ICI 164,384, a Pure Antagonist of Estrogen-Stimulated MCF-7 Cell Proliferation and Invasiveness1

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[CANCER RESEARCH 49. 6929-6934, December 15. I989| ICI 164,384, a Pure Antagonist of Estrogen-stimulated MCF-7 Cell Proliferation and Invasiveness1

Erik W. Thompson,2 Deborah Katz, Thomas B. Shima, Alan E. Wakeling, Marc E. Lippman, and Robert B. Dickson

Laboratory of Developmental Biology and Anomalies, NI DR, NI H, Belhesda, Maryland 20892 [E. W. T., T. B. S.]; Vincent T. Lombardi Cancer Center, Georgetown University Medical Center, 3800 Reservoir Rd., NW Washington, DC 20007 Â¡D. K., M. E. L., R. B. D.J; and ICI Pharmaceuticals. Alderly Park, Macclesfield, Cheshire, England [A. E. W.I

ABSTRACT ment and testing of new antiestrogens with the objective of achieving drugs with pure antagonist properties. Some success Estrogen is known to stimulate the proliferation and basement mem was seen with the benzothiophene antiestrogens, particularly brane Â¡nvasivenessofthe MCF-7 human breast cancer cell line. We have compared the new steroidal antiestrogen ICI 164,384, the triphenyleth- LY 117018, which exhibits similar activities to OHT in block ylene 4-hydroxytamoxifen (OHT), and the benzothiophene LY 117018, ing estrogen-induced proliferation of MCF-7 cells (22), and for their effects on the proliferation and invasiveness of the MCF-7 cell show less partial agonism of estrogen effects on MCF-7 cells line and its antiestrogen-resistant variant LY-2. While all three anties- (4, 23), or on progesterone receptor expression in, or growth trogens blocked the proliferative effects of 17/J-estradiol on MCF-7 cells, of, the immature rat uterus (24). Although this molecule pro OHT and LY 117018, but not ICI 164,384 stimulated their proliferation vides a good tool for the study of estrogen action in vitro, its in the absence of estrogen. The proliferative effects of OHT and LY use in the clinic is severely limited by poor pharmacokinetics 117018 were blocked by ICI 164,384. Basement membrane Â¡nvasiveness (25, 26). ICI 164,384 is a new steroidal antiestrogen which of MCF-7 cells was stimulated by 17/3-estradiol and OHT, but not LY exhibits no estrogenic effect on uterine growth in immature rats 117018 or ICI 164,384. Both ICI 164,384 and LY 117018 were able to and mice, and reverses estrogen-induced proliferation of cul block the invasiveness induced by either 17/9-estradiol or OHT. The LY- tured MCF-7 cells (27-29). It binds to partially purified estro 2 antiestrogen-resistant variant of the MCF-7 cell line showed increased basal proliferation, and responded only slightly to estrogen. ICI 164,384, gen receptor, but unlike estrogen, does not stimulate the affinity but not OHT or LY 117018 antagonized the effects of 17/3-estradiol, but of the receptor complex for DNA cellulose (30). did not reduce proliferation below control levels. The LY-2 line was not Recently, we have demonstrated that invasion of a reconsti resistant to the antiestrogenic effects of LY 117018 or ICI 164,384 on tuted basement membrane by MCF-7 cells in vitro is estrogen invasiveness, and was stimulated by LY 117018 for this parameter. Thus, stimulated (31), and that this activity is also stimulated by ICI 164,384 is a pure antiestrogen for MCF-7 cell proliferation and tamoxifen and OHT, presumably due to estrogen agonist prop invasiveness, and may offer clinical advantage over nonsteroidal anties- erties (32). Basement membranes surround epithelial tissues, trogens which can stimulate these activities in tumor models in vitro. nerves, and muscles, providing physical support to these tissues and segregating parenchymal and mesenchymal elements (33). INTRODUCTION Their major components, collagen IV, laminin, and heparan sulfate proteoglycan are common to all known basement mem Appreciation of the mechanism of action of estrogen in branes (34). The interaction of tumor cells with basement cellular systems, and of its importance in human breast cancer membranes is a critical step in metastasis, since the cells en growth, has led to the development and clinical application of counter and pass numerous basement membranes as they dis antiestrogens. Nonsteroidal antiestrogens like nafoxidine and seminate (35). Basement membrane-invasion initiates with tu clomifene have been studied in clinical trials, and tamoxifen, a mor cell attachment to laminin (36), which induces a protease substituted triphenylethylene, has been used successfully in the cascade terminating with active collagenase IV, specific for clinical management of advanced breast cancer for many years basement membrane collagen degradation (37, 38, 44). The (1, 2). Although clearly of clinical value, tamoxifen and its importance of these events to tumor cell metastasis has been active metabolite OHT' show evidence of partial estrogen agon- demonstrated with the dramatic reduction in lung colonization ism both in vivo and in vitro. They stimulate uterine growth in by metastatic melanoma cells injected into the nude mouse in ovariectomized rats and mice (3-5), and stimulate progesterone the presence of peptides which block the attachment of cells to receptor expression (6-10), morphological changes (11), and basement membranes (39) or in the presence of inhibitors of elevation of estrogen-dependent mRNA species (12-14) in cul collagenase type IV (40). tured estrogen-dependent human MCF-7 breast carcinoma In this study, we compared the effects of OHT, LY 117018 cells. Proliferation of MCF-7 cells in vitro is also stimulated by and ICI 164,384 on the basement membrane invasiveness and tamoxifen and OHT (15, 16), an effect which was previously proliferation of the MCF-7 cell line. We have similarly exam masked by the estrogenic nature of phenol red, the pH indicator ined the stable, estrogen receptor-positive, but antiestrogen- dye used for culture media ( 17). Although tamoxifen is generally resistant, MCF-7 cell variant LY-2 (41). The data provide tumoristatic to MCF-7 tumors grown in the nude mouse (18. further evidence that ICI 164,384 is a pure antiestrogen lacking 19), prolonged tamoxifen exposure can lead to tamoxifen- agonist activity, and suggest that such pure antiestrogens are resistant (20) and tamoxifen-stimulable (21) tumors. good candidates for clinical use. These considerations have fostered the continuous develop-

Rcceived 2/3/89; revised 8/28/89; accepted 9/21/89. MATERIALS AND METHODS 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. Cell Lines. MCF-7 cells were obtained from Dr. Marvin Rich (Mich 1This work. Erik W. Thompson, and Thomas B. Shima were supported in igan Cancer Foundation) and used at passage 23. MDA-MB-231 cells part by the Breast Cancer Study Group, National Cancer Institute. NIH. 2To whom requests for reprints should be addressed, at Vincent T. Lombardi were obtained from the ATCC (Rockville, MD). Both cell lines were Cancer Center. Georgetown University Medical Center. 3800 Reservoir Road. maintained in Costar T75 flasks with Richters modified Minimal NW. Washington. DC 20007. Essential Medium (IMEM; Biofluids, Rockville, MD) supplemented 'The abbreviation used is: OHT, 4-hydroxytamoxifen. with 2 HIMglutamine and 5% fetal bovine serum (GIBCO, NY; FBS/ 6929 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MCF-7 CELL INHIBITION WITH ICI I6-U84

MEM). LY-2 cells were obtained from Diane Bronzert, NCI, NIH, and for this study. Effects on MCF-7 cells were compared to the maintained under the same conditions as MCF-7 cells. To deplete cells effects on the antiestrogen-resistant LY-2 subclone of the MCF- of estrogens, they were passaged for I week in phenol red-free IMEM 7 cell line. supplemented with 5% estrogen-depleted calf serum (DCS/MEM) Proliferation Studies. Fig. 1 shows dose response data for the which was made by sequential treatment of calf serum (GIBCO, New York, NY) with sulfatase (Sigma, St. Louis, MO) and dextran-coated effect of ICI 164,384 on the proliferation of estrogen-dependent charcoal (Sigma, St. Louis, MO). NIH-3T3 cells, kindly provided by MCF-7 cells and estrogen-insensitive MDA-MB-231 cells after Dr. Stewart Aaronson, NIH, were maintained in Dulbecco's modified 9 days of treatment. This time point shows the maximum effects minimal essential medium (Biofluids, Rockville, MD) supplemented of ICI 164,384 on cell populations within the logarithmic with 5% FBS. growth phase. Although this experiment was performed in Estrogen and Antiestrogens. 17/S-Estradiol was obtained from Sigma, DCS/MEM, control cultures (ethanol alone) showed logarith St. Louis. MO, tamoxifen (TAM) and 4-hydroxytamoxifen (OHT) were mic growth for the 9-day experimental period (doubling time, obtained from ICI America, Inc., Wilmington, DE, LY 117108 was a 108 h), suggesting the presence of residual estrogens. On days gift from Eli Lilly Co., Indianapolis, IN. ICI 164,384 was synthesized 6 (not shown) and 9 (Fig. 1), MCF-7 cells were inhibited in a by Jean Bowler, ICI Pharmaceuticals, Cheshire, England. Stock solu tions of these agents in absolute ethanol were stored at â€”¿20Â°C,and dose-dependent manner by increasing concentrations of ICI 164,384, with maximal effect at 10~7M.MCF-7 cultures treated diluted lOOOx in the appropriate medium when required. Control with 10~7M ICI 164,384 remained at constant density after day cultures received the ethanol vehicle (0.1%) alone. Proliferation Studies. MCF-7 cells were carried in 5% FBS/MEM 3. Proliferation was stimulated approximately twofold by 10~9 and transferred to 5% DCS/MEM without phenol red approximately M 17/3-estradiol (not shown). Under similar conditions, the 1 week before experiments were begun. Cells were plated in six-well growth of estrogen receptor-negative MDA-MB-231 cells was dishes (Costar) at approximately 20,000 cells/well. One or 2 days after not affected by concentrations of ICI 164,384 up to 4 x 10~7M plating (Day 0) they were counted and the treatment media were added. on day 9, or at any other time point. These data indicate that At each data point (i.e.. days 3 and 6, etc.) the media on the remaining 10^7M ICI 164,384 is an effective concentration for the specific plates were changed and fresh treatment media readded. Cells were inhibition of MCF-7 cell proliferation, presumably through removed in PBS with 0.029o EDTA and counted in a cell counter (Particle Data, Inc.). competition with residual estrogens for the estrogen receptor. Estrogen Receptor Binding Studies. MCF-7 cells were plated in 12- ICI 164,384, OHT and LY 117018, were assessed for their well dishes at approximately 200,000 cells/well with 5% DCS/MEM capacity to stimulate MCF-7 cell proliferation in the absence without phenol red. ['H]17/i-Estradiol (95.4 Ci/mmol, 10~'M final of phenol red (Fig. 2). Control cultures remained at constant concentration, NEN Research Products, Dupont, Washington, DC) density after the initial 3-day period, indicating a true absence and unlabeled antiestrogens were diluted in DCS/MEM and added to each well. Cells were incubated for 1 or 2 h at 37Â°C.Each well was of estrogen(s) in the medium in this experiment. Stimulation was seen with either OHT or LY 117018 on days 6, 9, and 12, washed three times with PBS containing 1% DCS, and digested for 1 but not with ICI 164,384. Significantly larger stimulations were h with 1 ml of l N NaOH. The digests were neutralized with 250 ^1 4 seen with 10"10and 10~" M 17ÃŸ-estradiolas evidenced in Fig. N HC1, combined with a 0.5 ml H2O wash, and counted on a Beckman liquid scintillation counter. 3, which shows data from day 9 of the same experiment. We further compared the effects of ICI 164,384 (10~7ivi), LY Chemoinvasion Assay. The Boyden chamber chemoinvasion assay was performed as described previously (32, 42). Matrigel, a mixture of 117018 (10~7M), and OHT (10~7M), either alone or in combi basement membrane proteins (43), was kindly provided by Dr. Hynda nation with 17/3-estradiol, on the proliferation of wild type Kleinman, LDBA, NIDR, NIH, Bethesda, MD. Polycarbonate filters MCF-7 cells and the antiestrogen resistant variant LY-2 (Fig. (12 iim pore; PVP free. Neuroprobe, Rockville, MD) were coated with 3). Under these conditions, MCF-7 cell proliferation was stim 25 Â¿ig/filterof Matrigel which was reconstituted at 37Â°Cinto a solid ulated by 17/i-estradioI at 10'"'or 10"" M, and toa lesser extent even layer over the surface of the filter. Cells were cultured in FBS/ by LY 117018 (IO"7 M) and OHT (IO"7 M). ICI 164,384, MEM, harvested with trypsin, washed twice in IMEM containing 0.1% bovine serum albumin (BSA/IMEM) and added to the top of the however, did not stimulate MCF-7 cell proliferation. All three modified Boyden chamber (300,000 cells/chamber). Treatment media were added to the cells immediately after they were added to the chamber, such that the final concentration in the chamber would be that specified. Fibroblast conditioned medium, obtained by incubating confluent NIH-3T3 cells for 24 h with BSA/IMEM containing 0.05 mg/ml ascorbic acid (GIBCO, New York, NY), was used as the chemo- attractant. Chambers were incubated in a humidified incubator at 37Â°C in 5% CO; in air for 9 h. Cells which had traversed the Matrigel and attached to the lower surface of the filter were stained with Diff-Quick (American Scientific Products) and quantitated electronically with the Optomax V-HR image analyzer. Statistical Analysis. Combined data from pooled experiments were analyzed by one-way analysis of variance using the StatView 512 Apple Macintosh package software.

io-9 io-8 io7 io-Â« RESULTS [ICI 164, 384] M The steroidal antiestrogen ICI 164,384 was compared to the Fig. 1. Dose-response curve for If'I 164,384 effects on the proliferation of estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) cells in nonsteroidal antiestrogens OHT and LY 117018 in terms of DCS/MEM. MCF-7 (2 x 104/well) and MDA-MB-231 (1 x 104/well) cells were potency in antagonizing estradiol effects, degree of intrinsic plated overnight in 6-well dishes and treated the next day (day 0) with IO"1*M 17/i-estradiol (not shown, see text), various concentrations of ICI 164.384. or estrogenicity, and ability to reverse the estrogenic effects of 0. lr; ethanol (control). Cells from triplicate wells were harvested with EDTA on other antiestrogens. Proliferation and basement membrane in- days 3. 6. 9. and 12. Data, expressed as rt of control, represent cell counts on vasiveness in vitro were used as estrogen-sensitive parameters day 9. Standard deviations of the primary data were less than 10'V of the mean. 6930 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MCF-7 CELL INHIBITION WITH ICI 164..1H4

10V antiestrogens effectively blocked the effects of 10 " M 17ÃŸ- estradiol on wild type MCF-7 cells. IO'1" M 17^-estradiol- induced proliferation was completely blocked by LY 117018 and OHT, but only partially by ICI 164,384. These data suggest that ICI 164,384 is somewhat less potent than OHT or LY 117018, and relative binding affinities, compared in Fig. 4, IO4 support this observation (see below). However, ICI 164,384 is a superior antiestrogen in terms of absence of growth stimula I OHI I10 7M) tory effects. A LY 117018 110 'Ml The lower efficacy of ICI 164,384, compared to the other O ICI 164, 384 110'Ml antiestrogens, in blocking the proliferation effect of 10~'Â°M â€¢¿Control 17/tf-estradiol on MCF-7 cells (Fig. 3/1) indicated that ICI 103 164,384 may be less potent than the other antiestrogens studied. 6 12 We used competition experiments that examine the ability of Days each antiestrogen to compete with ['H]17/j-estradiol for estro Fig. 2. Effects of anticstrogens on MCF-7 cell proliferation. MCF-7 cells were cultured in DCS/MEM for 1 week, plated in Costar 6 clusters (2 X 104/well). gen receptor binding to confirm this (Fig. 4). A concentration of 3.4 x 10~8 M ICI 164,384 was required to achieve half allowed to attach overnight and treated the next day (day 0). Cells in triplicate wells Â»ereharvested with EDTA and counted electronically. Data represents the maximal competition compared to 6.3 x lO^1*Mfor OHT and mean Â±SEM values. Day 9 data from the same experiment, including estrogen LY 117018 and 1 x 10-9M for 17/J-estradiol. and antiestrogen combinations, are presented in Fig. 3. The effects of different concentrations of antiestrogen, either alone or in combination, on MCF-7 cell proliferation were also examined (Fig. 5). Significant stimulation of MCF-7 cell pro A. MCF-7 Cell liferation was seen with LY 117018 at IO"8 M, but not 10"" M. OHT was stimulatory at both 10s M and 10 '' M. and combi-

4.8 H Control 0 +107MOHT 120^ [ | +10'M LY 117018 4.0 1 I +10 7M ICI 164,384 100..

80 - 3.6 o x o 60 .. .0

-1 1 -10 -9 -a -7 -6 [E2] log 10 Fig. 4. Competition for the binding of 10 *M ['Hjn^-estradiol to MCF-7 cell monolayers with unlabelled antiestrogens and 17^-eslradiol. Lines were fitted with the "Sigma-PI.ol" scientific graphics software (Jandel Scientific. Sausalito. CA). Points, means from three different experiments where SEM was less than 10%.

Control 10 8M 10 9M 10 8M 10 9M 10 BM 10 9M LY 117 018 OHT LY 117 018 Fig. 3. Agonism/antagonism of 17,i-estradiol (//Â¿-Â¿'^-induced proliferation OHT of MCF-7 and LY-2 cells by different anticstrogens. MCF-7 cells (.-() and LY-2 Treatment cells (/f ) were treated as described in Fig. 2. U'fft'fid- These results depict the actual cell numbers (mean Â±SEM from triplicate dishes) harvested on day 9. Significant Fig. 5. Effects of different antiestrogens combinations on MCF-7 cell prolif differences; double symbol represents P < 0.01. single symbol represents P < eration. MCF-7 cells were cultured, treated and harvested on day 9 as descril>ed 0.05: *. compares control; $. compares 10~" \i I7,JE;; A. compares I0~'Â° M for Fig. 3, with the different treatment combinations specified. Significant differ 17,iEÂ¡;D. compares 10'" M HjJEi + L Y 117018; .compares 10~" M 17ffE2 + ences; double symbol represents P < 0.01; **. compares untreated control; \\. compares corresponding culture in the absence of IO"7 \i ICI I64.3X4. OHT. 6931 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MCF-7 CELL INHIBITION WITH ICI I64..W4 nations of OHT and LY 117018, (both at IO'8 or both at KT" A. MCF-7 Cell M) showed levels of stimulation similar to those achieved with either antiestrogen alone. In this experiment, ICI 164,384 ^B Control significantly reduced MCF-7 cell proliferation when added F"] +1(T7M LY 117018 alone, suggesting the presence of residual estrogen(s). Under [ 1 +10 7M ICI 164. 384 these conditions, ICI 164,384 blocked the growth-stimulatory effects of LY 117018 (IO"* M) and OHT (lO'8 M, 10~9M), and also inhibited MCF-7 cells treated with 10"" M LY 117018 (which alone did not stimulate proliferation) to levels below control. These data further implicate the estrogen receptor in mediating the growth stimulatory effects of LY 117018 and OHT, and demonstrate the broad antiestrogenic activity of ICI 164,384. The proliferation rate of the antiestrogen-resistant LY-2 cells under basal conditions was faster than that seen with the MCF- 7 parent cells (doubling times of 422 and 52 h, respectively), and the increases seen with 17/i-estradiol, LY 117018 and B. LY-2 Cell OHT, although significant, were less than those achieved with 3r the parent cells. As seen with the parent cells, ICI 164,384 did not stimulate LY-2 cell proliferation (Fig. 3B). Proliferation of LY-2 cells induced by 10" M 17/i-estradiol was not inhibited by either OHT or LY 117018 at 10~7M, but was significantly o 2 blocked by ICI 164,384. Similar effects were seen to a lesser degree with 10~'Â°M170-estradiol-induced proliferation, which was lower than that achieved with 10~" M 17,rf-estradiol, and was significantly reduced (P < 0.05) by either LY 117018 or ICI 164,384. Invasion of Artificial Basement Membrane. For this experi 10 9M 17/3-E2 ment, MCF-7 cells and LY-2 cells were cultured in FBS/MEM, Control 10 7M OHT harvested with trypsin and treated with estrogen and/or anties Treatment trogen directly in the Boyden chamber chemoinvasion assay for Fig. 6. Effects of estrogen and anticstrogens on the invasiveness of MCF-7 12 h (Fig. 6). As reported previously (32), invasiveness of wild cells (A) and LY-2 cells (B). Invaded cells Â«erequantified by image analy/cr and type MCF-7 cells under these conditions was significantly stim results are presented as the area of cells occupying a high power field. Bars represent mean Â±SEM for three experiments, each performed in duplicate. ulated by 170-estradiol and OHT, but not LY 117018. In Significant differences; double symbol represents P < 0.01. single symbol repre sents P < 0.05: *. compares untreated control; $, compares IO"' M l7fJE2; A, addition, ICI 164,384 did not stimulate MCF-7 cell invasive- compares 10"7M OHT. ness. MCF-7 cell invasiveness due to 17/U-estradiol or OHT was blocked by either LY 117018 or ICI 164,384 (Fig. 6A). In contrast to wild type cells, LY-2 cell invasiveness was stimulated the effects of 17fi-estradiol, OHT, and LY 117018 on MCF-7 cell proliferation, and the ability of 17ÃŸ-estradioland OHT to approximately twofold by LY 117018 (P < 0.01), and also stimulated slightly by ICI 164,384 (P < 0.05, Fig. 6B). In LY stimulate the invasiveness of these cells. The differential sensi 2 cells, ICI 164,384 did not significantly reduce 17/i-estradiol- tivity of proliferation and invasiveness to agonistic stimulation induced invasiveness at the concentrations used, but completely by LY 117018 suggests that these parameters are independently reversed that caused by OHT. regulated. This is further evidenced in the LY-2 subline, which proliferates at a basal rate similar to estrogen-treated wild-type cells, but resembles the wild-type cells in terms of baseline DISCUSSION invasiveness. Estrogenic and Antiestrogenic Activities of OUT and LY Lack of Intrinsic Estrogenic Activity of ICI 164,384. These 117018. Estrogenic effects of tamoxifen and OHT on MCF-7 studies were performed under conditions in which the stimula tory effects of estrogen (17/i-estradiol) on the proliferation and cell proliferation have been reported previously in vitro (15, 16) invasiveness of MCF-7 cells were clearly apparent. Under these and i/i vivo (21), but little such agonism has previously been conditions (phenol red-free DCS/MEM for proliferation and reported for LY 117018 (15). In the current study we found LY 0.1% BSA/IMEM for invasiveness), OHT and LY 117018 117018 to be a partial estrogen agonist for proliferation of the stimulated MCF-7 cell proliferation, and OHT also enhanced MCF-7 cells, and for both proliferation and basement mem- MCF-7 cell invasiveness. However, no stimulation of these brane-invasiveness of the LY-2 cells. Consistent with previous parameters seen with the recently developed steroidal antiestro reports, however, both OHT and LY 117018 were also very gen ICI 164,384. This 7Â«A'-methyltertiary amide is an estrogen effective antagonists of estradiol-stimulated MCF-7 prolifera analogue, which, in addition to its lack of agonism for the tion. Similarly, LY 117018, which stimulated LY-2 cell inva parameters examined here, shows no uterotrophic activity in siveness when added alone, antagonized estradiol and OHT- immature rats and mice or ovariectomized rats (28), and does induced LY-2 cell invasiveness. These data are not easily under not induce progesterone receptor expression in the rat uterus stood by a simple model of ligand occupancy of the estrogen (29). receptor. Perhaps the combination of estrogen plus antiestrogen Consistent with its previously demonstrated capacity to block occupancy of the estrogen receptor produces a different behav the stimulatory effects of either 170-estradiol or tamoxifen on ior of the receptor than that produced by occupancy by either uterine growth in the immature rat (28), ICI 164.384 blocked ligand alone. 6932 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MCF-7 CELL INHIBITION WITH ICI 164.384

Antiestrogenic Activities of ICI 164,384. By definition, anti- The apparently independent regulation of proliferation and estrogens act to limit the actions of estrogen through competi basement membrane invasiveness with regard to agonism and tive binding of the estrogen receptor, without functional acti antagonism by antiestrogen may have implications in tumor vation. In this study we have confirmed the antiestrogenic development in situ, particularly those tumors which develop nature of all three antiestrogens in terms of MCF-7 cell prolif antiestrogen-resistance. The current data with LY-2 cells sug eration and basement membrane invasiveness, since each com gest that these tumors could escape the antiproliferative effects pound blocked the effect of estradici on these parameters. In of antiestrogens while still under their positive influence for addition, ICI 164,384 blocked the stimulatory effects of OHT invasiveness. Furthermore, the LY 117018 stimulation of LY- and LY 117018 on MCF-7 cell proliferation, and that of OHT 2 cell invasiveness suggests that in the course of their progres on MCF-7 cell invasiveness. sion, breast cancer cells may acquire sensitivity to the stimula Although itself a partial agonist for MCF-7 cell proliferation, tory effects of these agents on invasiveness. Such a scenario LY 117018 was able to block the effects of OHT on invasive- could clearly result in increased metastatic dissemination of ness. This provides additional evidence that stimulation seen these tumors. The new antiestrogen ICI 164,384 shows no with OHT is due to partial estrogen agonism, mediated through intrinsic estrogenicity for proliferation and invasiveness of the estrogen receptor. It also indicates that the antiestrogenic MCF-7 or LY-2 cells, and is more effective than the nonsteroi capacity of ICI 164,384 is relatively broad. dal agents in antiestrogen-resistant cells. This antiestrogen may Antiestrogen-resistant LY-2 Cells. Antiestrogen resistance is offer advantages for therapeutic application, and warrants fur a major limitation for the use of antihormonal therapy in the ther study in the clinical management of human breast cancer, clinical management of human breast cancer. Although approx and other estrogen-related disorders. imately 30% of breast cancer patients have antiestrogen-responsive tumors at the time of diagnosis, most ultimately develop ACKNOWLEDGMENTS hormone independence. In many cases, estrogen receptor-pos itive tumors are antiestrogen resistant at the time of initial We thank Judy Wilson and Sherrell Gibson for typing assistance, treatment, perhaps representing an intermediate in the hypo and Drs. George Martin and Robert Clarke for critical reading of the thetical progression from estrogen dependence to an estrogen- manuscript. independent phenotype. 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Erik W. Thompson, Deborah Katz, Thomas B. Shima, et al.

Cancer Res 1989;49:6929-6934.

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