Antitumor Action of Physiological Estradiol on Tamoxifen- Stimulated Breast Tumors Grown in Athymic Mice1

2028 Vol. 6, 2028–2036, May 2000 Clinical Cancer Research

Antitumor Action of Physiological Estradiol on Tamoxifen- stimulated Breast Tumors Grown in Athymic Mice1

2 Kathy Yao, Eun-Sook Lee, David J. Bentrem, development of E2-stimulated regrowth. The mutation is Gale England, Jennifer I. MacGregor Schafer, associated with increased estrogen-like actions for the Ruth M. O’Regan, and V. Craig Jordan3 TAM-ER complex (A. S. Levenson et al., Br. J. Cancer, 77: 1812–1819, 1998), but we conclude that the mutant ER is not Department of Surgery [K. Y., D. J. B., G. E.], R. H. Lurie required for TAM resistance. On the basis of the new breast Comprehensive Cancer Center [E-S. L., J. I. M. S., V. C. J.], and the Division of Medical Oncology [R. M. O.], Northwestern University cancer models presented, we propose a cyclic sensitivity to Medical School, Chicago, Illinois 60611 TAM that may have important clinical implications: (a)itis possible that a woman’s own estrogen may produce an antitumor effect on the presensitized micrometastatic dis- ABSTRACT ease after 5 years of TAM. Long-term antitumor action The estrogen receptor (ER)-positive MCF-7 breast can- occurs because the drug is stopped, but resistance accumu- cer cell line can be transplanted into athymic mice and lates and tumors start to grow if adjuvant therapy is con- grown into tumors with estradiol (E2) support. Tamoxifen tinued; and (b) although in the clinic TAM-resistant tumors (TAM) blocks E2-stimulated tumor growth; however, con- respond to second-line therapies that cause estrogen with- tinuous TAM treatment results in transplantable tumors drawal, e.g., pure antiestrogens or aromatase inhibitors, within a year that will grow with either E2 or TAM (M. M. estrogen therapy may also be effective and return the tumor Gottardis and V. C. Jordan, Cancer Res., 48: 5183–5187, to TAM responsiveness. In this way, a hormone-responsive 1988). Although this model may represent the development tumor may be controlled longer in the patient with advanced of TAM resistance for the treatment of advanced breast disease. cancer, no laboratory model exists to study the exposure of breast cancer to 5 years of adjuvant TAM therapy. We have INTRODUCTION addressed this issue and report the development and char- 4 Adjuvant TAM has revolutionized breast cancer therapy, acterization of two tumor lines, MCF-7TAM and MT2, but the duration of treatment remains controversial. The recent which have been serially transplanted into TAM-treated overview analysis in 1998 (1) demonstrated that increasing athymic mice for 5 years. The MCF-7TAM tumor rapidly > adjuvant TAM therapy from 1 to 5 years is more effective in regresses in response to E and then about 50% of tumors 2 increasing survival, but a recent NSABP trial has shown that 5 regrow in response to E . Interestingly, tumor regression 2 years of TAM as adjuvant therapy for node-negative breast does not occur if TAM treatment is stopped, probably be- cancer carried a better disease-free and distant disease-free cause E levels are too low in ovariectomized athymic mice. 2 survival than Ͼ5 years of treatment (2). Large clinical trials will The development of the antitumor effect of E was 2 be needed to resolve this question, but it appears that some documented for MT2 tumors over a 1-year period; TAM- breast tumors acquire resistance to TAM after 5 years of ther- stimulated tumor growth was retained, but E caused pro- 2 apy. Drug resistance to TAM can be manifest in many ways, one gressively less of a stimulatory effect. Most importantly, of which is TAM-stimulated growth. There is some clinical E -stimulated tumors that regrew after initial tumor regres- 2 evidence that TAM-stimulated growth can occur during the sion in both MCF-7TAM and MT2 lines were again respon- treatment of advanced breast cancer (3, 4). In the laboratory, sive to TAM to block E -stimulated growth. Unlike MCF-7 2 long-term TAM treatment results in TAM-stimulated growth in tumors, the MT2 tumor line contains a single point muta- MCF-7 breast tumors (5, 6). After 1 year of continuous TAM tion, Asp351Tyr, in the ER, which was retained after the treatment, tumors grow in response to both TAM and E2 (MCF- 7TAM, a tamoxifen-stimulated MCF-7 tumor; Ref. 5). How- ever, no laboratory model is available that replicates the clinical use of 5 years of adjuvant TAM. We have addressed the issue Received 8/31/99; revised 1/31/00; accepted 2/16/00. and developed two transplantable MCF-7 tumor lines that have The costs of publication of this article were defrayed in part by the been exposed to TAM for Ͼ5 years by serially transplanting payment of page charges. This article must therefore be hereby marked TAM-stimulated tumors into athymic mice treated with TAM. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The two lines MCF-7TAM (7) and MT2 (an MCF-7 tamoxifen- 1 Supported by the Lynn Sage Foundation of Northwestern Memorial stimulated tumor with an Asp351Tyr mutant estrogen receptor; Hospital and the Avon Breast Cancer Fund. 2 Present address: Department of General Surgery, Korea University Ansan Hospital, 516 Kojan-Dong Ansan city Kyunggi-Do, 425-020, South Korea. 3 To whom requests for reprints should be addressed, at the R. H. Lurie 4 The abbreviations used are: TAM, tamoxifen; NSABP, National Sur-

Comprehensive Cancer Center, Northwestern University Medical gical Breast and Bowel Project; E2, estradiol; ER, estrogen receptor; School, 710 North Fairbanks, Olson 8258, Chicago, IL 60610. Phone: PEG, polyethylene glycol; SSCP, single-stranded conformational poly- 312-908-4148; Fax: 312-908-1372. morphism; DES, diethylstilbestrol.

Refs. 8 and 9) both grow in response to TAM, but we have used to mimic post- or premenopausal levels of E2.Inone discovered that E2 actually produces a tumoricidal action after experiment, an E2 pellet (1.5 mg) from Innovative Research of 4–5 years of TAM that is much more effective at reducing America (Toledo, OH) was used to replicate pharmacological tumor growth than stopping TAM treatment (7). However, a doses of E2. Serum levels of 1044 ng/ml were noted (13). The proportion of the tumors regrow in response to E2, and retrans- E2 and TAM capsules were replaced every 6–8 weeks (13). plantation has allowed us to examine the effectiveness of TAM Tumor Measurements. Tumor measurements were per- if it is again used as an antitumor agent. Additionally, we have formed weekly using Vernier calipers. Tumor cross-sectional noted previously that the MT2 tumor line had a single point area was calculated using the formula: length/2 ϫ width/2 ϫ␲. mutation in the ER, Asp351Tyr (9) that enhances the estrogen- Mean tumor area was plotted against time in weeks to monitor like properties of TAM (10). We, therefore, asked the question tumor growth. of whether the mutant ER was essential for the TAM-stimulated SSCP. Total RNA was prepared from tumors using the response of the tumors by an examination of Asp351Tyr in TRIzol reagent (Life Technologies, Inc.). SSCP was performed TAM-stimulated tumors and then when they had been converted using the methods originally described by Orita et al. (14, 15) to E2-stimulated tumors. but with minor modifications (16). Tumor total RNA (1 ␮g) was This unique observation of a tumoricidal effect of E2 on reverse transcribed in a 20-␮l reaction containing 50 mM Tris- two independent TAM-stimulated MCF-7 tumor lines raises the HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.5 mM each dATP, possibility that the clinical effectiveness of long-term TAM may dCTP, dGTP, and dTTP, 10 mM DTT, 3 ␮M oligo(dT)12–14, not result from indefinite treatment but from the cessation of 100 units of placental RNase inhibitor, and 200 units of Molo- therapy at the appropriate time. A woman’s own estrogen may ney murine leukemia virus reverse transcriptase. The DNA then produce a tumoricidal action on the supersensitized cells fragment containing the 351 codon was generated by PCR from after TAM is cleared from the body. Additionally, these data in 5 ␮l of reverse transcription mixture using two primers, 5Ј- the laboratory also suggest that estrogen treatment of recurrent GAGACATGAGAGCTGCCAAC-3Ј and 5Ј-GGGTGCTGGA- breast cancer after the failure of long-term TAM (on the pre- CAGAAATGTG-3Ј. Control amplification was carried out on sumed development of ER-positive, TAM-stimulated tumors) 50 pg of double-stranded DNA, coding for either a wild-type ER may result in a tumor regression that returns the breast cancer (HEGO) or a mutant ER (HETO), which contains a single cells to estrogen responsiveness. G-to-T point mutation at nucleotide 1559. The PCR cycles and conditions have been described previously (9). 32 MATERIALS AND METHODS An aliquot of P-labeled DNA was digested with XbaI The MCF-7 tumors used in these experiments were derived restriction endonuclease. After restriction digestion, an aliquot by inoculation of 1 ϫ 107 MCF-7 cells (originally obtained from of cut and uncut DNA was diluted 1:4 with 0.1% SDS, 10 mM Dr. Dean Edwards, University of Texas, San Antonio, TX) into EDTA. Half of each diluted sample was mixed 1:1 with non- estrogenized athymic mice as described previously (8, 11). denaturing loading buffer (50% glycerol, 0.05% bromphenol MCF-7, MCF-7TAM, and MT2 breast tumors were maintained blue, and 0.05% xylene cyanol), and the remaining half was as serially passaged solid tumors in ovariectomized BALB/c mixed 1:1 with denaturing loading buffer (95% formamide, 20 athymic nude mice (Harlan Sprague Dawley, Madison, WI), mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). The samples were electrophoresed on a 6% neutral polyacryl- 4–5 weeks of age, implanted with either E2 or TAM capsules (8, 12). Tumors were routinely passaged by removing a Ͼ1.0-cm- amide gel with 14–16 W constant power for 4 h. diameter tumor from an animal and mincing the remaining Statistical Analysis. Differences in mean tumor area be- viable tumor into ϳ1-mm3 pieces in a bath of cold RPMI 1640. tween groups were measured using ANOVA, followed by un- Tumor pieces were then implanted with a 13-gauge trocar into paired Student’s t test. the axillary mammary fat pads of the mice. At the time of tumor transplantation, all animals were also implanted s.c. with a RESULTS silastic capsule containing either TAM or E2, depending on the specific experiment. A MCF-7TAM tumor, which had been passaged in TAM- treated animals for 5 years and then frozen in PEG solution For tumor harvests, animals were killed by CO2 inhalation and cervical dislocation, and tumors were removed and snap- before storage in liquid nitrogen, was thawed and implanted into frozen in liquid nitrogen. Frozen tumor specimens were stored at 4–5-week-old athymic mice and treated with TAM (2-cm cap- 2 Ϫ80°C until use. sule) until the mean tumor size reached ϳ0.4-cm . At this point, Hormone Treatments. TAM was administered either the TAM capsule was removed, and the animals were random- p.o. or s.c. (via a 2-cm silastic capsule filled with TAM), ized into two groups of drug treatment. One group was im- depending on the type of experiment. TAM was administered planted with an E2 (1 cm) capsule that delivers E2 levels of 379 p.o. at doses ranging from 500 to 1500 ␮g as a suspension of pg/ml serum (13) and the other half a TAM (2 cm) capsule

10% PEG 400/Tween 80 (99.5% PEG/0.5% Tween 80) and (30–40 ng/ml). The serum levels of E2 and TAM were within 0.9% carboxymethyl cellulose (0.05 ml). The capsule produced the ranges documented in premenopausal women (17) and those blood levels of TAM of 30–40 ng/ml (12). E2 was administered taking TAM as adjuvant therapy (17, 18). After 2 weeks, the s.c. via either a 0.3- or 1-cm silastic capsule filled with E2, tumors began to regress in the E2-treated mice, and after 5 implanted s.c. into the animal’s back. The capsules produced weeks, the tumors were ϳ0.1 cm2 in size (Fig. 1a). Interest- serum levels of 83.8 and 379 pg/ml, respectively (13), and were ingly, after 6 weeks of E2 treatment, 8 of 18 tumors started to

Fig. 2 E2 response of MCF-7TAM versus MCF-7TAM2. Mean of 10 tumors/group. Two groups were bitransplanted with MCF-7TAM and

MCF-7TAM2 and treated with either E2 (1-cm capsule; premenopausal levels) or TAM (1.5 mg/day). Both MCF-7TAM and MCF-7TAM2 tumors grew in response to TAM, but only MCF-7TAM2 grew in Ͻ response to E2 (P 0.0001).

site specificity of TAM in breast and endometrial tumors (19). Two groups of 10 mice were bitransplanted with MCF-7TAM and MCF-7TAM2 (a newly TAM-stimulated MCF-7 tumor)

and treated with either E2 (1-cm capsule; premenopausal levels) or TAM (1.5 mg/day). Both MCF-7TAM and MCF-7TAM2 tumors grew in response to TAM, but only MCF-7TAM2 grew

in response to E2 (Fig. 2). Thus, early TAM-stimulated tumors can use either E2 or TAM to grow, but long-term TAM-stimulated tumors can only use TAM. However, do MCF-7TAME

tumors that regrow during E2 treatment respond to TAM treat- Fig. 1 a and b, E2-induced regression of late-passage MCF-7TAM ment? tumors (a mean of 20 tumors/group). a, MCF-7TAM tumors implanted To determine the growth characteristics of these newly into athymic mice were grown up to ϳ0.4 cm2 in size with a 2-cm TAM capsule, at which time the TAM capsule was removed and replaced with E2-responsive tumors (MCF-7TAME), experiments were performed to study the effect of E and TAM. In the first experi- a 1-cm E2 capsule in half the animals (arrow). After 2 weeks of E2 2 treatment, tumors begin to regress, and after 5 weeks, the mean tumor ment, athymic mice were implanted with MCF-7TAME tumors size was 0.1 cm2. After 8 weeks of E treatment, 6 of 18 tumors regrew. 2 and divided into the following four groups: E2 capsule (1 cm; The tumors of the TAM-treated group continued to grow, reaching a premenopausal levels); E capsule plus TAM, 0.5 mg/day p.o.; mean tumor size of 0.78 cm2 at 20 weeks. b, as above, with the addition 2 of a no-treatment group, at 6 weeks after stopping TAM treatment or E2 capsule plus TAM, 1.5 mg/day p.o.; and vehicle treated (Fig.

starting treatment with E2, the no-treatment group of tumors was sig- 3a). The TAM doses were based on studies published previously Ͻ nificantly larger than the E2-treated tumors at week 11 (P 0.0001). (6, 13). Tumor growth was observed in the E2-treated group, but Symbols, average tumor size at a specified time of treatment; bars, SE. both doses of TAM (0.5 and 1.5 mg/day) blunted E2-stimulated growth. The higher dose of TAM was more effective in blunting growth. In the second experiment, the mice were divided into

grow in the E2 group. We designated this line MCF-7TAME (a the same four groups as above, but an E2 (0.3 cm; premeno- newly estrogen-responsive MCF-7TAM tumor). pausal levels) capsule was used (Fig. 3b). Our goal was to

To ensure that E2 treatment of MCF-7TAM tumors was not establish that a lower dose of E2 could be blocked more effec- equivalent to stopping TAM alone, we repeated the first exper- tively by TAM. Tumor growth in the E2 group was similar to iment but included a no-treatment group (Fig. 1b). Six weeks that seen with the E2 (1-cm) capsule group, and tumor size 2 after stopping TAM treatment or starting treatment with E2, the reached a mean tumor size of 1.3 cm at 9 weeks. Similar treatment groups readily displayed a divergence of growth char- growth rates as the first experiment were seen in the E2 plus acteristics. The no-treatment group of tumors was significantly TAM 0.5 mg/day and E2 plus TAM 1.5 mg/day groups, and

larger than the E2-treated tumors at week 11 of the experiment once again the higher dose of TAM (1.5 mg/day) was more (week 6 of treatment; Fig. 1b). effective in suppressing E2-stimulated growth. It appears that

The observation that E2 caused tumor regression rather the MCF-7TAME tumors have evolved from exclusive TAM than tumor growth in the MCF-7TAM tumor raised the question dependence to E2 responsiveness with TAM now acting as an that the animals rather than the tumor had altered over the past antiestrogen again. 10 years (5). To exclude this possibility, we used a bitransplan- The MT2 tumor line was developed in 1993 and had been tation technique we used previously to demonstrate the target serially passaged into TAM-treated animals since that time. We

for 10 weeks (Fig. 4c). Tumor growth was observed in the TAM-treated group, and at 10 weeks the mean tumor size was 2 0.35 cm , whereas no tumor growth was observed in the E2- treated group, even at 10 weeks (0.08 cm2; P ϭ 0.0005). The difference between these two groups was statistically signifi-

cant. It was clear that E2 had gradually lost its ability to stimulate tumor growth, although TAM still maintained its

stimulatory effect. By the end of the third experiment, E2 suppressed tumor growth, a phenomenon observed previously only in the MCF-7TAM tumors.

We wanted to establish the level of E2 needed to inhibit MT2 tumors. All previous studies with MT2 tumors had used 2-cm TAM sustained-release capsules that delivered serum levels of ϳ40 ng/ml to maintain tumor growth (12). MT2 tumors were implanted into athymic mice and divided into the follow-

ing groups: TAM (2-cm capsule); E2 (0.3- and 1-cm capsule; post- and premenopausal levels, respectively); or an E2 pellet (pharmacological estrogen), and treated for 11 weeks (Fig. 5).

We reported previously the E2 levels produced by these different E2 regimens: 83, 379, and 1044 pg/ml, respectively (13). As expected, tumors grew with the TAM capsule, reaching a mean 2 tumor size of 0.6 cm at 11 weeks. However, all E2-treated groups exhibited little or no tumor growth. However, like the

MCF-7TAM tumors, some MT2 tumors regrew in the E2 1-cm capsule (premenopausal levels) group after 13 weeks of E2 treatment. The fact that only tumors from the E2 1-cm capsule group grew indicated that the phenomenon of selection was

random rather then related to the concentration of E2. Two additional experiments were conducted to evaluate the

Fig. 3 a and b, dose-response graphs of E2-stimulated MCF-7TAME hormonal sensitivity of this newly MT2E (newly estrogen- tumors to TAM and E2 (a mean of 20 tumors/group). a, MCF-7TAME responsive MT2) tumor using different doses of E2 and one dose E2-stimulated tumors were implanted into athymic mice, and mice were ϩ of TAM (0.5 mg/day). Our aim was to compare and contrast the divided into four groups: E2 1-cm capsule; E2 capsule TAM 0.5 ϩ actions of the compounds with the previously established MCF- mg/day; E2 capsule TAM 1.5 mg/day; and no treatment. Mice were treated for 8 weeks. The mean tumor sizes at 8 weeks were as follows: 7TAME tumors (Fig. 2). In the first experiment, athymic mice 2 ϩ 2 E2 capsule, 1.1 cm ;E2 capsule TAM, 0.5 mg/day, 0.6 cm ; and E2 were implanted with MT2E tumors and treated with an E 1-cm capsule ϩ TAM, 1.5 mg/day, 0.4 cm2. b, the mice were divided into the 2 capsule (premenopausal levels), an E2 1-cm capsule plus with same groups as the previous experiment, but a 0.3-cm E2 capsule was used instead of a 1-cm capsule. The mean tumor sizes at 9 weeks were TAM 0.5 mg/day, and TAM 0.5 mg/day for 6 weeks (Fig. 6a). 2 ϩ as follows: E2 0.3-cm capsule, 1.3 cm ;E2 capsule TAM 0.5 mg/day, Tumor growth was observed in the E2-treated alone group, and 0.58 cm2; and E capsule ϩ TAM 1.5 mg/day, 0.23 cm2. Symbols, mean 2 TAM 0.5 mg/day blocked E2-stimulated growth at 6 weeks, tumor size at a specified time of treatment; bars, SE. although this effect did not reach statistical significance (0.7 versus 0.45 cm2; P ϭ 0.11). TAM alone stimulated some tumor growth (0.2 cm2) at 6 weeks, demonstrating that TAM is a weak noted previously that the tumors are both E2 and TAM respon- agonist in this system. To evaluate the effect of a lower dose of sive for growth (8). However, after 4 years of serial passage in E2, athymic mice were implanted with MT2E tumors and di- TAM-treated animals, the growth response to E2 changed during vided into the same treatment groups as the previous experi- the fifth year. Three consecutive experiments illustrate the de- ment, but a 0.3-cm E2 capsule (postmenopausal levels) was used velopment of supersensitivity of the MT2 tumors to the tumori- (Fig. 6b). Tumor growth was observed in the E2-treated group, cidal effect of E2. In the first experiment, TAM and E2 both and TAM 0.5 mg/day significantly suppressed this E2-stimu- stimulated tumor growth, and no significant difference in tumor lated tumor growth but more effectively than seen with the E2 ϭ 2 ϭ size was seen between the E2- and TAM-treated groups (P 1-cm capsule (0.19 versus 0.4 cm , respectively; P 0.002).

0.97) after 13 weeks (Fig. 4a). TAM-treated MT2 tumors were TAM blocks the stimulatory effect of E2; however, higher doses retransplanted into new athymic mice and once again treated of E2 can reverse the antiestrogenic effect of TAM. The MT2 with TAM, E2, or vehicle for 11 weeks (Fig. 4b). Both E2 and tumors had evolved from pure TAM dependence to E2 respon- TAM stimulated growth; however, there was a difference in siveness, as the MCF-7TAME tumors did. However, the MT2 mean tumor size at 11 weeks between the TAM- and E2-treated tumors provided an opportunity to observe clonogenic changes 2 ϭ groups (1.02 versus 0.58 cm , respectively; P 0.08). How- in response to E2. ever, this did not reach statistical significance. In the third MT2 tumors are known to contain a mutant ER experiment, TAM-treated tumors were retransplanted into new (Asp351Tyr) in contrast to MCF-7TAM tumors, which contain athymic mice and treated with the same doses of TAM and E2 wild-type ER (9). This mutant ER has been shown to enhance

Fig. 5 Dose-response graph of MT2 tumors to TAM and E2 (a mean of 20 tumors/group). MT2 tumors were implanted into athymic mice and

divided into five treatment groups: 2-cm TAM capsule; E2 0.3-cm capsule; E2 1-cm capsule; E2 pellet; and no treatment. Mean tumor sizes for the groups at 13 weeks were as follows: TAM 2-cm capsule, 0.95 2 2 2 cm ;E2 0.3-cm capsule, 0.2 cm ;E2 1-cm capsule; 0.2 cm ; and E2 pellet, 0.2 cm2. At 19 weeks, the mean tumor sizes were as follows: 2 2 TAM 2-cm capsule, 1.35 cm ;E2 0.3-cm capsule, 0.2 cm ;E2 pellet, 0.2 2 2 cm ; and E2 1-cm capsule, 0.49 cm . Symbols, mean tumor size at a specified time of treatment; bars, SE.

the estrogenic properties of TAM (9, 10). To determine what

role this mutation played in E2 responsiveness or whether it disappeared in E2-responsive tumors, SSCP was carried out on the MCF-7TAM and MT2 tumors. Because MT2E tumors had regained sensitivity to TAM as an antiestrogen, our hypothesis was that a clone of cells grew into tumors that had lost their mutation and thus lost the TAM-stimulated phenotype. SSCP

was carried out on the newly E2-responsive MT2 tumors and compared with tumors that were still receiving TAM treatment as well as on MCF-7TAM and MCF-7TAME tumors. Tumor

samples from MT2 tumors in the E2 and TAM group produced bands that comigrated with those produced by amplification of the Asp351Tyr mutant ER cDNA (HETO; Fig. 7), demonstrating that the MT2 tumors had retained their mutated ER during clonal regrowth. On the other hand, tumor samples from MCF-

7TAM tumors in the E2 and TAM groups produced bands that comigrated exactly with those produced by amplification of the wild-type ER cDNA (HEGO). We conclude that the MT2 tumors retained their ER mutation, despite a phenotypic change in their responsiveness to TAM.

DISCUSSION We have presented data to show that breast tumors in the laboratory progress through different stages of hormonal de- Fig. 4 Serial study of the MT2 tumors (a mean of 20 tumors/group). pendency over 5 years. During the first few years of the acqui- MT2 tumors were implanted into athymic mice and divided into three sition of a TAM-stimulated phenotype, the MCF-7 ER-positive treatment groups: TAM 0.5 mg/day; E2 1-cm capsule; and no treatment. The same experiment was repeated in succession three times. a, the tumors respond to E2 and TAM equally (5). However, we 2 mean tumor size of the E2 group and the TAM group was 1.19 cm at discovered that serially transplanted tumors appear to acquire a 13 weeks. b, the mean tumor sizes of the E2- and TAM-treated groups paradoxical supersensitivity to physiological E administration were 0.59 and 1.0 cm2, respectively, at 11 weeks. c, the mean tumor 2 2 after stopping TAM (7). E2 causes a dramatic regression of sizes of the E2- and TAM-treated groups were 0.35 and 0.08 cm , respectively, at 10 weeks. Symbols, mean tumor size at a specified time tumors that is more effective than stopping TAM alone. How-

of treatment; bars, SE. ever, tumor growth can be reactivated by E2 in a proportion of

were considered the hormonal treatment of choice for advanced breast cancer in postmenopausal women (20–25). Estrogens, such as DES and ethinyl estradiol, were found to induce tumor regression (20–25), and this effect was dose dependent, with higher doses producing higher regression rates (23). Doses from 1.5 to 1500 mg/day of DES were used; therefore, the antitumor action of estrogen was pharmacological not physiological. How- ever, with the introduction of TAM, several clinical studies compared TAM to DES for the treatment of advanced breast cancer in postmenopausal women (20, 23, 24), and all found the two agents equal in efficacy. However, patients suffered more side effects with DES, and thus it was felt that TAM was superior to DES for the treatment of advanced breast cancer. Most laboratory research has focused on the mechanisms of estrogen-stimulated breast tumor growth, and surprisingly, there is little information about the mechanism of estrogen- induced tumoricidal actions. One exception is the T61 breast tumor model established by Brunner et al. (26–29). Unlike the MCF-7 tumor cell line, which was derived from a pleural effusion (30), the T61 tumor was derived from a primary breast cancer. T61 tumor growth in athymic mice is ovarian independ-

ent, and both TAM and E2 inhibit its growth (29). In these studies, tumor inhibition is dose dependent and varies with the specific doses of estrogen ranging from pharmacological to physiological (29). In our study, only physiological doses were needed to induce tumor regression or prevent tumor growth. We suggest that the repeated transplantation of our tumors in TAM- treated animals has resulted in the selection of an MCF-7 tumor that is now supersensitive to the cytotoxic effects of estrogen. It is, however, interesting to note that whatever the mechanisms, our MCF-7TAM tumors regress after ϳ2 weeks of E treatment, Fig. 6 a and b, dose-response graphs of E2-stimulated MT2E tumors to 2 TAM and E2 (a mean of 20 tumors/ group). a, MT2 E2-stimulated a time course noted by Brunner et al. (26) with the T61 tumors. tumors are implanted into athymic mice and divided into four groups: E2 However, MCF-7TAM and MT2 tumors progress through a ϩ 1-cm capsule; E2 capsule TAM 0.5 mg/day; TAM 0.5 mg/day; and no cycle of hormonal sensitivity, and the studies reported here have treatment. The mean tumor sizes at 6 weeks were: E2 1-cm capsule, 0.7 2 ϩ 2 2 not been done previously with the T61 tumor. In addition, there cm ;E2 TAM 0.5 mg/day, 0.46 cm ; and TAM 0.5 mg/day, 0.18 cm . b, the mice are divided into the same groups as in the previous exper- was no evidence that TAM could stimulate the growth of the iment, but a 0.3-cm E2 capsule was used. The mean tumor sizes at 6 T61 tumors. 2 ϩ weeks were as follows: E2 capsule, 0.7 cm ;E2 capsule TAM 0.5 It is important to point out that the mutant ER found in the 2 2 mg/day, 0.2 cm ; and TAM 0.5 mg/day, 0.19 cm . Symbols, mean tumor MT2 tumor is retained in the E -selected tumor subline MT2E size at a specified time of treatment; bars, SE. 2 (Fig. 7). Mutated ERs have been associated with increased estrogenicity of TAM (9, 10). The MT2 tumors retain the Asp351Tyr mutation, despite their phenotypic change from tumors, and TAM again acts as an antitumor agent to prevent TAM-stimulated growth (MT2) to E2-stimulated growth and E2-stimulated tumor growth. We have found that a second TAM-inhibited growth (MT2E). We must, therefore, conclude TAM-stimulated transplantable tumor, the MT2, which contains that other cellular factors are essential for the development of a mutant ER (9), is also susceptible to the tumoricidal effect of the MT2 TAM-stimulated phenotype. The fact that wild-type

E2 after 4 years of continuous TAM treatment. Similarly, some ER predominates in the MCF-7TAM-stimulated tumor suggests MT2 tumors regrew after E2-induced regression, but TAM again that a mutant receptor is only one of the many potential sup- blocked this E2-stimulated growth. We therefore conclude that porting mechanisms for drug resistance to TAM. the hormone-responsive breast cancer cell line MCF-7 can pro- An examination of the interaction of TAM and E2 illus- gress cyclically during 5 years of TAM treatment. We propose trates an important point about the therapeutic effectiveness of the following stages of hormone sensitivity: (a) TAM acts as an TAM as a competitive inhibitor of E2 action. We have reported antiestrogen by blocking tumor growth; (b) TAM stimulated the competition between E2 and TAM in vivo previously (12), tumors, which can grow with either TAM or E2;(c) TAM and it is important clinically when considering TAM therapy in exclusively stimulates tumor growth, but E2 causes a dramatic premenopausal women (17, 31). High doses of E2 can poten- regression of tumor size; and (d)E2 stimulates the regrowth of tially render the antitumor activity of TAM less effective. In the some tumors, but again TAM blocks E2-stimulated growth. We experiments evaluating TAM action in the second generation of have summarized this working model in Fig. 8. MCF-7TAME tumors, higher doses of TAM (1.5 versus 0.5

Interestingly, before the introduction of TAM, estrogens mg/day) were more effective in suppressing E2-stimulated

Fig. 7 SSCP of MCF-7TAM

and MT2 E2-stimulated recurrent and TAM-treated tumors.

TAM, TAM-treated group; E2, E2-treated group; HEGO, wild- type ER; HETO, mutant ER. Arrow, the presence of the mutant ER.

Fig. 8 Cyclical model of tumor sensitivity to TAM over a 5-year period. MCF-7 breast tumors are ER positive and re-

spond to E2, but TAM blocks this E2-stimulated growth. Af- ter 1 year of continuous TAM treatment, however, the tumors

respond to both E2 and TAM (MCF-7TAM). After 4 years of TAM treatment, the tumors are exclusively TAM dependent,

and 2 weeks of E2 treatment results in complete tumor regression. After 6–8 weeks of

E2 treatment, some tumors will regrow, and these have reverted back to the original MCF-7

phenotype, with E2 stimulating growth and TAM blocking this

E2-stimulated growth. Numbers in parentheses, references.

growth (Fig. 4, a and b). In the experiments evaluating TAM the reproducible change from TAM-stimulated growth to E2- action in MT2E tumors (Fig. 6, a and b), in which the TAM dose inhibited growth. Some breast cancer cells then revert back to

was held constant but the E2 dose changed, a 1-cm E2 capsule estrogen-stimulated growth, and TAM, once again, blocks es- (premenopausal levels) was more effective in reversing the trogen-stimulated tumor growth.

antitumor effect of TAM than a 0.3-cm E2 capsule (postmeno- Stoll (34) has described previously the regression of tumors pausal levels). This is powerful evidence that the effectiveness during high-dose estrogen therapy, but the tumor eventually of the antiestrogenic effect of TAM is dependent on the relative regrows, only to regress again when estrogen is removed. Pa-

concentration of E2 and TAM. A 1-cm E2 capsule produces E2 tients can be palliated by intermittent estrogen and withdrawal levels (379 pg/ml) equivalent to that of a premenopausal woman over many years. Our laboratory results are a variation of this

(150–350 pg/ml; Ref. 17), whereas a 0.3-cm E2 capsule pro- clinical observation, because the physiological estrogen is key to duces levels (83.8 pg/ml) equivalent to that of a postmenopausal tumor regression after TAM failure. Estrogen treatment is su- woman (13). Perhaps TAM would be more effective in a pre- perior to simply withdrawing TAM treatment (Figs. 1b and 5). menopausal woman if estrogen levels were lowered. Some Another potentially important point is the return of the resistant evidence to support this position has been obtained recently by tumor to TAM sensitivity. It is possible that patients could be comparing the action of TAM with TAM plus a luteinizing maintained on TAM, with episodic periods of estrogen treat- hormone-releasing hormone agonist to produce a medical oo- ment. However, this hypothesis can only be validated through phorectomy. The theoretically combined therapy is superior the clinical trials process.

(32), and a preliminary clinical report supports this view (33). The question of a mechanism to explain the actions of E2 We believe it is appropriate to advance a cyclic model of and TAM as antitumor agents is not a simple issue to answer. hormone dependency in breast cancer that is based on our The mechanism of TAM-stimulated tumor growth must involve experiences with transplantable MCF-7 breast tumors over the two components: a selection of cells that grow in response to the past decade (Fig. 8). The transition from estrogen- to TAM- partial agonist actions of TAM; and a second requirement for stimulated tumor growth occurs in some tumors within a year of estrogen-like angiogenesis to support tumor growth. TAM-stim- TAM treatment, but the important observation we now report is ulated tumors have an increased activation of the VEGF gene

(35), which is an important component for angiogenesis. The the ligand binding domain. Breast Cancer Res. Treat., 31: 129–138, 1994. mechanism of E2-induced tumor cell death is also unclear. Indeed, a mechanism for the antitumor action of pharmacolog- 10. Levenson, A. S., Tonetti, D. A., and Jordan, V. C. The oestrogen ical doses of estrogen has never been solved. like effect of 4-hydroxytamoxifen on induction of transforming growth factor ␣ mRNA in MDA-MB-231 breast cancer cells stably expressing The recent discovery of a second ER referred to as ER ␤ the oestrogen receptor. Br. J. Cancer, 77: 1812–1819, 1998. (36) introduces a new dimension for the understanding of es- ␤ 11. Gottardis, M. M., Robinson, S. P., and Jordan, V. C. Estradiol- trogen action. ER has a structural organization similar to ER stimulated growth of MCF-7 tumors implanted in athymic mice: a ␣, the classical ER (36), but there are important differences in model to study the tumoristatic action of tamoxifen. J. Steroid Biochem., the activating functions (37, 38) and the ligand-binding domains 30: 311–314, 1988. (39–42). Most importantly, ER ␤ and ER ␣ are able to activate 12. Iino, Y., Wolf, D. M., Langan-Fahey, S. M., Johnson, D. A., an AP-1 signal transduction pathway with TAM (43–45), which Ricchio, M., Thompson, M. E., and Jordan, V. C. Reversible control of might be able to amplify the agonist actions of TAM to induce oestradiol-stimulated growth of MCF-7 tumors by tamoxifen in the athymic mouse. Br. J. Cancer, 64: 1019–1024, 1991. TAM-stimulated growth. However, E inhibits ER ␤ AP-1 2 13. O’Regan, R. M., Cisneros, A., England, G. M., MacGregor, J. I., pathways (44, 45); therefore, it is plausible that this could be a Muenzner, H. D., Assikis, V. J., Bilimoria, M. M., Piette, M., Dragan, mechanism for the tumoricidal actions of E2. Long-term TAM- Y. P., Pitot, H. C., Chatterton, R., and Jordan, V. C. Effects of anties- stimulated tumors, such as MCF-7TAM and MT2, could contain trogens tamoxifen, toremifene, and ICI 182,780 on endometrial cancer cells selected for the ER ␤ AP-1 pathways. It is difficult to test growth. J. Natl. Cancer Inst., 90: 1552–1558, 1998. this hypothesis directly without reliable monoclonal antibodies 14. Orita, M., Suzuki, Y., Sekiya, T., and Hakashi, K. Rapid and to quantitate ER ␤, but it is possible to test the hypothesis sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics, 5: 874–879, 1989. experimentally. The pure antiestrogen ICI 182,780 stimulates 15. Orita, M., Iwahana, H., Kanasawa, H., Hayashi, K., and Sekiya, K. ␤ the ER AP-1 pathway (43–45); therefore, if the pathway is Detection of polymorphisms of human DNA by gel electrophoresis as critical for TAM-stimulated tumors after 5 years of treatment, single-stranded conformation polymorphisms. Proc. Natl. Acad. Sci. then ICI 182,780 should stimulate and not block tumor growth. USA, 86: 2766–2770, 1989. Only about half of the ER ␣-positive tumors that fail TAM 16. Bilimoria, M. M., Assikis, V. J., Muenzner, H. D., Wolf, D. M., respond to ICI 182,780 with tumor regression (46, 47). Perhaps, Satyaswaroop, P. G., and Jordan, V. C. An analysis of tamoxifen stimulated human carcinomas for mutations in the AF-2 region of the tumor growth is supported by an activated AP-1 pathway in the estrogen receptor. J. Steroid Biochem. Mol. Biol., 58: 479–488, 1996. tumors that progress. We are currently testing our hypothesis. 17. Jordan, V. C., Fritz, N. F., Langan-Fahey, S., Thompson, M., and Tormey, D. C. Alteration of endocrine parameters in premenopausal women with breast cancer during long term adjuvant therapy with REFERENCES tamoxifen as a single agent. J. Natl. Cancer Inst., 83: 1488–1491, 1991. 1. Early Breast Cancer Trialists’ Collaborative Group. Tamoxifen for 18. Langan-Fahey, S. M., Tormey, D. C., and Jordan, V. C. Tamoxifen early breast cancer: an overview of the randomized trials. Lancet, 351: metabolites in patients on long-term adjuvant therapy for breast cancer. 1451–1467, 1998. Eur. J. Cancer, 26: 883–888, 1990. 2. Fisher, B., Dignam, J., Bryant, J., DeCillis, A., Wickerjam, D. L., 19. Gottardis, M. M., Robinson, S. P., Satyaswaroop, P. G., and Jordan, Wolmark, N., Constantino, J., Redmond, C., Fisher, E. R., Bowman, V. C. Contrasting actions of tamoxifen on endometrial and breast tumor D. M., Deschenes, L., Dimitrov, N. V., Margolese, R. G., Robidoux, A., growth in the athymic mouse. Cancer Res., 48: 812–815, 1988. Shibata, H., Terz, J., Paterson, A. H. G., Feldman, M. I., Farrar, W., 20. Ingle, J. N., Ahmann, D. L., Green, S. J., Edmonson, J. H., Bisel, Evans, J., and Lickley, L. Five years versus more than 5 years of H. F., Kvols, L. K., Nichols, W. C., Creagan, E. T., Hahn, R. G., Rubin, tamoxifen therapy for breast cancer patients with negative lymph nodes and estrogen receptor positive tumors. J. Natl. Cancer Inst., 88: 1529– J., and Frytak, S. Randomized clinical trial of diethylstilbestrol versus 1542, 1996. tamoxifen in postmenopausal women with advanced breast cancer. N. Engl. J. Med., 304: 16–21, 1981. 3. Canney, P. A., Griffiths, T., Latief, T. N., and Priestman, T. J. Clinical significance of tamoxifen withdrawal response. Lancet, i: 36, 21. Kennedy, B. J., and Nathanson, I. T. Effect of intensive sex steroid 1987. hormone therapy in advanced breast cancer. J. Am. Med. Assoc., 152: 1135–1141, 1953. 4. Howell, A., Dodwell, D. J., Anderson, H., and Redford, J. Response after withdrawal of tamoxifen and progesterone in advanced breast 22. Ostrowski, M. J., and Jackson, A. W. Polyestradiol phosphate: a cancer. Ann. Oncol., 3: 611–617, 1992. preliminary evaluation of its effect on breast carcinoma. Cancer Treat. 5. Gottardis, M. M., and Jordan, V. C. Development of tamoxifen- Rep., 63: 1803–1807, 1979. stimulated growth of MCF-7 tumors in athymic mice after long term 23. Carter, A. C., Sedransk, N., Kelley, R. M., Ansfield, F. J., Ravdin, antiestrogen administration. Cancer Res., 48: 5183–5187, 1988. R. G., Talley, R. W., and Potter, N. R. Diethylstilbestrol: recommended 6. Osborne, C. K., Coronado, E. B., and Robinson, J. P. Human breast dosages for different categories of breast cancer patients. Report of the cancer in the athymic nude mouse: cytostatic effects of long-term Cooperative Breast Cancer Group. J. Am. Med. Assoc., 237: 2079– antiestrogen therapy. Eur. J. Cancer Clin. Oncol., 23: 1189–1196, 1987. 2080, 1977. 7. Wolf, D. M., and Jordan, V. C. A laboratory model to explain the 24. Beex, L., Pieters, G., Smals, A., Koenders, A., Benraad, T., and survival advantage observed in patients taking adjuvant tamoxifen ther- Kloppenborg, P. Tamoxifen versus ethinyl estradiol in the treatment of apy. In: H. J. Senn, A. Goldhirsch, and R. Gelber (eds.), Recent Results postmenopausal women with advanced breast cancer. Cancer Treat. in Cancer Research, Vol. 127, pp. 22–27. Heidelberg: Springer-Verlag, Rep., 65: 179–185, 1981. 1993. 25. Matelski, H., Greene, R., Huberman, M., Lokich, J., and Zipoli, T. 8. Wolf, D. M., and Jordan, V. C. Characterization of tamoxifen- Randomized trial of estrogen vs. tamoxifen therapy for advanced breast stimulated MCF-7 tumor variants growth in athymic mice. Breast Can- cancer. Am. J. Clin. Oncol., 8: 128–133, 1985. cer Res. Treat., 31: 11–27, 1994. 26. Brunner, N., Spang-Thomsen, M., and Cullen, K. The T61 human 9. Wolf, D. M., and Jordan, V. C. The estrogen receptor from a breast cancer xenograft: an experimental model of estrogen therapy of tamoxifen-stimulated MCF-7 tumor variant contains a point mutation in breast cancer. Breast Cancer Res. Treat., 39: 87–92, 1996.

27. Brunner, N., Spang-Thomsen, M., Vindelov, L., and Nielsen, A. 38. Hall, J. M., and McDonnell, D. P. The estrogen receptor ␤-isoform Effect of 17␤-oestradiol on growth curves and flow cytometric DNA (ER␤) of the human estrogen receptor modulates ER␣ transcriptional distribution of two human breast carcinomas grown in nude mice. Br. J. activity and is a key regulator of the cellular response to estrogens and Cancer, 47: 641–647, 1983. antiestrogens. Endocrinology, 140: 5566–5578, 1999. 28. Brunner, N., Bastert, G. B., Poulsen, H. S., Spang-Thomsen, M., 39. Brzozowski, A. M., Pike, A., and Dauter, Z. Molecular basis of Engelholm, S. A., Vindelov, L., Nielsen, A., Tommerup, N., and Elling, agonism and antagonism in the oestrogen receptor. Nature (Lond.), 389: F. Characterization of the T61 human breast carcinoma established in 753–758, 1997. nude mice. Eur. J. Cancer Clin. Oncol., 21: 833–843, 1985. 40. Pike, A. Brzozowski, A. M., Hubbard, R. E., Bonn, T., Thorsell, 29. Brunner, N., Spang-Thomsen, M., Vindelov, L., Nielsen, A., En- A., Engstrom, O., Ljunggren, J., Gustafsson, J., and Carlquist, M. ␤ gelholm, S. A., and Svenstrup, B. Dose-dependent effect of 17 estra- Structure of the ligand-binding domain of oestrogen receptor ␤ in the diol determined by growth curves and flow cytometric DNA analysis of presence of a partial agonist and a full antagonist. EMBO J., 18: a human breast carcinoma (T61) grown in nude mice. Exp. Cell Biol., 4608–4618, 1999. 53: 220–232, 1985. 41. Kuiper, G. G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., 30. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan, M. A Nilsson, S., and Gustafsson, J. A. Comparison of ligand binding spec- human cell line from a pleural effusion derived from a breast carcinoma. ificity and transcript tissue distribution of estrogen receptor ␣ and ␤. J. Natl. Cancer Inst., 51: 1409–1416, 1973. Endocrinology, 138: 863–870, 1997. 31. Sawka, C. A., Pritchard, K. I., Paterson, A. H., Sutherland, D. J., 42. Barkhem, T., Carlsson, B., Nilsson, Y., Enmark, E., Gustafsson, Thomson, D. B., Shelley, W. E., Myers, R. E., Mobbs, B. G., Malkin, J. A., and Nilsson, S. Differential response of estrogen receptor ␣ and A., and Meakin, J. W. Role and mechanism of action of tamoxifen in ␤ premenopausal women with metastatic breast carcinoma. Cancer Res., estrogen receptor to partial estrogen agonists/antagonists. Mol. Phar- 46: 3152–3156, 1986. macol., 54: 105–112, 1998. 32. Jonat, W. Luteinizing hormone-releasing hormone analogues–the 43. Paech, K., Webb, P., Kuiper, G., Nilsson, S., Gustafsson, J. A., rationale for adjuvant use in premenopausal women with early breast Kushner, P. J., and Scanlan, T. S. Differential ligand activation of ␣ ␤ cancer. Br. J. Cancer, 78: 5–8, 1998. estrogen receptors ER and ER at AP-1 sites. Science (Washington DC), 277: 1508–1510, 1997. 33. Boccardo, F., Blamey, R. W., Klijn, T., Tominaga, T., Duchateau, L., and Sylvester, R. LHRH-agonist ϩ tamoxifen versus LHRH agonist 44. Webb, P., Lopez, G. N., Uht, R. M., and Kushner, P. J. Tamoxifen alone in premenopausal women with advanced breast cancer: results of activation of the estrogen receptor/AP-1 pathway: potential origin for a meta analysis of four trials. Proc. Am. Soc. Clin. Oncol. Annu. Meet., the cell-specific estrogen-like effects of antiestrogens. Mol. Endocrinol., 18: 110, 1999. 9: 443–456, 1995. 34. Stoll, B. A. Palliation by castration or by hormone administration. 45. Webb, P., Nguyen, P., Valentine, C., Lopez, G. N., Kwok, G. R., In: B. A. Stoll (ed.), Breast Cancer Management, pp. 133–146. London: McInerney, E., Katzenellenbogen, B. S., Enmark, E., Gustafsson, J. A., William Heineman, 1977. Nilsson, S., and Kushner, P. J. The estrogen receptor enhances AP-1 35. Lee, E-S., Horiguchi, J., Liu, H., Takei, H., and Jordan, V. C. activity by two distinct mechanisms with different requirements for Regulation of angiogenesis in tamoxifen-stimulated breast tumors by the receptor transactivation functions. Mol. Endocrinol., 13: 1672–1685, pure antiestrogen ICI182,780. Proc. Am. Assoc. Cancer Res., 41: 26, 1999. 2000. 46. Howell, A., DeFriend, D. J., Robertson, J., Blamey, R., and Walton, 36. Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and P. Response to a specific antioestrogen (ICI 182780) in tamoxifen- Gustafsson, J. A. Cloning of a novel estrogen receptor expressed in rat resistant breast cancer. Lancet, 345: 989–990, 1995. prostate and ovary. Proc. Natl. Acad. Sci. USA, 93: 5925–5930, 1996. 47. Howell, A., DeFriend, D. J., Robertson, J. F., Blamey, R. W., 37. McInerney, E. M., Weis, K. E., Sun, J., Mosselman, S., and Kat- Anderson, L., Anderson, E., Sutcliffe, F. A., and Walton, P. Pharmaco- zenellenbogen, B. S. Transcription activation by the human estrogen kinetics, pharmacological and antitumor effects of the specific anties- receptor subtype ␤ (ER ␤) studied with ER ␤ and ER ␣ receptor trogen ICI 182780 in women with advanced breast cancer. Br. J. Cancer, chimeras. Endocrinology, 139: 4513–4522, 1998. 74: 300–308, 1996.

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