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Department of Internal Medicine (W.Y., J.-P.W., Y.L., R.J.S.) and Division of Biostatistics and Epidemiology (M.C.), University of Virginia, Charlottesville, Virginia 22908; and Department of Surgery (S.M.), Tokyo Dental College, Ichikawa City, Chiba 272, Japan
Address all correspondence and requests for reprints to: Wei Yue, Department of Internal Medicine, P.O. Box 801416, University of Virginia, Charlottesville, Virginia 22903. E-mail: wy9c{at}virginia.edu.
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Abstract |
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, and demonstrated a 2–3 log enhancement of sensitivity to E2. PD98059 abrogated the TGF-induced effect, indicating that MAPK activation is responsible for E2 hypersensitivity. To determine the level at which MAPK activation enhanced E2 sensitivity, we examined the dose-response effects of E2 on several transcriptional readouts, including ERE-reporter activity and the levels of progesterone receptor and pS2. Wild-type and LTED cells exhibited nearly identical responses to E2, suggesting that mechanisms downstream of estrogen receptor-mediated transcription are involved in inducing hypersensitivity. In support of this possibility, LTED and TGF-treated wild-type cells were hypersensitive to the effects of E2 on the key cell cycle regulator, E2F1. |
Introduction |
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As a model system, we cultured MCF-7 human breast carcinoma cells in estrogen-free medium to mimic the effects of primary endocrine therapy. This process is called long-term E2 deprivation, and the adapted cells are termed long-term estrogen-deprived (LTED) cells. Under these conditions, (i.e. phenol-red-free media with 5% charcoal stripped serum), the E2 concentration is reduced to 10-13 M or less. In response to E2 deprivation, MCF-7 cells initially stop growing but then, 3–6 months later, adapt and grow as rapidly as wild-type MCF-7 cells maximally stimulated with E2. This biologic effect seems to be attributable to the development of hypersensitivity to E2 in LTED cells. We demonstrated previously that 10-15 M exogenous E2 caused maximal stimulation of proliferation of LTED cells when grown under serum-free and completely estrogen-free conditions (2). When cultured in the medium containing charcoal stripped serum, growth of the LTED cells can be inhibited by pure antiestrogens, suggesting that residual estrogen in the culture environment is sufficient to stimulate proliferation of LTED cells. Our in vivo study also showed that LTED cell xenografts are hypersensitive, because they grew more rapidly than the wild-type MCF-7 cells in response to low doses of E2 in nude mice (3).
We have reported that LTED cells exhibit enhanced MAPK activity and rapid proliferation in vitro and in vivo (3, 4). Elevated MAPK activation was also found in other breast cancer cell lines deprived of estrogen (5). These results suggest that the MAPK pathway is activated during the process of adaptation of breast cancer cells to a low estrogen environment. Though enhanced MAPK activation and hypersensitivity to E2 are associated phenomena, it is not clear that the increments in activated MAPK actually cause hypersensitivity to E2.
The current studies employed LTED and wild-type MCF-7 cells to examine the hypothesis that MAPK activation causally induces hypersensitivity to the mitogenic effect of E2. We used exogenous TGF to increase the levels of activated MAPK in wild-type MCF-7 cells and showed that this caused a marked enhancement in the sensitivity to the proliferative effects of E2. In these proof of principle experiments, the effect could be blocked by a MAPK inhibitor, showing that hypersensitivity did not represent a specific TGF effect acting independently of MAPK. Unexpectedly, this effect on hypersensitivity did not represent a generalized enhancement of estrogen receptor (ER)-mediated transcription but more likely represented a cell-cycle-mediated event. These studies highlight the importance of interactions between the MAPK and ER pathways in mediating cell proliferation.
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Materials and Methods |
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was obtained from Discovery Labware (Bedford, MA) and PD98059 from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Anti-active MAPK monoclonal antibodies were obtained from Sigma (St. Louis, MO). Anti-p44/p42 MAPK (total MAPK) antibody was purchased from Zymed Laboratories, Inc. (South San Francisco, CA). Anti-E2F1 monoclonal antibody (KH95) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary antibodies conjugated with horseradish peroxidase were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Cell culture medium [improved MEM Zn2+ option (Richter’s modification) (IMEM)], fetal bovine serum (FBS), glutamine, and trypsin were purchased from Life Technologies, Inc. (Gaithersburg, MD). All chemicals were obtained from Sigma.
Cell culture
MCF-7 cells (kindly provided by Dr. R. Bruggemeier, Ohio State University, Columbus, OH) were grown in IMEM containing 5% FBS. LTED cells were routinely grown in phenol-red-free IMEM containing 5% dextran-coated charcoal-stripped FBS (DCC-FBS). To reverse LTED cells to the wild-type phenotype, LTED cells were cultured in IMEM containing phenol red and 5% FBS.
Growth assay
Cells were plated in 6-well plates at the density of 60,000 cells/well in their culture media. Two days before treatment, the medium was replaced with phenol-red- and serum-free IMEM. The cells were then treated in phenol-red- and serum-free IMEM containing vehicle or treatment compound, for 5 d, with medium change on d 3. The final concentration of vehicle (ethanol or dimethylsulfoxide) was 0.1–0.2%. At the end of treatment, cells were rinsed twice with saline. Nuclei were prepared by sequential addition of 1 ml HEPES-MgCl2 solution (0.01 M HEPES and 1.5 mM MgCl2) and 0.1 ml ZAP solution [0.13 M ethylhexadecyldimethylammonium bromide in 3% glacial acetic acid (vol/vol)] and counted using a Coulter counter.
Quantitative measurement of PgR and pS2
Wild-type and LTED MCF-7 cells were cultured in 60-mm dishes. Two days before treatment, cells were shifted to phenol-red-free IMEM without serum and were treated for 48 h with E2 in the same medium. After treatment, the cells were rinsed with cold PBS, scraped off from the dish, and homogenized in homogenization buffer (10 mM Tris, 1.5 mM EDTA, 5.0 mM sodium molybdate, and 1 mM monothioglycerol). The homogenate was centrifuged at 61,524 x g for 1 h at 4 C. The cytosol (supernatant) was kept at -80 C until analysis. The protein concentration of the cytosol was determined using Lowry’s method (6). PgR was measured by a solid-phase enzyme immunoassay using PgR-EIA monoclonal kit (Abbott Laboratories, Park, IL). pS2 content in the cytosol was quantitated by a solid-phase immunoradiometric assay using an ELISA-pS2 kit from CIS-Bio International (Gif-Sur-Yvette Cedes, France).
Immunoblotting of MAPK and E2F1
Cells grown in 60-mm dishes were washed with ice-cold PBS. To each dish was added 0.5 ml lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1% Triton X-100, 1 mM ß-glycerophosphate, 1 µg/ml leupeptin and aprotinin, and 1 mM phenylmethylsulfonylfluoride] or RIPA buffer [20 mM Tris (pH 8.0), 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 20 µM leupeptin, and 0.15 u/ml aprotinin]. The dishes were incubated on ice for 5 min before collection. Cells were then pulse sonicated and centrifuged at 14,000 rpm for 10 min. Cell lysates were stored at -80 C until analysis. Fifty micrograms of total protein were loaded and separated on 10% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was probed with antibodies against MAPK or E2F1. Secondary antibody, conjugated with horseradish peroxidase (1:2000), was then applied. After reacting with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL), targeted protein bands were visualized by exposing the membrane to x-ray film. Bands of MAPK and E2F1 were scanned and quantitated using a Molecular Dynamics, Inc. (Sunnyvale, CA) scanner and ImageQuant program.
Statistical analysis
A four-parameter logistic model (7) was used for fitting curves to cell growth to allow estimation of the EC50 stimulatory doses of E2. This method allows us to precisely quantitate the levels of sensitivity to E2 and to combine the results of multiple experiments to enhance precision. The curve fitting model allowed for different asymptotes across different experiments to account for small deviations in initial numbers of cells plated. Standard errors for the parameters in the models were computed using the covariance matrix of Zhang et al. (8).
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Results |
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). In contrast, serum starvation dramatically reduced the amount of activated MAPK in wild-type MCF-7 cells. These results suggest that activation of MAPK in LTED cells does not reflect increased sensitivity to the growth factors present in the media.
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in wild-type MCF-7 cells to induce MAPK activation.
We then examined the growth of wild-type MCF-7 cells in response to various concentrations of E2 with or without TGF. In the presence of TGF, a two-log shift to the left in the dose-response curve (i.e. enhancement of sensitivity to E2) was observed when compared with cells not receiving this growth factor. The EC50 of E2 was 2.0 x10-15 M in the presence of TGF (P < 0.01) and 3.16 x10-13 M in its absence (Fig. 3). We reasoned that the effects of TGF to enhance E2 sensitivity represented an increase in MAPK rather than some other specific action of TGF. To demonstrate that this was the case, we coadministered the MAPK/ERK kinase inhibitor PD 98059 along with TGF and reversed the hypersensitivity (Fig. 4). The EC50 of E2 was 3.2 x 10-15 M in the absence of PD98059 and 2.5 x 10-12 M in its presence, respectively (P < 0.001). These data served as proof of the principle that enhancement of MAPK activation could induce E2 hypersensitivity.
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) and pS2 (Fig. 5C), and estrogen response element-chloramphenicol acetyltransferase (ERE-CAT) reporter activity (Fig. 5D) and cell number (Fig. 5A). Growth of LTED cells displayed enhanced sensitivity to E2 stimulation, compared with wild-type MCF-7 cells. The estimated EC50 is 7.2 x 10-14 M for LTED cells and 3.6 x 10-11 M for wild-type MCF-7 cells (P < 0.001). However, we observed no enhancement of E2 sensitivity on any of these transcriptional responses when comparing LTED with wild-type MCF-7 cells. The presence of ICI 182,780 did not alter the pattern of the responses to E2 (data not shown). The results suggest that the E2 hypersensitivity of LTED cells did not occur at the level of ER-mediated gene transcription.
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). The sensitivity of E2F1 expression to E2 stimulation in wild-type cells could also be increased by coadministration of TGF (Fig. 6B). Each experiment shown in Fig. 6 was repeated three times. The results are highly consistent. These data suggest that activation of the MAPK pathway results in enhanced sensitivity to E2 and involves E2F1. These results indicate that the mechanism inducing hypersensitivity acts downstream of ER-mediated transcription and probably involves events occurring in the cell cycle.
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Discussion |
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The MAPK pathway is an amplification system that links extracellular mitogenic signals to cell proliferation. Prior data indicate that this pathway can act in concert with or independently of E2-mediated events in breast cancer cells. Independent proliferative effects result from overexpression of growth factors (11) and/or growth factor receptors (12, 13) in breast cancer cells. Cells stably transfected with EGF receptor and erbB-2 or various growth factor genes (13, 14, 15, 16) become tumorigenic in nude mice without supplementation of estrogen. Constitutive activation of Raf-1 kinase, a kinase downstream of growth factor receptor, allows for growth of MCF-7 cells in the absence of estrogen (17). Activation of the MAPK pathway in hormone-dependent breast cancer may explain aggressive progression and loss of hormone dependency.
The MAPK pathway can also act in concert with the ER pathway. In hormone-responsive breast cancer cells, growth factors and E2 synergistically stimulate cell proliferation (18). Mechanistic studies indicated that interaction between the MAPK and ER signaling pathways potentiates ER-mediated transcription (19, 20, 21) through enhanced ER phosphorylation by MAPK (22) or its downstream kinase (23). This is a generally accepted mechanism that explains synergistic interaction between the two signal pathways.
Our studies indicate that activation of the MAPK pathway is involved in E2 hypersensitivity in LTED cells. This is supported by the observed association between the levels of activated MAPK and the sensitivity to E2. Proof-of-principle studies, using TGF as a means of acutely increasing the levels of MAPK, provide further evidence of the causative relationship between the MAPK pathway and E2 hypersensitivity. TGF is known to stimulate the growth of breast cancer cells independently of E2. Thus, it is possible that its effect to increase the sensitivity to the proliferative actions of E2 might reflect intrinsic properties of TGF and not its MAPK-enhancing effect. This possibility is considered highly unlikely, because the MAPK inhibitor, PD 98059, completely abrogated the effects of TGF on shifting E2 dose response curves to the left.
Based on the known effects of MAPK on ER-mediated transcription, we expected that hypersensitivity would be mediated by transcriptional effects in LTED cells. We further assumed that if this was the case, expression of all E2-regulated genes should exhibit enhanced sensitivity to E2 in LTED cells. We chose to measure E2-induced expression of proteins that are unrelated to growth, such as PgR and pS2, and transcriptional activation of ERE-reporter gene. All reactions were dose-dependently stimulated by E2 in both wild-type and LTED cells. Surprisingly, none of these reactions showed enhanced sensitivity to E2 in LTED cells. Three separate transcriptional readouts exhibited great contrast to the data of growth response to E2, suggesting that enhanced E2 sensitivity in LTED cells does not occur at the level of ER-mediated transcription. However, we recognize the possibility that we have not yet measured the appropriate estrogen-responsive genes that might be critical for cell proliferation. An exhaustive study of estrogen-responsive genes and their level of sensitivity to E2 in wild-type and hypersensitive cells will be necessary to rule out this possibility.
Cell cycle progression is the common and final pathway for cell proliferation induced by steroid hormones, growth factors, and other mitogens (24, 25, 26). The commitment of mammalian cells, in late G1, to replicate the genome and divide in response to mitogens is controlled by cyclin D-associated cyclin-dependent kinases and their inhibitors. The stimulation of G1-phase CDK activity leads to phosphorylation of Rb and Rb-related proteins, thereby releasing Rb-sequestered E2F. The E2F family of transcription factors plays a pivotal role in the regulation of cell cycle entry and progression by regulating expression of genes involved in cell cycle control, initiation of replication, and DNA synthesis. These include dihydrofolate reductase, thymidine kinase, ribonucleotide reductase, DNA polymerase , Cdc6, and a component of the origin recognition complex (27, 28, 29, 30, 31).
In breast cancer cells, E2 regulates expression of cyclins and activities of cyclin-dependent kinases (32). Though alteration in expression of certain cyclins could provide important information on enhanced sensitivity to the mitogenic effect of E2, it would not reflect the integrated effect of E2 on cell cycle progression, because some of the effects are indirect [e.g. through c-Myc (33) or MAPK-pathway- (34)] or cell-cycle-independent (35). This might be more important for LTED cells, because both c-myc expression (36) and MAPK activation (4) are elevated in these cells.
Based on the above concern, we decided to examine a more downstream factor, E2F1, which is known to be regulated by E2 (9) and growth factors (10) and represents cell cycle progression. We found that LTED cells are more sensitive to E2 stimulation of E2F1 protein, as shown by the left shift of the dose-response curve (Fig. 6A
). In an additional proof-of-principle experiment, we also directly demonstrated that an increase in MAPK activity caused a hypersensitive response of E2F1 to E2 in wild-type MCF-7 cells. Taken together, these observations suggest an effect of MAPK acting at a level downstream of ER-mediated transcription and probably at the level of the cell cycle. Though the mechanism remains unclear, these results provide an insight into the molecules involved in E2 hypersensitivity. It has been reported that the oncogenes Myc and Ras act collaboratively to induce cyclin-E-dependent kinase activity, activation of E2F, and S-phase entrance (37). This collaborative action of Myc and Ras may also occur in LTED cells. As a result, LTED cells become more sensitive to the mitogenic effect of E2.
Enhancement of MAPK is not an in vitro phenomenon that is confined to breast cancer cells in culture. Increased MAPK expression and/or activation has been reported in human breast cancer tissues (38) and carcinogen-induced mammary tumors in animals (39). MAPK activity was higher in primary tumors from patients with positive lymph nodes than in those without lymph node tumor spread. Higher MAPK activity in primary breast cancer is correlated with a shorter period of disease-free survival in these patients (40). Whether these biologic effects reflect the action of MAPK on ER transcription, on more downstream events, or represent ER- independent actions is not currently known. Nonetheless, these observations serve to emphasize the importance of MAPK in human breast cancer growth.
Interpretation of experiments with LTED cells has always been confounded by their rapid growth rate in the absence of exogenous E2. The ability to block their growth with antiestrogens and rescue this inhibition with exogenous estrogen suggested the presence of residual estrogen in the culture media. Recent data suggest that the putative residual estrogen in the culture medium may reflect weak estrogens leached out of the plastic culture ware. Chemicals such as bisphenol-A, released from plastic tissue culture flasks, are estrogenic (41, 42) and can cause growth of MCF-7 cells in culture. The findings under these conditions are very similar to those observed several years ago before phenol red was found to contain an estrogenic substance. Specifically, MCF-7 cells grown in the presence of phenol red grew in the absence of E2 and stopped growing when an antiestrogen was added. Exogenous E2 stimulated further growth minimally unless an antiestrogen was added to the media. Based on this analogy and recent findings regarding plastic ware, we add antiestrogen to both wild-type and LTED cells to block the effects of residual E2 in experiments where appropriate. As published previously, LTED cells respond to three-logs-lower concentrations of E2 than do wild-type cells under these conditions (43). The use of ICI then obviates problems resulting from variable amounts of contaminating E2 in the culture media. Therefore, ICI 182,780 (10-9–10-8 M) was used, when appropriate, to block the effect of residual E2 when examining the effect of added E2 in LTED cells.
In summary, breast cancer cells become hypersensitive to the proliferative effects of E2 after long-term deprivation of estrogen. Mechanistically, the induction of hypersensitivity seems to involve up-regulation of MAPK. Interactions between the estrogen-stimulated and MAPK pathways occur at the level of the cell cycle to enhance the level of the transcription factor E2F1. These interactive events seem to explain hypersensitivity to the proliferative effects of E2.
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Acknowledgments |
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Footnotes |
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Abbreviations: E2, Estradiol; ER, estrogen receptor; ERE-CAT, estrogen response element-chloramphenicol acetyltransferase; FBS, fetal bovine serum; IMEM, improved MEM Zn2+ option (Richter’s modification); LTED, long-term estrogen-deprived (cells); PgR, progesterone receptor.
Received February 14, 2002.
Accepted for publication May 9, 2002.
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on the growth of MCF-7 human breast cancer cells in vitro and in vivo. Mol Endocrinol 3:372–380-induced but not estrogen-induced growth of hormone-dependent human breast cancer. Mol Endocrinol 2:1064–1069-Shc association and Shc pathway activation. Mol Endocrinol 16:116–127This article has been cited by other articles:
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