived of estrogen in phenol red-free IMEM, containing 5% dextran-coated charcoal-stripped FBS (DCC-FBS), for a period of 4 –24 months. The DCC-FBS was prepared according to methods described

previously (6), with minor modification. Basically, stripped serum was ultracentrifuged twice at 35,000 rpm for 1 h and then filtered through 0.2-mm, then 0.1-mm, filters (Gelman Sciences, Ann Arbor, MI) to remove residual charcoal in the stripped serum. After a period of proliferative quiescence, these cells began to regrow in the absence of exogenous estrogen. Tissue culture reagents were obtained from Gibco/BRL, Gaithersburg, MD. To evaluate the effect of estrogen and antiestrogen on reporter activity, gene expression, and cellular growth, studies were designed to compare wild-type and LTED cells. Wild-type MCF-7 cells were deprived of estrogen by stepping down into phenol red-free IMEM, containing 5% DCC-FBS and media devoid of phenol red, for 3–7 days before the addition of exogenous E2 (17-b estradiol) and/or antiestrogen ICI 182,780. LTED cells were plated, grown for 1–3 days in medium identical to that for the wild-type cells, and then exposed to estrogens and/or antiestrogens. For growth analysis, MCF-7 wild-type cells were deprived of estrogen for 3 days and plated onto 6-well plates at the same time as LTED cells in phenol red-free IMEM containing 5% DCC-FBS. The next day (day 1), cells were rinsed and counted using a coulter counter (13). Cell counts were performed for 10 days for time course experiments. For growth assay, using ICI 182,780, LTED cells were plated in phenol red-free IMEM containing 5% DCC-FBS. ICI 182,780, at indicated concentrations, was then added the next day for 6 days. For cell counts, cell monolayers were rinsed with isotonic saline (0.9% NaCl) in situ twice and lysed in buffer containing 0.01 m HEPES, 1.5 mm MgCl2, and 0.13 m ZAP (ethylhexadecyldimethylammonium bromide from Kodak, Rochester, NY) at room temperature for 5 min. The released nuclei were counted in isoton (Coulter Corporation, Miami, FL) on a model Z1 Coulter Counter. Cell counts were done in duplicate wells, and results were calculated as the mean 6 se.

Whole-cell PgR assay MCF-7 cells were deprived of estrogen for a period of 3–7 days and plated onto 24-well plates in IMEM (phenol red-free) containing 5% DCC-FBS. Seventy-two hours later, cells were treated with various concentrations of E2 for an additional 72 h. [3H]R5020 (5 nm; NEN Life Science Products, Inc., Boston, MA), in the presence and absence of 1000-fold excess cold R5020, were then added to determine the [3H]R5020-bound PgR, according to the standard MacIndoe method (14).

Transfection and CAT (chloramphenicol acetyl transferase) assay Cells were plated in 6-well plates and transfected with plasmids containing 1.5 mg pERE1-tk-CAT consisting of one copy of an ERE (estrogen response element) derived from the vitellogenin 2A gene and the tk (thymidine kinase) promoter derived from herpes simplex virus linked to the reporter gene for CAT or ERE2-tk-CAT or two copies of the ERE and a simple TATA box, pERE2-E1b-CAT, and 2 mg pCMVbgal, using the calcium phosphate method, as described previously (15). Six hours after transfection, medium was removed; and cells were incubated with 10% glycerol for 3 min. Fresh media, containing compounds, were then added for an additional 2 days. Similar results were obtained

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FIG. 1. Increased basal growth rate and inhibition by ICI 182,780 in LTED cells, as compared with wild-type MCF-7 cells. A, Wild-type cells were deprived of estrogen for 3 days and plated onto 6-well plates, at the same time as LTED cells, at a density of 6 3 104 cells per well. The next day (day 1), cells were lysed, and cell nuclei were then counted, every day for the next 10 days, using Coulter Counter. B, LTED cells were plated onto 6-well plates at a density of 1 3 105 cells/well. ICI 182,780, at various concentrations, was added the next day for 6 days. Media were changed every other day, and cells were lysed, and cell nuclei were then counted using Coulter Counter. Medium was changed every other day. Data are presented as mean 6 SE from duplicate wells and SE when error bars were sufficiently large to visualize. whether or not the 5% DCC-FBS was added into the medium after transfection. Cytosols were collected and assayed for CAT activity using the same amount of b-galactosidase unit (16, 17). Data were presented as the mean 6 se of duplicate samples. The experiments were repeated at least five times. Representative experiments were shown.

Messenger RNA (mRNA) analysis Cells were plated in 15 cm-diameter dishes and treated with compounds for 2 days after step-down conditions were established, as described above (18, 19). Poly(A)1 RNA was prepared and subjected to Northern analysis using 32P-labeled probes, as previously described (20). Complementary DNA (cDNA) for human c-myb (21) (2.2 kb EagI fragment) and human c-myc (1.6 kb EcoRI-HindIII fragment from exon III) were also used as the probes. For ER mRNA analysis, one band on Northern analysis corresponded with the full-length ER message, whereas two other smaller variants were detected. To assess which coding exons were contained in the smaller variants, oligonucleotide hybridization probes were used to detect the presence of Exons I, III, V, VII, and VIII (22). Oligonucleotide hybridization probes were synthesized to correspond to human ER mRNA, exon I (59-GACCATGACCATGACGGTCCAGACC-39), exon III (59-GGTGCACTGGTTGGTGGCTGGACACATATAG-39), exon V (59-AGGAGCAAACAGTAGCTTCACTGGGTC-39), exon VII (59-CTCATGTGCCTGATGTGG39), and exon VIII (59-GGAAACCCTCTGCCTCC-CCCGTGATGTAATAC-39). Probes ranged from 18 –32 bases, with calculated melting temperatures of 68 –73 C. In each case, oligonucleotides were hybridized to Northern blots, which were stripped and rehybridized with the entire coding region of human ERa cDNA to verify the position of hybridizing mRNAs.

Western blot analysis MCF-7 cells were deprived of estrogen, and cytosols were collected at the same time as for LTED cells. c-myc Western blot analysis was performed according to methods previously described (23). For ER Western blot analysis, cell monolayers were rinsed with cold PBS and lysis buffer initially containing 10 mm Tris (pH 7.5), 1.5 mm EDTA, 10 mm b-mercaptoethanol, and 0.6 m NaCl. In a subsequent experiment, 1 mm dithiothreitol, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 100 mg/ml phenylmethlysulfonylflouride were added as protease inhibitors. Cells were homogenized using an eppendorf pestle, and lysate was

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FIG. 2. ER mRNA expression in wild-type and LTED cells. Wild-type MCF-7 cells were deprived of estrogen and treated with or without 10210 M E2 for 2 days. Poly(A)1 RNA was collected, and Northern analysis was performed using 32P-labeled ER cDNA and 32P-labeled glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) probes. collected after spinning at 14,000 3 g for 15 min at 4 C. Cytosol protein (measured with Bio-Rad Protein Assay kit, Bio-Rad Laboratories, Hercules, CA) was loaded per lane, separated by electrophoresis on 10% polyacrylamide gels containing 1% SDS, and transferred onto nitrocellulose transfer membrane (MSI, Westboro, MA). Membrane was stained with Ponseau S solution (Sigma Diagnostics, St. Louis, MO) to visualize the loading and transfer efficiency. Equal loading was visualized. Membranes were blocked in 10% nonfat dry milk in Tris-buffered saline containing 0.1% polyoxyethylene sorbitan monolaurate (Tween-20) (TBS-T) for 1 h at room temperature. Five different anti-ER antibodies (C-314, H226, D547, H222, or D75) were used, each of which recognized a different epitope site (see Fig. 3A) (24 –27). C314 is an affinity-purified mouse monoclonal antibody. H226, D547, H222, and D75 are affinitypurified rat monoclonal antibodies. Membranes were incubated with first antibodies (1 mg/ml) in TBS-T containing 5% nonfat dry milk for 1 h and washed with TBS-T three times. Subsequently, membranes were incubated with 1:5,000 diluted peroxidase-conjugated goat antirat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or 1:1,000 diluted peroxidase-conjugated rabbit antimouse IgG (New England Biolabs, Inc., Beverly, MA) in TBS-T containing 5% nonfat dry milk for 1 h and washed three times with TBS containing 0.3% Tween 20, followed by an additional three washes with TBS containing 0.1% Tween 20. Membranes were then incubated with LumiGLO (New England Biolabs, Inc.) for 1 min and exposed to x-ray films to visualize the bound proteins. All incubations were performed at room temperature. C-314 ER antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other ER antibodies (H226, D547, H222, and D75) were kindly provided by Dr. Geoffrey L. Greene. The epitope map is shown in Fig. 3A (24 –27).

Results Cellular proliferation and ER functionality

We initially confirmed that human breast cancer cells adapt to long-term estrogen deprivation with an increase in growth rate in the absence of exogenous estrogen. Figure 1A demonstrates that LTED cells proliferate in the absence of exogenous E2, whereas wild-type MCF-7 cells become quiescent during short-term (1–2 weeks) estrogen deprivation. Furthermore, antiestrogen ICI 182,780 inhibited the increased cell proliferation rate in LTED cells (Fig. 1B). We then examined ER expression, by Northern analysis. Levels of full-length ER mRNA were increased in LTED cells (1.3-fold increase), compared with wild-type cells under basal con-


ditions (Fig. 2) and were down-regulated by exogenous estrogen. Smaller-molecular-weight mRNA bands were also increased in LTED cells. The three ER mRNA species all hybridized to individual oligonucleotides representing exons I, III, V, VII, and VIII (data not shown). Thus, the various mRNAs are unlikely to encode grossly different ER proteins. This view is supported by data from immunoblots using ER antibodies to various epitopes (Fig. 3, B–D), as described below. Given the long 39 untranslated region of human ER mRNA (28, 29), the different mRNA species could arise from differential use of polyadenylation sites in this region.

FIG. 3. ER protein expression in wildtype and LTED cells. Wild-type MCF-7 cells were deprived of estrogen and treated with or without 10210 M E2 for 2 days. Cell cytosols were collected, and 50 mg protein (B and C) or 100 mg protein for MCF-7 and 300 mg protein for LTED cells (D) were loaded per lane and separated by electrophoresis on 10% polyacrylamide gels containing 1% SDS and transferred onto nitrocellulose membranes. Western blot analysis was performed as mentioned in Materials and Methods. ER antibody C-314 was used in panel B and ER antibodies H226, D547, H222, and D75 were used in panels C and D. An epitope map of these antibodies is shown in panel A. Regions A–F and exons 1– 8 are marked. Numbers marked above each antibody were the estimated amino acid ranges for the epitope locations. Ponseau S staining of the nitrocellulose membranes, after transfer of proteins, showed equal loading of proteins for all lanes.

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We next examined ER protein expression, by Western blot analysis (Fig. 3, B–D) using different affinity purified antibodies to identify epitopes (Fig. 3A) (24–27) at points spanning the full-length receptor. Detection of ER with each would indicate the presence of the full-length receptor. Each antibody demonstrated substantially increased immunoreactive receptor in the LTED cells. In addition, E2 was able to down-regulate the ER protein in LTED cells to 67% of control level (Fig. 3B). Smaller-molecular-weight protein bands were observed (Fig. 3C), suggesting the possibility of proteolysis of ER into smaller fragments. Repeat of this experiment with the use of protease

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inhibitors revealed complete loss or elimination of the smallermolecular-weight bands (Fig. 3D). This indicated that several of the smaller bands resulted from proteolysis of the wild-type ER protein. In addition, a greater increase in ER was observed in LTED cells with protease inhibitors. With respect to the amount of functional receptor, prior data from our laboratory has demonstrated that the number of binding sites per cell was increased from 1.8 3 104 site/cell in wild-type cells to 9.5 3 104 sites/cell in LTED cells (6). We next determined whether increased ER expression was associated with a concomitant increase in functionality. Accordingly, we examined transactivation by the ER, choosing reporter gene constructs linked to a consensus ERE. Both wild-type and LTED cells were transfected and analyzed for reporter activity. Constructs containing one copy of the ERE, pERE1-tk-CAT; two copies of the ERE and a minimal promoter from tk gene, pERE2-tk-CAT; and two copies of the

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ERE and a simple TATA box, pERE2-E1b-CAT were used to evaluate the ER transactivation function. We found that the LTED cells, in comparison with wild-type cells, had elevated basal ER transactivation activity (mean increase: 5 6 3) when examined using all three different reporter constructs (Fig. 4A) and transfection performed at the same time. The mean increase of the basal ER transactivation activity was 15 6 8 if we only consider the transfection performed using pERE1tk-CAT. The pure antiestrogen ICI 182,780 was able to block and inhibit the basal transactivation activity dramatically in LTED cells but had minimal effect on the basal transactivation activity in wild-type cells (Fig. 4B). Both wild-type and LTED cells demonstrated similar response patterns to exogenous E2, with increased activation of reporter construct (Fig. 4C); and E2 increased ERE-CAT activity to 15.7-fold over control level in wild-type cells and to 4.9-fold over control level in LTED cells.

FIG. 4. ER transactivation function in wild-type and LTED cells. A, Increased basal transcriptional activation of ER target genes in LTED cells, as compared with wild-type MCF-7 cells. Wild-type MCF-7 and LTED cells were deprived of estrogen and transfected with pERE-tk-CAT (a), pERE2-tk-CAT (b), or pERE-E1b-CAT (c) reporter plasmids in conjunction with pCMV-bgal plasmid as internal control. Two days later, cell cytosols were collected and assayed for CAT activities using the same amount of b-galactosidase units. B, ICI 182,780 inhibited the basal level of ER target gene transcriptional activation in LTED cells, as compared with wild-type MCF-7 cells. Cells were deprived of estrogen and transfected with pERE-tk-CAT and pCMV-bgal plasmids and treated with various doses of ICI 182,780 for 42 h. Cell cytosols were then collected and assayed for CAT activities using the same amount of b-galactosidase units. C, E2 transactivated ER target gene in both wild-type and LTED cells. Cells were deprived of estrogen and transfected with pERE-tk-CAT and pCMV-bgal plasmids and treated with E2 at different doses for 42 h. Cell cytosols were then collected and assayed for CAT activities using the same amount of b-galactosidase units.

INCREASED ER AFTER ESTROGEN DEPRIVATION Expression of endogenous ER target genes

Transfected reporter genes represent artificial constructs that do not contain all promoter elements upstream of the transcriptional complexes and may use, as in our studies, response elements restricted to those considered canonical or consensus. For this reason, we conducted further experiments to examine expression of endogenous genes thought to be regulated by the ER (30). We detected elevations of the steady-state mRNA levels for c-myb and c-myc under basal conditions in LTED (approximately 2-fold increase), as compared with the wild-type cells (Fig. 5). However, basal expression of TGFa steady-state mRNA was similar in both cell types (Fig. 5). To further study the involvement of ER signaling pathway in the elevated basal expression of c-myb and c-myc in LTED cells, we examined the effect of antiestrogens, and we performed estrogen time course and dose-response experiments to determine the kinetics of gene regulation in response to E2. Basal levels of c-myb and c-myc were markedly reduced by ICI 182,782 in LTED cells, indicating a role for the ER in their enhanced expression (Fig. 6A). Additionally, we found that E2 was able to stimulate the expression of c-myb and c-myc in a dose-responsive fashion in wild-type MCF-7 cells (2-fold increase) (Fig. 6A). Notably, the peak stimulated levels observed in wild-type cells equaled values seen basally in LTED cells (Fig. 6B). However, the LTED cells still responded to exogenous estrogen with further elevations in c-myb and c-myc mRNA levels (2- to 4-fold increase), which exceeded those in wild-type cells under maximal estrogen stimulation. As evidence of a direct effect of estradiol without need for intervening steps, the induction of c-myb and c-myc mRNA levels by E2 occurred in less than 1 h after E2 addition (Fig. 6, C and D). We also observed a 7-fold increase in basal levels of c-myc protein in LTED cells, as compared with wild-type cells (Fig. 7). E2 was able to increase further the c-myc protein level (2-fold, as compared with control level) in both cell

FIG. 5. Differential elevation of steady-state mRNA levels in LTED cells. Wild-type (WT) MCF-7 and LTED cells were deprived of estrogen, and poly(A)1 RNAs were then prepared, subjected to Northern analysis, and probed with either [32p]-labeled TGFa, c-myb, c-myc, b-actin, or GAPDH probes.

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types. As further support that the regulation of c-myb and c-myc mRNA levels was indeed ER mediated, a rescue experiment was performed with increasing concentrations of E2 in combination with the pure antiestrogen ICI 182,780. In LTED cells, we found that the inhibition of basal expression of c-myb and c-myc mRNA by ICI 182,780 was reversed by an increasing concentration of E2 (Fig. 8). Interestingly, while the expression of c-myb and c-myc was increased in the LTED cells, that of other ER-regulated genes was not. The mRNAs for TGFa (Fig. 5), TGFb1, and TGFb3 (data not shown) were present at similar levels in both LTED and wild-type MCF-7 cells. Basal PgR levels were also not substantially increased with 0.5 3 104 sites/cell in LTED cells and 1.1 3 104 in wild-type (Table 1). The lack of a basal increase in PgR in LTED cells was not caused by unresponsiveness of the ER-ERE mechanisms to estradiol. E2, at a concentration of 10210 m, increased the PgR level to 8.8 3 104 sites/cell in wild-type cells and to 8.2 3 104 sites/cell in LTED cells, respectively. Steroid receptor coactivator SRC-1 involvement

SRC-1 (31) has been shown to enhance the ER transactivational activity when cotransfected into Hela cells. We speculated that SRC-1 might be overexpressed in LTED cells as a mechanism for specificity of basal ER transactivation or of its increase. To determine whether SRC-1 was involved, we compared the expression of SRC-1 in LTED and wild-type MCF-7 cells. Interestingly, rather than an elevation of SRC-1 expression in LTED cells, we observed that the expression of SRC-1 was somewhat lower in LTED cells, as compared with wild-type MCF-7 cells (Fig. 9). Discussion

The mechanisms that mediate sequential responses to hormonal therapy in patients with breast cancer are not well understood. We postulated that adaptive mechanisms involving ER-mediated transactivation may be involved, and we directly examined the role of ER expression and target gene activation in this process. We used a model system in which breast cancer cells initially stop proliferating in response to estrogen deprivation but later regrow without added estrogen and are inhibited with pure antiestrogens. We demonstrated that adaptation does occur and is characterized by enhanced basal ER expression and target gene transcriptional activation in LTED cells. Interestingly, the marked increase in expression of certain estrogen target genes, such as c-myb and c-myc, was selective, with minimal or no increase in PgR or TGFa expression. Increased gene expression was inhibited by the pure antiestrogen ICI 182,780 and rescued by coaddition of estradiol. Taken together, these observations provide direct evidence that breast tumor cells can adapt to conditions of long-term estradiol deprivation by increasing ER-mediated processes that mechanistically relate to cell proliferation. These observations provide a potential means of explaining how patients with hormone-dependent breast cancers respond to secondary hormonal therapies such as pure antiestrogens. Our observations in LTED cells are remarkably similar to findings in LNCaP prostate cancer cells deprived of andro-

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FIG. 6. Estrogen regulation of c-myb and c-myc mRNA levels. Wild-type MCF-7 and LTED cells were deprived of estrogen and treated with E2 at various concentration or ICI 182,780 at a concentration of 1027 M for 2 days (A and B) or treated with E2 at a concentration of 10210 M for various times (C and D). Panel B was plotted using c-myb data presented at panel A. Poly(A)1 RNAs were then prepared and subjected to Northern analysis and probed with either [32P]-labeled c-myb, c-myc, or GAPDH probes. Phosphoimager analysis or densitometer analysis were performed for data quantitation (B and D).

gen, for a long term, in tissue culture (32). Kokontis et al. reported that androgen-deprived LNCaP cells exhibit a 10fold increase in androgen receptor levels, enhanced growth rate in the absence of added androgens, increased basal c-myc mRNA levels, increased sensitivity to exogenous androgens, and enhanced androgen receptor-mediated transactivation, both basally and in response to androgen. Observations in LNCaP cells, coupled with ours in LTED cells, suggest that adaptive mechanisms, in response to hormone deprivation, may be a common phenomenon and may involve similar mechanisms. We and other investigators have demonstrated that the ER was up-regulated after long-term estrogen deprivation (Table 1). We believe that the increased ER expression at both

mRNA and protein levels in LTED cells during the adaptive process may provide advantage for cells to escape the requirement of estrogen for cell proliferation. The increase of steady-state level of ER mRNA can occur via either transcriptional or posttranscriptional regulation or a combination of these two processes. The regulation of ER mRNA has been shown to occur at both transcriptional (11, 33) and posttranscriptional (34 –36) levels. Our current studies did not address this issue, which will be explored in future experiments. The observation of three ER mRNAs in LTED cells has prompted us to examine the potential identity of these two smaller ER mRNAs. The increase in ER protein seen with every antibody roughly corresponds with the increase in ER


FIG. 7. Increased c-myc protein expression in LTED cells, as compared with wild-type MCF-7 cells. Wild-type and LTED cells were deprived of estrogen and treated with 1029 M E2 (E2), 1027 M ICI 182,780 (IC), or a combination of both (IC 1 E2) for 48 h. Total lysates (200 mg each) from LTED (lanes 1– 4) or wild-type MCF-7 cells (lanes 5– 8) were resolved onto a 7% SDS-PAGE and immunoblotted with an anti-c-myc Ab (clone 9E10.3, Neomarkers, Inc., Fremont, CA) (upper panel). As an internal control, the lower portion of the above blot was immunoblotted with an anti-actin Ab (Sigma, lower panel). The position of the 68-kDa marker is shown on the left.

mRNA seen overall with all species in Fig. 2. The observation that these three ER mRNA species all hybridized to individual oligonucleotides, representing exons I, III, V, VII, and VIII (data not shown), suggest that the various mRNAs are unlikely to encode grossly different ER proteins. We also tested the original hybridization profile of the ER oligonucleotides to various types of RNA and DNA to ensure specificity. Observations with multiple positive and negative controls demonstrated the specificity of binding of the probes used. The intensity of binding of the oligos to the three mRNA species was quite equivalent, much as the cDNA was. These results do not suggest the presence of the ER variants published previously (37–39). It is certainly possible that there could be a population of mRNAs of similar size in any one band; and in some respects, this has been shown to be true. For example, single exon deletions of human ERa mRNA occur routinely, but the changes are too small to result in different sized bands (i.e. a few hundred bp, compared with 6.4 kb). It is possible that more than one RNA species is contained in the bands shown and that at least one species of each band had the various exons. The cited references all cleanly showed that C-terminal cDNA did not bind. After careful analysis, by comparing the epitope recognition sites, we conclude that the smaller ER immunoreactive bands do not resemble any exon-deleted ER protein products. Exon deletion variants, as previously described, would correspond to 54 (exon 4), 51–52 (exon 7), or 41 (exon 5) kDa. All would bind H226, and D547 would bind the 51- and 41-kDa proteins (not seen). Binding of D75 to smaller proteins shows they are not deletion variants, which would not have this exon. Clone 24 (39) would produce a 37-kDa protein and should bind only ER21 and H226 but not C314. A protein of similar size is seen in lanes with D547 and D75, but they contain exons 4 – 8 (not seen in the published clones). Clone 4 should produce a 24-kDa protein and be seen only with H226; it seems too small for the immunopositive protein seen. We do not rule out the possibility that, within the cell population, there is a small fraction of cells that could express

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these variant proteins. However, the relative ratio of wildtype ER protein to these smaller ER immunoreactive proteins did not reflect the equal intensity of the three ER mRNAs detected in LTED cells. Therefore, these smaller ER immunoreactive proteins, if they are specific, may not contribute to the adaptive process caused by their low level, as compared with the wild-type ER protein level. The enhanced ERE-mediated transactivation observed in LTED breast cancer cells could reflect increased receptor number exclusively or, in addition, modulation of processes distal to receptor binding. Thus, an increased concentration of ER could be the sole explanation for the enhanced ERmediated transactivation observed. Other adaptive processes could also be taking place in the LTED cells. Increased phosphorylation of the ER or of coactivator proteins could serve to enhance ER-mediated transactivation. This process could involve up-regulation of PKC, PKA, MAP kinase, or other pathways. Increased activity or up-regulation of integrator proteins, such as CBP or coactivator proteins other than SRC-1, could also increase ER-mediated transactivation. A reduction of suppressor proteins, such as SMRT (40), NCoR (41), or ERE-binding inhibitory proteins (42), could also induce the same effect. These additional processes could act to amplify the effects of increased ER number in enhancing ERE-mediated transcription. Kokontis et el (32) concluded from studies in LNCaP cells that deprivation of androgen induces both an increased number of receptors and of factors acting distal to the receptor. This was based upon their observation of a 5-fold increase in androgen receptor protein, which was not considered sufficient to account for a 21-fold induction of prostate-specific antigen mRNA. We also detected greater enhancement of ER target gene activation (15-fold) than expected from the increase in ER levels. Taking into consideration the studies of Kokontis et al. and our own, we postulate that postreceptor mechanisms may play a role in the adaptive process in LTED cells. However, further studies are required to identify specific mechanisms that may be involved. We have previously provided evidence that LTED breast cancer cells exhibit increased sensitivity to estradiol and that residual estrogen, present in supposedly estrogen-free culture media, could contribute to their enhanced rate of growth (6). Residual estrogen could also contribute to enhanced ERE transactivation observed under basal conditions in LTED cells. Kokontis et al. also suggested that the regrowth phenomenon in the LNCaP prostate cancer cells may reflect small amounts of residual androgen. These investigators postulated that hypersensitivity to residual androgen allows small amounts of ligand to activate important pathways involved in stimulating cellular proliferation. The alternate explanation (rather than residual estrogens in the media) for receptor-mediated transactivation in the absence of hormone is that unliganded receptors can bind to their respective response elements and initiate transcription. Ligand-independent transactivation, however, cannot provide the sole explanation for the observations in our study. With enhanced receptor-mediated transactivation, one would expect a generalized increase in expression of all hormone responsive genes; and yet, responses were selective. Why should increased ER transactivation cause incre-

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FIG. 8. Reversal of the ICI 182,780 inhibited c-myb and c-myc mRNA levels by increased E2 concentration. LTED cells were treated with various concentrations of E2 with or without 1027 M ICI 182,780. Poly(A)1 RNAs were then prepared and subjected to Northern analysis and probed with either [32P]labeled c-myb, c-myc, or GAPDH probes.

FIG. 9. Expression of SRC-1 mRNA in both wild-type MCF-7 and LTED cells. Cells were deprived of estrogen, and poly(A)1 RNAs were prepared for Northern analysis. [32P]-labeled SRC-1 and GAPDH cDNAs were used as the probes.

ments in c-myb and c-myc levels without altering TGFa and PgR levels substantially? We thus suggest that additional (but unknown) processes might alter the specificity of the transcriptional process whereby some, but not other, endogenous estrogen responsive genes are stimulated. Alternatively, ligands similar to, but slightly modified from, estradiol might exert effects on estrogen target genes that differ from those of estradiol (43, 44). Studies with both estradiol and antiestrogen analogs demonstrate differential regulation of ERE-related transcription (45). Thus, residual estrogens in the culture media could differentially stimulate genes involved in cell proliferation but not differentiation. ER can differentially activate ER-responsive genes by binding to various nonconsensus ER sequences that are under the influence of various enhancers (46 – 48). It is possible that cells adapted to long-term estradiol depletion might up-regulate enhancers of transcription that use nonconsensus ERE sequences. These possibilities are speculative but provide testable explanations for our observations. Other model systems exist that demonstrate preferential responses of some genes and not others to receptor-mediated hormonal stimulation. For example, in studies of highly inbred mice, some strains respond to androgens with substan-

tial stimulation of the androgen-responsive genes, such as ornithine decarboxylase, whereas others exhibit minimal increase (49 –52). Further, the dose-response curves of stimulation of androgen-responsive proteins are markedly shifted, often in opposite directions, in specific genetic strains. Thus, biologic events, distal to the binding of hormone-to-receptor, can influence the magnitude of response to a given stimulus, its sensitivity to that stimulus, and the selectivity of responses observed. The recent demonstration of the enhancement of ER target gene activation by steroid receptor coactivators in the presence of estrogen (31, 53) had raised the possibility that MCF-7 cells may have adapted to the low estrogen environment with an overexpression of SRC-1 that subsequently caused an elevation of and differential specificity of ER target gene activation. The observation that the expression of SRC-1 mRNA was even lower than the wild-type MCF-7 cells suggests that SRC-1 is not involved in this regrowth process. However, overexpression of other coactivators (54 –57) or reduced expression of corepressors (40) could also be responsible to this regrowth process. It is also possible that the determination of the expression of SRC-1 at mRNA level is not sufficient to explain the function of SRC-1, where phosphorylation may take place to influence its functionality. Recent data provide insight into the complexity of the ER-mediated effects that need to be considered when interpreting our studies. A recent publication, indicating a differential regulation by estrogens of growth and PRL synthesis in pituitary cells, suggests that only a small pool of ERs is required for growth (58). This is analogous to our observation that growth response sensitivity does not parallel reporter gene sensitivity or target gene regulation. In addition, the ER pathway influences a wide variety of cellular processes by regulating gene expression (59). This effect is mediated both by ERa and ERb, which recognize and bind to sequence-specific enhancers to regulate the rate of transcription of hormone responsive genes (60). One of these effects involves EREs, and the other involves AP-1-responsive sites (61). Via one pathway, the binding of estrogen to its receptor induces the ligand-binding domain to undergo


conformational changes, allows receptor to bind to DNA and to coactivator and integrator proteins, and subsequently to stimulate gene expression. Via the AP-1 pathway, ligandbound receptor binds to the FOS/JUN complex involved in the AP-1 site of DNA. Regulation of ER target gene expression, through either of these pathways, has been postulated to play an important role in estrogen-induced cell proliferation. Further studies will be required to assess the role of AP-1 in the adaptive responses observed in LTED cells. Our studies assume that the protooncogenes c-myb and c-myc represent direct estrogen-responsive target genes. Evidence supporting the estradiol responsiveness of c-myc is strong. Estradiol increases c-myc levels within 15 min, and antiestrogens block this response. Inhibitors of protein synthesis do not prevent the acute response of c-myc to estradiol, a finding demonstrating the direct regulation of c-myc by estradiol without need for an intermediary protein. c-myc seems to be involved in estradiol-stimulated proliferation, because c-myc antisense administration blocks estradiolinduced proliferation. Similar data support the notion that c-myb is an estradiol-inducible protein (Figs. 5 and 6, and unpublished data from Drs. Timothy Bender and Eric H. Westin regarding inhibition of c-myb expression). In other model systems, c-myb and c-myc (32, 62) expression correlates with rate of cell proliferation. Thus, it is possible that basal increments in c-myb and c-myc in the LTED cells may merely reflect enhanced proliferation and not specifically an ER-mediated effect. We consider this unlikely because the pure antiestrogen ICI 182,780 can lower the levels of these oncogenes in LTED cells under basal conditions, and exogenous estrogen rescues this effect. More importantly, these observations directly implicate the ER in the adaptation process. More direct proof of the estrogen dependence of c-myb and c-myc in our cells awaits experiments that can completely dissociate proliferation from expression of these oncogenes. In summary, our experiments demonstrate that long-term estradiol deprivation causes adaptive changes in breast cancer cells that involve increased receptor expression, reporter gene activation, and c-myb and c-myc (but not TGFa) expression. These changes could partially explain the ability of LTED cells to regrow in the absence of added estrogen. Similar mechanisms occurring in women treated for breast cancer could explain secondary responses to pure antiestrogens or to potent aromatase inhibitors. Acknowledgments We would like to thank Drs. Bert O’Malley, Ming Tsai, John A. Cidlowski, Gunther Schutz, and Michael E. Williams for the supply of plasmids; Dr. Alan Wakeling for ICI 182,780; Dr. Geoffrey L. Greene for ER antibodies; Dr. Ji-Ping Wang for preparing the charcoal-stripped serum; and Miss Anne C. Eischeid for performing some of the cell-count experiments.

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