Comparison of the Effects of a
`Pure Steroidal Antiestrogen
`With Those of Tamoxifen in a
`
`Model of Human Breast Cancer
`
`C. Kent Osborne, Ester B.
`
`Coronado-Heinsohn, Susan G.
`
`Hilsenbeck, Bryant L. McCue,
`Alan E. Wakeling. Richard A.
`McClelland, David L. Manning,
`Robert I. Nicholson*
`
`non-
`a
`Tamoxifen,
`Background:
`is the
`steraidal estrogen antagonist,
`most prescribed drug for the treatment
`of breast cancer. The use of tamoxifen
`
`is limited, however, by the development
`of resistance to this compound in most
`patients. Although tamoxifen behaves
`primarily as an estrogen antagonist, it
`has agonist (or growth-stimulatory) ac-
`tivity as well. ICI 182,780 is a 7ot-alltyl-
`sulfinyl analogue of estradiol lacking
`agonist activity. The absence of agonist
`activity may make this steroidal an-
`tiestrogen superior to tamoxifen in sup-
`pressing tumor cell growth. Purpose:
`We compared the inhibitory effects of
`[Cl 182,780,
`tamoxifen, and estrogen
`withdrawal on the growth of estab-
`lished tumors and on tumorigenesis in
`a model system that uses estrogen-de-
`pendent, human MCF-7 breast tumor
`cells growing in athymlc nude mice.
`We also studied the hormonal respon-
`siveness of tumors that became resis-
`
`tant to the two estrogen antagonists
`and the effects of
`these drugs on
`estrogen-regulated gene
`expression.
`Methods: MCF-7 cells were injected
`subcutaneously into the
`flanks of
`castrated,‘female nude mice. The ef-
`. fects of repeated doses of tamoxifen
`and ICI 182,780 (500 pg and 5 mg,
`respectively) on the growth of estab-
`lished tumors (8-10 mm in size) were
`determined after supplemental estro-
`gen was removed. The effects of anti-
`estrogen treatments on the process of
`tumorigenesis, in the absence of estro-
`gen supplementation, were determined
`by initiating drug administration on
`
`the same day as tumor cell inoculation.
`To evaluate the hormonal responsive-
`ness of tumors resistant to tamoxifen
`and [CI 182,780, 1-mm’ segments of
`the tumors were transplanted onto the
`flanks of new recipient mice, which
`were then treated with estrogen or the
`antiestrogens—-alone or in combina-
`tion. Tumor growth was monitored by
`measuring tumor volumes
`twice a
`week. Expression of
`the estrogen-
`responsive genes, pLI‘V1 and p82,
`in
`the tumors of treated animals was
`
`analyzed using blots of total cellular
`RNA and complementary DNA probes.
`Results: Treatment with ICI 182,780
`suppressed the growth of established
`tumors twice as long as treatment with
`tamoxifen or
`estrogen withdrawal.
`Tumorigenesis, in the absence of sup-
`plemental estrogen, was delayed to a
`greater extent in ICI 182,780-treated
`mice than in tamoxifen-treated mice.
`ICI 182,780 was found to be more ef-
`fective than tamoxifen in reducing the
`expression of estrogen-regulated genes.
`Most tumors eventually became resis-
`tant to ICI 182,780 and grew inde-
`pendently of estrogen. Conclusions: ICI
`182,780 is a more effective estrogen an-
`tagonist than tamoxifen in the MCF-7
`tumor cell/nude mouse model system.
`[J Natl Cancer Inst 87:746-750, 1995]
`
`Tamoxifen, a nonstcroidal antiestro-
`gen, is the most prescribed drug for the
`treatment of breast cancer. When used in
`
`the adjuvant setting after surgery for
`primary breast cancer, about one fifth of
`the deaths at 10 years are avoided by 2
`years or more of treatment (I). Tamox-
`ifen is also effective in inducing remis-
`sions in women with estrogen receptor
`(ER)-positive metastatic breast cancer.
`Invariably, however, tumors become re-
`sistant to tamoxifen, and tumor progres-
`sion and death ensue. The evolution to
`tamoxifen resistance in metastatic breast
`
`cancer occurs after an average treatment
`duration of only l0-I2 months, severely
`limiting the usefulness of this approach.
`The mechanisms by which tumors ac-
`quire resistance to tamoxifen are poorly
`understood. Loss of ER from the tumor
`
`can occur by selection of an ER-negative
`clone or by suppression of receptor ex-
`pression. but
`this loss explains only a
`minority of cases (2). Growing experi-
`
`mental and clinical evidence suggests that
`resistance in some patients may be caused
`by the intrinsic estrogen agonist proper-
`ties of tamoxifen. Although tamoxifen is
`predominantly an estrogen antagonist in
`breast cancer cells, acquisition of increas-
`ingly dominant agonist activity over time
`may result in clinical resistance because
`of the acquired ability of the drug to
`stimulate, rather than to inhibit. tumor
`growth (3-7). The mechanisms for ta-
`moxifen-stimulated tumor growth are not
`clear, but
`these data suggest
`that an-
`tiestrogens with pure antagonist proper-
`ties might
`have
`superior
`antitumor
`activity.
`ICI 182,780 is a 7a-alltylsulfinyl ana-
`logue of estradiol that differs substantial-
`ly from tamoxifen in terms of
`its
`chemical. pharmacologic. and biologic
`properties. This agent has no intrinsic
`estrogen-agonist activity and,
`thus,
`is
`considered a “pure" antiestrogen (8.9). it
`has potent
`antiestrogenic
`activity in
`preclinical in vitro and in vivo model sys-
`tems (10). We recently reported (7) that
`-treating nude mice with ICI 182,780 in-
`hibits the growth of MCF-7 human breast
`tumor
`implants that had acquired ta-
`moxifen resistance through the mechanism
`of tamoxifen-stimulated growth. Similar
`results were
`obtained with
`another
`
`ICI 164.384, studied earlier
`analogue,
`(ll). These data suggest the possibility
`that pure steroidal antiestrogens may be
`effective
`in
`some
`tamoxifen-resistant
`patients.
`In the present study, we have inves-
`tigated the preclinical activity of ICI
`l82,780 in more detail. We compared the '
`inhibitory effects of ICI 182,780, tamox-
`ifen, and estrogen withdrawal on the
`
`‘Afliliarions of authors: C. K. Osborne, E. B.
`Coronado-Hcinsohn. S. G. Hilscnbeclz. B. L.
`McCue, Department of Medicine, Division of Medi-
`cal Oncology. The University of Texas Health
`Science Center at San Antonio. San Antonio. Tex.
`A.
`E. Walteling, hneca
`Pharmaceuticals,
`Mereside Alderley Park, Macclestield, Cheshire.
`England. U.K.
`R. A. McClelland. D. L. Manning. R. l. Nichol-
`son. Tenovus Cancer Research Center. University of
`Wales College of Medicine, Heath Park, Cardiff,
`Walcs.U.K.
`'
`Correspondence to: C. Kent Osborne. MD.,
`Department of Medicine. Division of Medical On-
`eulogy, The University of Texas Health Science
`Center at San Antonio, ‘I703 Floyd Curl Dr., San
`Antonio, TX 78284-7884.
`See “Notes” section foliowing “Ret'erenoes."
`
`746 REPORTS
`
`Journal of the National Cancer institute, Vol. 87, No. 10, May I7, 1995
`
`|nnoPharma Exhibit 10390001
`
`

`

`growth of established tumors and on
`tumorigenesis in a model system that uses
`estrogen-dependent,
`human MCF-7
`breast
`tumor cells growing in athymic
`nude mice. We also studied the hormonal
`
`responsiveness of tumors that had be-
`come resistant to the two estrogen an-
`tagonists and the effects of these drugs on
`estrogen-regulated gene expression.
`
`Materials and Methods
`
`Nude Mouse Model System
`
`ER-positive MCF-7 human breast cancer cells
`(passage I00-200) were cultured as described pre-
`viously (I2). The athymic nude mice used in these
`experiments were 4- to 5-week-old female castrated
`BALB/c-nu‘/nu‘ mice purchased from Harlan
`SpragueDawley_
`lnc.
`(Madison. Wis.). The
`methods for maintenance artd housing of the mice
`and for growing MCF-7 tumors from cell suspen-
`sions and from tumor transplants have been pub-
`lished in detail (3,7). Animal care was in accordance
`with institutional guidelines.
`Approximately 5 X 106 MCF-7 cells were in-
`jected subcutaneouslyinto the flanks, just under the
`forelimb. of female nude mice to initiate tumor for-
`mation. Estrogen supplementation was provided in
`the fomr of a 0.25-mg estradiol (E2) pellet (Innova-
`tive Research. Rocltville, Md.) placed subcutaneous-
`ly in the interscapular region of the mice. The
`effects of tamoxifen and ICI 182,780 on the growth
`of established tumors were studied after the tumors
`had reached a size of 8-H) mm (3-5 weeks). At this
`time, the animals were randomly allocated into four
`treatment groups: 1) continued estrogen supplemen-
`tation. 2) removal of the E; pellet. 3) removal of the
`E, pellet plus treaunent with 500-pg tamoxifen
`citrate (Zeneca Pharmaceuticals, Wilmington, Del.)
`in peanut oil (injected subcutaneously each day,
`Monday through Friday), or 4) removal of the E,
`pellet and treatment with the indicated doses of
`lCl l82.780 (Zeneca Pharmaceuticals, Maccleslield,
`England) in castor oil (subcutaneous injections once
`a week).
`Initial dose-response studies with ICI
`l82,780 were performed in the presence of con-
`tinucd estrogen supplementation. Tumor growth was
`assessed. and tumor volumes were measured twice a
`week as described previously (I2).
`In tumorigenesis experiments. various treatments
`were begun on the same day tumor cells were in-
`jected. Inoculated mice were randomly allocated im-
`mediately into four treatment groups: I) estrogen
`supplementation. 2) 500 pg tamoxifen once a day.
`Monday through Friday, 3) 5 mg [Cl l82,780 once a
`week. or 4) drug vehicle (peanut oil andlor castor
`oil). Tumor volumes were measured twice a week.
`To investigate the honnonal responsiveness of
`tumors that had become resistant to ICI 182,780,
`mice with resistant rumors were killed by cervical
`dislocation. and the tumors were resected and cut
`into l-mm’ fragments. The fragments were then
`uansplanted subcutaneously on the flank just under
`the forelimb of new 4- to 5-week-old recipient mice
`that were then treated with estrogen, tamoxifen. lCl
`l 82.780. or vehicle alone.
`
`Estrogen and Progesterone Receptor
`Assays
`ER content was determined in minors homo-
`
`genized in 0.4 M KCl—Tn's buffer, usingthe ER an-
`tibody kit (ER-EIA; Abbott Laboratories. North
`Chicago. 01.). Progesterone receptor (PgR) levels
`were measured by a ligand-binding, dextrarrcoated
`charcoal method (3).
`
`mg to 10.0 mg. Inhibitory activity was
`modest with doses of 0.5 mg or 1.0 mg.
`while more dramatic—but approximately
`equivalent--inhibitory effects were ob-
`served with 5.0-mg and 10.0-mg doses
`(data not shown). For subsequent experi-
`ments, a dose of 5.0 mg per mouse, given
`once a week, was used.
`
`Estrogen-Regulated Gene Expression
`
`Expression of the estrogen-responsive genes.
`pLlVl and pS2. was detennined by nonhcrn blot
`
`using comzplementary DNA (cDNA)
`analysis,
`probes labeled with [3 Pldeoxycytidine ltiphosphale
`(SOW Cilmmolz Amcrsham Lttl.. Amcrsham.
`England, U.K.) by the random-priming method as
`described previously (13). Briefly. total RNA was
`obtained from the ntrnors of treated mice by cell
`lysis in 4 M zflflnidinium thiocyanate and 1% 2-mer-
`captoethanol and ccntrifugation through 5.7 M
`caesium chloride (Beckman L-30 ultracentrifuge,
`SW50 rotor. 34000 rpm at 20 ‘C for I7 hours).
`Purified samples were stored in R.Nase-free water at
`-70 ‘C before electrophoresis (I0 ttgllane). blotting.
`and hybridization. Densitometric analysis of auto-
`radiographs was performed using a model 620 video
`densitometer
`(Bio-Rad Laboratories, Richmond.
`Calif.), and values obtained were con-acted for
`equivalence of RNA loading by comparison with the
`signals generated using a CDNA probe to human
`glyceraldeyhyde
`3-phosphate
`dehydrogenase
`(G3PDH) (Clontech Laboratories. Inc., Palo Alto.
`Calif.).
`Recorded densitometry values represent the area
`of peak values obtained. following background sub-
`traction. from equivalently exposed autoradiographs
`(where x = band width in mm and y = optical den-
`sity value). Hybridizations of each set of filters in
`the study were carried out simultaneously with the
`same labeled probes. The reported values represent
`means of groups, and at
`least
`two separate
`hybridization: of different filters were performed for
`each probe (stripping the previous probe with high-
`stringency washes and checking for clearance by
`autoradiography).
`
`Statistical Analysis
`
`Analyses were performed using either the Knis-
`kal-Wallis one-way analysis of variance (when
`there were more than two groups) or the Wilcoxon
`signed rank test for two samples. All statistical tests
`were two-sided.
`
`Results
`
`ICI 182,780 Dose-Response
`
`lCl l82,780 inhibited estrogen-induced
`growth of MCF-7 tumors in a dose-de-
`pendent manner. Estrogen-supplemented
`mice with established MCF-7 tumors
`
`were randomly allocated to receive either
`continued estrogen treatment or estrogen
`treatment plus injections of ICI l82,780
`once a week in doses ranging from 0.5
`
`Effect of Estrogen Withdrawal,
`Tamoxifen, and [CI 182,780 on
`MCF-7 Tumor Growth
`
`Treatment of mice by removal of the
`F4 pellet alone or with tamoxifen or ICI
`182,780 significantly inhibited MCF-7
`tumor growth (Fig. 1). In this experiment,
`tumor volumes remained stable for nearly
`100 days after estrogen withdrawal before
`progression ensued.
`In contrast,
`tumor
`volumes decreased slightly with tamoxi-
`fen and ICI 182.780 treatment, and tumor
`size remained stable for variable periods
`of time. A consistent observation was the
`
`delayed time to progression that was evi-
`dent
`in mice treated with ICI 182,780.
`With estrogen withdrawal alone or with
`tamoxifen, tumors developed resistance,
`and progression was evident in all mice
`after 3-4 months of treatment (median, 97
`and 104 days, respectively). However, the
`median time to progression was nearly
`twice as long with ICI 182,780. and the
`growth of some tumors remained con-
`trolled for extended periods of time
`(median, 200 days). In fact, two of the 10
`tumors from ICI 182,780-treated mice
`still had not progressed after 11 months
`and one small tumor (4 mm diameter)
`completely regressed and did not reap-
`pear during the course of the experiment
`(data not shown).
`
`Effect of [CI 182,780 on Tumorigenesis
`
`ICI l82,780 also had a greater impact
`on tumor formation in mice in which drug
`treatments were begun on the day of
`tumor cell
`inoculation (Fig. 2). Tumors
`grew rapidly in mice treated with es-
`trogen. Tumor growth was substantially
`delayed in mice treated with tamoxifen,
`but after 2 months. the growth rate in-
`creased. Tumors grew very slowly. or not
`at all, in mice treated with ICI 182.780-
`similar to the growth pattern observed in
`estrogen-deprived mice (12). By day 70,
`barely measurable tumors were present in
`the majority of mice. In another experi-
`ment (data not shown), three of six mice
`
`Journal of the National Cancer Institute, Vol. 87, No. l0, May I7, 1995
`
`REPORTS
`
`747
`
`|nnoPharma Exhibit 10390002
`
`

`

`1C1 182,780 were transplanted into new
`castrated, recipient mice that were then
`treated with estrogen,
`tamoxifen,
`ICI
`182,780, tamoxifen plus ICI 182,780, or
`vehicle alone. This experiment was con-
`ducted five times with different
`tumor
`
`transplants, and a representative result is
`shown in Fig. 3. Transplanted tumor frag-
`ments. grew well in all mice, even those
`treated with vehicle alone (-154), suggest-
`ing estrogen indepcndence. However, in
`four of five experiments, tumor growth
`was slightly increased by estrogen treat-
`ment
`(+E¢),
`indicating continued sen-
`sitivity to the hormone. As expected,
`growth of these transplanted 1C1 182,780-
`resistant
`tumors was also observed in
`
`recipient mice treated with [C1 182,780.
`Interestingly, in four of the five experi-
`ments, treatment of recipient mice with
`tamoxifen
`alone
`or
`tamoxifen
`plus
`ICI 182,780 resulted in a slight retarda-
`tion of tumor growth compared with
`treatment
`using
`vehicle
`alone
`or
`1C1 182,780 alone, although the observed
`differences in the individual experiments
`were modest and not statistically sig-
`nificant. A total of six of the 25 mice in
`
`.
`
`these experiments showed slower tumor
`growth with tamoxifen treatment, indicat-
`ing some heterogeneity among the trans-
`planted
`fragments
`in
`response
`to
`tamoxifen. However, most mice resistant
`
`to ICI 182,780 showed cross—resistance to
`tamoxifen.
`
`Resistance to ICI 182,780 was not due
`to a complete loss of tumor ER, although
`treatment with this drug reduced expres-
`sion of both ER and PgR. Tumors har-
`vested 4 weeks after initiating treatment
`with ICI 182,780 (ER = 37 1 3 fmol/mg
`protein; PgR = 27 :l: 7 fmol/mg protein)
`as well as those harvested at the time of
`resistance to 1C1 182,780 (ER = 16 :1: 4
`fmollmg protein; PgR = 17 :1: 8 fmol/mg
`protein) expressed both ER and PgR at
`markedly reduced levels compared with
`estrogen—treated controls (ER = 208 :1: 81
`fmol/mg protein; PgR = 103 :1: 20
`fmol/mg protein) (P = .024),
`Expression of two estrogen-responsive
`genes, pS2 and pL1Vl, was also mea-
`sured in these tumors (Table 1). pS2 and
`pL1Vl messenger RNA (mRNA) expres-
`sion was reduced by 20%-74% in tumors
`from tamoxifen-treated mice (P = .013).
`It is interesting that pS2 and pL1Vl ex-
`pression remained suppressed even after
`
`Fig. 1. Effects of estrogen (estradiol [E2]) withdrawal. tamoxifen and 1C! 182.780 on MCF-7 tumor growth.
`Estrogen-supplemented mice were inoculated with MCF-7 cells. On day 36 when tumor: had formed. mice
`were randomly allocated to treatment by withdrawal of estrogen (-152: -3-): Withdrawal Of esttlozcn and
`treatment with 500 pg tamoxifen given once a day. Monday through Friday (-0-); or 5 mg 1C1 182,780
`given once it week (-I-). Tumor volumes were determined at the times shown. it = 10 mice per group; means
`iSE.
`
`
`
`
`
`‘rumorVolume(mm3)
`
`101 182,780
`
`Tomoxllon
`
`Fig. 2. Effect of estrogen. tamoxifen, and 1C1 182,780 on MCF-7 tumorigenesis. Mice were inoculated with
`MCF-7 cells on day 0 and randomly allocated immediately to receive treatment with a 175 est:-adio1(Bz) pel-
`let (+E2; - —): 500 ttg tamoxifen given once a day. Monday through Friday (-0-); or 5 mg 1C1 182,780
`given once a week (-I-). Tumor volumes were determined at the times shown. n = 8 mice per group: means
`:tSE.
`
`treated with 1C1 182,780 failed to grow
`measurable tumors even after 6 months of
`treatment.
`
`ICI 182,780-Resistant Tumors
`
`As indicated above, tumor resistance
`eventually occurred in most, but not all,
`
`mice treated with 1CI 182,780. This resis-
`tance was manifested by regrowth of
`tumors. usually after many months of
`treatment. To investigate the hormonal
`sensitivity of these resistant tumors, frag-
`ments of a tumor that had progressed
`after months
`of
`treatment with
`
`748 REPORTS
`
`Journal of the National Cancer Institute. Vol. 87, No. 10, May 17, 1995
`
`|nnoPharma Exhibit 10390003
`
`I
`
`101 182,780
`
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`Tlflloalfenolcll 02,780
`Tamoxifen
`
`than treatment by estrogen withdrawal
`alone or with tamoxifen. Finally, expres-
`sion of the estrogen—regulated genes p82
`and pLIVl was nearly abolished by neat-
`ment with ICI 182,780.
`
`Previous reports by us and by other in-
`vestigators (7,1l,l4-I6) have also shown
`that the growth of tumors with acquired
`tamoxifen resistance can be inhibited or
`
`blocked by treatment with a pure an-
`tiestrogen such as ICI 182,780, suggest-
`ing that the pure antiestrogens work by a
`different mechanism of
`action
`than
`
`tamoxifen and other similar antiestrogens. A
`Tamoxifen resistance in our model sys-
`tem is associated with drug—induced
`tumor growth stimulation that occurs
`after an initial period of growth suppres-
`sion (3). The ability of tamoxifen alone to
`stimulate the growth of these tumors is
`less than that of estrogen. Interestingly,
`when combined with estrogen, tamoxifen
`can
`still
`inhibit
`estrogen-stimulated
`growth,
`indicating that
`it continues to
`possess both estrogen-agonist and an-
`tagonist properties (7). The increasingly
`dominant agonist properties of tamoxifen
`that develop after prolonged treatment
`can be blocked by the addition of pure
`antiestmgens
`(7,ll).
`Evidence
`for
`tamoxifen-stimulated tumor growth as a
`mechanism for acquired tamoxifen resis-
`tance in patients has also been presented
`(5,6.17). On the basis of these preclinical
`studies, it has been suggested that treat-
`ment with lCl
`|82,780 might
`induce
`tumor regression in some patients who
`have developed tamoxifen resistance.
`One recent study (18) has shown that
`short-term lCl
`182,780 treatment of
`patients who have ER-positive minors
`causes statistically significant reductions
`in the Ki67 labeling index and reductions
`in the expression of estrogen-regulated
`genes such as PgR and pS2. In addition,
`remissions have now been reported in
`tamoxifen-resistant patients treated with
`this drug (19).
`Although ICI 182,780 controls MCF-7
`tumor growth for longer durations than
`tamoxifen, eventual
`resistance to this
`agent
`is common. MCF-7 tumors that
`progress after prolonged treatment are
`estrogen-independent (grow in the ab-
`sence of estrogen supplementation) al-
`though they are still estrogen-sensitive
`(growth is enhanced by estrogen). The
`mechanisms
`by which resistance
`to
`
`Fig. 3. Hormonal sensitivity of ICI l82,780-resistant tumors. Fragments of a tumor that had developed resis-
`tance in a mouse treauzd with ICI l82.780 were transplanted into new recipient ferrule castrated nude mice.
`The recipient mice were then randomly allocated to receive vehicle alone (--E2; 45! -). an esttadiol (E2) pellet
`(-r-E2; -9-); tamoxifen alone (-A-); [Cl l82.780 alone (-Q). or I combination of tamoxifen plus ICI 182,780
`(-I-). Tumor volume were calculated on the days shown. it = 6 mice per group; means :tSE.
`
`evolution to tamoxifen resistance when
`the drug was stimulating tumor growth
`(3). In fact, p82 was significantly lower
`in tamoxifen-resistant
`tumors than in
`
`tamoxifen-sensitive tumors (P = .012).
`This finding suggests that the agonist ac-
`tivity of tamoxifen,
`if responsible for
`tamoxifen-stimulated tumor growth, may
`be specific to genes associated with cell
`proliferation, while its antagonist activity
`continues to suppress the activity of
`genes less crucial for tumor survival. in
`contrast
`to the results obtained with
`
`tamoxifen, mRNA expression was nearly
`
`Table 1. Exprusion of estrogen-satsitive genes‘
`
`Treatment group
`(No. of blots analyzed)
`
`Gene. relative
`mRNA level
`
`pS2
`
`pl.lVl
`
`Estrogen (4)
`Tamoxifen-sensitive (5)
`Tamoxifen—tesistant (5)
`lCl-sensitive (5)
`ICI-resistant (8)
`
`I22 1 0.7
`9.8 :t 0.5
`3.2 1 0.4
`0.3 :l: 0.05
`0.6 10.23
`
`12.2 1 0.6
`6.0 1 l .5
`7.5 :t I .8
`0 t 0
`2.3 1 L3
`
`‘rnRNA expession was meuured by northern
`blot analysis of total RNA extracted from MCF-7
`tumors taken from mice treated with estrogen (con-
`trols). tamoxifen for 3 weeks (tarnoxifen-sensitive).
`tamoxifen until
`the time of tumor progression
`(tamoxifen-resistant), ICI I82. 780 for 4 weeks (ICI-
`sensitive). or [Cl
`l82,780 until tumor progression
`(ICI-resistant). Values shown are the means 1 SE of
`scanning densitometry units corrected for RNA
`loading.
`
`abrogated by treatment with ICI 182,780
`(P<.004), and there was no difference be-
`tween sensitive and resistant tumors. It is
`
`unlikely, therefore, that ICI 182,780 resis-
`tance is caused by metabolic conversion
`of the drug to E2, since expression of
`these estrogen-regulated genes remained
`low.
`
`Discussion
`
`Clinical data demonstrate that, in some
`patients, the current endocrine therapies
`for breast cancer result
`in temporary
`tumor regression or growth stabilization.
`followed by tumor
`regrowth, usually
`within 6-18 months of treatment. We
`
`have developed an experimental in vivo
`model that mimics this clinical scenario.
`
`Our data suggest that, in this experimen-
`tal model system, ICI 182.780 possesses a
`greater ability to suppress estrogen—sensi-
`tive gene expression and greater an-
`titumor activity than the partial estrogen
`antagonist tamoxifen. In addition, MCF-7
`tumorigenesis was significantly delayed
`by [C1
`l82,780 when compared with
`tamoxifen. Moreover, a proportion of
`treated mice failed to develop tumors
`even after prolonged follow-up, an event
`rarely encountered in our experience
`treating mice with tamoxifen. lCl 182,780
`also suppressed growth of established
`tumors for a significantly longer duration
`
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`References
`
`mary breast cancer. Eur J Cancer 29A:l462-
`I468. I993
`(14) Briinner N. Frandsen TL. Holst-Hansen C. et
`al: MCP7/LCC2: a 4-hydroxytamoxifen resis-
`tant human breast cancer variant that retains
`sensitivity to the steroidal antiestrogen ICl
`182.780. Cancer Res 53:3229-3232. i993
`(I5) Lyltkesfeldt AE. Sotettsen EK: Effect of
`estrogen and amiestrogens on cell proliferation
`and synthesis of secreted proteins
`in the
`human breast cancer cell lines MCF-7 and a
`tamoxifen resistant variant subline. AL-l.
`ActaOncoI3l:l3l-l38.l992
`(I6) Parker MG: Action of pure antiestrogens iii in-
`hibiting estrogen receptor action. Breast Can-
`cer Res Treat 26: l 3|-I37. 1993
`(I7) Wiebe VJ. Osborne CK. Fuqun SA. et al:
`Tamoxifen resistance in breast cancer. Crit
`Rev Oncol Hemalol 14:173-188. 1993
`(I8) DeFt-lend DJ. Howell A. Nicholson Rl. et al:
`Investigation of a new pure antiestrogen (ICI
`182780) in women with primary breast cancer.
`Cancer Res 54:408-414. I994
`(19) DeFr-iend DJ. Blarney RW. Robertson JF. et
`al: Response to the pure antiestrogen ICl
`182780 after tamoxifen failure in advanced
`breast cancer. Breast Cancer Res Treat I7: 136.
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`(20) Gibson MK. Nemmels LA. Beelurutn WC Jr.
`et al: The mechanism of ICI
`I6-1.384 an-
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`receptor
`in uterine tissue. Endocrinology
`l29:2000-2010, I99!
`(2!) Love RR. Mazess RB. Bastien HW. et al: Ef-
`fects of tamoxifen on bone mineral density in ‘
`postmenopausal women with breast cancer
`[see comment citation in Medlitte]. N Engl J
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`(22) Love RR. Newcombe PA. Wiebe DA. et al:
`Efiects of tamoxifen‘ therapy on lipid and
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`(23) Fomander T. Hellsubm AC. Moberger B:
`Descriptive clinicopathologic study of
`I7
`patients with endometrial cancer during or
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`
`Notes
`
`ICI 182.780 develops are not clear. but
`reduced levels of ER and reduced expres-
`sion of estrogen-regulated genes (com-
`pared with tamoxifen-sensitive or with
`tamoxifen-resistant tumors) are evident.
`Reduced ER levels have also been seen in
`
`treated with
`from patients
`tumors
`ICI 182,780.
`in cultured breast cancer
`cells; and in mouse uterine tissue follow-
`ing the administration of the prototype
`pure antiestrogen ICI 164,384 (18-20).
`Other data suggest
`that
`the pure anti-
`estrogen-ER complex may be more
`fragile and more susceptible to receptor
`degradative pathways (16).
`in contrast,
`ER levels are high in tamoxifen-resistant
`tumors obtained with our model system
`(3). On the basis of our data. we would
`predict
`that most
`patients
`with
`ICI 182,780-resistant tumors would not
`respond well
`to subsequent
`treatment
`with tamoxifen.
`
`Even if pure antiestrogens are shown to
`have
`superior
`antitumor
`activity
`in
`women with breast cancer, they may not
`be the optimal antiestrogens for clinical
`use. The estrogenic properties of ta-
`moxifen in bone and on blood lipids may
`help to reduce bone loss and prevent car-
`diovascular disease. which are added
`benefits when treating breast cancer
`patients
`for prolonged periods
`after
`surgery for primary tumors or for breast
`cancer prevention (21,22). The effect of
`ICI l82,78O on these parameters is not
`yet known, but
`it might be deleterious
`given its lack of estrogenic qualities.
`However.
`treatment with [Cl 182.780
`might not be associated with the in-
`creased risk of endometrial cancer recent-
`ly attributed to tamoxifen (23). Further
`clinical study of pure antiestrogens in
`tamoxifen-resistant
`and in tamoxifen-
`
`(1) Systemic treatment of early breast cancer by
`hormonal. cytotoxic. hr immune therapy. I33
`randomised trials involving 3l.000 recurrences
`and 24,000 deaths among 75.000 women.
`Early Breast Cancer TI-ialists' Collaborative
`Group [see comment citations in Medline].
`Laneet339:l-15.71-85. 1992
`(Z) Encamacion CA. Cioeca DR. McGuire WL. et
`al: Measurement of steroid hormone receptors
`in breast cancer patients on tamoxifen. Breast
`Cancer Res Treat 26:237-246. I993
`(3) Osborne CK. Coronado E. Allred DC. et a1:
`Acquired tamoxifen resistance: correlation
`with reduced breast tumor levels of tamoxifen
`and isomeriution of trans-4-hydroxytnmoxi-
`fen [see comment citation in Medline]. J Natl
`Cancer Inst 83: l477- I482. I99!
`(4) Gottardis MM. Jordan VC: Development of
`tamoxifen-stimulated
`growth
`of MCF-7
`tumors in athymic mice after long-term anti-
`estrugen administration. Cancer Res 48:5l83-
`Sl87. 1988
`(5) Pritchttrd Kl. Thomson DB. Myers RE. el al:
`Tamoxifen therapy in ptemenopausal patients
`with metastatic breast cancer. Cancer Treat
`Rep 64:787-796. I980
`(6) Hoogstratut B. Gad-el-Mawla N. Maltmey TR.
`et alt Combined modality therapyforfirst recur-
`rence of breast cancer. A Southwest Oncology
`Group Study. Cancer 542248-2256. I984
`(7) Osborne CK. Jsrman M, Mccague R. et al:
`The importance of tamoxifen metabolism in
`tamoxifen-stimulated breast
`tumor growth.
`Cancer Chemother Phatrnacol 34:89-95. -I994 '
`(8) Wakeling AE. Bowler J: Novel antioesttogens
`without partial agonist activity. J Steroid
`Biochem 3| :645-653. I988
`(9) Walreling AB. Dukes M. Bowler J: A potent
`specific pure anliestrogen with clinical poten-
`tial. Cancer Res 5| 5867-3873. 1991
`(10) Wakeling AE: steroidal pure antiestrogens. In
`Regulatory Mechanisms
`in Breast Cancer
`(Lippman M. Dickson R, eds). Boston: Kluwer
`Acad Pttbl. t99t. pp 239.257
`(/1) Gotutrdis MM. Jiang SY. Jeng MH. et al: In-
`hibition of tamoxifen-stimulated growth of an
`MCF-7 tumor variant
`in athymic mice by
`novel
`steroidal antiestrogens. Cancer Res
`49:4090-4093. I989
`(I2) Osborne CK. Hobbs K. Clark CM: Effect of
`estrogens and antiestrogens on growth of
`human breast cancer cells in athytnic nude
`Supported in part by Public Health Service grants
`CA3025l. CA30l95. C/£58183. and CA54l74 from
`mice. Cancer Res 45:584-590. I985
`the National Cancer Institute. National Institutes of
`(13) Manning DL. McClelland RA. Cree JM. et sl:
`Health. Department of Health and Human Services.
`The role of four oestrogen-responsive genes.
`Manuscript received September 19. 1994; revised
`pLIVl. p52. pSYD3 and pSYD8, in predicting
`naive patients is clearly indicated.
`December 27. I994; accepted March 6. I995.
`responsiveness to endocrine therapy in pri-
`
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`750 REPORTS
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`Journal of the National Cancer Institute. Vol. 87. No. l0. May 17. I995
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