differential regulation of growth and invasiveness of mcf-7 breast cancer cells by...

(CANCER RESEARCH 48, 6764-6768, December 1. 1988]

Differential Regulation of Growth and Invasiveness of MCF-7 Breast Cancer Cellsby Antiestrogens1

Erik W. Thompson, Reuven Reich, Thomas B. Shima, Adriana Albini, Jeannette Graf, George R. Martin,Robert B. Dickson, and Mare E. LippmanLaboratory of Developmental Biology and Anomalies, National Institute of Dental Research Â¡E.W. T., R. R., T. B. S., A. A., J. G., G. R. M.], and Medical Breast CancerSection, Medicine Branch, National Cancer Institute [R. B. D., M. E. L.], NIH, Bethesda, Maryland 20892

ABSTRACT

Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through anartificial basement membrane. In studying the response of MCF-7 cellsto various antiestrogens, we found that 4-hydroxytamoxifen and tamoxi-fen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018and LY1S6758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agentson invasion correlated with changes in production of collagenase IV.while no significant change was seen in the chemotactic activity of thecells. Invasiveness was increased by 17/3-estradiol or 4-hydroxytamoxifenafter a few hours of treatment and was rapidly lost when 17/3-estradiolwas withdrawn. Stimulation of invasiveness with 17/J-estradiol wasblocked by the antiestrogen, LY117018. Cells from the MDA-MB-231line which lacks estrogen receptors were not affected by estrogen orantiestrogen in terms of proliferation or invasion. These studies indicatethat the invasiveness of MCF-7 cells is regulated by antiestrogensthrough the estrogen receptor and may be mediated by collagenase IVactivity. Antiestrogens which reduce both the proliferation and invasive-ness of these cells may be interesting new candidates for clinical application.

INTRODUCTION

The MCF-7 cell line, derived from a pleural effusion of amalignant breast cancer (1), is a widely studied model forhormone-dependent human breast cancer. These cells containfunctional estrogen receptors and show a pleiotropic responseto estrogen (2-5). Estrogen induces MCF-7 cells to form tumors

in the nude mouse (6, 7), and it stimulates the proliferation ofthese cells in vitro (2, 8-10). In contrast, the MDA-MB-231cell line provides a model for human breast cancer whichexhibits an estrogen-independent state and does not expressestrogen receptors (11). These latter characteristics are associated with advanced disease.

An appreciation of the necessity of estrogen for the growthof some breast cancers led to the clinical use of antiestrogens,particularly tamoxifen, to suppress the growth of these tumors(12-15). However, a number of studies in model systems attribute partial estrogenic activity to tamoxifen and to its activemetabolite OHT. For example, under certain conditions, tamoxifen stimulates the proliferation of estrogen-dependent human breast cancer cell lines in culture (10, 16-18) as well as inthe nude mouse (7,19, 20), and tamoxifen induces an estrogen-like morphological change in cultured MCF-7 cells (21). Agonistic activities of tamoxifen and OHT on MCF-7 cell proliferation have previously been masked by the estrogenic natureof phenol red, the pH indicator dye commonly used in cell

Received 7/5/88; revised 8/31/88; accepted 9/6/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work, T. B. S., and E. W. T. were supported by the Breast Cancer Study

Group, Medical Breast Cancer Section, Medicine Branch, National Cancer Institute, NIH.

2The abbreviations used are: OHT, 4-hydroxytamoxifen; IMEM, Richtersmodified minimal essential medium; FBS, fetal bovine serum; DCC, dextran-coated charcoal; BSA, bovine serum albumin; DCS, depleted calf serum.

culture medium (10, 22). Like estrogen, tamoxifen inducesuterine growth in ovariectomized rats and mice (23-25), andboth tamoxifen and 4-hydroxytamoxifen show partial estrogenic activity in inducing progesterone receptors in culturedMCF-7 cells (3, 26-30). May and Westley (31) have recentlyreported that tamoxifen and 4-hydroxytamoxifen show someability to stimulate two mRNA species which are also estrogeninducible. Thus, a variety of estrogen-regulated functions existwhich are similarly regulated by tamoxifen.

Recent studies have demonstrated that estrogen-treatedMCF-7 cells are more capable of traversing a reconstitutedbasement membrane barrier in vitro than their estrogen-deprived counterparts (32). Such activity is thought to relate tothe invasive and metastatic potential of tumor cells (33). Theaim of this study was to examine the effect of tamoxifen and 4-hydroxytamoxifen, as well as the structurally distinct benzothiophene antiestrogens LY 117018 and LY 156758 (25, 34-36) on the invasiveness of breast cancer cells. The resultsindicate that, while tamoxifen and hydroxytamoxifen stimulatethe invasiveness of estrogen-dependent MCF-7 breast cancercells at concentrations which are growth inhibitory, benzothiophene antagonists inhibit both the invasiveness and proliferation of these cells.

MATERIALS AND METHODS

Cell Lines. MCF-7 cells and MDA-MB-231 cells were obtained fromthe American Type Culture Collection (Rockville, MD) and weremaintained in T75 flasks (Costar) in IMEM (Biofluids, Rockville, MD)supplemented with 2 DIM glutamine and 10% fetal bovine serum(GIBCO, New York, NY; FBS/IMEM). To deplete estrogen, the cellswere passaged for at least 2 wk in IMEM supplemented with 5% calfserum (GIBCO) which had been treated sequentially with sulfatase(Sigma, St. Louis, MO) and DCC (Sigma) to remove endogenousestrogen (DCS/MEM; Ref. 2).

Estrogen and Antiestrogens. 17/3-Estradiol was obtained from Sigma;tamoxifen and OHT were obtained from ICI America, Inc., Wilmington, DE; LY 117018 and LY 156758 were gifts from Eli Lilly Co.,Indianapolis, IN. Stock solutions (lOOOx) in absolute ethanol (MidwestSolvents Co., Perkin, IL) were stored at â€”¿�20Â°Cand applied directly to

the culture dish.Estrogen and Antiestrogen Treatment. Cells were trypsinized, re-

seeded into tissue culture dishes (1 x IO6cells/10-cm-diameter Falcondish), and allowed to adhere overnight in a humidified incubator (37Â°C,

5% CO2:95% air). The cells were treated the next day with either 17/3-estradiol (10"' M), one of the antiestrogens (10~7 M hydroxytamoxifen,IO"6M tamoxifen, 10~7M LY 117018, 10~7M LY 156758), or ethanol

alone (0.1% final concentration). Four days later, the cells were harvested with trypsin (GIBCO), washed twice in IMEM containing 0.1%bovine serum albumin (BSA/IMEM), counted with a Coulter CellCounter, and tested for chemotaxis and chemoinvasion activities. Insome experiments, confluent MCF-7 cells were harvested from culturewith trypsin, washed twice in BSA/IMEM, counted, and treated directlyin the chemoinvasion assay.

Chemoinvasion Assay. The Boyden chamber chemoinvasion assaywas performed as described previously (32, 37). Matrigel, a mixture ofbasement membrane components (38), was kindly provided by Dr.Hynda Kleinman, NIH. Polycarbonate filters (12-fjm pore, polyvinyl-

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ANTIESTROGENS AND MCF-7 CELL INVASIVENESS

pyrrolidone-free, Nucleopore) were coated with matrigel (25which was dried and then reconstituted at 37Â°Cinto a solid, even layer

over the surface of the filter. Fibroblast-conditioned medium, obtainedby incubating confluent NIH-3T3 cells for 24 h with IMEM, was usedas the chemoattractant. Cells were harvested with trypsin, washed twicewith BSA/IMEM, and added to the top chamber (300,000 cells/chamber). Chambers were incubated in a humidified incubator at 37Â°C

in 5% CO2 in air for 6, 9, or 12 h. The cells which had traversed thematrigel and attached to the lower surface of the filter were stainedwith Diff-Quick (American Scientific Products) and quantitated electronically with the Optomax V image analyzer.

Chemotaxis Assay. Chemotaxis assays were performed as describedfor the chemoinvasion studies with the single exception that the filtersurfaces were coated with 5 /ig of collagen IV (kindly provided by Dr.Roy Ogle, NIH) instead of the layer of matrigel. This coats the interstices of the filter but does not form a barrier over the surface. Chemotaxis assays were performed in parallel to the chemoinvasion assaysusing the same cells and conditioned medium. Duplicate or triplicatefilters were used in each experiment. One-way analysis of variance wasperformed on the data from each experiment using the Stat WorksPackage with the Apple Macintosh II computer.

Type IV Collagenase Assay. Collagenase IV activity was measuredusing a modified solid phase radioimmunoassay (39). Briefly, collagenIV isolated from EHS tumor tissue (40) was iodinated by the Bolton-Hunter method, and a solution of the labeled collagen (10,000 to 20,000cpm) was allowed to bind to microtiter plates (Removawell; Dynatech)overnight. Serial dilutions of medium from the Boyden chambers werethen incubated in the microtiter plates for 24 h at 37Â°Cand the

collagenase activity was determined from the dilution within the linearrange which gave half-maximal degradation of '"I-collagen IV from

the solid phase in the presence of serine proteinase inhibitors.

RESULTS

Studies were carried out comparing the effects of estrogenwith various antiestrogens on the proliferation, invasiveness,chemotactic responsiveness, and collagenase production of human breast cancer cells.

Hormonal Effects on Invasion and Proliferation. Fig. 1 compares the invasiveness and chemotactic activity of MCF-7 cellsand MDA-MB-231 cells after culture in either complete serum-containing medium (FBS/IMEM) or estrogen-depleted medium (DCS/IMEM). In confirmation of our previous studies,the MCF-7 cells harvested from medium supplemented with

complete serum (containing endogenous estrogen) were moreinvasive than those cultured in estrogen-depleted medium.MDA-MB-231 cells, which lack estrogen receptors, were considerably more invasive than the MCF-7 cells regardless ofwhich medium the cells were cultured in prior to testing. No

300r

INVASION CHEMOTAXIS

MCF-7

INVASION CHEMOTAXIS

MDA-MB-231

Fig. 1. Comparison of invasiveness and Chemotaxis of MCF-7 cells and MDA-MB-231 cells passaged continuously in FBS/IMEM or cultured for 2 wk prior toassay in DCS/IMEM. The assay was carried out for 9 h with the MCF-7 cellsand for 6 h with the MDA-MB-231 cells to reflect differences of the cells inmotility. This experiment was performed 3 times, and data (mean Â±SEM) arefrom a representative experiment. *, P < 0.05 comparing FBS/IMEM to DCS/IMEM.

difference was noted in the treated cells' ability to show directed

movement (chemotaxis) towards fibroblast-conditioned medium as a source of chemoattractants.

Since estrogen increases the invasiveness of the MCF-7 cells,it was of interest to study the response of these cells to variousantiestrogens. Subconfluent cultures of MCF-7 cells grown inFBS/IMEM were treated with 17/3-estradiol, an an tÂ¡estrogen.or the ethanol vehicle (Fig. 2). Under these conditions, 17/3-estradiol had no effect on the proliferation of the cells or oninvasiveness. Treatment with tamoxifen or 4-hydroxytamoxifenmarkedly reduced proliferation but significantly increased invasiveness. The benzothiophene antiestrogens LY 117018 andLY156758 also reduced proliferation but did not stimulate theinvasiveness of the cells. No significant differences were seenin the chemotactic activities of these cells after the differenttreatments.

We further examined the effects of 17/3-estradiol and theseantiestrogens on the invasive, chemotactic, and proliferarneactivities of the MDA-MB-231 cell line, which lacks estrogenreceptors (Fig. 3). In these studies, MDA-MB-231 cells werecultured in FBS/IMEM and treated for 4 days with estrogenor antiestrogen as described for MCF-7 cells in Fig. 2. As

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PROLIFERATION

CHEMOTAXIS

lull. â€¢¿� INVASION

.111,E2 OHT TAM LY117 LY166

TREATMENT

Fig. 2. Effect of 17/3-estradiol and antiestrogens on proliferation, chemotaxis,and invasiveness of MCF-7 cells cultured continuously in medium containing10% fetal bovine serum (FBS/IMEM). Cells were treated with the ethanol vehicle(Control, 0.1%), 17/3-estradiol (E2, 10"' M), or one of the antiestrogens (10"' M

final) 4-hydroxytamoxifen (OHT), LY 117018 (LYI17), or LY 156758 (LY]56)for 4 days, before harvest, counting, and assay for invasion. Values were pooledfrom 4 experiments and are expressed as the percentage of the control response(mean Â±SEM). *,P< 0.05 compared to control. TAM, tamoxifen (IO"6 M).

CNTRL E2 OHT

TREATMENT

LY117

Fig. 3. Effect of 17/S-estradiol and antiestrogens on proliferation and invasive-ness of MDA-MB-231 cells. MDA-MB-231 cells were cultured continuously inFBS/IMEM and treated for 4 days with 17/3-estradiol (E,, 10"' M), 4-hydroxytamoxifen (OHT, 10~7M), LY117018 (LY117, IO"7 M), or ethanol vehicle alone

(Control, 0.1 %) as described for Fig. 2. Cells were harvested with trypsin, counted,and assayed for 6 h. Data are expressed as mean Â±SEM from a representativeexperiment.




expected, their proliferation was not affected by either 17/3-estradiol or 4-hydroxytamoxifen. Furthermore, neither theirinvasiveness nor chemotactic responsiveness (data not shown)was altered by these treatments.

The effects of 17/3-estradiol and antiestrogens were seen evenmore clearly with MCF-7 cells which had been cultured inestrogen-depleted medium (Fig. 4). Under these conditions,added 17/3-estradiol caused a much greater stimulation ofgrowth while the antiproliferative effects of the antiestrogenswere less evident (data not shown). 17/3-Estradiol and 4-hydroxytamoxifen dramatically stimulated MCF-7 cell invasive-ness (Fig. 4), while the benzothiophene compounds LY117018and LY156758 did not (not shown). The effects of 17/Ã®-estradiol(IO"9 M) on both proliferation and invasion under these conditions were blocked by LY117018 at 10~7 M (data not shown).

The same trends were seen in experiments performed withMCF-7 cells cultured in the absence of phenol red (10, 22) for1 to 2 wk prior to treatment and assay (data not shown).Preliminary results from a similar experiment using the estrogen-dependent human mammary carcinoma T47D cell lineshowed similar effects of 17/3-estradiol and OHT on proliferation, invasiveness, and chemotaxis. These data suggest that theeffects of 17/3-estradiol and 4-hydroxytamoxifen on the invasiveness and proliferation of MCF-7 cells are mediated via theestrogen receptor.

In some studies, the cells were pretreated with 17/8-estradiolor 4-hydroxytamoxifen in culture, but the treatment was omitted from the subsequent chemoinvasion assay (Fig. 4, withdrawn group). In this case, the increased invasiveness causedby 17/3-estradiol was lost during the 9-h assay period. In contrast, cells treated with 4-hydroxytamoxifen under the sameconditions maintained a high level of invasiveness. Such observations suggest that the stimulation of invasiveness by 17/3-estradiol involves a rapidly reversible process. Perhaps differences in the retention of 4-hydroxytamoxifen account for itslonger duration of action. These effects also have a rapid onset,and a kinetic analysis showed that MCF-7 cells exposed to 4-hydroxytamoxifen in the assay chamber for as little as 9 hshowed increased invasiveness (Fig. 5). Under these conditions,the maximal effect of hydroxytamoxifen was observed at 10~8

M (Fig. 6).Effects on Collagenase IV Production and the Chemotactic

Responsiveness of the Cells. To explore possible mechanismsby which estrogen and certain antiestrogens induced invasive-ness, we measured the chemotactic response of the cells andtheir production of type IV collagenase (Table 1). These agents

1C/3

I

CONTROL E2 OHT

TREATMENT

Fig. 4. Effect of 17/3-estradiol and 4-hydroxytamoxifen on the invasiveness ofMCF-7 cells which were cultured for 2 wk in medium containing 5% estrogen-depleted calf serum (DCS/IMEM) and then treated for 4 days with the ethanolvehicle (Control, 0.1%), 17/3-estradiol (E2,10~9 M), or 4-hydroxytamoxifen (OHT,

10 M). In some cases treatment was not continued in the assay chamber whichresulted in a rapid loss of invasiveness in the estrogen-treated group. The invasiondata are expressed as the area occupied by invading cells on the bottom of thefilter (mean Â±SEM). *, P < 0.05 compared to control.

6 hrs 9 hrs

INVASION

9 hrs

CHEMOTAXIS

Fig. 5. Effects of 4-hydroxytamoxifen (OHT) added directly to the assaychamber. MCF-7 cells were transferred to DCS/IMEM in the absence of phenolred for 2 wk. Cells were treated directly in the assay chamber with either ethanolalone (Control, 0.1%) or 4-hydroxytamoxifen (OHT, 10"' M), and duplicate

chambers were harvested after 6 or 9 h. Chemotaxis chambers were run in parallelfor 9 h. Data are expressed as mean Â±SEM from a representative experiment. *,

P < 0.05 compared to control.

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OHT DOSE(M)

Fig. 6. Dose response of OHT on MCF-7 cells treated directly in the assaychamber for 9 h as described for Fig. 5.

Table 1 Effect of estrogen and antiestrogens on chemotaxis and collagenaseproduction by MCF-7 cells

MCF-7 cells were cultured in DCS/IMEM and treated for 4 days prior toassay.

Control 17/3-Estradiol OHT LY 117018Collagenase"Chemotaxis1*

0.46 Â±0.20*

12.40 Â±1.20

2.05 Â±0.56C

10.40 Â±1.19

1.60Â±0.53C

8.77 Â±0.600.50 Â±0.30

15.20Â± 1.70"Collagenase levels are expressed as cpm x 10 ' released from the solid-

phase assay as described in "Materials and Methods."4 Mean Â±SEM.c Significantly higher than control levels (P < 0.05).'' Data are expressed as area of migrated cells/field X 10 ' in a representative

experiment.

did not cause any significant change in the chemotactic responseof the cells, suggesting that the increased invasiveness was notdue to an enhanced migratory capacity. Since the only difference between the invasion and chemotactic assays was thepresence of a layer of basement membrane components on topof the porous filter in the Boyden chamber, we also assayed forcollagenase IV, the enzyme activity that degrades basementmembranes. These studies showed that both 17/3-estradiol and4-hydroxytamoxifen significantly increased collagenase IV levels in the medium collected from the upper compartment of theBoyden chamber at the conclusion of the assay. These dataindicate that an increased production of collagenase IV in cellstreated with either 17/3-estradiol or 4-hydroxytamoxifen couldmediate their increased ability to invade through basementmembrane.

DISCUSSION

As noted previously, 17|8-estradiol enhances the ability ofMCF-7 cells to invade through a reconstituted basement membrane gel as well as to proliferate (32). We have extended theseresults here to show that the effects of estrogen on invasivenessare rapidly achieved and also rapidly lost when estrogen isremoved. In addition, the invasive responses of the MCF-7 cellsto antiestrogens were striking. The antiestrogens studied are




separable into two classes, substituted triphenylethylenes (ta-moxifen and 4-hydroxytamoxifen, which have appreciable agonist activity) and the benzothiophenes (LY117018 andLY156758, which show less agonist activity). All antiestrogenswere effective in reducing the proliferation of the MCF-7 cells,

particularly when applied in the presence of complete serum(containing estrogen). Tamoxifen and 4-hydroxytamoxifen,however, increased the invasiveness of the MCF-7 cells, whichunder some conditions even exceeded the response induced by17/3-estradiol. The other two antiestrogens studied (LY 117018and LY 156758) had no effect on the invasiveness of the cells.

The reversibility of 17/3-estradiol-induced invasiveness with10~7M LY117018 and the similar responsiveness seen with the

T47D cell line suggest a role for the estrogen receptor inmediating these effects. Consistently, no effects of 17/8-estradiolor antiestrogens were seen on the invasiveness of the MDA-MB-231 breast cancer cell line or on its proliferation. Thisparticular cell line lacks estrogen receptors and would not beexpected to respond to signals mediated in this way. The MDA-MB-231 cell line is more invasive and may exhibit a constitu-tively elevated expression of malignant parameters, attainedduring its progression. A similar situation may also exist with\-ras" transfected MCF-7 cells, which form hormone-inde

pendent tumors (41, 42) and show increased invasiveness inthis assay (32). While the estrogen receptor is clearly implicatedin the increased invasiveness caused by OHT, the fact that OHTeffects were sometimes stronger than those of estrogen andtheir relative persistence suggest something more complex thanpartial agonism. Distinct antiestrogen binding sites are knownto exist in the cytosol of these cells (43-45), and these couldmake some contribution.

Recent studies suggest that the degradation of the collagenIV network in the reconstituted basement membrane representsthe critical step in the passage of the tumor cells (46). Lamininincreases collagenase IV production by malignant tumor cells(47), and 17/3-estradiol as well as \-ras" transfection appear to

regulate laminin receptor expression in the breast cancer cells(32). The effects of antiestrogens on invasion, mediated via theestrogen receptor, could also involve the laminin receptor.

A proteolytic cascade, including plasminogen activator, plasmin, and collagenases I and IV, is also required for the degradation of various extracellular matrix barriers by tumor cells(46, 48). Type I collagenase production by another estrogen-dependent human breast cancer cell line (ZR75-1) is not stimulated by estrogen (49), but the enzyme is activated by anestrogen-sensitive plasminogen activator (50, 51). The possibility exists that increased production of plasminogen activator,or some other protease cascade component, may result in thegeneration of active proteases which facilitate the increasedinvasiveness observed in response to estrogen. Although theproduction of plasminogen activator by MCF-7 cells is stimulated by estrogen (52, 53), it is not stimulated by tamoxifen orhydroxytamoxifen (54). It should also be noted that many cellssecrete inhibitors of collagenases, plasminogen activator, andother proteases, and a reduction in the production of suchinhibitors could also increase the degradation of matrix barriers.Taken together, the data suggest that alterations at some levelof this protease cascade might account for the increased invasiveness seen in this study.

The relevance of our observations on the enhancement of thein vitro invasive activity of the breast cancer cells by tamoxifenand 4-hydroxytamoxifen to the in vivo behavior of estrogen-dependent breast cancer is unclear. Antiestrogens, includingtamoxifen and 4-hydroxytamoxifen, have been extraordinarilyuseful in the treatment of breast cancer (55, 56). There are,

however, some epiphenomena associated with tamoxifen treatment, particularly "tumor flare," which involves a transient

increase in tumor size (57). In addition, estrogenic effects oftamoxifen are thought to cause partial reduction in gonadotro-pin levels in postmenopausal women, changes in vaginal cytology, increased circulatory levels of sex hormone binding globulin, and decreased antithrombin II levels (58-61). It is possiblethat the estrogen agonism of tamoxifen and hydroxytamoxifencould increase a variety of activities of breast cancer cells invivo and could eventually contribute to the escape of the tumorfrom antihormonal control.

Antiestrogens from the benzothiophene class appear to lacksuch activity, and for this reason they may be more effective ininhibiting both the growth and invasive spread of breast cancer.It should be noted, however, that LY 156758 compared poorlyto tamoxifen in terms of its capacity to reduce tumor appearance in the nitrosomethylurea-induced rat mammary tumormodel (62), apparantly due to poor pharmacokinetics. Tamoxifen is generally tumoristatic to MCF-7 tumors in the athymicmouse (63, 64), but prolonged exposure can lead to antiestro-gen-resistant tumors (19, 20), perhaps analogous to the escapefrom antihormonal control often seen in human treatment.Interestingly, progesterone receptor expression, another tamox-ifen-sensitive, estrogen-dependent function, was found to beelevated in MCF-7 cells from these tumors, indicating thattamoxifen recognition persists in these cells for certain functions. TamoxilVn stimulated invasiveness could thus figureprominently in the subsequent progression of these tumors,since proliferation and invasiveness are shown in the currentstudy to be independently regulated by antiestrogens. The extension of these studies into in vivo models of human breastcancer cell invasion and metastasis, perhaps using antiestrogen-resistant clones, should help to shed light on the relevance ofour findings to the clinical situation.

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1988;48:6764-6768. Cancer Res Erik W. Thompson, Reuven Reich, Thomas B. Shima, et al. Breast Cancer Cells by AntiestrogensDifferential Regulation of Growth and Invasiveness of MCF-7

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