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Clinical Cancer Research Vol. 11, 315-322, January 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

TAS-108, a Novel Oral Steroidal Antiestrogenic Agent, Is a Pure Antagonist on Estrogen Receptor {alpha} and a Partial Agonist on Estrogen Receptor ß with Low Uterotrophic Effect

Yasuji Yamamoto1, Jiro Shibata1, Kazuhiko Yonekura1, Kouji Sato1, Akihiro Hashimoto1, Yoshimi Aoyagi1, Konstanty Wierzba1, Shingo Yano1, Tetsuji Asao1, Aman U. Buzdar2 and Tadafumi Terada1

1 Hanno Research Center, Taiho Pharmaceutical, Co. Ltd., Saitama, Japan, and2 The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Yasuji Yamamoto, Hanno Research Center, Taiho Pharmaceutical Co. Ltd., 1-27 Misugidai, Hanno, Saitama 357-8527, Japan. Phone: 81-42-972-8900; Fax: 81-42-972-0034; E-mail: yas-yamamoto{at}taiho.co.jp.


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Investigators are currently conducting phase II trials on TAS-108, a novel oral steroidal antiestrogenic agent. The purpose of this study is to investigate the molecular and pharmacologic properties of TAS-108 comparedwith other antiestrogenic agents such as tamoxifen,raloxifene, and fulvestrant.

Experimental Design: The antagonistic or agonistic activities of these agents against both estrogen receptors (ER) {alpha} and ß were compared in the reporter assay systems. Their effects on the uterus were evaluated in ovariectomized rat models. The antitumor activity of TAS-108 given p.o. was evaluated in both dimethylbenzanthracene-induced mammary tumor model and human breast cancer MCF-7 cell line xenografts.

Results: TAS-108 inhibited the transactivation of ER{alpha} under the presence of 17ß-estradiol (E2) and did not induce the transactivation of ER{alpha} in the absence of E2, unlike the agonistic activity of tamoxifen. On the other hand, it exhibited the most agonistic activity on ERß among the antiestrogenic agents tested. When given p.o. in the ovariectomized rat, TAS-108 showed a much weaker estrogenic effect on utterine weight compared to tamoxifen, or with similar levels of raloxifene, a selective estrogen receptor modulator. Also, TAS-108 strongly inhibited tumor growth in dimethylbenzanthracene-induced mammary carcinomain the rat, the endogenous E2 model, at a dosage of 1 to 3 mg/kg/day. It also inhibited high exogenous E2, inducing tumor growth against MCF-7 xenografts at a dosage of 1 mg/kg/day without any toxic manifestation.

Conclusions: Taken together, p.o. treatment with TAS-108 has a novel mode of action on ERs and inhibits E2-dependent tumor growth with little uterotrophic effect.

Key Words: antiestrogen • breast cancer • estrogen receptor • SERM


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In North America, there are 212,600 estimated new cases of breast cancer, with more than 40,000 deaths reported (1), a figure which is also increasing in many Asian and South American countries. Many chemotherapeutic and hormone therapies have been used to treat, as well as to prevent, breast cancer in the neoadjuvant and adjuvant setting. In the case of estrogen receptor (ER)–positive breast cancer, tamoxifen is often the first drug of choice for postmenopausal women (2, 3).

Tamoxifen acts not only as an antagonist but also as an ER agonist depending on the tissues involved (4, 5). This is because ER-estrogen or its other ligand complex exerts a variety of physiologic effects in different tissues (6). The ER-agonistic properties of tamoxifen induce additional clinical benefits, such as the prevention of osteoporosis and cardiac disease, by controlling rapid bone resorption and the increase of blood cholesterol in women with decreased ovarian function (4, 7). However, its ER-agonistic activity can also be associated with an increased risk for endometrial cancer(8)(9). Moreover, tamoxifen is effective in only 30% of patients with ER-positive breast cancer (10), some of whom suffer relapse (2).

Much effort has been made to overcome the clinical limitations of tamoxifen. Aromatase inhibitors, pure antiestrogens such as fulvestrant, and selective estrogen receptor modulators such as raloxifene have been developed as a result of these efforts. Raloxifene has been approved for the prevention and treatment of osteoporosis in postmenopausal women (11). It exhibits estrogen-like action on the bone tissue as an estrogen agonist but acts as anantagonist in the uterus, which enables it to become an antiosteoporosis drug without the risk of endometrial cancer (12).However, selective estrogen receptor modulators including raloxifene have thus far not been able to prove its superiority over tamoxifen in breast cancer therapy (13).

On the other hand, some aromatase inhibitors have shown improved results as the first-line of treatment for ER-positive metastatic breast cancer in an adjuvant setting (14–17), and fulvestrant was found to be as effective as anastrozole, an aromatase inhibitor, in second-line treatment for postmenopausal women with advanced breast cancer progressing on prior endocrine therapy (18).

These drugs do not have any agonistic activities on ER and therefore do not have the risk of endometrial cancer associated with tamoxifen (10, 19). At the same time, however, it is unclear whether these drugs have the clinical benefit of tamoxifen's agonistic properties, such as the prevention of osteoporosis and cardiac disease. Aromatase inhibitors cannot be used in premenopausal women without complete estrogen blockade with luteinizing hormone–releasing hormone.

Thus, there has been no agent that is superior to tamoxifen with its clinical benefits and added beneficial effects in the treatment of osteoporosis and cardiac disease. In addition, although some clinical improvements have been made with these new types of hormonal agents, there will still be patients with disease refractory to these agents. Additionally, the treatment of metastatic and advanced breast cancers demands for new types of endocrine therapies. To address these issues, we screened for an endocrine drug that has little uterotrophic effect while retaining the beneficial effects of tamoxifen. As a result, the oral steroidal antiestrogenic compound TAS-108 (SR16234; Fig. 1) was found to be effective against breast cancer with low uterotrophic effect based on its novel molecular mechanisms.



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  		Fig. 1 Chemical structure of TAS-108.

 

   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals, Cells, and Animals
TAS-108 (Fig. 1), raloxifene, and fulvestrant were synthesized in our institute. For the ER binding assay and cell proliferation assay, raloxifene, and fulvestrant were purchased from Sigma Chemicals (St. Louis, MO) and Tocris Cookson (Bristol, United Kingdom),respectively. Tamoxifen citrate salt (TAM) and 4-hydroxytamoxifen were purchased from Sigma Chemicals. 17ß-Estradiol (E2) was purchased from Wako Pure Chemicals (Osaka, Japan). 7, 12-Dimethylbenzanthracene (DMBA) was purchased from Tokyo Chemicals (Tokyo, Japan). [6,7-3H]-Estradiol [(3H)-E2; 1.92 TBq/mmol] was purchased from Amersham Biosciences (Piscataway, NJ). For xenograft study, estrogen pellets were made according to the method by Wieder and Shimkin (20). Estrogen and antiestrogens were first dissolved in DMSO to prepare stock solutions, then diluted with the buffer or culture media for thein vitro experiments. For thein vivo experiments, a suspensionof the drug in a vehicle for oral administration (0.5% hydroxypropylmethylcellulose) was prepared.

The MCF-7 cell line was obtained from American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 medium, supplemented with 10–9 mol/L E2 and 5% (v/v) fetal bovine serum. Forin vivo experiments, MCF-7 cells were maintained by serial s.c. transplantation in the subaxillary region of female athymic nude mice.

Female Sprague-Dawley rats and BALB/c (nu/nu) mice were respectively purchased from Japan SLC, Inc. (Hamamatsu, Japan) and Charles River Japan, Inc. (Yokohama, Japan). The animals were housed according to institutional guidelines in a protected environment, and maintained on a 12-hour light-dark cycle at a temperature of 20°C to 26°C. Food and water were made availablead libitum. All animal experiments were done in accordance with institutional guidelines for animal welfare.

ER Binding Assay
Recombinant ER{alpha} and ERß (Invitrogen, San Diego, CA) were mixed with 10 nmol/L [3H]-E2 and test compounds in a binding buffer [10 mmol Tris-HCl, 1 mmol EGTA, 10% glycerol, 10 mg/mL {gamma}-globulin, 0.5 mmol phenylmethylsulfonyl fluoride, 0.02 mmol leupeptin (pH 7.4)] thoroughly. Each mixture was prepared as shown below and measurement was carried out intriplicate.

Each mixture was kept for 1 hour at room temperature. One hundred microliters of a dextran-coated charcoal suspension (0.04% dextran, 0.4% activated charcoal in PBS) was added to each well. Each mixture was kept on ice for 10 minutes and the radioactivity of [3H]-E2 in the supernatant was measured with a TopCount (Perkin Elmer Inc., Wellesley, MA):

Based on the percentage of bound values, the IC50 value, which is the concentration necessary for 50% reduction of ER-specific binding of radioactive E2, was calculated. The relative binding affinity (RBA) was calculated using the following formula:

Cell Proliferation Assay
Cells were plated in 96-well multiplates containing RPMI 1640 (phenol red–free) medium supplemented with 10% (v/v) charcoal dextran–treated fetal bovine serum (HyClone, Logan, UT). After cell adhesion, the cells were treated with different concentrations of each antiestrogen for 6 days. The cells were then fixed with glutaraldehyde and stained with crystal violet (21). The following formula was used to assess the number of cells (T/C%), based on dye concentration extracted from stained cells and measured by a spectrophotometer ({lambda} = 540 nm):


The IC50 value were determined based on the experimental data derived from experiments repeated six times.

Luciferase Assay and Mammalian Two-Hybrid Assay
ER{alpha} and ERß expression vectors (HEG0, HEG19, pcDNA3ERß), VP16 fused TIF2 expression vector (VP-TIF2), and reporter constructs (EREx3-Luc, 17m8-luc) have been described previously (22–25). The ligand binding domain (LBD) regions of ERß were inserted into the pM vector (Clontech, Franklin Lakes, NJ) to generate GAL-ERß LBD.

For transfections, 293T cells were seeded in 12-well plates in phenol red–free DMEM (Invitrogen) supplemented with 10% charcoal dextran–treated fetal bovine serum andL-glutamine. At 50% to 60% confluence, cells were transfected with the following plasmids: for luciferase assays, 100 ng EREx3-Luc plasmid was cotransfected with 25 ng full-length ER expression vectors (HEG0, pcDNA3ERß) or A/B region activation function (AF-1) deleted ER{alpha} expression vector (HEG19) by LipofectAMINE 2000 (Invitrogen); for mammalian two-hybrid assays, 1 µg 17m8-luc vector was cotransfected with 250 ng GAL-ERßEF constructs in combination with 250 ng VP-TIF2 plasmids or the mock plasmid VP by Lipofectin (Invitrogen).

As a reference plasmid to normalize for transfection efficiency, 5 ng phRL-cytomegalovirus vector (Promega, Madison, WI) for luciferase assay or 2.5 ng pRL-cytomegalovirus vector (Promega) for mammalian two-hybrid assay was cotransfected in all experiments. Six hours after transfection, media were replaced with fresh medium containing 10% fetal bovine serum. At this time, E2 and antiestrogens were added and cells incubated for an additional 24 hours. Preparation of cell extracts and dual luciferase assays were done following the manufacturer's protocols (Promega). Individual transfections, each consisting of triplicate wells, were repeated at least thrice. In mammalian two-hybrid assays, specific recruitment of TIF2 to ERß was evaluated by subtracting the mean measure of the wells transfected with VP-TIF2 by that for VP.

Uterotrophic and Antiuterotrophic Assay in Ovariectomized Rats
Four-week-old female Sprague-Dawley rats wereovariectomized, and randomly assigned to treatment groups (n=7)2weeks after operation. TAS-108, tamoxifen, or raloxifenewere prepared in 0.5% hydroxypropyl methylcellulose. Oral treatment by gavage was based on a total volume of 10 mL/kg body weight. E2 was prepared in 100% ethanol as a 100-fold stock solution (0.3 mg/mL) and diluted in sesame oil with sonication on the treatment day. Subcutaneous treatment was based on a total of 100 µl/body. Animals were treated with a ligand for 3 days (26). On day 4, animals were sacrificed and uteri were removed and weighed.

DMBA Chemical-Induced Tumor Model
Mammary tumors were induced in 50 ± 1-day-old female Sprague-Dawley rats by a single p.o. dose of 20 mg DMBA in 1.0 mL of sesame oil (26, 27). Drug treatment was started when the tumors reached adiameter of about 10 mm. TAS-108 was given p.o. daily for 4 weeks.

The size of the tumors was recorded once a week, using the two perpendicular dimensions, and the tumor volume (TV) was calculated from the following formula:

The relative tumor volume (RTV) was then calculated as the ratio of the TV on day n to that on day 1, according to following formula:

Xenograft Study in Nude Mice
A 2-mm3 tumor fragmentwas implanted into the right flank of a female nude mouse. Estrogen supplementation was provided by the s.c. implantation of an E2 pellet into the left flank of the mouse. When a tumor grew to the size of a diameter of about 6 mm, mice were allocated to the experimental groups and given TAS-108 p.o. daily for 3 weeks.

The size of the tumors was recorded on the day following the last administration (day 22), using the two perpendicular dimensions, the TV was calculated from the following formula:

The RTV was then calculated as the ratio of the TV on day 22 to that of day 1, according to following formula:


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Binding Affinity of TAS-108 to ER{alpha} and ERß
To estimate the binding affinity of TAS-108 to ERs in comparison with other known antiestrogenic agents, competition assay using radiolabeled E2 was carried out. RBA was calculated from the ratio between the concentrations of test compounds and nonradioactive E2 necessary for 50% reduction of ER-specific binding. TAS-108 exhibited high binding affinity to both ER{alpha} and ERß, and the RBAs were 80% and 98%, respectively, which were much higher than tamoxifen and were similar to that of 4-hydroxytamoxifen, an active metabolite of tamoxifen, and fulvestrant (Table 1). On the other hand, the binding affinity of raloxifene against ER{alpha} showed similar levels to TAS-108, but its binding affinity against ERß was almost 10 times weaker than ER{alpha}.


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  		Table 1 Binding affinity of TAS-108 and various antiestrogens to ERs

 
In vitro Anti-Proliferating Activity of TAS-108 Against ER-Positive MCF-7 Cell Line
The antiproliferative activity of TAS-108 on ER-positive/estrogen-responsive MCF-7 cells, growing in the presence of E2, was determined and compared withantiestrogens. TAS-108 inhibited the estrogen-dependent cell growth of MCF-7 with an IC50 value of 34 nmol/L (Fig. 2). Tamoxifen weakly inhibited the proliferation of MCF-7 cells at an IC50 value of 1.5 µM. Fulvestrant, raloxifene, and 4-hydroxytamoxifen exhibited equally strong growth inhibitory activities with IC50 values of 3.6, 4.2, and 5.5 nmol/L, respectively (Fig. 2).



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  		Fig. 2 TAS-108 suppresses the estrogen-dependent growth of a breast cancer cell line. The effect of TAS-108 on MCF-7 cell proliferation was studied using increasing concentrations of tamoxifen [TAM ({blacksquare})], 4-hydroxytamoxifen [OHT ({square})], raloxifene [RAL ({blacklozenge})], fulvestrant [FUL ({blacktriangleup})], and TAS-108 ({circ}) in the presence of 1 nmol/L E2. Mean ± SD of six separate experiments, expressed as the percentage of absorption with respect to the nontreatment control group.

 
Agonistic/Antagonistic Activity of TAS-108 on ER{alpha} and ERß in the Reporter Assay System
The antagonistic and agonistic activities of TAS-108 on ER{alpha} and ERß were compared with other known antiestrogenic agents. First, to compare the antagonistic activities, ligand-induced transactivation of ER{alpha} and ERß was examined using a transient expression assay. An ERE reporter plasmid (EREx3-Luc) and an ER{alpha} or ERß expression vector were cotransfected into 293T cells, and then cells were incubated in the presence of E2 with or without agents. As shown in Fig. 3A, TAS-108 inhibited the transactivation of ER{alpha} stronger than 4-hydroxytamoxifen, such as fulvestrant, a pure antiestrogen, and raloxifene, a selective estrogen receptor modulator. Interestingly, the antagonistic potencies of these agents against ERß were more varied (Fig. 3B). Fulvestrant effectively antagonized ERß similar to ER{alpha}. Although TAS-108 and raloxifene inhibited transactivation of ERß, when compared with the case of ER{alpha}, its antagonistic activities were much weaker. On the other hand, OHT inhibited the transactivation ofERß more prominent than that of ER{alpha}.



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  		Fig. 3 TAS-108 and raloxifene exhibits weaker antagonistic activity on ERß than ER{alpha}. Dose responses of antiestrogens [4-hydroxytamoxifen (OHT), TAS-108, fulvestrant (FUL), raloxifene (RAL)] on the transcriptional activity of ER{alpha} (A) and ERß (B) in the presence of 1 nmol/L E2 were studied using the EREx3-Luc reporter plasmid and the expression plasmid for ERs in 293T cells. Points show the percentages of transcriptional activity in the presence of E2 alone (set at 100%). The basal untreated level of ERs is also shown. Results represent the average of at least three independent experiments, error bars indicate ± SD.

 
Next, we compared the agonistic activity of these agents against ERs. Only 4-hydroxytamoxifen activated transcription of ER{alpha} (Fig. 4A); however, other agents such as TAS-108, did not exhibit transactivation of ER{alpha}. In the ER{alpha} mutant deleted AF-1 domain, 4-hydroxytamoxifen showed no agonistic activity. We previously reported that the agonistic activity of tamoxifen came from AF-1 activation (25). Strikingly, in contrast with ER{alpha}, TAS-108 exhibited an agonistic activity against the transactivation of ERß similar to 4-hydroxytamoxifen (Fig. 4B). Fulvestrant and raloxifene did not exhibit agonistic activity against ERß similar to ER{alpha}.



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  		Fig. 4 TAS-108 induces ERß-mediated transactivation without transactivation on ER{alpha}. 293T cells were transfected plasmids for transient reporter assays and incubated for 24 hours in the presence (+) or absence (–) of E2 (10 nmol/L), 4-hydroxytamoxifen (OHT, 100 nmol/L), TAS-108 (100 nmol/L), fulvestrant (FUL, 100 nmol/L) or raloxifene (RAL, 100 nmol/L).A, EREx3-Luc reporter and full-length ER{alpha} (ER{alpha}) or AF-1 domain deleted ER{alpha} mutant (ER{alpha}{Delta}AF-1) expression plasmids were cotransfected and luciferase activities were evaluated in the presence or absence of E2 or antagonists, respectively. Mean ± SD of three separate experiments expressed as the fold response over basal levels of ER{alpha} or ER{alpha}{Delta}AF-1 without E2 or antagonists, which was arbitrarily set at 1.B, EREx3-Luc reporter and full-length ERß (ERß) expression plasmid were cotransfected and luciferase activities were evaluated in the presence or absence of E2 or antagonists, respectively. Mean ± SD of three separate experiments expressed as the fold response over basal levels of ERß without E2 or antagonists, which was arbitrarily set at 1.C, binding between ERß and TIF2 was examined using the mammalian two-hybrid system. 17m8-Luc reporter, GAL4-fused ERß-LBD (GAL-ERß-LBD) and VP16-fused COOH-terminal region of TIF2 including the NR interaction domains (VP-TIF2) expression plasmids or empty VP16 vector (VP) were cotransfected and luciferase activities were evaluated in the presence or absence of antagonists, respectively. Results represent the average of at least three independent experiments. The specific recruitment of TIF2 to ERß was evaluated by subtracting the mean measure of the wells transfected with VP-TIF2 by that for VP. Bars, fold-change of subtracted value in the absence of ligand.

 
It has been well established that ERs exhibit two types of transactivation, AF-1 and AF-2, and they are characterized by several coregulator binding ERs (28, 29). To clarify which type of transactivation or which co-activator affects the agonistic activity of TAS-108 against ERß, we examined the transactivation activity of ERß by TAS-108 using mammalian two-hybrid assay between GAL4-fused ERß-LBD and VP16-fused TIF2, a major co-activator in AF-2.Figure 4C clearly showed that TAS-108 induced the strongest ERß transactivation among the antiestrogenic agents, which means that TAS-108 induces the AF-2 transactivation of ERß by recruiting coactivator TIF2 to ERß. Onthe other hand, 4-hydroxytamoxifen induced only weak transactivation in this system in spite of exhibiting agonistic activity against full-length ERß. Its agonistic activity may depend on AF-1 as we have previously shown in the case of ER{alpha} (25). The partial agonistic property of TAS-108 to ERß, with the addition ofa pure antagonistic feature against ER{alpha}, is the unique mode ofaction of ERs that have never been observed in other antiestrogenic agents.

Agonistic/Antagonistic Activity Of TAS-108 as Determined in the Model of Ovariectomized Rat Uterus
To test theuterotrophic and antiuterotrophic activity of TAS-108, the uterine response in wet weight of ovariectomized rats was tested in the presence and absence of E2, respectively.

Tamoxifen alone exhibited estrogenic activity on uterine weights in ovariectomized rats under the non-E2 supplement condition (Fig. 5A), and a little antisetrogenic activity under the E2supplement condition (Fig. 5B). Raloxifene exhibited little estrogenic activity in the absence of E2 (Fig. 5A), and exhibited antiestrogenic activity in the presence of E2 (Fig. 5B). Like raloxifene, TAS-108 significantly antagonized the E2 action on uterus with little agonistic activity (Fig. 5A andB). TAS-108 seemed more potent as an antagonist in the uterine response than raloxifene in the absence of E2. Thus, unlike tamoxifen, TAS-108 acted as a potent antagonist in the uterus and had low risk for uterotrophy similar to raloxifene.



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  		Fig. 5 TAS-108 exhibits little uterotrophic activity in ovariectomized rats. The effect of TAS-108 on uteri of ovariectomized rats was studied using various concentrations of tamoxifen [TAM ({bullet})], raloxifene [RAL ({triangleup})] and TAS-108 ({blacksquare}), in the absence (A) and presence (B) of E2. Uterine wet weights are shown as the mean ± SD for seven rats per group. The ranges of uterine wet weight in E2-treated ({04-1320fx01}) and nontreated ({04-1320fx02}) animals are indicated.

 
In vivo Antitumor Activity in the DMBA-Induced Rat Mammary Tumor Model and in the MCF-7 Human Mammary Tumor Xenograft
To determine the antitumor activity, TAS-108 was given p.o. in the DMBA-induced rat mammary tumor model. This model does not require exogenous E2 and tumor growth depends on endogenous estrogen. This was confirmed in ovariectomized rats. In ovariectomized rats, the growth of DMBA-induced mammary tumor was completely suppressed by depletion of endogenously produced estrogen (Fig. 6).



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  		Fig. 6 TAS-108 suppresses tumor growth on the DMBA-induced rat tumor model. The effect of TAS-108 in the DMBA-induced rat mammary tumor model was studied using the indicated concentrations of TAS-108. Tumor volume was measured once a week. Relative tumor volumes against TV on the first dosing day are shown as the mean ± SD.*,P < 0.05 versus control;**,P < 0.01 versus control (Dunnett'st test).

 
TAS-108 given p.o. also markedly inhibited tumor growth in a dose-dependent manner at the dose range from 0.3 to 30 mg/kg/day (Fig. 6). There was no obvious toxic manifestation (data not shown). It is noteworthy that this dose range was similar to that used in the uterotrophic assay in Fig. 5. This means that TAS-108 inhibits E2-dependent tumor growth within the dose range exhibiting little uterotrophic activity. Next, the antitumor effect of TAS-108 was studied in the MCF-7 human breast cancer xenografts models, high exogenous E2 supplement model. TAS-108 also exhibited significant tumor growth inhibition from thedose of 1 mg/kg/day in a dose-dependent manner (Fig. 7A). The antitumor activity of TAS-108 was similar or potentially higher than tamoxifen, both compounds did not affect the body weight of nude mice (Fig. 7B). Thus, TAS-108 efficiently inhibited E2-dependent tumor growth under both low endogenous and high exogenous E2 levels without risk of uterotrophy.



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  		Fig. 7 TAS-108 suppresses in vivo tumor growth on the mouse xenograft model. The effect of TAS-108 in the nude mouse MCF-7 xenograft was studied using the indicated increasing concentrations of TAS-108. A, TVs were measured on the next day of the last administration day (day 22). Bars, T/C% mean ± SD.**,P < 0.01 versus control (Dunnett's t test). B, body weights were measured twice a week. Results, mean ± SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tamoxifen has played a major role in the hormonal therapies for both early and advanced ER-positive breast cancers (1, 2). However, the drawback of tamoxifen therapy is that it is associated with an increased risk of developing endometrial cancer, cataracts, and thromboembolic events based on its agonistic activity on ERs (8, 9). Some estrogen depletion strategies such as aromatase inhibitors or pure antiestrogens have overcome these drawbacks and exhibited some improvement for metastatic and advanced breast cancer therapy (16–18). However, these drugs have no agonistic activity on ERs and lack the clinical benefits of tamoxifen's agonistic properties, such as the prevention of osteoporosis and cardiac disease. On the other hand, raloxifene has gained approval for the prevention and treatment of osteoporosis, but it has not exhibited superiority over tamoxifen in advanced cancer therapy (13). Thus, there has been no anticancer agent which is superior to the clinical benefits of tamoxifen while continuing to provide favorable effects in the treatment of osteoporosis and cardiac disease treatment. Here we have shown that TAS-108 given p.o., with its novel mode of action on ERs, exhibited antitumor activities against E2-dependent tumor growth of MCF-7 xenografts and DMBA-induced rat mammary carcinoma models with little uterotrophic effect.

TAS-108 had high binding affinity to ER{alpha} (Table 1) and inhibited its transactivation without any agonistic properties (Figs. 3 and 4), which is in striking contrast to the agonistic property of tamoxifen (Fig. 4A). This is because tamoxifen can inhibit AF-2situatedin COOH-terminal E domain of ER{alpha} but induces AF-1 activation, situated in NH2-terminal A/B domain, whereas TAS-108 inhibits both AF-1 and AF-2 transactivation by promoting the recruitment of the SMRT co-repressor as we have previously reported (25). The transactivation of ER{alpha} AF-1 by tamoxifen is thought to be the mechanism for the agonistic property of tamoxifen leading to its resistance (28, 30, 31), and therisk of endometrial cancers in patients with breast cancer (8, 28, 30). TAS-108 is expected to overcome at least some type of tamoxifen resistance without the risk of endometrial cancer (25).

In fact, TAS-108 given p.o. showed little uterotrophic effect on ovariectonized rats under the non-E2 supplement condition at the dose level exhibiting significant tumor growth inhibition (Figs. 5 and 6), and it exhibited more potent antagonistic activity on rat uterus than that of tamoxifen under the E2 supplement condition (Fig. 5B). Recently, this beneficial property in the uterus of TAS-108 has been examined in phase I clinical trials (32).

The action on ERß of TAS-108 was another unique characteristic among antiestrogen agents. As shown in Fig. 4B and C, it induced transactivation of ERß such as tamoxifen and dramatically induced the AF-2 transactivation of ERß much more than any other antiestrogenic agent. This clearly indicates that TAS-108 acts as a partial agonist of ERß through the recruitment of TIF2 coactivator in contrast to its pure antagonistic action on ER{alpha}. The biological function of ERß is still not well understood and controversial (33, 34), and we have not had enough data to link the agonistic action of TAS-108 and ERß to its beneficial effect. However, reports have indicated that ERß is expressed in many tissue organs and has an important biological function in several tissues such as bones, lungs, or others (35–37). Also, there have been reports that ERß acts as an inhibitor of ER{alpha} function (38), and the amount of ERß expression is reported to be negatively correlated with the malignancy of breast cancer (33, 39). For example, genistein, an ERß-selective agonist, is reported to prevent osteoporosis (40). Therefore, we expect that the prominent agonistic property TAS-108 on ERß may beneficially affect some organs such as bone or cardiovascular systems similar to the agonistic property of tamoxifen on ER{alpha}. We are now examining the effects of TAS-108 on bone metabolisms. Preliminary data shows that TAS-108 prevents osteoporosis in ovariectomized rats or mice (data not shown).

The characteristic summaries of the unique mode of action of TAS-108, when compared with other known antiestrogenic agents are follows: in comparison to tamoxifen, TAS-108 is a pure antagonist on ER{alpha} unlike tamoxifen (25). On ERß, it acts as a partial agonist similar to tamoxifen but both modes of action are different. Tamoxifen induces no AF-2 transactivation of ERß, whereas TAS-108 markedly induces AF-2 transactivation by promoting the recruitment of TIF-2, a co-activator (Fig. 4). In the comparison to raloxifene, a selective estrogen receptor modulator, although raloxifene is a pure antagonist of ER{alpha}, it acts as an agonist on D351Y mutant ER{alpha} derived from a tamoxifen-resistant breast cancer cell line. TAS-108 acts as an ER{alpha} antagonist even in this mutant (25). The antagonistic activity of raloxifene on ERß is much weaker than that on ER{alpha} (Fig. 3A and B). This is because its binding affinity to ERß is 10times weaker than that of ER{alpha} (Table 1; ref. 41). On the other hand, TAS-108 binds both ERs with the same affinity (Table 1). In comparison, fulvestrant acts asa pure antiestrogen on both ER{alpha} and ERß via down-regulation of ERs and binding inhibition of ERs to DNA (42). This is completely different from the mode of action of TAS-108 which modulates the recruitment of coregulators to ERs and exhibits agonistic properties on ERß in the absence of E2 (25).

Finally, TAS-108 given p.o. significantly inhibited tumor growth against DMBA-induced rat mammary carcinoma, physiologic endogenous E2 models, at a dosage of 1 to 3 mg/kg/day (Fig. 6). This dosing level is similar to that used in the uterotrophic test of Fig. 5. TAS-108 also inhibited E2-dependent tumor growth against MCF-7 xenograft, exogenous high E2 supplement models, at a dosage of 1 mg/kg/day. This indicates that TAS-108 can inhibit tumor growth even under high E2 concentrations in the blood at approximately the same dosage level range inhibiting tumor growth in low E2 concentrations (43–46). These will also be important results when its clinical usage is considered because tamoxifen is currently one of the few choices for hormonal therapy in premenopausal women with advanced breast cancer. Aromatase inhibitors cannot be used in premenopausal women without complete estrogen blockade with luteinizing hormone–releasing hormone. If TAS-108 can inhibit ER{alpha} transactivation in patients with premenopausal breast cancer in high E2 concentration in the blood, it would provide patients another choice for treatment with low risk for endometrial cancer.

In summary, TAS-108 has a novel characteristic for ER modulation, a pure antagonist of ER{alpha} and a partial agonist of ERß exhibiting strong antitumor effects with little uterotrophic effects. It will be expected that TAS-108 becomes an ideal endocrine therapy which has efficacy for advanced breast cancer, safety for uteri, and agonistic benefits for osteoporosis and cardiac disease via its novel mode of action on ERs. We are now developing phase II clinical trials for advanced postmenopausal and premenopausal breast cancer.


   ACKNOWLEDGMENTS
 
We thank Drs. Shigeaki Kato of the University of Tokyo for kindly providing reporter assay systems and helpful technical advice, and Masato Tanabe of SRI International for stimulating suggestions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/ 6/04; revised 9/ 9/04; accepted 10/18/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Jemal A, Murray T, Samuels A, et al. Cancer statistics, 2003.CA Cancer J Clin2003;53:5–26.[Abstract/Free Full Text]
  2. Jaiyesimi IA, Buzdar AU, Decker DA, Hortobagyi GN. Use of tamoxifen for breast cancer: twenty-eight years later.J Clin Oncol1995;13:513–29.[Abstract/Free Full Text]
  3. Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists' Collaborative Group. Lancet 1998;351:1451–67.[CrossRef][Medline]
  4. McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW. Analysis of estrogen receptor functionin vitro reveals three distinct classes of antiestrogens.Mol Endocrinol1995;9:659–69.[Abstract]
  5. Love RR, Mazess RB, Barden HS, et al. Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer.N Engl J Med1992;326:852–6.[Abstract]
  6. Kauffman RF, Bryant HU. Selective estrogen receptor modulators.Drug News Perspect1995;8:531–9.
  7. Frolik CA, Bryant HU, Black EC, Magee DE, Chandrasekhar S. Time-dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and alendronate.Bone1996;18:621–7.[Medline]
  8. Kedar RP, Bourne TH, Powles TJ, et al. Effects of tamoxifen on uterus and ovaries of postmenopausal women in a randomised breast cancer prevention trial.Lancet1994;343:1318–21.[CrossRef][Medline]
  9. Fisher B, Costantino JP, Redmond CK, et al. Endometrial cancer in tamoxifen-treated breast cancer patients: findings from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14.J Natl Cancer Inst1994;86:527–37.[Abstract/Free Full Text]
  10. Baum M. Tamoxifen and the breast.Eur J Cancer1998;34:S7–8.
  11. Heringa M. Review on raloxifene: profile of a selective estrogen receptor modulator.Int J Clin Pharmacol Ther2003;41:331–45.[Medline]
  12. Black LJ, Sato M, Rowley ER, et al. Raloxifene (LY139481 HCI) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats.J Clin Invest1994;93:63–9.
  13. Buzdar AU, Marcus C, Holmes F, Hug V, Hortobagyi G. Phase II evaluation of Ly156758 in metastatic breast cancer.Oncology1988;45:344–5.[Medline]
  14. Gradishar WJ, Jordan VC. Clinical potential of new antiestrogens.J Clin Oncol1997;15:840–52.[Abstract/Free Full Text]
  15. Barker S. Anti-estrogens in the treatment of breast cancer: current status and future directions.Curr Opin Investig Drugs2003;4:652–7.[Medline]
  16. Nabholtz JM, Bonneterre J, Buzdar A, Robertson JF, Thurlimann B. Anastrozole (Arimidex) versus tamoxifen as first-line therapy for advanced breast cancer in postmenopausal women: survival analysis and updated safety results.Eur J Cancer2003;39:1684–9.
  17. Mouridsen H, Gershanovich M, Sun Y, et al. Phase III study of letrozole versus tamoxifen as first-line therapy of advanced breast cancer in postmenopausal women: analysis of survival and update of efficacy from the International Letrozole Breast Cancer Group.J Clin Oncol2003;21:2101–9.[Abstract/Free Full Text]
  18. Bross PF, Baird A, Chen G, et al. Fulvestrant in postmenopausal women with advanced breast cancer. Clin Cancer Res 2003;9:4309–17.[Abstract/Free Full Text]
  19. Meegan MJ, Lloyd DG. Advances in the science of estrogen receptor modulation.Curr Med Chem2003;10:181–210.[Medline]
  20. Wieder R, Shimkin M. An improved method of producing hormone-cholesterol pellets.J Natl Cancer Inst1964;32:957–8.
  21. Saotome K, Morita H, Umeda M. Cytotoxicity test with simplified crystal violet staining method using microtitre plates and its application to injection drugs.Toxicol In Vitro1989;3:317–21.
  22. Tora L, Mullick A, Metzger D, et al. The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties.EMBO J1989;8:1981–6.[Medline]
  23. Watanabe M, Yanagisawa J, Kitagawa H, et al. A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor a coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA.EMBO J2001;20:1–12.[CrossRef][Medline]
  24. Yanagisawa J, Yanagi Y, Masuhiro Y, et al. Convergence of transforming growth factor-ß and vitamin D signaling pathways on SMAD transcriptional coactivators.Science1999;283:1317–21.[Abstract/Free Full Text]
  25. Yamamoto Y, Wada O, Takada I, et al. Both N- and C-terminal transactivation functions of DNA-bound ER{alpha} are blocked by a novel synthetic estrogen ligand. Biochem Biophys Res Commun 2003;312:656–62.[CrossRef][Medline]
  26. Toko T, Shibata J, Sugimoto Y, et al. Comparative pharmacodynamic analysis of TAT-59 and tamoxifen in rats bearing DMBA-induced mammary carcinoma.Cancer Chemother Pharmacol1995;37:7–13.[Medline]
  27. Huggins C, Grand LC, Brillantes FP. Mammary cancer induced by a single feeding of polymucular hydrocarbons, and its suppression.Nature1961;189:204–7.[CrossRef][Medline]
  28. Kato S. Estrogen receptor-mediated cross-talk with growth factor signaling pathways.Breast Cancer2001;8:3–9.[Medline]
  29. Warnmark A, Treuter E, Wright AP, Gustafsson JA. Activation functions 1 and 2 of nuclear receptors: molecular strategies for transcriptional activation.Mol Endocrinol2003;17:1901–9.[Abstract/Free Full Text]
  30. Furr BJ, Jordan VC. The pharmacology and clinical uses of tamoxifen.Pharmacol Ther1984;25:127–205.[CrossRef][Medline]
  31. Yamamoto Y, Wada O, Suzawa M, et al. The tamoxifen-responsive estrogen receptor {alpha} mutant D351Y shows reduced tamoxifen-dependentinteraction with corepressor complexes. J Biol Chem 2001;276:42684–91.[Abstract/Free Full Text]
  32. Blakely LJ, Buzdar A, Chang HY, et al. A phase I and pharmacokinetic study of TAS-108 in postmenopausal female patients with locally advanced, locally recurrent inoperable, or progressive metastatic breast cancer. Clin Cancer Res 2004;10:5425–31.[Abstract/Free Full Text]
  33. Speirs V, Carder PJ, Lane S, et al. Oestrogen receptor ß: what it means for patients with breast cancer.Lancet Oncol2004;5:174–81.[CrossRef][Medline]
  34. Weihua Z, Andersson S, Cheng G, et al. Update on estrogen signaling.FEBS Lett2003;546:17–24.[CrossRef][Medline]
  35. Gustafsson JA. Estrogen receptor ß—a new dimension in estrogen mechanism of action.J Endocrinol1999;163:379–83.[CrossRef][Medline]
  36. Lindberg MK, Weihua Z, Andersson N, et al. Estrogen receptor specificity for the effects of estrogen in ovariectomized mice.J Endocrinol2002;174:167–78.[Abstract]
  37. Seidlova-Wuttke D, Becker T, Christoffel V, Jarry H, Wuttke W. Silymarin is a selective estrogen receptor beta (ERß) agonist and has estrogenic effects in the metaphysis of the femur but no or antiestrogenic effects in the uterus of ovariectomized (ovx) rats.J Steroid Biochem Mol Biol2003;86:179–88.[CrossRef][Medline]
  38. Lindberg MK, Moverare S, Skrtic S, et al. Estrogen receptor (ER)-ß reduces ER{alpha}-regulated gene transcription, supporting a "ying yang" relationship between ER{alpha} and ERß in mice.Mol Endocrinol2003;17:203–8.[Abstract/Free Full Text]
  39. Iwao K, Miyoshi Y, Egawa C, Ikeda N, Noguchi S. Quantitative analysis of estrogen receptor-ß mRNA and its variants in human breast cancers.Int J Cancer2000;88:733–6.[CrossRef][Medline]
  40. Albertazzi P. Purified phytoestrogens in postmenopausal bone health: is there a role for genistein? Climacteric2002;5:190–6.[Medline]
  41. Barkhem T, Carlsson B, Nilsson Y, et al. Differential response of estrogen receptor {alpha} and estrogen receptor ß to partial estrogen agonists/antagonists.Mol Pharmacol1998;54:105–12.[Abstract/Free Full Text]
  42. Jones SE. Fulvestrant: an estrogen receptor antagonist that downregulates the estrogen receptor.Semin Oncol2003;30:14–20.
  43. Sturgeon SR, Potischman N, Malone KE, et al. Serum levels of sex hormones and breast cancer risk in premenopausal women: a case-control study (USA).Cancer Causes Control2004;15:45–53.[CrossRef][Medline]
  44. Endogenous Hormones and Breast Cancer Collaborative Group. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies.J Natl Cancer Inst2002;94:606–16.[Abstract/Free Full Text]
  45. Shafie SM, Grantham FH. Role of hormones in the growth and regression of human breast cancer cells (MCF-7) transplanted into athymic nude mice.J Natl Cancer Inst1981;67:51–6.
  46. Finkelman RD, Bell NH, Strong DD, Demers LM, Baylink DJ. Ovariectomy selectively reduces the concentration of transforming growth factor beta in rat bone: implications for estrogen deficiency-associated bone loss.Proc Natl Acad Sci U S A1992;89:12190–3.[Abstract/Free Full Text]



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