Hong Liu, Eun-Sook Lee, Csaba Gajdos, Sandra Timm Pearce, Bin Chen, Clodia Osipo, Jessica Loweth, Kevin McKian, Alexander De Los Reyes, Laura Wing, V. Craig Jordan
Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL (HL, CG, STP, BC, CO, JL, KM, ADLR, LW, VCJ); National Cancer Center, Goyang, Gyeonggi, Korea (ESL).
V. Craig Jordan, OBE, PhD, DSc, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, 8258 Olson, 303 E. Chicago Ave., Chicago, IL 60611; E-mail: vcjordan@northwestern.edu
Abstract
Background: Resistance to tamoxifen, a selective estrogen receptor modulator (SERM), involves changes that prevent apoptosis and enhance cell proliferation and survival. Para-doxically, estrogen treatment inhibits the growth of longterm tamoxifen-treated breast tumors. Because of the increasing use of raloxifene, another SERM, to prevent osteoporosis and potentially reduce breast cancer risk, some women will develop raloxifene-resistant breast cancer. We developed a raloxifene-resistant MCF-7 cell model (MCF-7/ Ral) and investigated the nature of raloxifene-resistant breast cancer and its response to estradiol. Methods: Raloxifene resistance and hormone responsiveness were assessed by proliferation assays and cell cycle analysis in parental MCF-7 and MCF-7/Ral cells. Nuclear factor KB (NF-KB) activity was investigated with a transient transfection assay. Apoptosis was investigated by annexin V staining, mRNA was measured by real-time polymerase chain reaction, and protein was measured by western blotting. Tumorigenesis was studied by injecting MCF-7 or MCF-7/Ral cells into ovariectomized athymic mice (10 per group) and monitoring tumor size weekly. All statistical tests were two-sided. Results: Basal NF-KB activity was higher in MCF-7/Ral cells (1.6 U, 95% confidence interval [CI] = 1.2 to 2.0 U) than in MCF-7 cells (0.8 U, 95% CI = 0.4 to 1.1 U; P = .004). When cultured with 1 μM raloxifene, MCF-7/Ral cells grew statistically significantly (P<.001) faster than MCF-7 cells. Estradiol treatment of MCF-7/Ral cells arrested cells in G2/M phase of the cell cycle, decreased NF-KB activity (0.2 U, 95% CI = 0.2 to 0.3 U; P<.001), increased expression of Fas protein and mRNA (4.5-fold, 95% CI = 2.8-to 6.3-fold versus 0.5-fold, 95% CI = 0.3-to 0.8-fold for control treatment; P<.001), and induced apoptosis. Treatment with either raloxifene or tamoxifen stimulated MCF-7/Ral tumor growth, suggesting that such tumors were resistant to both drugs. When a 9-week raloxifene or tamoxifen treatment was followed by a 5-week estradiol treatment, estradiol statistically significantly reduced the size of tumors stimulated by raloxifene or tamoxifen (at week 14, P = .004 for raloxifene and P< .001 for tamoxifen). Conclusions: Growth of raloxifene-resistant MCF-7/Ral cells in vitro and in vivo is repressed by estradiol treatment by a mechanism involving G2/M-phase arrest, decreased NF-KB activity, and increased Fas expression to induce apoptosis. [J Natl Cancer Inst 2003;95:1586-97]
Introduction
Selective estrogen receptor modulators (SERMs) are a novel class of compounds that have multiple applications in women’s health. Tamoxifen, the first clinically useful SERM, has anties-trogenic effects in breast tissue and can be used to treat (1) or prevent (2) breast cancer. SERMs also have estrogen-like activ-ity in bone and can be used to treat and prevent osteoporosis (3). Raloxifene is the first SERM approved for the treatment and prevention of osteoporosis in postmenopausal women (4). In a randomized clinical trial of patients with osteoporosis, raloxifene treatment was found to be associated with a statistically significant decrease in the incidence of breast cancer (5,6). Consequently, raloxifene is currently being evaluated to determine whether it is an effective chemopreventive agent in women who were determined by the Gail model to be at high risk for breast cancer (7). The Study of Tamoxifen and Raloxifene (STAR) trial is recruiting 19 000 high-risk postmenopausal women to establish whether raloxifene is more or less effective than tamoxifen, the current standard of care, as a chemopreven-tion agent for breast cancer but with fewer side effects.
Resistance to tamoxifen, in the form of tamoxifen-stimulated growth, is well documented in the laboratory and the clinic (8-10). In contrast, there is only limited information about the development of resistance to raloxifene. The widespread use of raloxifene to prevent osteoporosis, or perhaps in the future to prevent breast cancer, means that increasing numbers of women will be developing raloxifene-exposed breast cancer. Thus, it is important to elu-cidate the mechanism of raloxifene resistance in breast cancer and to develop an effective therapy for raloxifene-resistant tumors.
Resistance to SERMs results from a complex series of changes that prevent apoptosis and thus enhance cell prolifera-tion and survival. Alterations in several signal transduction pathways have been described for tamoxifen resistance, includ-ing enhanced activity of the activating protein 1 (AP1) (11-13) and phosphatidylinositol 3-kinase/protein kinase B (PKB or AKT) pathways (14,15) and altered expression of protein kinaseCα(16), erbB-2 (17-19), and insulin-like growth factor I (20); all of these events have been linked to the activation of nuclear factor KB (NF-KB) (21-24). Increased NF-KB activity is also important for hormone-independent growth and for ICI 182,780 (fulvestrant) resistance (25,26). Thus, NF-KB pathways may also play a central role in raloxifene resistance. After resistance to one SERM appears, the breast tumors often exhibit cross-resistance to other antiestrogens (27-31), which limits the effectiveness of secondary endocrine therapy. However, the process that causes resistance to SERMs appears to begin with a stage in which both estrogen and the SERM stimulate growth through the estrogen receptor (ER) (8) and then progress to a stage in which cell proliferation is dependent on the SERM or on prolonged estrogen deprivation but is inhibited by estrogen. Physiologic levels of estrogen inhibit the growth of long-term tamoxifen-stimulated breast tumors (32,33), T47D cells stably transfected with protein kinaseCα(T47D:protein kinaseCαcells) inoculated into athymic mice (16), long-term estrogen-deprived T47D and MCF-7 cells in culture (34-36), and raloxifene-resistant ECC1 endometrial cancer cells (37). Therefore, we propose that a patient’s own estrogens might ultimately destroy SERM-resistant tumor cells after the SERM therapy is stopped.
In this article, we report the development of a reproducible raloxifene-resistant cell model, termed MCF-7/Ral. We used this model to investigate raloxifene resistance in breast cancer and to provide a preclinical basis for possible therapy for raloxifene-resistant tumors. We also used this model to explore the mech-anism for raloxifene resistance and responsiveness to 17β-estradiol (hereafter estradiol), with particular attention to the regulation of NF-KB activity and apoptosis initiated through Fas, a member of the tumor necrosis factor receptor family (38,39).
Materials and methods
Breast Cancer Cells
MCF-7 cells used in this study (40) were cloned from an ERα-positive human MCF-7 breast cancer cells originally obtained from the American Type Culture Collection (Man-assas, VA). They were maintained in full serum medium composed of RPMI-1640 medium, 10% fetal bovine serum, 2 mM glutamine, penicillin at 100 U/mL, streptomycin at 100 μg/mL, 1X nonessential amino acids (all from Invitrogen, Carlsbad, CA), and bovine insulin at 6 ng/mL (Sigma-Aldrich, St. Louis, MO). Raloxifene-resistant MCF-7 (termed MCF-7/Ral) cells were derived by culturing MCF-7 cells for more than 12 months in 1 πM raloxifene in estrogen-free medium composed of phenol red-free minimal essential medium, 5% calf serum (treated three times with dextran-coated charcoal), 2 mM glutamine, bovine insulin at 6 ng/mL, penicillin at 100 U/mL, streptomycin at 100 μg/mL, and 1X nonessential amino acids. 4-Hydroxytamoxifen and estradiol were purchased from Sigma-Aldrich. Raloxifene used in cell cultures was a generous gift from Lilly Research Laboratories (Indianapolis, IN). Raloxifene (Evista) tablets used in animal studies were commercially available (Lilly Research Laboratories). ICI 182,780 (fulvestrant) and tamoxifen were provided by AstraZeneca Pharmaceuticals (Macclesfield, U.K.).
Proliferation Assays
MCF-7 or MCF-7/Ral cells were cultured in estrogen-free medium for 4 days before beginning the proliferation assay (day 0) by plating 1.5 X 104 cells in 1 mL of estrogen-free medium per well in 24-well plates. Medium containing the appropriate test compound was added on day 1. All compounds were dissolved in 100% ethanol (i.e., vehicle) and added to the medium at a 1 : 1000 dilution. Compound-containing medium was changed on days 3 and 5, and the experiment was stopped on day 7. The DNA content of cells was measured as described previously (41) with a VersaFluor fluorometer (Bio-Rad Laboratories, Hercules, CA).
To measure cell proliferation rates, 7.5 X 104 MCF-7 or MCF-7/Ral cells were plated into T-25 flasks in 5 mL of me-dium. Medium containing the appropriate test compound was added on day 1, and the cell number was counted each day with a hemacytometer.
To determine the effect of extended estradiol treatment on the proliferation of MCF-7/Ral cells, the cells were grown in estrogen-free medium without raloxifene for 4 days before start-ing experiments. For passage 0, cultures containing 2.5 X 105 cells were treated with vehicle control, 1 πM raloxifene, 1 nM estradiol, or a combination of 1 πM raloxifene and 1 nM estra-diol for 6 days. After trypsinization, the cells in a single-cell suspension were counted with a hemacytometer. For passage 1, 2.5 X 105 cells from each treatment group were plated and continuously treated with the respective treatment for another 6 days. The cells were trypsinized and counted. This procedure was repeated for passages 2 and 3.
Cell cycle analysis was performed in the flow cytometry core facility (Robert H. Lurie Comprehensive Cancer Center, North-western University, Chicago, IL), as described (42,43). Briefly, single-cell suspensions of MCF-7 or MCF-7/Ral cells were washed with phosphate-buffered saline and resuspended at 1 X 106 cells per milliliter, and 1 mL of cell suspension was mixed with 1 mL of a solution containing propidium iodide at 50 μg/mL, RNase at 180 U/mL, 0.1% Triton X-100, and 3% polyethylene glycol in 3.36 mMsodium citrate (pH 7.8). After a 20-minute incubation at room temperature in the dark, 1 mL of a solution containing propidium iodide at 50 μg/mL, 0.1% Triton X-100, 3.56 MNaCl, and 0.3% polyethylene glycol was added. The tubes were incubated at 4 °C in the dark for a minimum of 6 hours before flow cytometry analysis (Beckman Coulter Epics XL-MCL; Beckman Coulter, Miami, FL). Data were analyzed with Modfit (version 5.2; Verity Software House, Topsham, ME).
Apoptosis
Approximately 2 X 105 cells from a treatment group were stained with an annexin V fluorescein isothiocyanate kit (Beck-man Coulter), and the number of apoptotic cells was determined by flow cytometry. To visualize apoptotic cells, the cells were grown on cover slides and stained with annexin V. Micrographs were taken with a Zeiss LSM510 confocal microscope (Carl Zeiss, Thornwood, NY). Phase-contrast live cell images were also taken with a Zeiss Invertoskop inverted microscope (Carl Zeiss).
Western Blot Analysis
Whole-cell lysates were extracted in protein extraction buffer, as described previously (44). Protein concentration was measured with the Bio-Rad Protein Assay kit (Bio-Rad Labora-tories). Antibodies against ERα (product G-20) and Fas (products B-10 and C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against Fas ligand (FasL; product F37720) and retinoblastoma protein (product 554136) were from BD Biosciences (San Jose, CA). Antibody against NF-KB p65 (product 06-418) was from Upstate Biotechnology (Waltham, MA). Antibody against p-actin (product AC-15; Sigma-Aldrich) was used to standardize loading. The appropriate secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) was used to visualize bands with an enhanced chemiluminescence (ECL) visualization kit (Amersham, Arlington Heights, IL).
Transient Transfection and Luciferase Assays
MCF-7 or MCF-7/Ral cells were grown in the estrogen-free medium 4 days before the transient transfection assay. Approx-imately 5 X 106 cells were mixed with 1μg of a reporter plasmid, either VitA2-ERE3-luciferase (where VitA2 is vitellogenin A2 and ERE3 is three estrogen response elements) (45) or pNF-KB-Luc (where Luc is luciferase; product 6053-1; BD Biosciences Clontech, Palo Alto, CA), and 0.2 μg of pCMV-β-galactosidase (where CMV is cytomegalovirus). Cells were electroporated (950 μF, 320 V) with a Bio-Rad Gene Pulser II (Bio-Rad Laboratories). Luciferase and β-galactosidase activities were measured as previously described (45).
Approximately 1 X 107 MCF-7 or MCF-7/Ral cells sus-pended in saline solution (product 20012027; Invitrogen) were bilaterally inoculated into mammary fat pads of ovariectomized BALB/c nu/nu mice (Harlan Sprague Dawley, Madison, WI) as described previously (46). Inoculated mice were randomly di-vided into groups of 10 and were treated with estradiol, tamox-ifen, or raloxifene or were not treated. For the estradiol treat-ment, silastic estradiol capsules [1 cm long (47)] were implanted subcutaneously in the mouse’s back on the day of cell inocula-tion and replaced after 8-10 weeks of treatment. These capsules produced a mean serum estradiol level of 380 pg/mL (48). Preparation of tamoxifen and raloxifene were described in detail previously (31). Tamoxifen and raloxifene were administered orally by gavage at 1.5 mg/day per mouse for 5 days a week for 9 or 14 weeks, as indicated. Tumors were measured weekly with vernier calipers. The cross-sectional tumor area was calculated by multiplying the length (l) by the width (w) and π and dividing by 4 (i.e., lwπ/4).
The Animal Care and Use Committee of Northwestern Uni-versity approved all of the procedures involving animals.
Real-Time Polymerase Chain Reaction
Total RNA (100 ng) was reverse transcribed by use of ran-dom hexamers (TaqMan Reverse Transcription Reagents; Ap-plied Biosystems, Foster City, CA). Primers and probes for Fas and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed with Primer Express 1.5 software (Applied Biosystems). The sequences for all primers and probes are as follows: GAPDH forward primer, 5 '-GAAGGTGAAGGTCGGAGTCA-3'; GAPDH reverse primer, 5'-GAAGATGGTGATGGGATTTC-3'; GAPDH probe, 5'-FAM-CAAGCTTCCCGTTCTCAGCC-QSY7-3'; Fas forward primer, 5 '-TGGAAGGCCTGCAT-CATGA-3'; Fas reverse primer, 5'-CAGTCCCTAGCTTTCC-TTTCACC-3'; and Fas probe, 5'-FAM-CCAATTCTGCC-ATAAGCCCTGTCCTCC-3 '. All probes were labeled with 6-carboxyfluorescein (FAM) as reporter and with QSY7 (a non-fluorescent diarylrhodamine derivative) as quencher. The prim-ers and probes were synthesized by MegaBases (Evanston, IL). The TaqMan polymerase chain reaction (PCR) Core Reagent Kit (Applied Biosystems) was used for PCR. A 25-μL reaction mixture contained 2 μL of the cDNA, probe at 100 nM, and each primer at 200 nM. PCRs were performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems). The PCR conditions were 50 °C for 2 minutes and 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. Human GAPDH was used as an internal control, and each total RNA sample was normalized to the content of GAPDH mRNA.
Statistical Analysis
All data are expressed as the mean (with 95% confidence interval [CI]) of at least three determinations, unless stated otherwise. Paired t test was used when only two groups were compared. The interaction between estradiol and raloxifene was determined with a two-way analysis of variance (SPSS software; SPSS, Chicago, IL). When the interaction was statistically sig-nificant, each pairwise comparison was made with a one-way analysis of variance followed by Tukey’s honestly significant difference. All statistical tests were two-sided.
Results
Raloxifene Resistance of MCF-7 Cells In Vitro
Growth characteristics of raloxifene-resistant MCF-7/Ral cells and parental MCF-7 cells were investigated. When cultured with 1 μM raloxifene, MCF-7/Ral cells (at day 6, 152 X 104 cells, 95% CI = 130 X 104 to 174 X 104 cells; P = .006) grew statistically significantly faster than MCF-7 cells (at day 6, 55 X 104 cells, 95% CI = 44 X 104 to 64 X 104 cells) (Fig. 1, A). In fact, raloxifene at 1 μM statistically significantly (on day 4, P = .001; on day 5 and day 6, P<.001) inhibited the growth of cultured MCF-7 cells (Fig. 1, A), as reported previously (49), and cell cycle analyses demonstrated that such MCF-7 cells were arrested in G0/G1 phase (Fig. 1, B).
Figure 1. Proliferation of MCF-7 and MCF-7/Ral cells in vitro. A) Proliferation rate. Approximately 7.5 X 104 MCF-7 or MCF-7/Ral cells were plated in T-25 flasks. MCF-7 cells were treated with ethanol vehicle (MCF-7 Control) or 1 μM raloxifene (MCF-7 + 1 μM Ral) in full serum medium. MCF-7/Ral cells were grown in estrogen-free medium alone [MCF-7/Ral (-Ral)] or containing 1 μM raloxifene (MCF-7/Ral). Three samples were counted every day. Data are the means; error bars are 95% confidence intervals. Statistically significant differences compared with MCF-7 treated with 1 μM raloxifene cells are as follows: a, P<.001; b,P = .041; c,P = .006. The experimentwas repeatedtwice. B) Cell cycle analysis. MCF-7 cells were grown in full serum medium containing ethanol vehicle (Control) or 1 p M raloxifene (Ral) for 4 days, and MCF-7/Ral cells were grown in medium containing vehicle (Control) or 1 p M raloxifene for 4 days. * P<.001 compared with the corresponding control. All statistical tests were two-sided.
Because the ERα plays an important role in the development and growth of breast cancer (3), raloxifene resistance could be a consequence of changes in the expression and/or transactivation activity of ERα. We measured the level of ERα protein by western blot analysis in MCF-7 and MCF-7/ Ral cells and found that ERα protein levels were slightly lower in MCF-7/Ral cells than in MCF-7 cells (Fig. 2, A). In both cell lines, addition of 1 μM raloxifene had essentially no effect on the level of ERα protein, whereas addition of 1 nM estradiol decreased the level of ERα protein dramatically. Both cell lines had similar basal ERE3-luciferase activities. Addition of 1 nM estradiol statistically significantly increased ER transcriptional activity (P<.001), and addition of 1 μM raloxifene inhibited the estradiol-induced ER transactivation activity in both cell lines (Fig. 2, B). Addition of estradiol also induced expression of endogenous ER-regulated genes, such as cyclin D1, p53, and BCL2 in both MCF-7 and MCF-7/Ral cells (data not shown). Because MCF-7/Ral cells appeared to express a functional ERα, as do MCF-7 cells, and raloxifene did not increase ER transactivation activity, en-hanced estrogenic activity of the raloxifene-ERα complex is probably not the primary mechanism of raloxifene resistance in MCF-7 cells.
Figure 2. . Expression of functional estrogen receptor a (ERα) in MCF-7 and MCF-7/Ral cells. A) Western blot analysis for ERα in MCF-7 and MCF-7/Ral cells grown in estrogen-free medium. The cells were treated with ethanol vehicle (lanes C), 1 μM raloxifene (lanes Ral), 1nM estradiol (lanes E2), or a combination of 1 μM raloxifene and 1 nM estradiol (lanes Ral + E2) for 4 days. p-Actin was used as the loading control. The blot is representative of three experiments, all with similar results. B) Transcriptional activity of the estrogen receptor in MCF-7 and MCF-7/Ral cells, as measured by VitA2-ERE3-luciferase transient transfection assays (where VitA2 is vitellogenin A2 and ERE3 is three copies of estrogen response elements). RLU = relative light units. *P<.001 compared with the corresponding control groups. All statistical tests were two-sided.
Enhanced NF-KB Activity in MCF-7/Ral Cells
We next investigated whether NF-KB activity plays a role in raloxifene resistance. Short-term treatment (up to 48 hours) with estradiol or raloxifene had essentially no effect on NF-KB activity in either cell line, as measured with NF-KB reporter activity (data not shown). However, as shown in Fig. 3, statistically significantly higher basal NF-KB activity was detected in MCF-7/Ral cells (1.6 U, 95% CI = 1.2 to 2.0 U) than in MCF-7 cells (0.8 U, 95% CI = 0.4 to 1.1 U; P = .004). In addition, a higher level of NF-KB p65 protein, as measured by western blot analysis, was found in MCF-7/Ral cells than in MCF-7 cells (Fig. 3, inset). Thus, enhanced NF-KB activity may play a role in raloxifene resistance in MCF-7 cells.
Figure 3. Basal activity of nuclear factor-KB (NF-KB) in MCF-7 and MCF-7/Ral cells. Activity was measured by NF-KB-luciferase transient transfection assays. MCF-7 and MCF-7/Ral cells were grown in estrogen-free medium for 4 days before the assays. Inset) Protein levels of NF-KB p65 measured by western blot analysis in MCF-7 (lane 1) and MCF-7/Ral (lane 2) cells. RLU = relative light units. *P = .004 compared with MCF-7 cells. All statistical tests were two-sided.
Inhibitory Effect of Estradiol in MCF-7/Ral Cells
To investigate potential cross-resistance to other SERMs, we used DNA assays to measure the proliferation in MCF-7 and MCF-7/Ral cells treated with vehicle control, 1 nM estradiol, 1 μM raloxifene, or 1 μM 4-hydroxytamoxifen for 6 days. As shown in Fig. 4, A, proliferation of MCF-7 cells was statistically significantly higher in estradiol-treated cultures than in untreated control cell cultures (P<.001; Fig. 4, A). Both raloxifene and 4-hydroxytamoxifen inhibited estradiol-induced increased proliferation of MCF-7 cells (data not shown). MCF-7/Ral cells proliferated more rapidly than MCF-7 cells (P<.001) in estrogen-free medium. Although a 6-day estradiol treatment did not appreciably affect the proliferation of MCF-7/Ral cells (Fig. 4, A), cells were larger in estradiol-treated cultures than in untreated cultures, and apoptotic-like and multinucleated cells were observed in estradiol-treated cultures (Fig. 4, B). In contrast, MCF-7/Ral cultures treated for 6 days with a combination of raloxifene and estradiol contained normal-sized cells (Fig. 4, B). Thus, estradiol may be involved in the regulation of cell proliferation and apoptosis in raloxifene-resistant cells.
Figure 4. Hormone-dependent growth of MCF-7 and MCF-7/Ral cells. All cells were grown in estrogen-free medium for 4 days before the assays. Cells were treated with ethanol vehicle (C or control), 1 nM 17β-estradiol (E2), 1 μM raloxifene (Ral), or 1 μM 4-hydroxytamoxifen (4-OHT) for 6 days. A) DNA assay. For MCF-7 cells, values were as follows: control = 2.0 pg of DNA per well (95% CI = 1.6 to 2.3 pg); E2 = 10.0 pg (95% CI = 5.8 to 14.3 pg); Ral = 2.2 pg (95% CI = 1.5 to 3.0 pg); and 4-OHT = 1.5 pg (95% CI = 1.1 to 1.9 pg). For MCF-7/Ral cells, values were as follows: control = 16.1 pg (95% CI = 14.1 to 18.2 pg); E2 = 19.6 pg (95% CI = 15.6 to 23.6 pg); Ral = 19.8 pg (95% CI = 16.7 to 22.9 pg); and 4-OHT = 21.7 pg(95%CI = 19.3 to 24.1 pg). * P<.001 compared with the MCF-7 control cells. B) Cell morphology. Micrographs are phase-contrast images. Open arrow = apoptotic-like cells; solid arrow = double nuclei. Scale bar = 50 pm. C) Estradiol inhibition of MCF-7/Ral cell growth after an extended treatment in vitro. Approximately 2.5 X 104 cells were grown in estrogen-free medium for each passage (0-3) in the absence (C) or presence of 1 μM raloxifene (Ral), 1 nM estradiol (E2), or the combination of raloxifene and estradiol (E2 + Ral). Experiments were repeated five times. The statistical interaction of estradiol and raloxifene was determined with a two-way analysis of variance. The interaction was statistically significant in passages 1 (P = .022), 2 (P = .023), and 3 (P = .029). All six pairwise comparisons among the four groups were calculated with Tukey’s honestly significant difference. *Statistically significant compared with the values in raloxifene treatment group as follows: for passage 1, P<.001; for passage 2, P = .006; and for passage 3, P = .001. D) Estradiol treatment and nuclear factor KB (NF-KB) activity in MCF-7 and MCF-7/Ral cells. MCF-7 or MCF-7/Ral cells were grown in estrogen-free medium for 4 days before the NF-KB-luciferase transient transfection assay. MCF-7 + E2 or MCF-7/Ral + E2 cells were treated with 1 nM estradiol for 6 days and then grown in estrogen-free medium for 4 days before the transient transfection assay. * P<.001 compared with MCF-7/ Ral cells. All statistical tests were two-sided.
To study long-term effects of estradiol, MCF-7/Ral cells were treated with vehicle control, 1 μM raloxifene, 1 nM estradiol, or a combination of raloxifene and estradiol for four passages (passages 0-3). As shown in Fig. 4, C, treatment with estradiol statistically significantly inhibited the proliferation of MCF-7/Ral cells after 12-24 days compared with cells in raloxifene-treated cultures (for example, for passage 3, P = .001). Inter-estingly, extended estradiol treatment of MCF-7/Ral cells also statistically significantly inhibited NF-KB activity (0.2 U, 95% CI = 0.2 to 0.3 U, compared with 1.6 U, 95% CI = 1.2 to 2.0 U for baseline control; P<.001) (Fig. 4, D) without affecting the level of NF-KB p65 protein (data not shown). This result suggests that elevated NF-KB activity plays a role in raloxifene resistance in MCF-7 cells. In contrast, estradiol treatment did not statistically significantly affect NF-KB activity in MCF-7 parental cells (estradiol = 0.6 U, 95% CI = 0.4 to 0.8 U; control = 0.8 U, 95% CI = 0.5 to 1.1 U; P = .225).
Effect of Estradiol on the Cell Cycle and Apoptosis in MCF-7/Ral Cells
Because a decrease in cell number can result from decreased cell proliferation, increased cell death, or both, we first evaluated the effect of estradiol on the cell cycle in MCF-7 and MCF-7/Ral cells. In parental MCF-7 cells, estrogen deprivation blocked the transition from G1 phase to S phase, whereas treatment with 1 nM estradiol relieved the block as shown by the decreased number of cells in G0/G1 phase and the increased number in S phase (for both, P<.001), apparently by inducing phosphorylation of the retinoblastoma protein (Rb) (Fig. 5, A). The estradiol-mediated Rb phosphorylation was blocked by raloxifene. In contrast, a 6-day estradiol treatment did not affect the cell cycle of MCF-7/Ral cells (data not shown), but a treatment of 2 weeks or longer with 1 nM estradiol statistically significantly increased the percentage of cells in G2/M phase to 12.5% (95% CI = 10.6% to 14.3%) compared with 7.6% for vehicle control treatment (95% CI = 5.1% to 10.0%; P = .003), 7.7% for raloxifene (95% CI = 5.6% to 9.8%; P = .004), and 7.6% for estradiol plus raloxifene (95% CI = 4.9% to 10.2%; P = .003). A treatment of 2 weeks or longer with 1 nM estradiol also statistically significantly decreased the percentage of cells in G0/G1 phase to 69.7% (95% CI = 65.0% to 74.4%) compared with 80.1% for vehicle control treatment (95% CI = 74.2% to 85.9%; P = .006), 77.5% for raloxifene (95% CI = 72.1% to 82.8%; P = .041), and 77.3% for estradiol plus raloxifene (95% CI = 72.7% to 82.0%; P = .045) (Fig. 5, B). Rb was phosphorylated in raloxifene-treated MCF-7/Ral cells, and addition of estradiol did not induce additional phosphorylation of Rb (Fig. 5, B, lower panel).
Figure 5. Hormone-dependent effects on the cell cycle in MCF-7 (A) and MCF- 7/Ral (B) cells. Cell cycle analysis is shown in the upper panels, and western blot analysis for the retinoblastoma protein (Rb) and phosphorylated Rb (ppRb) is shown in the lower panels. MCF-7 cells (A) and MCF-7/Ral cells (B) were treated with vehicle control (C), 1 μM raloxifene (Ral), 1 nM 17β-estradiol (E2), or 1 μM raloxifene plus 1 nM 17β-estradiol (E2 + Ral) for 6 days. Because there was no statistically significant difference in results from MCF-7/Ral cells in passages 1-3, the results from these passages were pooled for analysis.
Estradiol induces apoptosis in long-term estrogen-deprived cells (36) and, in fact, we observed apoptotic cells inMCF-7/Ral cultures treated with estradiol for 6 days (Fig. 4, B). To confirm that apoptotic cells were present in estradiol-treated MCF-7/Ral cultures, we used annexin V binding. Untreated control MCF-7/Ral cultures or MCF-7/Ral cultures treated with 1 μM raloxifene for up to 24 days had a minimal number of apoptotic cells (Fig. 6, B), an observation that reflects enhanced survival of MCF-7/Ral cells (Fig. 3) that may be mediated by their high activity of NF-KB, a known survival factor (50-54). After a 6-day estradiol treatment (passage 0), the percentage of apoptotic MCF-7/Ral cells was not statistically significantly different than that in untreated control cultures or cultures treated with 1 μM raloxifene or a combination of estradiol and raloxifene (P = .063). Extended estradiol treatment (passages 1-3; 12-24 days), however, increased the percentage of apoptotic cells, and the addition of raloxifene essentially blocked this effect(Fig. 6, B). Apoptosis was observed in untreated control and raloxifene-treated MCF-7 cell cultures, and estradiol treatment decreased the percentage of apoptotic MCF-7 cells (Fig. 6, B). Thus, estradiol treatment inhibited apoptosis in parental MCF-7 cells, and estradiol treatment promoted apoptosis in raloxifene-resistant MCF-7/Ral cells.
Figure 6. . Hormones and apoptosis in MCF-7 and MCF-7/Ral cells. C = vehicle control; Ral = raloxifene; E2 = estradiol. A) Confocal images of annexin V staining in MCF-7/Ral cells. Upper panel = annexin V staining; lower panel = phase-contrast image. Scale bar = 50 pm. B) Apoptosis as measured by annexin V staining and flow cytometry. For panels MCF-7 and MCF-7/Ral (passage 0), cells were treated for 6 days with vehicle control, 1 μM raloxifene, 1 nM estradiol, or 1 μM raloxifene plus 1 nM estradiol. For panel MCF-7/Ral, cells 1-3 were pooled. C) Western blot analysis for Fas and FasL in MCF-7 and MCF-7/Ral cells treated as indicated for two passages. p-Actin was used as the loading control. D) Real-time polymerase chain reaction analysis of Fas mRNA expression. MCF-7 cells were treated as indicated for 6 days, and MCF-7/Ral cells were treated as indicated for passages 0-3 (12-24 days). * P<.001 compared with any other group of MCF-7/Ral cells. All statistical tests were two-sided.
Estradiol and the Induction of Fas Expression
Estradiol regulates FasL expression in a variety of cells and tissues (55-59), including long-term estrogen-deprived MCF-7 cells (36). Untreated MCF-7 cells express a low or nondetectable level of Fas receptor (36,60), but long-term estrogen deprivation of MCF-7 cells increases Fas expression (36).
We investigated whether estradiol plays a role in the regula-tion of the Fas/FasL system in MCF-7/Ral and MCF-7 cells by determining their levels of Fas and FasL expression. Both MCF-7 and MCF-7/Ral cells expressed FasL but in an estradiol-independent manner. Although more apoptotic cells were ob-served in untreated control and raloxifene-treated MCF-7 cul-tures (Fig. 6, B), Fas protein was barely detected in MCF-7 cells. Estradiol treatment of parental MCF-7 cells did not appreciably affect Fas expression (Fig. 6, C) but did prevent apoptosis (Fig. 6, B), indicating that apoptosis in MCF-7 cells does not appear to be mediated by the Fas/FasL pathway. Fas protein was not detected in untreated control or raloxifene-treated MCF-7/Ral cells (Fig. 6, C). Estradiol treatment dramatically increased the level of Fas protein and mRNA in MCF-7/Ral cells (Fig. 6, C and D). For example, Fas mRNA levels increased 4.5-fold (95% CI = 2.8-to 6.3-fold) after estradiol treatment compared with 0.5-fold (95% CI = 0.3-to 0.8-fold) after control treatment, 0.8-fold (95% CI = 0.7-to 1.0-fold) after 1 μM raloxifene, and 0.7-fold (95% CI = 0.4-to 1.1-fold) after raloxifene plus estradiol (all P<.001). Addition of raloxifene blocked estradiol-induced increases in Fas protein and mRNA levels (Fig. 6, C and D). In MCF-7/Ral cells, the effects of estradiol on Fas expression correspond to those on apoptosis and indicate that estradiol-induced apoptosis might be mediated by the Fas/FasL pathway in these cells.
Hormone-Dependent Growth of MCF-7/Ral Cells In Vivo
To further study raloxifene resistance and examine the unique inhibitory effect of estradiol on the growth of SERM-sensitized breast cancer cells, we examined the ability of MCF-7/Ral cells and parental MCF-7 cells to form tumors in athymic mice. As previously reported (8,61), treatment with estradiol, but not with raloxifene, stimulated the growth of MCF-7 cell tumors in ovariecto-mized athymic mice (Fig. 7, A). MCF-7/Ral tumors, in contrast, required treatment with a SERM to grow; both raloxifene and tamoxifen, but not estradiol, statistically significantly stimulated the growth of MCF-7/Ral tumors in athymic mice (Fig. 7, B). Furthermore, if SERM treatment of the tumor-bearing athymic mice was stopped after 9 weeks and replaced by estradiol treatment for 5 weeks, tumors decreased in size in both groups of mice (P = .004 for raloxifene and P<.001 for tamoxifen), and some tumors disappeared altogether. These data are consistent with the effect of estradiol on the MCF-7/Ral cells in vitro.
Figure 7. Growth of MCF-7 (A) and MCF-7/Ral (B) tumors in ovariectomized athymic mice. Each group had 10 mice. The mice were treated with a 1-cm estradiol (E2) capsule, 1.5 mg of tamoxifen (Tam), 1.5 mg of raloxifene (Ral), or vehicle control. At week 9 (arrow), raloxifene- and tamoxifen-stimulated MCF- 7/Ral tumors were split into two arms: 1) continue raloxifene or tamoxifen treatment or 2) stop raloxifene or tamoxifen treatment and start estradiol treatment for 5 weeks (i.e., Stop Ral + E2 or Stop Tam + E2). For both treatments in the second arm, tumors at 14 weeks were statistically significantly decreased in size in both groups of mice (P = .004 for raloxifene and P<.001 for tamoxifen). Results are shown as mean tumor size with 95% confidence interval (only the upper confidence limit for each point is shown).
Discussion
The increased use of SERMs to prevent breast cancer (with tamoxifen) or osteoporosis (with raloxifene) has added a new therapeutic dimension to clinical practice. The results of clinical trials now underway may establish SERMs as multifunctional medicines for the prevention of osteoporosis, coronary heart disease, and breast cancer (3). However, the clinical use of these agents must be accompanied by rigorous laboratory studies to elucidate the mechanisms involved in the development of SERM-resistant breast cancer. The process of tamoxifen-resistant breast cancer development is well documented, but it is important to examine the potential for drug resistance to SERMs in general. We now report data from a new study of the evolu-tion of raloxifene-resistant breast cancer in the laboratory that builds on our previous studies with tamoxifen (8,29,30,33,62). Understanding the mechanism of raloxifene resistance should contribute important information for effective breast cancer therapy. We have confirmed and extended our original observation that low concentrations of estrogen shift the survival of SERM-resistant breast cancer cells by initiating apoptosis. Unlike our previous work that was entirely in vivo (32,33,63), we now report results from both in vitro and in vivo studies using a new model of raloxifene-resistant breast cancer cells, MCF-7/Ral cells. We have defined the shift from cell survival induced by raloxifene in breast cancer to cell death induced by a modest concentration (1 nM) of estradiol in pre-sensitized cells.
Most research on SERM-resistant breast cancer has previ-ously focused on tamoxifen. Apparent increased expression of ERα (64,65) and decreased expression of the nuclear receptor corepressor (i.e., N-CoR) (66) were detected in tamoxifen-resistant cells and tumors. An altered balance between ER co-activators and corepressors has been suggested as the mecha-nism for tamoxifen resistance (67,68). Consequently, we initially examined the expression of ERα, ER coactivators (such as steroid receptor coactivators SRC-1, SRC-2, and SRC-3, and p300/CBP [cAMP response element-binding protein binding protein]), and ER corepressors (N-CoR and silencing mediator for retinoid and thyroid receptors) in MCF-7 and MCF-7/Ral cells with real-time PCR. We found no statistically significant differences in the expression levels of ERα and the ERα co-regulators in MCF-7 cells and MCF-7/Ral cells grown in estrogen-free medium or treated with 1 μM raloxifene for 24 or 48 hours (data not shown). Therefore, we conclude that changes in the expression of ERα or ER coregulators are not the primary mechanism for raloxifene resistance.
NF-KB is a key transcriptional factor for cell growth (69) and survival (50-54). Increased NF-KB activity is involved in drug resistance in breast cancer (70-72), and ERα represses NF-KB activity in the presence of estradiol (52-54,73,74). Because raloxifene is an antiestrogen, we anticipated that raloxifene would block estradiol-mediated repression of NF-KB activity and thus increase NF-KB activity. In fact, we found higher levels of NF-KB p65 protein and activity in MCF-7/Ral cells than in MCF-7 cells (Fig. 3). Thus, the NF-KB signal transduction pathway could be involved in the survival of raloxifene-resistant MCF-7/Ral cells.
The finding that raloxifene-resistant MCF-7/Ral cells are also resistant to tamoxifen has important therapeutic implications. Tamoxifen is used to treat all stages of ER-positive breast cancer and is used as a chemopreventive agent in women at high risk for breast cancer (2). In vitro (Fig. 4, A) and in vivo (Fig. 7) data show that cells and/or tumors resistant to raloxifene are also resistant to tamoxifen and indicate that tamoxifen should not be given to patients with raloxifene-exposed breast cancer. The converse is also true (31). It is perhaps prudent to consider treatment with an aromatase inhibitor or fulvestrant (i.e., Faslodex), a pure antiestrogen, in patients who have been exposed to raloxifene.
However, the most important observation for the outcome of long-term SERM-resistant breast cancer is the effectiveness of estrogen to induce apoptosis. Antitumor activity of estradiol has been reported for estrogen-deprived breast cancer cells in vitro (34-36), tamoxifen-stimulated breast tumors in vivo (16,32,33), and raloxifene-resistant ECC1 endometrial cancers in vivo (37). More important, these findings have been extended to clinic settings, where estrogen is an effective treatment for breast cancers that have developed resistance to successive antiestrogenic therapies (75). In this study, we found that a 6-day estradiol treatment (passage 0) did not inhibit MCF-7/Ral cell growth (Figs. 4, A, and 6, B), whereas an extended estradiol treatment (passages 1-3) of 12-24 days statistically significantly inhibited MCF-7/Ral cell growth (Fig. 4, C). Although the causes of the delayed inhibitory effect of estradiol are still not known, estradiol treatment also caused a delayed decrease in NF-KB activity (Fig. 4, D) without changing the level of NF-KB p65 protein (data not shown). Estradiol binds to ERα, and the estradiol-ERα complex suppresses NF-KB activity by inhibiting the binding of NF-KB to DNA or by competing with p300/CBP (52,53,76,77), independent of new protein synthesis. However, because a 24-or 48-hour estradiol treatment did not inhibit NF-KB activity in MCF-7 and MCF-7/Ral cells (data not shown), the delayed inhibitory effect of estradiol on NF-KB activity may require the synthesis of new protein(s). This hypothesis is consistent with the report that estradiol inhibits NF-KB activity by increasing the expression of p105, the precursor of NF-KB (78). However, further studies are necessary to understand the mechanism through which estradiol induces the delayed inhi-bition of NF-KB activity and growth in MCF-7/Ral cells.
If NF-KB plays an important role in raloxifene resistance, the decreased NF-KB activity induced by estradiol should increase the number of cells undergoing apoptosis. In fact, we observed that an extended estradiol treatment statistically significantly increased the number of apoptotic MCF-7/Ral cells (Fig. 6, A and B), as reported in long-term estrogen-deprived MCF-7 cells (36) and in T47D:protein kinaseCαcells (16). Song et al. (36) suggested that estradiol induces apoptosis in long-term estrogen-deprived MCF-7 cells via the Fas/FasL pathway, and they noted that the level of Fas is elevated in the long-term estrogen-deprived MCF-7 cells and that treatment with estradiol increases the expression level of FasL and induces apoptosis through the elevated level of Fas. Consequently, we examined the expres-sion of Fas and FasL in MCF-7 and MCF-7/Ral cells in response to estradiol treatment. We found that estradiol did not induce expression of FasL in MCF-7 or MCF-7/Ral cells or the expres-sion of Fas in MCF-7 cells but did induce the expression of Fas protein and mRNA in MCF-7/Ral cells (Fig. 6, D). This result is perplexing because, to our knowledge, there are no reports that estrogen stimulates the expression of Fas; however, increased Fas expression is observed in tamoxifen-resistant MCF-7 cells treated with estradiol, which presages apoptosis in vivo (63).
In MCF-7/Ral cells, estradiol treatment induced cell cycle arrest at G2/M phase (Fig. 5, B) and generated multinucleated cells (Fig. 4, B), although the mechanism is currently unclear. Estradiol stimulates the expression of genes that are involved in both inhibiting and promoting G2/M-phase arrest. In MCF-7 cells, estrogen induces the estrogen-responsive finger protein (79,80), which targets the 14-3-3 CT protein for proteolysis. [14-3-3CT is a p53-regulated gene that, along with p21Cip-1/Waf-1, plays a role in p53-mediated cell cycle arrest at G2/M phase (81,82).] Because estradiol also induces p53 (83), the estrogen-responsive finger protein and p53 might be involved in the estradiol-mediated G2/M-phase arrest observed in MCF-7/Ral cells. We are currently investigating this hypothesis.
In summary, the MCF-7/Ral model is the first reproducible raloxifene-resistant breast cancer grown in athymic mice. Using this model, we demonstrated that breast tumors and/or cells resistant to tamoxifen are also resistant to raloxifene, and so tamoxifen would not be an appropriate therapy for breast cancer after raloxifene resistance is diagnosed. More importantly, we have established that estradiol treatment causes tumor regression in SERM-sensitized cells by inducing G2/M-phase arrest and apoptosis. Although the mechanism is not completely elucidated, the balance between survival and apoptosis may be mediated through the Fas/FasL pathway by the increasing Fas expression and the decreasing NF-KB activity. Overall, these data, and those in the companion study (63) using a tamoxifen-resistant model in vivo, suggest that it is possible for a patient’s own estrogen to act as an anticancer agent in SERM-resistant breast cancers. Clearly, a clinical strategy to use an aro-matase inhibitor after SERM resistance may have some short-term benefit for patients, but it is possible that a novel strategy of briefly treating patients with estrogen before re-instituting estrogen-deprivation therapy may benefit certain women. Indeed, preliminary studies (75) have demonstrated the beneficial effects of estrogen in patients for whom repeated estrogen-deprivation treatments have failed.
Notes
Supported by Specialized Programs of Research Excellence (SPORE) grant CA89018-01 (to V. C. Jordan) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, grant DAMD 17-00-1-0386 (to V. C. Jordan) from the Department of Defense, and the Avon Foundation. We thank Dr. Joan Lewis for her valuable discussion and Dr. Alfred Rademaker for his statistical advice. Manuscript received February 10, 2003; revised August 21, 2003; accepted August 29, 2003.
References
Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet 1998;351:1451-67.
Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M, Cronin WM, et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 1998;90:1371-88.
Jordan VC, Gapstur S, Morrow M. Selective estrogen receptor modulation and reduction in risk of breast cancer, osteoporosis, and coronary heart disease. J Natl Cancer Inst 2001;93:1449-57.
Ettinger B, Black DM, Mitlak BH, Knickerbocker RK, Nickelsen T, Genant HK, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators [published erratum appears in JAMA 1999;282: 2124]. JAMA 1999;282:637-45.
Cummings SR, Eckert S, Krueger KA, Grady D, Powles TJ, Cauley JA, et al. The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. Multiple Outcomes of Raloxifene Evaluation. JAMA 1999;281:2189-97.
Cauley JA, Norton L, Lippman ME, Eckert S, Krueger KA, Purdie DW, et al. Continued breast cancer risk reduction in postmenopausal women treated with raloxifene: 4-year results from the MORE trial. Multiple Outcomes of Raloxifene Evaluation. Breast Cancer Res Treat 2001;65:125-34.
Gail MH, Brinton LA, Byar DP, Corle DK, Green SB, Schairer C, et al. Projecting individualized probabilities of developing breast cancer for white females who are being examined annually. J Natl Cancer Inst 1989;81:1879-86.
Gottardis MM, Jordan VC. Development of tamoxifen-stimulated growth of MCF-7 tumors in athymic mice after long-term antiestrogen adminis-tration. Cancer Res 1988;48:5183-7.
Howell A, Dodwell DJ, Anderson H, Redford J. Response after withdrawal of tamoxifen and progestogens in advanced breast cancer. Ann Oncol 1992;3:611-7.
Schafer JM, Lee ES, O’Regan RM, Yao K, Jordan VC. Rapid development of tamoxifen-stimulated mutant p53 breast tumors (T47D) in athymic mice. Clin Cancer Res 2000;6:4373-80.
Dumont JA, Bitonti AJ, Wallace CD, Baumann RJ, Cashman EA, Cross-Doersen DE. Progression of MCF-7 breast cancer cells to antiestrogen-resistant phenotype is accompanied by elevated levels of AP-1 DNA-binding activity. Cell Growth Differ 1996;7:351-9.
Johnston SR, Lu B, Scott GK, Kushner PJ, Smith IE, Dowsett M, et al. Increased activator protein-1 DNA binding and c-Jun NH2-terminal kinase activity in human breast tumors with acquired tamoxifen resistance. Clin Cancer Res 1999;5:251-6.
Schiff R, Reddy P, Ahotupa M, Coronado-Heinsohn E, Grim M, Hilsen-beck SG, et al. Oxidative stress and AP-1 activity in tamoxifen-resistant breast tumors in vivo. J Natl Cancer Inst 2000;92:1926-34.
Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem 2001;276:9817-24.
Sun M, Paciga JE, Feldman RI, Yuan Z, Coppola D, Lu YY, et al. Phosphatidylinositol-3-OH kinase (PI3K)/AKT2, activated in breast can-cer, regulates and is induced by estrogen receptor alpha (ERαlpha) via interaction between ERαlpha and PI3K. Cancer Res 2001;61:5985-91.
Chisamore MJ, Ahmed Y, Bentrem DJ, Jordan VC, Tonetti DA. Novel antitumor effect of estradiol in athymic mice injected with a T47D breast cancer cell line overexpressing protein kinase Calpha. Clin Cancer Res 2001;7:3156-65.
Pietras RJ, Arboleda J, Reese DM, Wongvipat N, Pegram MD, Ramos L, et al. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene 1995;10:2435-46.
Kurokawa H, Lenferink AE, Simpson JF, Pisacane PI, Sliwkowski MX, Forbes JT, et al. Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells. Cancer Res 2000;60:5887-94.
Chung YL, Sheu ML, Yang SC, Lin CH, Yen SH. Resistance to tamoxifen-induced apoptosis is associated with direct interaction between HER2/neu and cell membrane estrogen receptor in breast cancer. Int J Cancer 2002;97:306-12.
Parisot JP, Hu XF, DeLuise M, Zalcberg JR. Altered expression of the IGF-1 receptor in a tamoxifen-resistant human breast cancer cell line. Br J Cancer 1999;79:693-700.
Vertegaal AC, Kuiperij HB, Yamaoka S, Courtois G, van der Eb AJ, Zantema A. Protein kinase C-alpha is an upstream activator of the IkappaB kinase complex in the TPA signal transduction pathway to NF-kappaB in U2OS cells. Cell Signal 2000;12:759-68.
Zhou BP, Hu MC, Miller SA, Yu Z, Xia W, Lin SY, et al. HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappaB pathway. J Biol Chem 2000;275:8027-31.
Pianetti S, Arsura M, Romieu-Mourez R, Coffey RJ, Sonenshein GE. Her-2/neu overexpression induces NF-kappaB via a PI3-kinase/Akt path-way involving calpain-mediated degradation of IkappaB-alpha that can be inhibited by the tumor suppressor PTEN. Oncogene 2001;20:1287-99.
Bhat-Nakshatri P, Sweeney CJ, Nakshatri H. Identification of signal trans-duction pathways involved in constitutive NF-kappaB activation in breast cancer cells. Oncogene 2002;21:2066-78.
Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ Jr, Sledge GW Jr. Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol Cell Biol 1997;17:3629-39.
Gu Z, Lee RY, Skaar TC, Bouker KB, Welch JN, Lu J, et al. Association of interferon regulatory factor-1, nucleophosmin, nuclear factor-kappaB, and cyclic AMP response element binding with acquired resistance to Faslodex (ICI 182,780). Cancer Res 2002;62:3428-37.
Brunner N, Boysen B, Jirus S, Skaar TC, Holst-Hansen C, Lippman J, et al. MCF7/LCC9: an antiestrogen-resistant MCF-7 variant in which acquired resistance to the steroidal antiestrogen ICI 182,780 confers an early cross-resistance to the nonsteroidal antiestrogen tamoxifen. Cancer Res 1997;57: 3486-93.
McLeskey SW, Zhang L, El-Ashry D, Trock BJ, Lopez CA, Kharbanda S, et al. Tamoxifen-resistant fibroblast growth factor-transfected MCF-7 cells are cross-resistant in vivo to the antiestrogen ICI 182,780 and two aro-matase inhibitors. Clin Cancer Res 1998;4:697-711.
Lee ES, Schafer JM, Yao K, England G, O’Regan RM, De Los Reyes A, et al. Cross-resistance of triphenylethylene-type antiestrogens but not ICI 182,780 in tamoxifen-stimulated breast tumors grown in athymic mice. Clin Cancer Res 2000;6:4893-9.
Schafer JM, Lee ES, Dardes RC, Bentrem D, O’Regan RM, De Los Reyes A, et al. Analysis of cross-resistance of the selective estrogen receptor modulators arzoxifene (LY353381) and LY117018 in tamoxifen-stimulated breast cancer xenografts. Clin Cancer Res 2001;7:2505-12.
O’Regan RM, Gajdos C, Dardes RC, De Los Reyes A, Park W, Rademaker AW, et al. Effects of raloxifene after tamoxifen on breast and endometrial tumor growth in athymic mice. J Natl Cancer Inst 2002;94:274-83.
Wolf DM, Jordan VC. A laboratory model to explain the survival advan-tage observed in patients taking adjuvent tamoxifen therapy. Recent results in cancer research. Vol. 127. Heidelberg (Germany): Springer-Verlag; 1993. p. 23-33.
Yao K, Lee ES, Bentrem DJ, England G, Schafer JI, O’Regan RM, et al. Antitumor action of physiological estradiol on tamoxifen-stimulated breast tumors grown in athymic mice. Clin Cancer Res 2000;6:2028-36.
Fernandez P, Wilson C, Hoivik D, Safe SH. Altered phenotypic character-istics of T47d human breast cancer cells after prolonged growth in estrogen-deficient medium. Cell Biol Int 1998;22:623-33.
Liu H, Lee ES, De Los Reyes A, Jordan VC. Antitumor action of estradiol on estrogen-deprived or raloxifene-resistant human breast cancer cells. Breast Cancer Res Treat 2000;64:134.
Song RX, Mor G, Naftolin F, McPherson RA, Song J, Zhang Z, et al. Effect of long-term estrogen deprivation on apoptotic responses of breast cancer cells to 17beta-estradiol. J Natl Cancer Inst 2001;93:1714-23.
Dardes R, Liu H, Gajdos C, O’Regan R, De Los Reyes A, Jordan V. Raloxifene stimulated endometrial cancer grown in athymic mice: cross-resistance with ICI 182,780 and antitumor action of estradiol. Breast Cancer Res Treat 2001;69:288.
Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991;66:233-43.
Oehm A, Behrmann I, Falk W, Pawlita M, Maier G, Klas C, et al. Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily sequence identity with the Fas antigen. J Biol Chem 1992;267:10709-15.
Pink JJ, Jordan VC. Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancercell lines. Cancer Res 1996;56:2321-30.
Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980; 102:344-52.
Vindelov LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983; 3:323-7.
Robinson JK, Rademaker AW, Goolsby C, Traczyk TN, Zoladz C. DNA ploidy in nonmelanoma skin cancer. Cancer 1996;77:284-91.
Schafer JM, Liu H, Bentrem DJ, Zapf JW, Jordan VC. Allosteric silencing of activating function 1 in the 4-hydroxytamoxifen estrogen receptor com-plex is induced by substituting glycine for aspartate at amino acid 351. Cancer Res 2000;60:5097-105.
Catherino WH, Jordan VC. Increasing the number of tandem estrogen response elements increases the estrogenic activity of a tamoxifen ana-logue. Cancer Lett 1995;92:39-47.
Gottardis MM, Robinson SP, Jordan VC. Estradiol-stimulated growth of MCF-7 tumors implanted in athymic mice: a model to study the tumori-static action of tamoxifen. J Steroid Biochem 1988;30:311-4.
Robinson S, Jordan V. Antiestrogenic action of toremifene on hormone-dependent,-independent, and heterogeneous breast tumor growth in the athymic mouse. Cancer Res 1989;49:1758-62.
O’Regan RM, Cisneros A, England GM, MacGregor JI, Meunzner HD, Assikis VJ, et al. Effects of the antiestrogens tamoxifen, toremifene, and ICI 182,780 on endometrial cancer growth. J Natl Cancer Inst 1998;90:1552-8.
Thompson EW, Reich R, Shima TB, Albini A, Graf J, Martin GR, et al. Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens. Cancer Res 1988;48:6764-8.
Sonenshein GE. Rel/NF-kappa B transcription factors and the control of apoptosis. Semin Cancer Biol 1997;8:113-9.
Schwartz SA, Hernandez A, Mark Evers B. The role of NF-kappaB/ IkappaB proteins in cancer: implications for novel treatment strategies. Surg Oncol 1999;8:143-53.
Galien R, Garcia T. Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kappaB site. Nucleic Acids Res 1997;25:2424-9.
Harnish DC, Scicchitano MS, Adelman SJ, Lyttle CR, Karathanasis SK. The role of CBP in estrogen receptor cross-talk with nuclear factor-kappaB in Hep G2 cells. Endocrinology 2000;141:3403-11.
Evans MJ, Eckert A, Lai K, Adelman SJ, Harnish DC. Reciprocal antag-onism between estrogen receptor and NF-kappaB activity in vivo. Circ Res 2001;89:823-30.
Nilsen J, Mor G, Naftolin F. Estrogen-regulated developmental neuronal apoptosis is determined by estrogen receptor subtype and the Fas/Fas ligand system. J Neurobiol 2000;43:64-78.
Mor G, Kohen F, Garcia-Velasco J, Nilsen J, Brown W, Song J, et al. Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen. J Steroid Biochem Mol Biol 2000;73:185-94.
Amant C, Holm P, Xu SH, Tritman N, Kearney M, Losordo DW. Estrogen receptor-mediated, nitric oxide-dependent modulation of the immunologic barrier function of the endothelium: regulation of fas ligand expression by estradiol. Circulation 2001;104:2576-81.
Mor G, Munoz A, Redlinger R Jr, Silva I, Song J, Lim C, et al. The role of the Fas/Fas ligand system in estrogen-induced thymic alteration. Am J Reprod Immunol 2001;46:298-307.
Sapi E, Brown WD, Aschkenazi S, Lim C, Munoz A, Kacinski BM, et al. Regulation of Fas ligand expression by estrogen in normal ovary. J Soc Gynecol Investig 2002;9:243-50.
Yan Y, Haas JP, Kim M, Sgagias MK, Cowan KH. BRCA1-induced apoptosis involves inactivation of ERK1/2 activities. J Biol Chem 2002; 277:33422-30.
Osborne CK, Hobbs K, Clark GM. Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice. Cancer Res 1985;45:584-90.
Gottardis MM, Jiang SY, Jeng MH, Jordan VC. Inhibition of tamoxifen-stimulated growth of an MCF-7 tumor variant in athymic mice by novel steroidal antiestrogens. Cancer Res 1989;49:4090-3.
Osipo C, Gajdos C, Liu H, Chen B, Jordan VC. Paradoxical action of fulvestrant in estradiol-induced regression of tamoxifen-stimulated breast cancer. Natl Cancer Inst 2003;95:000-000.
Speirs V, Malone C, Walton DS, Kerin MJ, Atkin SL. Increased expression of estrogen receptor beta mRNA in tamoxifen-resistant breast cancer pa-tients. Cancer Res 1999;59:5421-4.
Speirs V, Kerin MJ. Prognostic significance of oestrogen receptor beta in breast cancer. Br J Surg 2000;87:405-9.
Lavinsky RM, Jepsen K, Heinzel T, Torchia J, Mullen TM, Schiff R, et al. Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc Natl Acad Sci U S A 1998;95:2920-5.
Graham JD, Bain DL, Richer JK, Jackson TA, Tung L, Horwitz KB. Thoughts on tamoxifen resistant breast cancer. Are coregulators the answer or just a red herring? J Steroid Biochem Mol Biol 2000;74:255-9.
Ratajczak T. Protein coregulators that mediate estrogen receptor function. Reprod Fertil Dev 2001;13:221-9.
Tantini B, Pignatti C, Fattori M, Flamigni F, Stefanelli C, Giordano E, et al. NF-kappaB and ERK cooperate to stimulate DNA synthesis by inducing ornithine decarboxylase and nitric oxide synthase in cardiomyocytes treated with TNF and LPS. FEBS Lett 2002;512:75-9.
Keane MM, Rubinstein Y, Cuello M, Ettenberg SA, Banerjee P, Nau MM, et al. Inhibition of NF-kappaB activity enhances TRAIL mediated apopto-sis in breast cancer cell lines. Breast Cancer Res Treat 2000;64:211-9.
Bhoumik A, Ivanov V, Ronai Z. Activating transcription factor 2-derived peptides alter resistance of human tumor cell lines to ultraviolet irradiation and chemical treatment. Clin Cancer Res 2001;7:331-42.
Weldon CB, Burow ME, Rolfe KW, Clayton JL, Jaffe BM, Beckman BS. NF-kappa B-mediated chemoresistance in breast cancer cells. Surgery 2001;130:143-50.
Pelzer T, Neumann M, de Jager T, Jazbutyte V, Neyses L. Estrogen effects in the myocardium: inhibition ofNF-kappaB DNA binding by estrogen receptor-alpha and-beta. Biochem Biophys Res Commun 2001;286:1153-7.
Sharma RV, Gurjar MV, Bhalla RC. Selected contribution: estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappaB activa-tion in VSM cells from female rats. J Appl Physiol 2001;91:2400-6.
Lonning PE, Taylor PD, Anker G, Iddon J, Wie L, Jorgensen LM, et al. High-dose estrogen treatment in postmenopausal breast cancer patients heavily exposed to endocrine therapy. Breast Cancer Res Treat 2001;67:111-6.
Ray P, Ghosh SK, Zhang DH, Ray A. Repression of interleukin-6 gene expression by 17 beta-estradiol: inhibition of the DNA-binding activity of the transcription factors NF-IL6 and NF-kappa B by the estrogen receptor. FEBS Lett 1997;409:79-85.
Speir E,Yu ZX, Takeda K, Ferrans VJ, Cannon RO 3rd. Competition for p300 regulates transcription by estrogen receptors and nuclear factor-kappaB in human coronary smooth muscle cells. Circ Res 2000;87:1006-11.
Hsu SM, Chen YC, Jiang MC. 17 beta-estradiol inhibits tumor necrosis factor-alpha-induced nuclear factor-kappa B activation by increasing nu-clear factor-kappa B p105 level in MCF-7 breast cancer cells. Biochem Biophys Res Commun 2000;279:47-52.
Inoue S, Orimo A, Hosoi T, Kondo S, Toyoshima H, Kondo T, et al. Genomic binding-site cloning reveals an estrogen-responsive gene that encodes a RING finger protein. Proc Natl Acad Sci U S A 1993;90:11117-21.
Ikeda K, Orimo A, Higashi Y, Muramatsu M, Inoue S. Efp as a primary estrogen-responsive gene in human breast cancer. FEBS Lett 2000;472:9-13.
Urano T, Saito T, Tsukui T, Fujita M, Hosoi T, Muramatsu M, et al. Efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth. Nature 2002;417:871-5.
Taylor WR, Stark GR. Regulation of the G2/M transition by p53. Oncogene 2001;20:1803-15.
Qin C, Nguyen T, Stewart J, Samudio I, Burghardt R, Safe S. Estrogen up-regulation of p53 gene expression in MCF-7 breast cancer cells is mediated by calmodulin kinase IV-dependent activation of a nuclear factor kappaB/CCAAT-binding transcription factor-1 complex. Mol Endocrinol 2002;16:1793-809.