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Estrogen induces death of tamoxifen-resistant MCF-7 cells: Contrasting effect of the estrogen receptor downregulator fulvestrant
Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 193–198
Estrogen induces death of tamoxifen-resistant MCF-7 cells: Contrasting effect of the estrogen receptor downregulator fulvestrant Maricarmen D. Planas-Silva ∗ , Paul K. Waltz, Robin L. Kilker Department of Pharmacology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA Received 9 August 2005; accepted 5 October 2005
Abstract A common problem in breast cancer therapy is resistance to the antiestrogen tamoxifen. However, tamoxifen-resistant breast tumors can still respond to other hormonal therapies. In animal models of tamoxifen-resistant breast cancer cells, physiological levels of estrogen can induce tumor regression. Recently, the estrogen receptor downregulator fulvestrant was shown to promote tumor growth of tamoxifen-resistant cells when added in combination with physiological levels of estrogen. Here, we show, using a cell culture model, that continuous exposure of tamoxifen-resistant cells to physiological levels of estrogen leads to cell death. Addition of the estrogen receptor downregulator fulvestrant prevents estrogen-induced death in a dose-dependent manner. Our data indicate that endogenous levels of estrogen affect the response of tamoxifen-resistant cells to fulvestrant. These results suggest that failure of fulvestrant to inhibit tumor growth in some tamoxifen-resistant patients may be due to endogenous estrogen levels. Moreover, these studies support short-term treatment with estrogen as a second-line hormonal therapy for tamoxifen-resistant breast cancer. © 2006 Elsevier Ltd. All rights reserved. Keywords: Estrogen receptor; Fulvestrant; Tamoxifen resistance; Breast cancer
1. Introduction Hormonal therapies have extended overall survival and quality of life in patients with hormone-dependent breast cancer. Nevertheless, in many patients, resistance to hormonal therapies develops eventually, limiting efficacy of such treatments. Several cell culture and animal models have been developed to elucidate the mechanisms leading to hormonal resistance [1,2]. These models have been used to develop specific ways to treat hormone-resistant breast cancer. In a preclinical model of tamoxifen-resistant breast cancer, treatment with physiological levels of estrogeninduced apoptosis and tumor regression [3]. Apoptosis in Abbreviations: CSS, charcoal-stripped fetal bovine serum; ER, estrogen receptor alpha; FBS, fetal bovine serum; MTR, MCF-7 tamoxifen-resistant cells; SRB, sulforhodamine B assay ∗ Corresponding author. Tel.: +1 717 531 4569; fax: +1 717 531 5013. E-mail address: [email protected] (M.D. Planas-Silva). 0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.10.003
response to physiological estrogen also occurs in cell culture models of breast cancer cells resistant to either raloxifene [4] or estrogen-deprivation [5–7]. A paradoxical effect of the antiestrogen and estrogen receptor (ER) downregulator fulvestrant (FaslodexTM , ICI 182,780), currently used in treating hormone-resistant breast cancer, was observed using an animal model of acquired tamoxifen resistance [8]. Treatment with either estrogen or fulvestrant blocked tumor growth, whereas simultaneous administration of estrogen and fulvestrant led to tumor progression. These results suggested that the effect of fulvestrant was affected by estrogen levels. Recently, we developed three independent tamoxifenresistant variants from parental MCF-7 cells. All three variants maintain expression of ER alpha and are growthinhibited by fulvestrant in the absence of estrogen supplementation [9]. Using this cell culture model, we studied the effect of estrogen and fulvestrant on the growth of these cells. We observed that physiological levels of
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estrogen-induced cell death whereas addition of fulvestrant blocked estrogen-dependent cell death. 2. Materials and methods 2.1. Cell culture MCF-7 cells were grown in phenol red-containing DME media (Irvine Scientific, Santa Ana, CA) supplemented with 5% fetal bovine serum (FBS) and antibiotics. Tamoxifenresistant cells were grown in phenol red-free DME media (Irvine Scientific) supplemented with 5% charcoal/dextranstripped fetal bovine serum (CSS) (Hyclone, Logan, UT), antibiotics and 1 M tamoxifen (Sigma, St. Louis, MO) [9]. Three tamoxifen-resistant variants derived from the same parental MCF-7 cells were used. These variants were selected by long-term treatment in the presence of 5% CSS supplemented with 1 M tamoxifen and their growth characteristics have recently been described [9]. 17-Estradiol and other chemicals were obtained from Sigma (St. Louis). The ER downregulator fulvestrant [10] {ICI 182,780; 7␣-[9-(4,4,5,5,5-pentafluoropentyl-sulphinyl) nonyl]estra-1,3,5(10)-triene-3,17-diol} was purchased from Tocris (Ellisville, MO). Fulvestrant was dissolved using ethanol as solvent to yield a concentrated stock solution of 5 mM. For dose–response curves, either fulvestrant or 17-estradiol were diluted further with ethanol as needed to
keep a constant concentration of ethanol (0.1%) when added to the media. 2.2. Cell proliferation assays Cells were plated at approximately 1 × 105 cells per well on six-well plates. At the start of the experiment (day = 0), cells were treated with 5% CSS media with or without the indicated hormones. Media was changed every 2–3 days until the end of the experiment. On each of the indicated days, samples were fixed in 10% trichloroacetic acid followed by sulforhodamine B assay (SRB) [11]. SRB staining was measured at 570 nm. To compare the growth of different variants, SRB values at different doses of hormone were divided over control (5% CSS) for each variant. In order to control for potential differences in growth rate and plating of the same variant, at different passage numbers, SRB values obtained at the end of the experiment, with the different doses of estrogen, were divided by the SRB value from the same cells at the start of the experiment (day = 0). Experimental values represent an average of triplicates ±S.E. 2.3. Statistics Significance of difference between means was determined using analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test.
Fig. 1. Estrogen-induced death in tamoxifen-resistant cells. Growth curves of MCF-7 cells and tamoxifen-resistant cells (MTR) in the presence of physiological levels of estrogen (5 nM 17-estradiol) or 1 M tamoxifen. Cells were plated in six-well plates at 1–1.2 × 105 cells per well. At the start of the experiments, cells were switched to media containing 5% charcoal-stripped fetal bovine serum (CSS) and the indicated hormones. Cells were fed every 2 or 3 days. Representative experiment is shown. Data represents average values (triplicate wells) ±S.E. Similar data were obtained in several independent experiments with either 1 or 5 nM 17-estradiol.
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3. Results MCF-7 cells and tamoxifen-resistant variants called MTR (MCF-7: tamoxifen-resistant) proliferated in the presence of physiological levels of 17-estradiol (5 nM) (Fig. 1). However, continuous treatment with 17-estradiol led to cell death of tamoxifen-resistant, but not parental, cells (Fig. 1). Dose–response studies indicated that physiological levels of 17-estradiol (1 nM) induced cell death of tamoxifen-resistant cells, whereas pharmacological levels (1 to 10 M = 10−6 to 10−5 M) were generally less effective in inducing cell death (Fig. 2). We observed that the kinetics and extent of estrogen-dependent cell death were influenced by the length of time that tamoxifen-resistant cells had been cultured in the presence of tamoxifen. Thus, we evaluated estrogen dose–response curves of the same tamoxifen-resistant variant at different passages. Considering that cells growing in the presence of tamoxifen for longer periods could be proliferating faster, we assessed relative growth of MTR-1 cells at different passages. Tamoxifen-resistant cells cultured for four additional months showed decreased sensitivity to estrogen-mediated killing, despite comparable growth rates (Fig. 3). Thus, these results suggest that tamoxifen-resistant cells are more sensitive to the killing actions of estrogen during the initial phase of their acquisition of tamoxifen resistance. It is possible that the prolonged growth of tamoxifenresistant cells allow them to acquire additional survival mechanisms that counteract estrogen-induced cell death. Since fulvestrant treatment in the presence of physiological estrogen levels led to tumor progression in an animal model of tamoxifen resistance [8], we evaluated the effect of fulvestrant on estrogen-induced cell death. Phase contrast microscopy showed that MTR cells were killed by physiological levels of estrogen (Fig. 4A). Co-treatment with fulvestrant prevented cell death in a dose-dependent manner (Fig. 4A), indicating that estrogen-dependent cell death depends on ER function and is not due to a cytotoxic effect
Fig. 3. Comparative estrogen dose–response curves of MTR-1 cells at different passages. MTR-1 cells differing in length of time of exposure to tamoxifen in culture (passage 21 vs. passage 40) were evaluated for their sensitivity to estrogen. For proper comparison between cells at different passages, cell growth measurements obtained at the specific doses were divided by day 0 values to obtain a growth ratio. Experimental values represent average ± S.E. from a representative experiment.
of estrogen independent of ER. When estrogen and fulvestrant were given simultaneously at 1 nM, estrogen-induced death of tamoxifen-resistant cells ensued (Fig. 4B). Nevertheless, when fulvestrant concentration was 10 nM or higher, within the therapeutic range [10,12], the “effective estrogen dose” was reduced to a growth-promoting range leading to partial or complete inhibition of cell death. These results suggest that tumor response and clinical outcome depend on the “effective estrogen dose” determined by the ratio between estrogens and antiestrogens. To determine whether continuous activation of ER function was required to cause cell death, 1 M fulvestrant was added to tamoxifen-resistant cells at different days after treatment with 1 nM 17-estradiol was started. Simultaneous addition of fulvestrant at the start of the experiment completely blocked estrogen-dependent cell death (Fig. 4C). Addition of fulvestrant during the proliferative phase partially blocked estrogen-dependent cell death. However, when fulvestrant was added at day 8 or later, fulvestrant was unable to inhibit cell death. Thus, a week of treatment with physiological levels of estrogen commits tamoxifen-resistant cells to cell death. These results suggest that a short pulse of estrogen treatment may induce regression of some tamoxifen-resistant tumors.
4. Discussion
Fig. 2. Estrogen dose–response curves of tamoxifen-resistant cells. Cell proliferation was measured using the sulforhodamine B assay after 12 days of treatment. Values obtained for each variant at the indicated doses were divided by their respective control values (5% CSS). Experimental values represent average from triplicate wells ±S.E.
Our studies show that breast cancer cells that acquire resistance to tamoxifen in culture are killed by continuous exposure to physiological levels of estrogen. Clinical evidence has shown that in advanced breast cancer tamoxifen-withdrawal alone can lead to tumor regression [13–16]. This withdrawal response seems to be more frequent in tumors that had an objective response to tamoxifen [17]. It is possible that the
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Fig. 4. Effect of fulvestrant on estrogen-induced death of tamoxifen-resistant cells. (A) Phase contrast micrographs of MTR-3 cells cultured for 14 days in the specified media (magnification ×200). (B) Values represent 14 days of treatment with 1 nM 17-estradiol and the indicated dose of fulvestrant compared to control (5% CSS). Bars represent standard error (S.E.). * Significantly different from treatment with estrogen alone (p < 0.001 by ANOVA and post hoc testing). (C) At the indicated days, fulvestrant (1 M) was added to MTR-3 cells exposed to 1 nM E2 since day 0. Effect of fulvestrant addition at different days of estrogen treatment was assessed at day 14 and compared to control (5% CSS). Experimental values represent average values obtained from triplicate samples ±S.E.
withdrawal response is due to high levels of estrogen in situ after tamoxifen withdrawal that induce cell death leading to tumor regression. Those tumors whose “effective estrogen dose” was decreased successfully by tamoxifen to a growthinhibiting range acquired resistance by adapting to the lower “effective estrogen dose” (estrogen/antiestrogen ratio). Tamoxifen withdrawal may induce death when the adapted tumor cells are re-exposed to the previous growth-promoting estrogen dose. However, not all patients experience a withdrawal response. Our studies suggest that the withdrawal response depends on in situ estrogen levels needing to reach a death-promoting range. In addition, the kinetics of tamoxifen clearance in situ, as well as the length of time elapsed between acquisition of hormonal-resistant growth and drug withdrawal, may affect withdrawal response. Our studies and
those of others suggest that the longer that hormone-resistant cells are maintained in culture under selection the less sensitive they become to estrogen killing [6]. These data may explain why other breast cancer cells selected for tamoxifen resistance in culture have not shown estrogen-induced cell death [1]. It is possible that continuous selection in culture upregulates additional survival pathways that allow cells to resist estrogen-dependent killing. Nevertheless, short-term additive estrogen therapy after early signs of recurrence may benefit some patients with tamoxifen-resistant breast cancer. Physiological levels of estrogen also induce cell death in breast cancer cells resistant either to the antiestrogen raloxifene [4] or to long-term estrogen deprivation [5,6]. However, the ability of estrogen to induce cell death can be affected by components present in the serum [7]. Collectively, these
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studies indicate that estrogen-dependent death is common after adaptation to different ablative hormonal therapies that reduce “effective estrogen dose” in situ. These data suggest that patients whose tumors acquire resistance to ablative hormonal therapy may benefit from additive estrogen therapy [18–20]. In the clinics, as well as in animal models, tumors that adapt to additive estrogen therapy may subsequently respond to therapy withdrawal or to rechallenge with tamoxifen or other hormonal therapies [21–25]. To determine the optimal type, dose and length of therapy for each patient, new non-invasive imaging techniques [26–28] are needed to assess the “effective estrogen dose” driving growth of a particular tumor. Evidence supporting the need for a more individualized hormonal therapy was provided in a clinical trial of additive estrogen therapy [29]. Perhaps intrinsic resistance to specific hormonal therapies arises, in some cases, from inadequate therapy or inappropriate dose for that particular tumor [30]. Some patients who develop tamoxifen-resistant tumors can benefit clinically from treatment with fulvestrant [12,31,32]. Thus, the paradoxical effect of fulvestrant could be explained by its antiestrogenic effect blocking estrogendependent death in tumors that may have acquired estrogensensitivity and their local levels of estrogen, after tamoxifen clearance, may be sufficient for that affect. It is possible that a brief treatment with additive estrogen therapy before fulvestrant administration will induce cell-death in tumor cells sensitized to estrogen. Tumor cells that survive estrogen therapy may be those that are more sensitive to fulvestrant. The recent report of decreased breast cancer risk in women receiving estrogen replacement therapy suggests that physiological levels of estrogen may also prevent survival of primary breast tumors in some postmenopausal women [33]. Considering variability in estrogen status of postmenopausal women due to differences in biosynthesis and metabolism of estrogen or diet [34,35], it is possible that some primary breast cancer arising in postmenopausal women may have already adapted to grow at subphysiological estrogen levels. In these cases, estrogen replacement therapy may kill breast cancer cells before the tumor is detectable. To apply additive estrogen therapy successfully, surrogate biomarkers that can predict which tumors will regress upon estrogen therapy are needed.
Acknowledgments We gratefully acknowledge E. Vesell and K. Leitzel for comments on the manuscript. M.D.P.-S. is a recipient of a Career Developmental Award from the Department of Defense DAMD17-02-1-0540.
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[30] A. Howell, J.F.R. Robertson, P. Abram, M.R. Lichinitser, R. Elledge, E. Bajetta, T. Watanabe, C. Morris, A. Webster, I. Dimery, C.K. Osborne, Comparison of fulvestrant versus tamoxifen for the treatment of advanced breast cancer in postmenopausal women previously untreated with endocrine therapy: a multinational, double-blind, randomized trial, J. Clin. Oncol. 22 (2004) 1605– 1613. [31] A. Howell, D. DeFriend, J.F.R. Robertson, R.W. Blamey, P. Walton, Response to a specific antiestrogen (ICI 182,780) in tamoxifenresistant breast cancer, Lancet 345 (1995) 29–30. [32] A. Howell, J.F.R. Robertson, J.Q. Albano, A. Aschermannova, L. Mauriac, U.R. Kleeberg, I. Vergote, B. Erikstein, A. Webster, C. Morris, Fulvestrant, formerly ICI 182,780, is as effective as anastrozole in postmenopausal women with advanced breast cancer progressing after prior endocrine treatment, J. Clin. Oncol. 20 (2002) 3396–3403. [33] The Women’s Health Initiative Steering Committee, Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial, JAMA 291 (2004) 1701–1712. [34] G.A. Kuchel, C. Tannenbaum, S.L. Greenspan, N.M. Resnick, Can variability in the hormonal status of elderly women assist in the decision to administer estrogens? J. Women’s Health Gender-Based Med. 10 (2001) 109–116. [35] J.R. Pasqualini, The selective estrogen enzyme modulators in breast cancer: a review, Biochim. Biophys. Acta 1654 (2004) 123– 143.
Estrogen induces death of tamoxifen-resistant MCF-7 cells: Contrasting effect of the estrogen receptor downregulator fulvestrant Maricarmen D. Planas-Silva ∗ , Paul K. Waltz, Robin L. Kilker Department of Pharmacology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA Received 9 August 2005; accepted 5 October 2005
Abstract A common problem in breast cancer therapy is resistance to the antiestrogen tamoxifen. However, tamoxifen-resistant breast tumors can still respond to other hormonal therapies. In animal models of tamoxifen-resistant breast cancer cells, physiological levels of estrogen can induce tumor regression. Recently, the estrogen receptor downregulator fulvestrant was shown to promote tumor growth of tamoxifen-resistant cells when added in combination with physiological levels of estrogen. Here, we show, using a cell culture model, that continuous exposure of tamoxifen-resistant cells to physiological levels of estrogen leads to cell death. Addition of the estrogen receptor downregulator fulvestrant prevents estrogen-induced death in a dose-dependent manner. Our data indicate that endogenous levels of estrogen affect the response of tamoxifen-resistant cells to fulvestrant. These results suggest that failure of fulvestrant to inhibit tumor growth in some tamoxifen-resistant patients may be due to endogenous estrogen levels. Moreover, these studies support short-term treatment with estrogen as a second-line hormonal therapy for tamoxifen-resistant breast cancer. © 2006 Elsevier Ltd. All rights reserved. Keywords: Estrogen receptor; Fulvestrant; Tamoxifen resistance; Breast cancer
1. Introduction Hormonal therapies have extended overall survival and quality of life in patients with hormone-dependent breast cancer. Nevertheless, in many patients, resistance to hormonal therapies develops eventually, limiting efficacy of such treatments. Several cell culture and animal models have been developed to elucidate the mechanisms leading to hormonal resistance [1,2]. These models have been used to develop specific ways to treat hormone-resistant breast cancer. In a preclinical model of tamoxifen-resistant breast cancer, treatment with physiological levels of estrogeninduced apoptosis and tumor regression [3]. Apoptosis in Abbreviations: CSS, charcoal-stripped fetal bovine serum; ER, estrogen receptor alpha; FBS, fetal bovine serum; MTR, MCF-7 tamoxifen-resistant cells; SRB, sulforhodamine B assay ∗ Corresponding author. Tel.: +1 717 531 4569; fax: +1 717 531 5013. E-mail address: [email protected] (M.D. Planas-Silva). 0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.10.003
response to physiological estrogen also occurs in cell culture models of breast cancer cells resistant to either raloxifene [4] or estrogen-deprivation [5–7]. A paradoxical effect of the antiestrogen and estrogen receptor (ER) downregulator fulvestrant (FaslodexTM , ICI 182,780), currently used in treating hormone-resistant breast cancer, was observed using an animal model of acquired tamoxifen resistance [8]. Treatment with either estrogen or fulvestrant blocked tumor growth, whereas simultaneous administration of estrogen and fulvestrant led to tumor progression. These results suggested that the effect of fulvestrant was affected by estrogen levels. Recently, we developed three independent tamoxifenresistant variants from parental MCF-7 cells. All three variants maintain expression of ER alpha and are growthinhibited by fulvestrant in the absence of estrogen supplementation [9]. Using this cell culture model, we studied the effect of estrogen and fulvestrant on the growth of these cells. We observed that physiological levels of
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estrogen-induced cell death whereas addition of fulvestrant blocked estrogen-dependent cell death. 2. Materials and methods 2.1. Cell culture MCF-7 cells were grown in phenol red-containing DME media (Irvine Scientific, Santa Ana, CA) supplemented with 5% fetal bovine serum (FBS) and antibiotics. Tamoxifenresistant cells were grown in phenol red-free DME media (Irvine Scientific) supplemented with 5% charcoal/dextranstripped fetal bovine serum (CSS) (Hyclone, Logan, UT), antibiotics and 1 M tamoxifen (Sigma, St. Louis, MO) [9]. Three tamoxifen-resistant variants derived from the same parental MCF-7 cells were used. These variants were selected by long-term treatment in the presence of 5% CSS supplemented with 1 M tamoxifen and their growth characteristics have recently been described [9]. 17-Estradiol and other chemicals were obtained from Sigma (St. Louis). The ER downregulator fulvestrant [10] {ICI 182,780; 7␣-[9-(4,4,5,5,5-pentafluoropentyl-sulphinyl) nonyl]estra-1,3,5(10)-triene-3,17-diol} was purchased from Tocris (Ellisville, MO). Fulvestrant was dissolved using ethanol as solvent to yield a concentrated stock solution of 5 mM. For dose–response curves, either fulvestrant or 17-estradiol were diluted further with ethanol as needed to
keep a constant concentration of ethanol (0.1%) when added to the media. 2.2. Cell proliferation assays Cells were plated at approximately 1 × 105 cells per well on six-well plates. At the start of the experiment (day = 0), cells were treated with 5% CSS media with or without the indicated hormones. Media was changed every 2–3 days until the end of the experiment. On each of the indicated days, samples were fixed in 10% trichloroacetic acid followed by sulforhodamine B assay (SRB) [11]. SRB staining was measured at 570 nm. To compare the growth of different variants, SRB values at different doses of hormone were divided over control (5% CSS) for each variant. In order to control for potential differences in growth rate and plating of the same variant, at different passage numbers, SRB values obtained at the end of the experiment, with the different doses of estrogen, were divided by the SRB value from the same cells at the start of the experiment (day = 0). Experimental values represent an average of triplicates ±S.E. 2.3. Statistics Significance of difference between means was determined using analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test.
Fig. 1. Estrogen-induced death in tamoxifen-resistant cells. Growth curves of MCF-7 cells and tamoxifen-resistant cells (MTR) in the presence of physiological levels of estrogen (5 nM 17-estradiol) or 1 M tamoxifen. Cells were plated in six-well plates at 1–1.2 × 105 cells per well. At the start of the experiments, cells were switched to media containing 5% charcoal-stripped fetal bovine serum (CSS) and the indicated hormones. Cells were fed every 2 or 3 days. Representative experiment is shown. Data represents average values (triplicate wells) ±S.E. Similar data were obtained in several independent experiments with either 1 or 5 nM 17-estradiol.
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3. Results MCF-7 cells and tamoxifen-resistant variants called MTR (MCF-7: tamoxifen-resistant) proliferated in the presence of physiological levels of 17-estradiol (5 nM) (Fig. 1). However, continuous treatment with 17-estradiol led to cell death of tamoxifen-resistant, but not parental, cells (Fig. 1). Dose–response studies indicated that physiological levels of 17-estradiol (1 nM) induced cell death of tamoxifen-resistant cells, whereas pharmacological levels (1 to 10 M = 10−6 to 10−5 M) were generally less effective in inducing cell death (Fig. 2). We observed that the kinetics and extent of estrogen-dependent cell death were influenced by the length of time that tamoxifen-resistant cells had been cultured in the presence of tamoxifen. Thus, we evaluated estrogen dose–response curves of the same tamoxifen-resistant variant at different passages. Considering that cells growing in the presence of tamoxifen for longer periods could be proliferating faster, we assessed relative growth of MTR-1 cells at different passages. Tamoxifen-resistant cells cultured for four additional months showed decreased sensitivity to estrogen-mediated killing, despite comparable growth rates (Fig. 3). Thus, these results suggest that tamoxifen-resistant cells are more sensitive to the killing actions of estrogen during the initial phase of their acquisition of tamoxifen resistance. It is possible that the prolonged growth of tamoxifenresistant cells allow them to acquire additional survival mechanisms that counteract estrogen-induced cell death. Since fulvestrant treatment in the presence of physiological estrogen levels led to tumor progression in an animal model of tamoxifen resistance [8], we evaluated the effect of fulvestrant on estrogen-induced cell death. Phase contrast microscopy showed that MTR cells were killed by physiological levels of estrogen (Fig. 4A). Co-treatment with fulvestrant prevented cell death in a dose-dependent manner (Fig. 4A), indicating that estrogen-dependent cell death depends on ER function and is not due to a cytotoxic effect
Fig. 3. Comparative estrogen dose–response curves of MTR-1 cells at different passages. MTR-1 cells differing in length of time of exposure to tamoxifen in culture (passage 21 vs. passage 40) were evaluated for their sensitivity to estrogen. For proper comparison between cells at different passages, cell growth measurements obtained at the specific doses were divided by day 0 values to obtain a growth ratio. Experimental values represent average ± S.E. from a representative experiment.
of estrogen independent of ER. When estrogen and fulvestrant were given simultaneously at 1 nM, estrogen-induced death of tamoxifen-resistant cells ensued (Fig. 4B). Nevertheless, when fulvestrant concentration was 10 nM or higher, within the therapeutic range [10,12], the “effective estrogen dose” was reduced to a growth-promoting range leading to partial or complete inhibition of cell death. These results suggest that tumor response and clinical outcome depend on the “effective estrogen dose” determined by the ratio between estrogens and antiestrogens. To determine whether continuous activation of ER function was required to cause cell death, 1 M fulvestrant was added to tamoxifen-resistant cells at different days after treatment with 1 nM 17-estradiol was started. Simultaneous addition of fulvestrant at the start of the experiment completely blocked estrogen-dependent cell death (Fig. 4C). Addition of fulvestrant during the proliferative phase partially blocked estrogen-dependent cell death. However, when fulvestrant was added at day 8 or later, fulvestrant was unable to inhibit cell death. Thus, a week of treatment with physiological levels of estrogen commits tamoxifen-resistant cells to cell death. These results suggest that a short pulse of estrogen treatment may induce regression of some tamoxifen-resistant tumors.
4. Discussion
Fig. 2. Estrogen dose–response curves of tamoxifen-resistant cells. Cell proliferation was measured using the sulforhodamine B assay after 12 days of treatment. Values obtained for each variant at the indicated doses were divided by their respective control values (5% CSS). Experimental values represent average from triplicate wells ±S.E.
Our studies show that breast cancer cells that acquire resistance to tamoxifen in culture are killed by continuous exposure to physiological levels of estrogen. Clinical evidence has shown that in advanced breast cancer tamoxifen-withdrawal alone can lead to tumor regression [13–16]. This withdrawal response seems to be more frequent in tumors that had an objective response to tamoxifen [17]. It is possible that the
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Fig. 4. Effect of fulvestrant on estrogen-induced death of tamoxifen-resistant cells. (A) Phase contrast micrographs of MTR-3 cells cultured for 14 days in the specified media (magnification ×200). (B) Values represent 14 days of treatment with 1 nM 17-estradiol and the indicated dose of fulvestrant compared to control (5% CSS). Bars represent standard error (S.E.). * Significantly different from treatment with estrogen alone (p < 0.001 by ANOVA and post hoc testing). (C) At the indicated days, fulvestrant (1 M) was added to MTR-3 cells exposed to 1 nM E2 since day 0. Effect of fulvestrant addition at different days of estrogen treatment was assessed at day 14 and compared to control (5% CSS). Experimental values represent average values obtained from triplicate samples ±S.E.
withdrawal response is due to high levels of estrogen in situ after tamoxifen withdrawal that induce cell death leading to tumor regression. Those tumors whose “effective estrogen dose” was decreased successfully by tamoxifen to a growthinhibiting range acquired resistance by adapting to the lower “effective estrogen dose” (estrogen/antiestrogen ratio). Tamoxifen withdrawal may induce death when the adapted tumor cells are re-exposed to the previous growth-promoting estrogen dose. However, not all patients experience a withdrawal response. Our studies suggest that the withdrawal response depends on in situ estrogen levels needing to reach a death-promoting range. In addition, the kinetics of tamoxifen clearance in situ, as well as the length of time elapsed between acquisition of hormonal-resistant growth and drug withdrawal, may affect withdrawal response. Our studies and
those of others suggest that the longer that hormone-resistant cells are maintained in culture under selection the less sensitive they become to estrogen killing [6]. These data may explain why other breast cancer cells selected for tamoxifen resistance in culture have not shown estrogen-induced cell death [1]. It is possible that continuous selection in culture upregulates additional survival pathways that allow cells to resist estrogen-dependent killing. Nevertheless, short-term additive estrogen therapy after early signs of recurrence may benefit some patients with tamoxifen-resistant breast cancer. Physiological levels of estrogen also induce cell death in breast cancer cells resistant either to the antiestrogen raloxifene [4] or to long-term estrogen deprivation [5,6]. However, the ability of estrogen to induce cell death can be affected by components present in the serum [7]. Collectively, these
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studies indicate that estrogen-dependent death is common after adaptation to different ablative hormonal therapies that reduce “effective estrogen dose” in situ. These data suggest that patients whose tumors acquire resistance to ablative hormonal therapy may benefit from additive estrogen therapy [18–20]. In the clinics, as well as in animal models, tumors that adapt to additive estrogen therapy may subsequently respond to therapy withdrawal or to rechallenge with tamoxifen or other hormonal therapies [21–25]. To determine the optimal type, dose and length of therapy for each patient, new non-invasive imaging techniques [26–28] are needed to assess the “effective estrogen dose” driving growth of a particular tumor. Evidence supporting the need for a more individualized hormonal therapy was provided in a clinical trial of additive estrogen therapy [29]. Perhaps intrinsic resistance to specific hormonal therapies arises, in some cases, from inadequate therapy or inappropriate dose for that particular tumor [30]. Some patients who develop tamoxifen-resistant tumors can benefit clinically from treatment with fulvestrant [12,31,32]. Thus, the paradoxical effect of fulvestrant could be explained by its antiestrogenic effect blocking estrogendependent death in tumors that may have acquired estrogensensitivity and their local levels of estrogen, after tamoxifen clearance, may be sufficient for that affect. It is possible that a brief treatment with additive estrogen therapy before fulvestrant administration will induce cell-death in tumor cells sensitized to estrogen. Tumor cells that survive estrogen therapy may be those that are more sensitive to fulvestrant. The recent report of decreased breast cancer risk in women receiving estrogen replacement therapy suggests that physiological levels of estrogen may also prevent survival of primary breast tumors in some postmenopausal women [33]. Considering variability in estrogen status of postmenopausal women due to differences in biosynthesis and metabolism of estrogen or diet [34,35], it is possible that some primary breast cancer arising in postmenopausal women may have already adapted to grow at subphysiological estrogen levels. In these cases, estrogen replacement therapy may kill breast cancer cells before the tumor is detectable. To apply additive estrogen therapy successfully, surrogate biomarkers that can predict which tumors will regress upon estrogen therapy are needed.
Acknowledgments We gratefully acknowledge E. Vesell and K. Leitzel for comments on the manuscript. M.D.P.-S. is a recipient of a Career Developmental Award from the Department of Defense DAMD17-02-1-0540.
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