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A mechanism of drug resistance to tamoxifen in breast cancer
Journal of Steroid Biochemistry & Molecular Biology 83 (2003) 75–83
A mechanism of drug resistance to tamoxifen in breast cancer夽 Jennifer MacGregor Schafer a , David J. Bentrem c , Hiroyuki Takei d , Csaba Gajdos a , Sunil Badve b , V. Craig Jordan a,∗ a
Robert H. Lurie Comprehensive Cancer Center, The Feinberg School of Medicine, Northwestern University, Olson Pavilion 8258, 303 East Chicago Avenue, Chicago, IL 60611, USA b Department of Pathology, The Feinberg School of Medicine, Northwestern University, Olson Pavilion 8258, 303 East Chicago Avenue, Chicago, IL 60611, USA c Department of Surgery, The Feinberg School of Medicine, Northwestern University, Olson Pavilion 8258, 303 East Chicago Avenue, Chicago, IL 60611, USA d Division of Breast Surgery, Saitama Cancer Center, Saitama 362-0806, Japan
Abstract Drug resistance to tamoxifen (Tam) is a significant clinical problem but the mechanism through which this occurs remains elusive. We have developed a number of xenograft models of Tam-stimulated growth that model breast cancer progression using estrogen receptor positive MCF-7 or T47D breast cancer cells. When estrogen-stimulated T47D:E2 tumors are treated long term with Tam, Tam-stimulated tumors develop (T47D:Tam) that are stimulated by both estrogen and Tam. When HER-2/neu status is determined, it is clear that the T47D:Tam tumors express significantly higher levels of HER-2/neu protein by immunohistochemistry and mRNA as measured by real-time RT-PCR. The T47D:Tam tumors also express higher levels of estrogen receptor and progesterone receptor protein than their estrogen-stimulated T47D:E2 counterparts. We compared out results to the MCF-7 model of Tam-stimulated growth. The MCF-7:Tam ST (estrogen- and Tam-stimulated) and MCF-7:Tam LT (estrogen-inhibited, Tam-stimulated) were bilaterally transplanted to account for any mouse to mouse variation and characteristic growth patterns were observed. TUNEL staining was performed on MCF-7:Tam LT treated with either estrogen or Tam and it was concluded that estrogen-inhibited tumor growth was a result of increased apoptosis. Three phases of tumor progression are described that involve increases in HER-2/neu expression, de-regulation of estrogen receptor expression and increases in apoptosis which in concert determine the phenotype of drug resistance to Tam. © 2003 Published by Elsevier Science Ltd. Keywords: Antiestrogen; Breast cancer; Estrogen receptor; HER-2/neu; Drug resistance
1. Introduction Tamoxifen (Tam) is the endocrine therapy of choice for all stages of estrogen receptor positive breast cancer [29] and is currently prescribed for the prevention of breast cancer in high-risk women [10]. In addition, in patients with estrogen receptor positive disease, up to 5 years of adjuvant Tam treatment confers a long-term survival benefit that continues for at least an additional 5 years after Tam treatment ceases [7]. Unfortunately, greater than 5 years of Tam treatment may lead to the clonal selection of metastatic breast cancer that is resistant to Tam [11]. In fact, some of these tumors are stimulated by Tam as evidenced by the decreased 夽 Proceedings of the 15th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, “Recent Advances in Steroid Biochemistry and Molecular Biology”, Munich, Germany, 17–20 May 2002. ∗ Corresponding author. Tel.: +1-312-908-4148; fax: +1-312-908-1372. E-mail address: [email protected] (V.C. Jordan).
0960-0760/03/$ – see front matter © 2003 Published by Elsevier Science Ltd. PII: S 0 9 6 0 - 0 7 6 0 ( 0 2 ) 0 0 2 5 1 - 0
growth of tumors after Tam therapy is stopped [4,19,22]. Most of these Tam-stimulated tumors retain expression of the estrogen receptor [32] and, therefore, retain the ability to respond to second-line endocrine therapies such as the pure antiestrogen ICI182,780 [18] or an aromatase inhibitor such as anastrozole [3]. Following the failure of adjuvant Tam, anastrozole is currently available as a second-line therapy [3], indeed, a whole range of aromatase inhibitors are currently being evaluated [13]. The mechanism of action of aromatase inhibitors is contingent on whether Tam-resistant disease still requires estrogen to grow, so by reducing circulating estradiol and estrone, the tumor will lose its growth stimulus. Recent data presented from the ATAC trial provides evidence that the aromatase inhibitor anastrozole is superior to Tam in postmenopausal women with estrogen receptor positive early stage breast cancer [2]. The anatrozole-treated women showed a decrease in adverse effects and an increase in disease-free survival compared to Tam and, in addition,
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anastrozole may actually further prevent the incidence of contralateral breast cancer. These results are preliminary and further follow-up is needed to determine effects on bone mineral density and cognitive function. Because Tam is used extensively and the development of drug resistance is a concern, many laboratories are focusing on developing in vitro and in vivo models of drug resistance. The MCF-7 breast cancer cell line [35] is an ideal model because these cells express functional wild-type estrogen receptor and grow maximally in E2-containing media. When the MCF-7 cells are treated with antiestrogens, growth is dramatically reduced providing an excellent model to study the mechanism of antiestrogen action. Another interesting feature of this cell line is that it mimics the clinical progression of breast cancer including the development of antiestrogen resistance and the progression to hormone-independence. After long-term treatments with Tam, for example, adapted clones of MCF-7 cells are selected that are Tam-stimulated and retain expression of estrogen receptor [23]. When MCF-7 tumors are transplanted into ovariectomized athymic mice, estrogen supplementation is required for maximal tumor growth. The antiestrogen Tam, conversely, does not result in tumor growth and, in fact, is able to inhibit estrogen-stimulated growth [15] consistent with human breast cancer treatment. After long-term treatment with low dose Tam (approximately 6 months), tumors do not grow, however, treatment with estrogen rescues the growth demonstrating the tumoristatic not tumoricidal effects of Tam. When transplanted MCF-7 tumors are initially treated with estrogen to establish the tumor, then switched to Tam, for about 4 months, tumors regression is observed, however, by 8 months some tumors begin to grow [14]. These tumors are estrogen receptor positive and grow in response to estrogen when retransplanted. Interestingly, one of these tumors grew only in response to Tam demonstrating the progression of tumors from estrogen-stimulated to Tam-stimulated and the validity of this model. This model can also be used to study the effects of second-line endocrine therapies when Tam-resistant or -stimulated tumors arise. MCF-7 cells express wild-type p53 [5], and, therefore, only represent 50% of breast cancers [17]. A novel human breast xenograft model derived from T47D cells [24] which express a mutant p53 that is nonfunctional [25] has been developed [24]. After approximately 10 weeks of Tam treatment, T47D estrogenstimulated (T47D:E2) tumors become Tam-stimulated (T47D:Tam) and remain estrogen-responsive. This occurs at a significantly faster rate than MCF-7 tumors undergoing similar treatment therefore suggesting that the differences between these tumor lines directly impact the development of Tam-stimulated growth [24]. This model allows critical comparisons between cellular events that determine hormone responsiveness and drug resistance. Overexpression/amplification of the proto-oncogene receptor tyrosine kinase HER-2/neu (also known as c-erbB2) imparts an alternative growth stimulatory pathway and provides an additional therapeutic target in the treatment of
breast cancer. It has been known for almost a decade that amplification of the HER-2/neu gene correlates with shorter overall and disease-free survival [34] and that there is an inverse correlation between estrogen receptor expression and HER-2/neu overexpression [36]. In addition, constitutive activation of HER-2/neu signaling molecules has been shown to alter estrogen receptor expression [26] and the estrogen treatment of breast cancer cells in vitro results in down-regulation of HER-2/neu [31]. There are conflicting data and considerable debate about the predictive value of HER-2/neu overexpression and response to chemo or endocrine therapy in breast cancer (reviewed in [1]). Meta-analysis has shown that the HER-2/neu negative patient is more likely to respond to Tam therapy compared to their HER-2/neu positive counterparts [6] though prospective clinical trials are necessary to address this issue. Indeed, it has even been stated that HER-2/neu overexpression in breast cancer may be a better predictor of response to Tam than estrogen receptor status alone [9]. When HER-2/neu is overexpressed in breast cancer cells in vitro, the cells become Tam-resistant, however, blockade of the HER-2/neu signal transduction pathway restores the inhibitory effect of Tam on estrogen receptor mediated transcription and cell proliferation [21]. The mechanism of drug resistance to Tam is poorly understood and though there have been several theories proposed, for example, loss of estrogen receptor expression, thus far, there is no unifying mechanism. Here we propose a unifying theory for the mechanism of drug resistance based on laboratory models of the development of Tam resistance. Clearly, the estrogen receptor plays a pivotal role in Tam-stimulated growth because Tam-stimulated tumors always retain estrogen receptor expression [12,24]. We also show that HER-2/neu is consistently overexpressed in Tam-stimulated tumors, therefore, we believe that previously established cross-talk between the estrogen receptor and HER-2/neu may be the primary determinant of Tam-stimulated growth in our xenograft models of Tam-stimulated growth. 2. Materials and methods 2.1. Athymic mouse model The MCF-7 and T47D tumors (Fig. 1) used in these experiments were derived by bilateral inoculation of suspended human breast cancer cell lines into the mammary fat pads of 4–6 weeks old ovariectomized BALB/c nu/nu athymic mice (Harlan Sprague Dawley, Madison, WI). Ovarectomized athymic mice were injected bilaterally with 0.5 million T47D human breast cancer cells and supplemented with 17-estradiol (E2) pellets to create T47D:E2 tumors. When the T47D:E2 tumors reached approximately 0.8 cm2 (data not shown), a single tumor was passaged into athymic mice by retransplanting 1 mm3 tumor pieces with a 13 gauge trocar and they were treated long term with 1.5 mg Tam
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Fig. 1. Illustration of development of in vitro and in vivo models of acquired resistance to SERMs. (I) Breast cancer cells are treated with a SERM or deprived of estrogen for more than 1 year. These resistant cells are subsequently inoculated into athymic mice and continue SERM treatment producing SERM-resistant tumors. (II) SERM-resistant tumors are developed in vivo by treated estrogen-stimulated tumors for more than 1 year with a SERM.
to establish Tam-stimulated growth (T47D:Tam) (Table 1) [24]. To create MCF-7:Tam-stimulated tumors (Table 1), 10 million MCF-7 cells were inoculated and supplemented with E2 capsules to achieve estrogen-stimulated tumor growth (MCF-7:E2) [15]. Short-term Tam-stimulated tumor (MCF-7:Tam ST) growth was achieved after several weeks by retransplanting the growing estrogen-dependent MCF-7 tumors into new athymic mice and treating them with Tam [37]. Tumor measurements were performed weekly using Vernier calipers. The cross-sectional area was calculated using the formula: length × width/4π.
80). Ethanol was evaporated under nitrogen before use. Tam was administered orally, 1.5 mg per mouse, 5 days per week. 2.3. Statistical methods Comparisons in mean tumor between the animal groups were analyzed by analysis of variance (ANOVA) at each week and followed by unpaired Student’s t-test. The two tailed P-value of the last week of each experiment was reported using StatMost 2.5 (Datamost Corp., Salt Lake, UT). 2.4. Western blot analysis
2.2. Hormone and drug treatments Mice were divided into groups of 10 and were treated with E2 (Sigma), Tam (Sigma) or combination. Silastic E2 capsules (0.3 or 1 cm) were made as described previously [33], implanted subqutaneously on the same day as tumor transplantation and replaced every 8–10 weeks of treatment. The 0.3 cm E2 capsule produces a mean 83.9 pg/ml of serum E2 while 1.0 cm E2 capsules produces a mean 379.5 pg/ml of serum E2 as described previously [27]. Tam was dissolved in ethanol and suspended in a solution of 90% carboxymethylcellulose (1% carboxymethylcellulose in double-distilled water) and 10% polyethylene glycol 400/Tween 80 (99.5% polyethylene glycol and 0.5% Tween Table 1 Classification of breast xenografts based on hormone responsiveness Tumor
Derived from
E2 stimulated
Tam stimulated
T47D:E2 T47D:Tam MCF-7:Tam ST MCF-7:Tam LT
T47D cells T47D cells MCF-7 cells MCF-7 cells
(++) (++) (++) (−−)
(−−) (++) (++) (++)
References for the origin of different tumor models: T47D:E2 and T47D:Tam tumors (4); MCF-7:Tam ST tumors (3). MCF-7:Tam LT is a long-term Tam-stimulated model (more than 5 years Tam), is inhibited by E2 and stimulated by Tam.
Tumors were harvested and snap-frozen in liquid nitrogen and then stored at −80 ◦ C. Frozen tumors were homogenized by grinding in liquid nitrogen. Tumor extract was resuspended in protein extraction buffer (RIPA buffer: 1X PBS, 1% NP40, 0.5%Na deoxycholate, 0.1% SDS, protease inhibitor cocktail, NaF (1 M) and NaVO3 (250 mM)) and pelleted. Protein concentration of the supernatants was measured using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories Inc., Santa Cruz, CA) and equal amounts of protein (30 g) were analyzed according to a standard Western blot protocol [30]. The estrogen receptor primary antibody used was G-20 (Santa Cruz Biotechnology, Santa Cruz, CA), the PR primary antibody used was C-20 (Santa Cruz Biotechnology) and -actin antibody AC-15 (Sigma) was used to standardize loading. The appropriate secondary antibody conjugated to horseradish peroxidase (Amersham Corp., Arlington Heights, IL) was used to visualize bands using an ECL visualization kit (Amersham Corp.). The membrane was exposed to Kodak X-OMAT film. 2.5. Real-time RT-PCR Total RNA was extracted from the tumors using the Rneasy Mini Kit (Qiagen, Stanford Valencia, CA) according
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to the manufacturer’s instructions. Total RNA was reverse transcribed using TaqMan reverse transcription reagents (PE Applied Biosystems, Hayward, CA) with the use of random hexamers as the primers according to the manufacturer’s instructions. Primers and probes for human HER-2/neu were designed using Primer ExpressTM1.5 software. The sequences for the forward and reverse primers for human HER-2/neu: 5 -ACTGCAGAGGCTGCGGATT-3 and 5 -ACGGCCAGGGCATAGTTGT-3 . The sequence for the probe is FAM-5 -TGCGAGGCACCCAGCTCTTTGA-3’QSY7 (MegaBases Inc., Chicago, IL). The quantity of human GAPDH mRNA was also measured in each total RNA sample for normalization purposes. The probe and primers for GAPDH were purchased from Perkin-Elmer Applied Biosystems (PE Applied Biosystems, Stanford Valencia, CA). The PCR portion of the reaction was performed with the use of the TaqMan PCR Core Reagent Kit (PE Applied Biosystems). Real-time RT-PCR was performed using the ABI-Prism 7700 Sequence Detection System. The PCR conditions were 50 ◦ C for 2 min, 95 ◦ C for 10 min followed by 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. 2.6. Immunohistochemistry All immunohistochemistry is provided through the Robert H. Lurie Comprehensive Cancer Center, Core Pathology Department. Immunohistochemistry was performed according to manufacturer’s instructions. Antibodies used were: estrogen receptor (DAKO, Carpinteria, CA); HER-2/neu (DAKO, Carpinteria, CA). TUNEL stain was performed using the In Situ Cell Death Detection Kit (Boehringer Mannhein, Indianapolis, IN) according to manufacturer’s instructions. Briefly, 5 M sections from formalin-fixed, paraffin-embedded MCF-7:Tam LT xenografts were deparaffinized in xylenes (100% for 10 min three times), rehydrated in graded alcohols (100% (twice), 95, 90, 80, 70, 50%, 5 min each), rinsed with water (5 min) and PBS (5 min, twice). The samples were then permeablized with Triton-X 100 for 30 min, rinsed with PBS (5 min, twice), treated with 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity and then rinsed with PBS (5 min, twice). Samples were then blocked with bovine serum albumin and then incubated with terminal deoxynucleotidyl transferase and POD-conjugated antifluorescein antibody for 60 min in a humidified chamber at 37 ◦ C. Then samples were mounted with coverslips under glycerol/PBS and analyzed via light microscopy.
3. Results 3.1. T47D model of Tam-stimulated growth Using the estrogen receptor positive T47D human breast cancer cell line [20] that expresses a mutated nonfunctional p53 [25], we developed estrogen-dependent tumors
Fig. 2. Growth of (A) T47D:E2 or (B) T47D:Tam tumors passaged in athymic mice (mean ± S.E.). (A) Ten mice per group were treated for 12 weeks with 1 cm E2 capsule (Cap), 0.5 mg p.o. Tam or combination. E2 significantly stimulated growth compared to control (P = 0.003). There was no significant difference between control and Tam or combination. (B) Ten mice per group were treated for 12 weeks with 1 cm E2 capsule (Cap), 0.5 mg or 1.5 mg p.o. Tam. E2, 0.5 mg Tam and 1.5 mg Tam significantly stimulated growth compared to control (P < 0.005).
that are transplantable and only grow in the presence of E2 (T47D:E2; Fig. 2A) [24]. Treatment with low dose Tam (0.5 mg) does not result in tumor growth and, in addition, Tam is capable of acting as an antiestrogen in the model by inhibiting premenopausal level (1 cm E2 Cap) estrogen-stimulated growth. Interestingly, when these tumors are treated with high dose Tam (1.5 mg Tam), after approximately 10 weeks, Tam-stimulated tumors develop (T47D:Tam). These T47D:Tam tumors are transplantable and are more effectively stimulated by high dose (1.5 mg) Tam than low dose (0.5 mg) Tam [24] (Fig. 2B). 3.2. Classification of T47D tumor models We performed immunohistochemical analysis of T47D:E2 and T47D:Tam tumors to determine the HER-2/neu status. We chose three different tumor passages of each type to analyze in order to provide as much variation as possible. We determined that estrogen-stimulated T47D:E2 tumors were HER-2/neu− (1+) (Fig. 3). Parental T47D cells are known to express HER-2/neu [16] but the specific clone used in this study (T47D:A18) does not express high levels. Next, we compared this to the Tam-stimulated T47D:Tam
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Fig. 3. Immunohistochemical analysis of HER-2/neu in T47D:E2 tumors treated with 1 cm E2 Cap compared with T47D:Tam tumors treated with 1.5 mg Tam. Immunohistochemistry was performed on xenograft sections as described in Section 2. These photographs are representative of three independent experiments.
tumors which were HER-2/neu+ (3+) (Fig. 3). Because overexpression of protein can arise at many different levels, we confirmed our findings using real-time RT-PCR and measured levels of HER-2/neu mRNA in these tumor types with different treatments (Fig. 4). In the T47D:E2 tumors, 1 cm E2 Cap treatment resulted in low HER-2/neu mRNA production consistent with the immunohistochemistry. However, when the capsule was removed for 1 week before tumor harvest, HER-2/neu mRNA levels increased six-fold as expected. When similar measurements were undertaken in the T47D:Tam-stimulated tumors, E2 treatment again yielded low levels of HER-2/neu mRNA while Tam treatment resulted in approximately a 10-fold increase in HER-2/neu mRNA. Because of the proposed cross-talk between HER-2/neu and the estrogen receptor, we determined estrogen re-
Fig. 4. Relative expression of HER-2/neu mRNA expression as determined using real-time RT-PCR. Real-time RT-PCR was performed as described in Section 2. Control values of HER-2/neu were normalized with those of GAPDH. Each experiment was repeated three times.
ceptor levels and function by measuring progesterone receptor levels to ascertain any influence of estrogen receptor on this signal transduction pathway (Fig. 5). In the T47D:E2 tumors, classical type II regulation occurred [30] where estrogen receptor protein levels are induced by estrogen treatment. In the T47D:Tam tumors, the estrogen receptor levels are similar, suggesting that estrogen receptor production is de-regulated in the Tam-stimulated tumors though clearly, estrogen maintains its ability to promote progesterone receptor production and to a greater extent than the T47D:E2 counterpart. These data are consistent with our immunohistochemical findings (data not shown).
Fig. 5. Western blot screen of estrogen receptor ␣ and progesterone receptor protein expression in T47D:E2 or T47D:Tam tumors after E2, Tam or no treatment. Western blot analysis was performed as described in Section 2. -Actin protein was measured to ensure even loading. The Western blot shown is representative of three independent experiments.
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3.3. MCF-7 models of Tam-stimulated growth The development of Tam-stimulated growth in MCF-7 tumors occurs after about 25 weeks of growth in high dose Tam (1.5 mg) that comparably is significantly longer compared the 10 weeks for the development of Tam-stimulated growth in T47D tumors [24]. When Tam-stimulated MCF-7 tumors (MCF-7:Tam ST) do develop, these tumors are stimulated by both estrogen and Tam and do not grow without treatment [14]. MCF-7:Tam-stimulated tumors are unique in that after 5 years of Tam treatment, tumors progress to a different phenotype (MCF-7:Tam LT) where Tam continues to stimulate growth, but paradoxically, estrogen treatment inhibits growth [37]. So the question of differences between animals such as changes in ability to metabolize drug or differences in immune response arise. To address these issues, mice were bitransplanted with MCF-7:Tam ST tumors implanted into the left mammary fat pad and MCF-7:Tam LT tumors implanted in the right mammary fat pad (Fig. 6A). The MCF-7:Tam ST display classic Phase II Tam-stimulated growth, both estrogen and Tam result in tumor growth (Fig. 6B). However, the MCF-7:Tam LT tumors implanted in the right side of the same animal display Phase III growth where Tam stimulates growth but estrogen does not (Fig. 6C). 3.4. Characterization of E2-inhibited growth in MCF-7:Tam LT model
Fig. 6. (A) Illustration of two MCF-7 xenograft models bitransplanted into athymic mice. (B) MCF-7:Tam ST tumors were transplanted into the left mammary fat pad. (C) MCF-7:Tam LT tumors were transplanted into the right mammary fat and treated for 7 weeks with 1 cm E2 Cap or 1.5 mg Tam. The growth of the 1 cm E2 Cap and 1.5 mg Tam group in MCF-7:Tam ST tumors (B) were significantly different compared to control (P < 0.05). The growth of MCF-7:Tam LT tumors treated with 1.5 mg Tam was significantly different than the 1 cm E2 Cap group (P < 0.05).
In fact, not only does estrogen fail to stimulate tumor growth of the MCF-7:Tam LT, it actually shrinks these tumors initially grown with Tam then treated with estrogen (data not shown). This provides clues to the mechanism through which this occurs. When estrogen-stimulated tumors are treated with Tam, Tam has a tumoristatic effect, however, estrogen treatment actually reduces the size of the MCF-7:Tam LT tumors suggesting that estrogen is playing more of a tumoricidal role. To determine whether this decrease in size is attributable to an increase in apoptosis, we performed TUNEL staining to view any increase in apoptotic cells (Fig. 7). We compared the MCF-7:Tam LT treated with 1.5 mg Tam which promotes growth or 1 cm E2 Cap which inhibits growth. Clearly, the estrogen-treated group has a higher percentage of TUNEL stained cells compared to the Tam-treated counterpart which proves apoptosis as a mechanism for the decreasing tumor size after estrogen treatment.
4. Discussion We have described the T47D:E2, T47D:Tam, MCF-7:Tam ST and MCF-7:Tam LT tumors with preliminary characterization. These are only a subset of the models that we have developed to study the mechanism of drug resistance to Tam and other antiestrogens [12,14,15,24,28,37]. From these models, we have determined that there are three phases in the
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Fig. 7. Determination of apoptosis in MCF-7:Tam LT tumors treated with 1.5 mg Tam or 1 cm E2 Cap. TUNEL stains were performed on xenograft sections as described in Section 2. These photographs are representative of three independent experiments.
evolution of antiestrogen sensitivity and resistance (Fig. 8). Phase I encompasses the T47D:E2 and the MCF-7:E2 models where estrogen stimulates growth but Tam acts as an antiestrogen. Over the course of 10–25 weeks of Tam treatment, the previous models progress to Phase II where they are stimulated by both estrogen and Tam. Interestingly, only the MCF-7 model of drug resistance, the MCF-7:Tam LT is capable of progressing to Phase III resistance in which Tam continues to stimulate tumor growth but estrogen becomes a tumoricidal agent by inducing apoptosis (Fig. 7). The importance of HER-2/neu expression in the development of drug resistance has been well documented both
clinically [1,6,9] and in the laboratory [21]. With the development of Tam-stimulated growth, we consistently see increases in HER-2/neu mRNA and protein expression in all of the models described here. It has been known for a decade that HER-2/neu mRNA is down-regulated by estrogen [31], which is shown in Figs. 3 and 4, but interestingly in Tam-stimulated T47D:Tam tumors, there is a greater induction of HER-2/neu mRNA in the presence of Tam compared to the T47D:E2 tumors without estrogen. This is also the case in MCF-7 tumors [12]. While HER-2/neu may play an important role in drug resistance, the estrogen receptor is still required for growth. The Tam-stimulated
Fig. 8. Illustration of the evolution of SERM sensitivities in xenograft models of breast cancer [37]. The progression of drug resistance is separated into three stages. Phase I is typified by estrogen-stimulated tumors that are inhibited by SERMs. After long-term SERM treatment, Phase II resistance occurs which is characterized by both estrogen- and SERM-stimulated growth. Further progression to Phase III has, thus far, only been achieved using the MCF-7 xenograft model where tumors remain SERM-stimulated but become estrogen-inhibited.
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Fig. 9. Proposed model of the integrated mechanism for the target site specific action of SERMs. SERMs act as antiestrogens in surface silent cells (i.e. cells that express low levels of growth factor receptors) resulting in dominant co-repressor activity that retains the antiestrogenic activity of SERMs. However, when growth factor receptors are expressed at the cell surface resulting in dominant co-activator activity, the SERM:estrogen receptor complex becomes transcriptionally active.
tumors described here are all estrogen receptor positive and ligand-dependent, i.e. no selective estrogen receptor modulator (SERM) treatment results in no tumor growth. Therefore, we postulate that while these tumors are ligand:estrogen receptor-dependent, it is HER-2/neu that modulates the action of estrogen receptor complex by potential cross-talk or co-regulator modulation (Fig. 9). Therefore, both surface signaling by HER-2/neu and a functional estrogen receptor are required for the maintenance of Tam-stimulated growth. The recent data from the ATAC trial [2] also support the above hypothesis. Initial results show that Tam is less effective than anastrozole for disease-free survival in postmenopausal women with early stage breast cancer. One reason for this observation may be that Tam will likely fail in HER-2/neu positive patients because these patients are predisposed to developing Tam-resistance. In other words, active cell surface signaling ultimately modulates SERM:estrogen receptor function and overrides the ability of the estrogen receptor to block growth. Anastrozole, on the other hand, is equivalent to no treatment because presumably no ligands will be available to bind the estrogen receptor. Therefore, a critical component of this signal transduction pathway is compromised and, as a result silences growth. This conclusion was also reached in short-term studies with letrozole [8] where in postmenopausal patients with primary breast cancer that were estrogen receptor positive, HER-1 (EGFR) and/or HER-2/neu positive tumors responded better to letrozole than Tam again suggesting that HER-2/neu signaling occurs through cross-talk with the estrogen receptor and is ligand-dependent. The proposed mechanism detailing the involvement of HER-2/neu and the estrogen receptor is illustrated in Fig. 9. This model
does not explain estrogen’s antitumor effects in Phase III resistance, though we have shown that apoptosis plays a key role, the mechanism through which estrogen triggers this action is still unclear and requires further study.
Acknowledgements These studies were supported by the Department of Defense Breast Cancer Training Grant DAMD 17-94-J-4466, DAMD 17-96-1-6169, P30 CA60553-09, National Research Service Award T32 DK07169 (JMS), NIH SPORE in breast cancer 1P50 CA89018-02 (VCJ), Lynn Sage Breast Cancer Research Foundation of Northwestern Memorial Hospital and the Avon Products Foundation. References [1] J. Bange, E. Zwick, A. Ullrich, Molecular targets for breast cancer therapy and prevention, Nat. Med. 7 (2001) 548–552. [2] M. Baum, on behalf of the ATAC Trialists’ Group, The ATAC (Arimidex, Tamoxifen, alone or in combination) adjuvant breast cancer trial in post-menopausal (PM) women, Breast Cancer Res. Treat. 69 (2001) (Abstract 8). [3] A.U. Buzdar, W. Jonat, A. Howell, P.V. Plourde, ARIMIDEX: a potent and selective aromatase inhibitor for the treatment of advanced breast cancer, J. Steroid Biochem. Mol. Biol. 61 (1997) 145–149. [4] P.A. Canney, T. Griffiths, T.N. Latief, T.J. Priestman, Clinical significance of tamoxifen withdrawal response, Lancet 1 (1987) 36. [5] G. Casey, M. Lo-Hsueh, M.E. Lopez, B. Vogelstein, E.J. Stanbridge, Growth suppression of human breast cancer cells by the introduction of a wild-type p53 gene, Oncogene 6 (1991) 1791–1797. [6] M. De Laurentiis, A.R. Bianco, S. De Placido, A meta-analysis of the interaction between HER2 expression and response to endocrine
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[21] H. Kurokawa, A.E. Lenferink, J.F. Simpson, P.I. Pisacane, M.X. Sliwkowski, J.T. Forbes, C.L. Arteaga, Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells, Cancer Res. 60 (2000) 5887–5894. [22] S. Legault-Poisson, J. Jolivet, R. Poisson, M. Beretta-Piccoli, P.R. Band, Tamoxifen-induced tumor stimulation and withdrawal response, Cancer Treat Rep. 63 (1979) 1839–1841. [23] A.S. Levenson, V.C. Jordan, MCF-7: the first hormone-responsive breast cancer cell line, Cancer Res. 57 (1997) 3071–3078. [24] J. MacGregor Schafer, E.-S. Lee, R.M. O’Regan, K. Yao, V.C. Jordan, Rapid development of tamoxifen-stimulated mutant p53 breast tumors (T47D) in athymic mice, Clin. Cancer Res. 6 (2000) 4373–4380. [25] P.M. O’Connor, J. Jackman, I. Bae, T.G. Myers, S. Fan, M. Mutoh, D.A. Scudiero, A. Monks, E.A. Sausville, J.N. Weinstein, S. Friend, A.J. Fornace Jr., K.W. Kohn, Characterization of the p53 tumors suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents, Cancer Res. 57 (1997) 4285– 4300. [26] A.S. Oh, L.A. Lorant, J.N. Holloway, D.L. Miller, F.G. Kern, D. El-Ashry, Hyperactivation of MAPK induces loss of ERalpha expression in breast cancer cells, Mol. Endocrinol. 15 (2001) 1344– 1359. [27] R.M. O’Regan, A. Cisneros, G.M. England, J.I. MacGregor, H.D. Muenzner, V.J. Assikis, M.M. Billimoria, M. Piette, Y.P. Dragan, H.C. Pitot, R. Chatterton, V.C. Jordan, Effects of the antiestrogens tamoxifen, toremifene, and ICI 182,780 on endometrical cancer growth [see comments], J. Natl. Cancer Inst. 90 (1998) 1552– 1558. [28] R.M. O’Regan, E.S. Lee, R.C. Dardes, C. Gajdos, A.D. Reyes, S. Badve, V.C. Jordan, Characteristics of raloxifene-stimulated breast tumors in vivo, Breast Cancer Res. Treat. 69 (2001) (Abstract 443). [29] C.K. Osborne, Tamoxifen in the treatment of breast cancer, N. Engl. J. Med. 339 (1998) 1609–1618. [30] J.J. Pink, V.C. Jordan, Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancer cell lines, Cancer Res. 56 (1996) 2321–2330. [31] L.D. Read, D. Keith Jr., D.J. Slamon, B.S. Katzenellenbogen, Hormonal modulation of HER-2/neu protooncogene messenger ribonucleic acid and p185 protein expression in human breast cancer cell lines, Cancer Res. 50 (1990) 3947–3951. [32] J.F. Robertson, Oestrogen receptor: a stable phenotype in breast cancer, Br. J. Cancer 73 (1996) 5–12. [33] S.P. Robinson, V.C. Jordan, Antiestrogenic action of toremifene on hormone-dependent, -independent, and heterogeneous breast tumor growth in the athymic mouse, Cancer Res. 49 (1989) 1758–1762. [34] D.J. Slamon, G.M. Clark, S.G. Wong, W.J. Levin, A. Ullrich, W.L. McGuire, Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene, Science 235 (1987) 177–182. [35] H.D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, A human cell line from a pleural effusion derived from a breast carcinoma, J. Natl. Cancer Inst. 51 (1973) 1409–1416. [36] T. Watanabe, T. Fukutomi, H. Tsuda, I. Adachi, T. Nanasawa, H. Yamamoto, K. Abe, Determination of c-erbB-2 protein in primary breast cancer tissue extract using an enzyme immunoassay, Jpn. J. Cancer Res. 84 (1993) 1279–1286. [37] K. Yao, E. Lee, D.J. Bentrem, G.M. England, J. MacGregor Schafer, R.M. O’Regan, V.C. Jordan, Antitumor action of physiological estradiol on tamoxifen-stimulated breast tumors grown in athymic mice, Clin. Cancer Res. 6 (2000) 2028–2036.
A mechanism of drug resistance to tamoxifen in breast cancer夽 Jennifer MacGregor Schafer a , David J. Bentrem c , Hiroyuki Takei d , Csaba Gajdos a , Sunil Badve b , V. Craig Jordan a,∗ a
Robert H. Lurie Comprehensive Cancer Center, The Feinberg School of Medicine, Northwestern University, Olson Pavilion 8258, 303 East Chicago Avenue, Chicago, IL 60611, USA b Department of Pathology, The Feinberg School of Medicine, Northwestern University, Olson Pavilion 8258, 303 East Chicago Avenue, Chicago, IL 60611, USA c Department of Surgery, The Feinberg School of Medicine, Northwestern University, Olson Pavilion 8258, 303 East Chicago Avenue, Chicago, IL 60611, USA d Division of Breast Surgery, Saitama Cancer Center, Saitama 362-0806, Japan
Abstract Drug resistance to tamoxifen (Tam) is a significant clinical problem but the mechanism through which this occurs remains elusive. We have developed a number of xenograft models of Tam-stimulated growth that model breast cancer progression using estrogen receptor positive MCF-7 or T47D breast cancer cells. When estrogen-stimulated T47D:E2 tumors are treated long term with Tam, Tam-stimulated tumors develop (T47D:Tam) that are stimulated by both estrogen and Tam. When HER-2/neu status is determined, it is clear that the T47D:Tam tumors express significantly higher levels of HER-2/neu protein by immunohistochemistry and mRNA as measured by real-time RT-PCR. The T47D:Tam tumors also express higher levels of estrogen receptor and progesterone receptor protein than their estrogen-stimulated T47D:E2 counterparts. We compared out results to the MCF-7 model of Tam-stimulated growth. The MCF-7:Tam ST (estrogen- and Tam-stimulated) and MCF-7:Tam LT (estrogen-inhibited, Tam-stimulated) were bilaterally transplanted to account for any mouse to mouse variation and characteristic growth patterns were observed. TUNEL staining was performed on MCF-7:Tam LT treated with either estrogen or Tam and it was concluded that estrogen-inhibited tumor growth was a result of increased apoptosis. Three phases of tumor progression are described that involve increases in HER-2/neu expression, de-regulation of estrogen receptor expression and increases in apoptosis which in concert determine the phenotype of drug resistance to Tam. © 2003 Published by Elsevier Science Ltd. Keywords: Antiestrogen; Breast cancer; Estrogen receptor; HER-2/neu; Drug resistance
1. Introduction Tamoxifen (Tam) is the endocrine therapy of choice for all stages of estrogen receptor positive breast cancer [29] and is currently prescribed for the prevention of breast cancer in high-risk women [10]. In addition, in patients with estrogen receptor positive disease, up to 5 years of adjuvant Tam treatment confers a long-term survival benefit that continues for at least an additional 5 years after Tam treatment ceases [7]. Unfortunately, greater than 5 years of Tam treatment may lead to the clonal selection of metastatic breast cancer that is resistant to Tam [11]. In fact, some of these tumors are stimulated by Tam as evidenced by the decreased 夽 Proceedings of the 15th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, “Recent Advances in Steroid Biochemistry and Molecular Biology”, Munich, Germany, 17–20 May 2002. ∗ Corresponding author. Tel.: +1-312-908-4148; fax: +1-312-908-1372. E-mail address: [email protected] (V.C. Jordan).
0960-0760/03/$ – see front matter © 2003 Published by Elsevier Science Ltd. PII: S 0 9 6 0 - 0 7 6 0 ( 0 2 ) 0 0 2 5 1 - 0
growth of tumors after Tam therapy is stopped [4,19,22]. Most of these Tam-stimulated tumors retain expression of the estrogen receptor [32] and, therefore, retain the ability to respond to second-line endocrine therapies such as the pure antiestrogen ICI182,780 [18] or an aromatase inhibitor such as anastrozole [3]. Following the failure of adjuvant Tam, anastrozole is currently available as a second-line therapy [3], indeed, a whole range of aromatase inhibitors are currently being evaluated [13]. The mechanism of action of aromatase inhibitors is contingent on whether Tam-resistant disease still requires estrogen to grow, so by reducing circulating estradiol and estrone, the tumor will lose its growth stimulus. Recent data presented from the ATAC trial provides evidence that the aromatase inhibitor anastrozole is superior to Tam in postmenopausal women with estrogen receptor positive early stage breast cancer [2]. The anatrozole-treated women showed a decrease in adverse effects and an increase in disease-free survival compared to Tam and, in addition,
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anastrozole may actually further prevent the incidence of contralateral breast cancer. These results are preliminary and further follow-up is needed to determine effects on bone mineral density and cognitive function. Because Tam is used extensively and the development of drug resistance is a concern, many laboratories are focusing on developing in vitro and in vivo models of drug resistance. The MCF-7 breast cancer cell line [35] is an ideal model because these cells express functional wild-type estrogen receptor and grow maximally in E2-containing media. When the MCF-7 cells are treated with antiestrogens, growth is dramatically reduced providing an excellent model to study the mechanism of antiestrogen action. Another interesting feature of this cell line is that it mimics the clinical progression of breast cancer including the development of antiestrogen resistance and the progression to hormone-independence. After long-term treatments with Tam, for example, adapted clones of MCF-7 cells are selected that are Tam-stimulated and retain expression of estrogen receptor [23]. When MCF-7 tumors are transplanted into ovariectomized athymic mice, estrogen supplementation is required for maximal tumor growth. The antiestrogen Tam, conversely, does not result in tumor growth and, in fact, is able to inhibit estrogen-stimulated growth [15] consistent with human breast cancer treatment. After long-term treatment with low dose Tam (approximately 6 months), tumors do not grow, however, treatment with estrogen rescues the growth demonstrating the tumoristatic not tumoricidal effects of Tam. When transplanted MCF-7 tumors are initially treated with estrogen to establish the tumor, then switched to Tam, for about 4 months, tumors regression is observed, however, by 8 months some tumors begin to grow [14]. These tumors are estrogen receptor positive and grow in response to estrogen when retransplanted. Interestingly, one of these tumors grew only in response to Tam demonstrating the progression of tumors from estrogen-stimulated to Tam-stimulated and the validity of this model. This model can also be used to study the effects of second-line endocrine therapies when Tam-resistant or -stimulated tumors arise. MCF-7 cells express wild-type p53 [5], and, therefore, only represent 50% of breast cancers [17]. A novel human breast xenograft model derived from T47D cells [24] which express a mutant p53 that is nonfunctional [25] has been developed [24]. After approximately 10 weeks of Tam treatment, T47D estrogenstimulated (T47D:E2) tumors become Tam-stimulated (T47D:Tam) and remain estrogen-responsive. This occurs at a significantly faster rate than MCF-7 tumors undergoing similar treatment therefore suggesting that the differences between these tumor lines directly impact the development of Tam-stimulated growth [24]. This model allows critical comparisons between cellular events that determine hormone responsiveness and drug resistance. Overexpression/amplification of the proto-oncogene receptor tyrosine kinase HER-2/neu (also known as c-erbB2) imparts an alternative growth stimulatory pathway and provides an additional therapeutic target in the treatment of
breast cancer. It has been known for almost a decade that amplification of the HER-2/neu gene correlates with shorter overall and disease-free survival [34] and that there is an inverse correlation between estrogen receptor expression and HER-2/neu overexpression [36]. In addition, constitutive activation of HER-2/neu signaling molecules has been shown to alter estrogen receptor expression [26] and the estrogen treatment of breast cancer cells in vitro results in down-regulation of HER-2/neu [31]. There are conflicting data and considerable debate about the predictive value of HER-2/neu overexpression and response to chemo or endocrine therapy in breast cancer (reviewed in [1]). Meta-analysis has shown that the HER-2/neu negative patient is more likely to respond to Tam therapy compared to their HER-2/neu positive counterparts [6] though prospective clinical trials are necessary to address this issue. Indeed, it has even been stated that HER-2/neu overexpression in breast cancer may be a better predictor of response to Tam than estrogen receptor status alone [9]. When HER-2/neu is overexpressed in breast cancer cells in vitro, the cells become Tam-resistant, however, blockade of the HER-2/neu signal transduction pathway restores the inhibitory effect of Tam on estrogen receptor mediated transcription and cell proliferation [21]. The mechanism of drug resistance to Tam is poorly understood and though there have been several theories proposed, for example, loss of estrogen receptor expression, thus far, there is no unifying mechanism. Here we propose a unifying theory for the mechanism of drug resistance based on laboratory models of the development of Tam resistance. Clearly, the estrogen receptor plays a pivotal role in Tam-stimulated growth because Tam-stimulated tumors always retain estrogen receptor expression [12,24]. We also show that HER-2/neu is consistently overexpressed in Tam-stimulated tumors, therefore, we believe that previously established cross-talk between the estrogen receptor and HER-2/neu may be the primary determinant of Tam-stimulated growth in our xenograft models of Tam-stimulated growth. 2. Materials and methods 2.1. Athymic mouse model The MCF-7 and T47D tumors (Fig. 1) used in these experiments were derived by bilateral inoculation of suspended human breast cancer cell lines into the mammary fat pads of 4–6 weeks old ovariectomized BALB/c nu/nu athymic mice (Harlan Sprague Dawley, Madison, WI). Ovarectomized athymic mice were injected bilaterally with 0.5 million T47D human breast cancer cells and supplemented with 17-estradiol (E2) pellets to create T47D:E2 tumors. When the T47D:E2 tumors reached approximately 0.8 cm2 (data not shown), a single tumor was passaged into athymic mice by retransplanting 1 mm3 tumor pieces with a 13 gauge trocar and they were treated long term with 1.5 mg Tam
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Fig. 1. Illustration of development of in vitro and in vivo models of acquired resistance to SERMs. (I) Breast cancer cells are treated with a SERM or deprived of estrogen for more than 1 year. These resistant cells are subsequently inoculated into athymic mice and continue SERM treatment producing SERM-resistant tumors. (II) SERM-resistant tumors are developed in vivo by treated estrogen-stimulated tumors for more than 1 year with a SERM.
to establish Tam-stimulated growth (T47D:Tam) (Table 1) [24]. To create MCF-7:Tam-stimulated tumors (Table 1), 10 million MCF-7 cells were inoculated and supplemented with E2 capsules to achieve estrogen-stimulated tumor growth (MCF-7:E2) [15]. Short-term Tam-stimulated tumor (MCF-7:Tam ST) growth was achieved after several weeks by retransplanting the growing estrogen-dependent MCF-7 tumors into new athymic mice and treating them with Tam [37]. Tumor measurements were performed weekly using Vernier calipers. The cross-sectional area was calculated using the formula: length × width/4π.
80). Ethanol was evaporated under nitrogen before use. Tam was administered orally, 1.5 mg per mouse, 5 days per week. 2.3. Statistical methods Comparisons in mean tumor between the animal groups were analyzed by analysis of variance (ANOVA) at each week and followed by unpaired Student’s t-test. The two tailed P-value of the last week of each experiment was reported using StatMost 2.5 (Datamost Corp., Salt Lake, UT). 2.4. Western blot analysis
2.2. Hormone and drug treatments Mice were divided into groups of 10 and were treated with E2 (Sigma), Tam (Sigma) or combination. Silastic E2 capsules (0.3 or 1 cm) were made as described previously [33], implanted subqutaneously on the same day as tumor transplantation and replaced every 8–10 weeks of treatment. The 0.3 cm E2 capsule produces a mean 83.9 pg/ml of serum E2 while 1.0 cm E2 capsules produces a mean 379.5 pg/ml of serum E2 as described previously [27]. Tam was dissolved in ethanol and suspended in a solution of 90% carboxymethylcellulose (1% carboxymethylcellulose in double-distilled water) and 10% polyethylene glycol 400/Tween 80 (99.5% polyethylene glycol and 0.5% Tween Table 1 Classification of breast xenografts based on hormone responsiveness Tumor
Derived from
E2 stimulated
Tam stimulated
T47D:E2 T47D:Tam MCF-7:Tam ST MCF-7:Tam LT
T47D cells T47D cells MCF-7 cells MCF-7 cells
(++) (++) (++) (−−)
(−−) (++) (++) (++)
References for the origin of different tumor models: T47D:E2 and T47D:Tam tumors (4); MCF-7:Tam ST tumors (3). MCF-7:Tam LT is a long-term Tam-stimulated model (more than 5 years Tam), is inhibited by E2 and stimulated by Tam.
Tumors were harvested and snap-frozen in liquid nitrogen and then stored at −80 ◦ C. Frozen tumors were homogenized by grinding in liquid nitrogen. Tumor extract was resuspended in protein extraction buffer (RIPA buffer: 1X PBS, 1% NP40, 0.5%Na deoxycholate, 0.1% SDS, protease inhibitor cocktail, NaF (1 M) and NaVO3 (250 mM)) and pelleted. Protein concentration of the supernatants was measured using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories Inc., Santa Cruz, CA) and equal amounts of protein (30 g) were analyzed according to a standard Western blot protocol [30]. The estrogen receptor primary antibody used was G-20 (Santa Cruz Biotechnology, Santa Cruz, CA), the PR primary antibody used was C-20 (Santa Cruz Biotechnology) and -actin antibody AC-15 (Sigma) was used to standardize loading. The appropriate secondary antibody conjugated to horseradish peroxidase (Amersham Corp., Arlington Heights, IL) was used to visualize bands using an ECL visualization kit (Amersham Corp.). The membrane was exposed to Kodak X-OMAT film. 2.5. Real-time RT-PCR Total RNA was extracted from the tumors using the Rneasy Mini Kit (Qiagen, Stanford Valencia, CA) according
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to the manufacturer’s instructions. Total RNA was reverse transcribed using TaqMan reverse transcription reagents (PE Applied Biosystems, Hayward, CA) with the use of random hexamers as the primers according to the manufacturer’s instructions. Primers and probes for human HER-2/neu were designed using Primer ExpressTM1.5 software. The sequences for the forward and reverse primers for human HER-2/neu: 5 -ACTGCAGAGGCTGCGGATT-3 and 5 -ACGGCCAGGGCATAGTTGT-3 . The sequence for the probe is FAM-5 -TGCGAGGCACCCAGCTCTTTGA-3’QSY7 (MegaBases Inc., Chicago, IL). The quantity of human GAPDH mRNA was also measured in each total RNA sample for normalization purposes. The probe and primers for GAPDH were purchased from Perkin-Elmer Applied Biosystems (PE Applied Biosystems, Stanford Valencia, CA). The PCR portion of the reaction was performed with the use of the TaqMan PCR Core Reagent Kit (PE Applied Biosystems). Real-time RT-PCR was performed using the ABI-Prism 7700 Sequence Detection System. The PCR conditions were 50 ◦ C for 2 min, 95 ◦ C for 10 min followed by 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. 2.6. Immunohistochemistry All immunohistochemistry is provided through the Robert H. Lurie Comprehensive Cancer Center, Core Pathology Department. Immunohistochemistry was performed according to manufacturer’s instructions. Antibodies used were: estrogen receptor (DAKO, Carpinteria, CA); HER-2/neu (DAKO, Carpinteria, CA). TUNEL stain was performed using the In Situ Cell Death Detection Kit (Boehringer Mannhein, Indianapolis, IN) according to manufacturer’s instructions. Briefly, 5 M sections from formalin-fixed, paraffin-embedded MCF-7:Tam LT xenografts were deparaffinized in xylenes (100% for 10 min three times), rehydrated in graded alcohols (100% (twice), 95, 90, 80, 70, 50%, 5 min each), rinsed with water (5 min) and PBS (5 min, twice). The samples were then permeablized with Triton-X 100 for 30 min, rinsed with PBS (5 min, twice), treated with 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity and then rinsed with PBS (5 min, twice). Samples were then blocked with bovine serum albumin and then incubated with terminal deoxynucleotidyl transferase and POD-conjugated antifluorescein antibody for 60 min in a humidified chamber at 37 ◦ C. Then samples were mounted with coverslips under glycerol/PBS and analyzed via light microscopy.
3. Results 3.1. T47D model of Tam-stimulated growth Using the estrogen receptor positive T47D human breast cancer cell line [20] that expresses a mutated nonfunctional p53 [25], we developed estrogen-dependent tumors
Fig. 2. Growth of (A) T47D:E2 or (B) T47D:Tam tumors passaged in athymic mice (mean ± S.E.). (A) Ten mice per group were treated for 12 weeks with 1 cm E2 capsule (Cap), 0.5 mg p.o. Tam or combination. E2 significantly stimulated growth compared to control (P = 0.003). There was no significant difference between control and Tam or combination. (B) Ten mice per group were treated for 12 weeks with 1 cm E2 capsule (Cap), 0.5 mg or 1.5 mg p.o. Tam. E2, 0.5 mg Tam and 1.5 mg Tam significantly stimulated growth compared to control (P < 0.005).
that are transplantable and only grow in the presence of E2 (T47D:E2; Fig. 2A) [24]. Treatment with low dose Tam (0.5 mg) does not result in tumor growth and, in addition, Tam is capable of acting as an antiestrogen in the model by inhibiting premenopausal level (1 cm E2 Cap) estrogen-stimulated growth. Interestingly, when these tumors are treated with high dose Tam (1.5 mg Tam), after approximately 10 weeks, Tam-stimulated tumors develop (T47D:Tam). These T47D:Tam tumors are transplantable and are more effectively stimulated by high dose (1.5 mg) Tam than low dose (0.5 mg) Tam [24] (Fig. 2B). 3.2. Classification of T47D tumor models We performed immunohistochemical analysis of T47D:E2 and T47D:Tam tumors to determine the HER-2/neu status. We chose three different tumor passages of each type to analyze in order to provide as much variation as possible. We determined that estrogen-stimulated T47D:E2 tumors were HER-2/neu− (1+) (Fig. 3). Parental T47D cells are known to express HER-2/neu [16] but the specific clone used in this study (T47D:A18) does not express high levels. Next, we compared this to the Tam-stimulated T47D:Tam
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Fig. 3. Immunohistochemical analysis of HER-2/neu in T47D:E2 tumors treated with 1 cm E2 Cap compared with T47D:Tam tumors treated with 1.5 mg Tam. Immunohistochemistry was performed on xenograft sections as described in Section 2. These photographs are representative of three independent experiments.
tumors which were HER-2/neu+ (3+) (Fig. 3). Because overexpression of protein can arise at many different levels, we confirmed our findings using real-time RT-PCR and measured levels of HER-2/neu mRNA in these tumor types with different treatments (Fig. 4). In the T47D:E2 tumors, 1 cm E2 Cap treatment resulted in low HER-2/neu mRNA production consistent with the immunohistochemistry. However, when the capsule was removed for 1 week before tumor harvest, HER-2/neu mRNA levels increased six-fold as expected. When similar measurements were undertaken in the T47D:Tam-stimulated tumors, E2 treatment again yielded low levels of HER-2/neu mRNA while Tam treatment resulted in approximately a 10-fold increase in HER-2/neu mRNA. Because of the proposed cross-talk between HER-2/neu and the estrogen receptor, we determined estrogen re-
Fig. 4. Relative expression of HER-2/neu mRNA expression as determined using real-time RT-PCR. Real-time RT-PCR was performed as described in Section 2. Control values of HER-2/neu were normalized with those of GAPDH. Each experiment was repeated three times.
ceptor levels and function by measuring progesterone receptor levels to ascertain any influence of estrogen receptor on this signal transduction pathway (Fig. 5). In the T47D:E2 tumors, classical type II regulation occurred [30] where estrogen receptor protein levels are induced by estrogen treatment. In the T47D:Tam tumors, the estrogen receptor levels are similar, suggesting that estrogen receptor production is de-regulated in the Tam-stimulated tumors though clearly, estrogen maintains its ability to promote progesterone receptor production and to a greater extent than the T47D:E2 counterpart. These data are consistent with our immunohistochemical findings (data not shown).
Fig. 5. Western blot screen of estrogen receptor ␣ and progesterone receptor protein expression in T47D:E2 or T47D:Tam tumors after E2, Tam or no treatment. Western blot analysis was performed as described in Section 2. -Actin protein was measured to ensure even loading. The Western blot shown is representative of three independent experiments.
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3.3. MCF-7 models of Tam-stimulated growth The development of Tam-stimulated growth in MCF-7 tumors occurs after about 25 weeks of growth in high dose Tam (1.5 mg) that comparably is significantly longer compared the 10 weeks for the development of Tam-stimulated growth in T47D tumors [24]. When Tam-stimulated MCF-7 tumors (MCF-7:Tam ST) do develop, these tumors are stimulated by both estrogen and Tam and do not grow without treatment [14]. MCF-7:Tam-stimulated tumors are unique in that after 5 years of Tam treatment, tumors progress to a different phenotype (MCF-7:Tam LT) where Tam continues to stimulate growth, but paradoxically, estrogen treatment inhibits growth [37]. So the question of differences between animals such as changes in ability to metabolize drug or differences in immune response arise. To address these issues, mice were bitransplanted with MCF-7:Tam ST tumors implanted into the left mammary fat pad and MCF-7:Tam LT tumors implanted in the right mammary fat pad (Fig. 6A). The MCF-7:Tam ST display classic Phase II Tam-stimulated growth, both estrogen and Tam result in tumor growth (Fig. 6B). However, the MCF-7:Tam LT tumors implanted in the right side of the same animal display Phase III growth where Tam stimulates growth but estrogen does not (Fig. 6C). 3.4. Characterization of E2-inhibited growth in MCF-7:Tam LT model
Fig. 6. (A) Illustration of two MCF-7 xenograft models bitransplanted into athymic mice. (B) MCF-7:Tam ST tumors were transplanted into the left mammary fat pad. (C) MCF-7:Tam LT tumors were transplanted into the right mammary fat and treated for 7 weeks with 1 cm E2 Cap or 1.5 mg Tam. The growth of the 1 cm E2 Cap and 1.5 mg Tam group in MCF-7:Tam ST tumors (B) were significantly different compared to control (P < 0.05). The growth of MCF-7:Tam LT tumors treated with 1.5 mg Tam was significantly different than the 1 cm E2 Cap group (P < 0.05).
In fact, not only does estrogen fail to stimulate tumor growth of the MCF-7:Tam LT, it actually shrinks these tumors initially grown with Tam then treated with estrogen (data not shown). This provides clues to the mechanism through which this occurs. When estrogen-stimulated tumors are treated with Tam, Tam has a tumoristatic effect, however, estrogen treatment actually reduces the size of the MCF-7:Tam LT tumors suggesting that estrogen is playing more of a tumoricidal role. To determine whether this decrease in size is attributable to an increase in apoptosis, we performed TUNEL staining to view any increase in apoptotic cells (Fig. 7). We compared the MCF-7:Tam LT treated with 1.5 mg Tam which promotes growth or 1 cm E2 Cap which inhibits growth. Clearly, the estrogen-treated group has a higher percentage of TUNEL stained cells compared to the Tam-treated counterpart which proves apoptosis as a mechanism for the decreasing tumor size after estrogen treatment.
4. Discussion We have described the T47D:E2, T47D:Tam, MCF-7:Tam ST and MCF-7:Tam LT tumors with preliminary characterization. These are only a subset of the models that we have developed to study the mechanism of drug resistance to Tam and other antiestrogens [12,14,15,24,28,37]. From these models, we have determined that there are three phases in the
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Fig. 7. Determination of apoptosis in MCF-7:Tam LT tumors treated with 1.5 mg Tam or 1 cm E2 Cap. TUNEL stains were performed on xenograft sections as described in Section 2. These photographs are representative of three independent experiments.
evolution of antiestrogen sensitivity and resistance (Fig. 8). Phase I encompasses the T47D:E2 and the MCF-7:E2 models where estrogen stimulates growth but Tam acts as an antiestrogen. Over the course of 10–25 weeks of Tam treatment, the previous models progress to Phase II where they are stimulated by both estrogen and Tam. Interestingly, only the MCF-7 model of drug resistance, the MCF-7:Tam LT is capable of progressing to Phase III resistance in which Tam continues to stimulate tumor growth but estrogen becomes a tumoricidal agent by inducing apoptosis (Fig. 7). The importance of HER-2/neu expression in the development of drug resistance has been well documented both
clinically [1,6,9] and in the laboratory [21]. With the development of Tam-stimulated growth, we consistently see increases in HER-2/neu mRNA and protein expression in all of the models described here. It has been known for a decade that HER-2/neu mRNA is down-regulated by estrogen [31], which is shown in Figs. 3 and 4, but interestingly in Tam-stimulated T47D:Tam tumors, there is a greater induction of HER-2/neu mRNA in the presence of Tam compared to the T47D:E2 tumors without estrogen. This is also the case in MCF-7 tumors [12]. While HER-2/neu may play an important role in drug resistance, the estrogen receptor is still required for growth. The Tam-stimulated
Fig. 8. Illustration of the evolution of SERM sensitivities in xenograft models of breast cancer [37]. The progression of drug resistance is separated into three stages. Phase I is typified by estrogen-stimulated tumors that are inhibited by SERMs. After long-term SERM treatment, Phase II resistance occurs which is characterized by both estrogen- and SERM-stimulated growth. Further progression to Phase III has, thus far, only been achieved using the MCF-7 xenograft model where tumors remain SERM-stimulated but become estrogen-inhibited.
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Fig. 9. Proposed model of the integrated mechanism for the target site specific action of SERMs. SERMs act as antiestrogens in surface silent cells (i.e. cells that express low levels of growth factor receptors) resulting in dominant co-repressor activity that retains the antiestrogenic activity of SERMs. However, when growth factor receptors are expressed at the cell surface resulting in dominant co-activator activity, the SERM:estrogen receptor complex becomes transcriptionally active.
tumors described here are all estrogen receptor positive and ligand-dependent, i.e. no selective estrogen receptor modulator (SERM) treatment results in no tumor growth. Therefore, we postulate that while these tumors are ligand:estrogen receptor-dependent, it is HER-2/neu that modulates the action of estrogen receptor complex by potential cross-talk or co-regulator modulation (Fig. 9). Therefore, both surface signaling by HER-2/neu and a functional estrogen receptor are required for the maintenance of Tam-stimulated growth. The recent data from the ATAC trial [2] also support the above hypothesis. Initial results show that Tam is less effective than anastrozole for disease-free survival in postmenopausal women with early stage breast cancer. One reason for this observation may be that Tam will likely fail in HER-2/neu positive patients because these patients are predisposed to developing Tam-resistance. In other words, active cell surface signaling ultimately modulates SERM:estrogen receptor function and overrides the ability of the estrogen receptor to block growth. Anastrozole, on the other hand, is equivalent to no treatment because presumably no ligands will be available to bind the estrogen receptor. Therefore, a critical component of this signal transduction pathway is compromised and, as a result silences growth. This conclusion was also reached in short-term studies with letrozole [8] where in postmenopausal patients with primary breast cancer that were estrogen receptor positive, HER-1 (EGFR) and/or HER-2/neu positive tumors responded better to letrozole than Tam again suggesting that HER-2/neu signaling occurs through cross-talk with the estrogen receptor and is ligand-dependent. The proposed mechanism detailing the involvement of HER-2/neu and the estrogen receptor is illustrated in Fig. 9. This model
does not explain estrogen’s antitumor effects in Phase III resistance, though we have shown that apoptosis plays a key role, the mechanism through which estrogen triggers this action is still unclear and requires further study.
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