Responses to Pure Antiestrogens
`(ICI 164384, ICI 182780) in
`Estrogen-Sensitive and -Resistant
`Experimental and Clinical Breast
`Cancer“
`
`R. I. NICHOLSON,” J. M. w. GEE,” D. L. MANNING,”
`A. E. WAKELING,” M. M. MONTANO,d AND
`B. s. KATZENELLENBOGENd
`bTenovus Cancer Research Centre
`University of Wales College of Medicine
`Tenovus Building
`Heath Park
`
`Cardifi CF4 4XX, United Kingdom
`
`cZeneca Pharmaceuticals
`Macclesfield, United Kingdom
`dDepartment of Physiology and Biophysics
`University of Illinois
`Urbana, Illinois
`
`INTRODUCTION
`
`The last ten years has seen the emergence of a new class of pharmacological
`agents termed pure antiestrogens (reviewed in Refs. 1, 2). These compounds, which
`were originally discovered by ICI Pharmaceuticals Division (now Zeneca Pharmaceu-
`ticals) in the UK, have the unique property of binding to the estrogen receptor (ER),3
`producing a receptor complex which lacks estrogenic activity.“'5 They are of use in
`two important areas of breast cancer research. Firstly, as clinical agents, where it is
`hoped that their ability to induce total estrogen deprivation will improve the effective—
`ness of endocrine therapy. Secondly, as pharmacological probes to investigate the
`cellular and molecular actions of estrogens and tamoxifen. Inherent in each of these
`areas of research are questions associated with the impact pure antiestrogens may
`have on the therapy of endocrine-resistant states and whether resistance develops as
`a consequence of incomplete estrogen withdrawal; with tumor cells more efficiently
`utilizing either a reduced estrogenic pool or the agonistic activity of an antiestrogen,
`
`“The authors gratefully acknowledge the financial support of the Tenovus Organisation (RIN,
`JMWG, DLM), the Association for International Breast Cancer Research (RIN), the National Institutes
`of Health, and the Susan G. Komen Foundation (BSK).
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`or whether the resistant cells have completely circumvented the need for ER-mediated
`growth and hence sensitivity to pure antiestrogens.2
`Since pure antiestrogens are now entering clinical development, the current paper
`seeks to outline some of their basic cellular and antitumor properties on estrogen-
`responsive (MCF—7) human breast cancer cells in vitro, primarily using the lead
`compound ICI 164384. This information will be briefly compared with the properties
`exhibited by pure antiestrogens in endocrine-resistant variants of human breast cancer
`cells (see refs.
`in Ref. 2) and phase I and II trials of ICI 182780 in primarys'6
`and advanced7 breast cancer patients. Where possible examples will be given from
`immunohistochemical studies, since this technique is most readily applicable to
`clinical material and ultimately should facilitate an assessment of the degree to which
`pure antiestrogens are fulfilling their potential as complete antagonists of estrogen
`action in clinical breast cancer and thereby aid in defining the importance of estrogens
`in the regulation of breast cancer growth and development.
`
`Experimental Studies with Pure Antiestrogens
`
`Biological Consequences of Exposure of Breast Cancer Cells In Vitro to
`Pure Antiestrogens
`
`Evidence from breast cancer cells grown in culture suggests that pure antiestrogens
`may be highly efficient in counteracting the stimulatory effects exhibited by estrogens
`both on cell proliferation and on steroid hormone-regulated gene expression.
`One of the most important early observations arising from the functional dis—
`ablement of ER signalling by pure antiestrogens in estrogen—sensitive human breast
`cancer cell lines was that, in contrast to the stimulatory activity of estradiol, treated
`cells became efficiently growth arrested (FIG. 1A).8‘10 This action is reflected in the
`growth dynamics of the tumor cells, with several groups showing that while estradiol
`increases the tumor cell growth fraction and acts to stimulate the passage of cells
`through the cell cycle, pure antiestrogens promote a highly effective restriction of
`the cell cycle approximately 5 hours into 61 and hence cause a reduction of the
`proportion of cells undergoing DNA synthesis."’9 On continuous exposure to pure
`antiestrogens there is almost a complete loss of those nuclear antigens which mark
`cell proliferation (FIG, 1B,C)10 as a large proportion of the cells pass into a noncycling
`population.9 Such cells show a reduced RNA/DNA ratio, characteristic of Go (Nichol-
`son, Francis and Hoy, unpublished results). It is noteworthy that the growth-inhibitory
`activity of pure antiestrogens is not solely restricted to cytostatic activity; on continu-
`ous exposure there also appears to be a limited cytotoxic component.5
`The growth~inhibitory activity of pure antiestrogens on human breast cancer cell
`lines is characteristically preceded by changes in the expression of several estrogen-
`regulated genes,”H4 with, for example, the high levels of nuclear immunodetectable
`PR induced in estradiol—treated MCF—7 cells being rapidly reversed by a lOO-fold
`molar excess of ICI 164384 (FIG. 1D).5 Indeed, examination of the percentage of
`PR-positive cells throughout estradiol and ICI 164384 treatment shows that not only
`does the pure antiestrogen block estradiol-induced PR levels, but that it also obliterates
`all PR signalling after 4 days of culture. Such cells are as a consequence no longer
`responsive to progesterone.
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`ANNALS NEW YORK ACADEMY OF SCIENCES
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`A
`Cell numbers /1000
`
`1.000
`
`B
`96 K167+ cells
`
`‘00
`
`Oestradiol
`(10~9M)
`
`600
`
`400
`
` 800
`
`Oestradiol
`+
`ICI 164384
`(1o-7M)
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`200
`
`0
`
`80
`
`60
`
`40
`
`20
`
`O
`
`E2
`
`E2 +
`164
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`6
`
`Days in culture
`C
`Proliferafive Index
`
`Days in culture
`D
`% PgR-ICA + cells
`
`40
`
`30
`
`20
`
`1°
`
`o
`
`E2
`
`E2 +
`164
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`100
`
`80
`
`60
`
`40
`
`2°
`
`o
`
`E2
`
`E2 +
`164
`
`O
`
`1
`
`2
`
`3
`
`_
`4
`5
`
`6
`
`7
`
`8
`
`Days in culture
`
`Days in culture
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`Similarly, the substantial increase in cytoplasmic p82 immunostaining (a protein
`of unknown function in the breast) that is induced by estradiol in human breast cancer
`cells is largely absent following ICI 164384 treatments Any residual p52 staining
`tends to be present towards the outer cell membrane in small secretory vesicles.
`Once secreted, however, the cells remain negative, with no evidence of further p52
`production within the endoplasmic reticulum.
`Predictably, the decrease in estrogen-regulated proteins often parallels a highly
`significant fall in their mRNA levels,5'“’13‘l4 with, for example, p52 mRNA levels
`being undetectable following 5 days of ICI 164384 treatment.5 Indeed, even after
`reverse transcription PCR (30 cycles), the pS2 mRNA has been shown to be barely
`detectable in ICI 164384— and ICI 182780-treated cells,5 indicating that pure antiestro-
`gens can produce a rapid and complete shutdown of estrogen-regulated gene function.
`These actions contrast with the effect of both ICI 164384 and ICI 182780 on the
`
`estrogen-suppressed gene sequence pMGT—l, the expression of which is very signifi-
`cantly upregulated in their presence.15
`A number of the changes in gene expression may directly contribute to the
`mechanism of action of the drugs, with ICI 182780 promoting decreases in immunode—
`tectable TGFa, an estrogen-inducable mitogenic growth factor,16 and the bc1—2 protein,
`a factor which has been implicated in the protection of cells against programmed
`cell death.17 In each instance, while these proteins are readily detectable in a high
`proportion of cells treated by estradiol, their levels are lowered by estrogen withdrawal
`and further reduced by the pure antiestrogen.5 This is especially evident for the
`bcl—2 protein, with estradiol-related immunostaining being largely abolished by ICI
`182780. Indeed, bcl-2 positivity is a relatively rare event following the administration
`of pure antiestrogens and, in line with its role in cell survival, its absence is often
`associated with the presence of pyknotic tumor cell nuclei.5
`
`Comparison with Antiestrogens Exhibiting Partial Estrogen-like Activity In Vitro
`
`The inhibitory actions of pure antiestrogens, initially on estrogen—induced tran-
`scriptional events and subsequently on cell proliferation and survival, consistently
`exceed those effects which may be achieved by established antiestrogens with partial
`estrogen~like activity.
`A comparison of the potency and efficacy of tamoxifen, ICI 164384 and ICI
`182780 as inhibitors of the growth of MCF-7 cells showed that the pure antiestrogens
`are up to two orders of magnitude more 130tent,”'21’22 reflecting, in part, their higher
`
`‘——————-—-——-————————-—
`
`FIGURE 1. Growth and immunohistochemical characterization of MCF—7 cells. The cells
`were grOWn in multiwell dishes in white RPMI tissue culture medium with 5% DCC stripped
`FCS (medium A) containing estradiol 1' ICI 164384. (A) Cell numbers were assessed using
`a Coulter Counter; (B,C) K167 and (D) PR assays were performed according to the methods
`of Bouzubar et al.“3 and Press & Greene,19 respectively. The Ki67 proliferative index (c) was
`calculated as the proportion of cells showing intense nucleoplasmic and nucleolar staining
`pattems.20 The results are shown as the mean : SD of six replicates.
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`ANNALS NEW YORK ACADEMY OF SCIENCES
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`affinity for estrogen receptors.3’22 More significantly, flow cytometric analysis of the
`growth dynamics of the cultured cells showed that, although both classes of agent
`share the ability to block cell division in the G1 phase of the cell cycle, both ICI 164384
`and ICI 182780 were more effective than tamoxifen“22 or hydroxyclomipheneB in
`reducing the proportion of cells which remain able to synthesize DNA after prolonged
`exposure. These activities, which are specific for estrogen receptor signalling,10 are
`reflected in the tumor cell growth fractions“ with pure antiestrogens abrogating
`growth responses to tamoxifen.9
`These differences observed between pure and partial antiestrogens and the control
`of tumor cell growth have been ascribed to their interactions with other signalling
`pathways, with the partial agonistic activity of tamoxifen being amplified by the
`presence of growth factors.9 This appears particularly evident for the interaction of
`tamoxifen and insulin/IGF—l, where a modest growth response to the antiestrogen is
`considerably increased by the presence of these factors.""23 Such activity is much
`weaker for ICI 164384, with the compound being more effective than tamoxifen in
`inhibiting the stimulatory activities of IGF-I and TGFor.
`A further feature of the cellular actions of pure antiestrogens which may relate
`to their improved antitumor activity has recently been revealed by studying their
`effects on the expression of estrogen receptorss'w‘24 It has been observed that they
`are associated with a rapid loss of the receptor protein in estrogen receptor-positive
`cells, producing after relatively short periods of time cellular estrogen-receptor nega-
`tivity.5’1°‘24'25 This property contrasts with the increases in ER levels that are seen
`on either estrogen withdrawal or tamoxifen treatments‘10 Recent studies by Fawell
`et al.26 with ICI 164384 have shown that dimerization of the receptor is impaired
`by the pure antagonist and this may result in the pure antiestrogen receptor complex
`becoming more fragile and perhaps more sensitive to the normal processes involved
`in receptor degradation. Certainly, the half-life of the ICI 164384 receptor complex
`appears substantially shorter25 than the half—lives of the estrogen receptor and tamoxi~
`fen receptor complexes.26’27
`
`Comparison with Estrogen Withdrawal In Vitro
`
`Encouragineg and somewhat surprisingly, the effects of pure antiestrogens on
`the growth of estrogen receptor-positive breast cancer cells substantially surpass the
`effects of estrogen withdrawalm’21 This property has been demonstrated by several
`groups, with, for example, ICI 164384 severely impairing the growth of MCF-7 cells
`in phenol red-free medium where the 5% fetal calf serum has been extensively
`stripped of its endogenous estrogens by charcoal absorption, a procedure which
`reduces the level of endogenous estradiol to below 10'12 M.5'10 Once again changes
`in cell numbers correspond to their recorded growth dynamics, with pure antiestrogens
`decreasing tumor cell S-phase fraction and increasing the proportion of cells in Gol
`G1 relative to estrogen withdrawn cells,22
`The local production of hormones by breast cancer cells may play an important
`role in promoting some cell growth and gene expression in estrogen—withdrawn cells.
`The cells would potentially be highly sensitive to these, since estrogen receptor levels
`are elevated following oestrogen withdrawals” However, the actions of locally
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`produced steroids, through the estrogen receptor, would remain vulnerable to the
`antagonistic activity of pure antiestrogens.
`Thus, in charcoal-stripped serum it is possible that some estradiol is formed from
`polar precursors that are inefficiently adsorbed from the fetal calf serum, the most
`obvious candidate being estrone sulphate. Indeed, several groups have shown sulpha-
`tase activity in estrogen-sensitive human breast cancer cell lines which can initiate
`the conversion of estrone sulphate to estradiol.23'30 Such conversions can occur at
`physiological concentrations of estrone sulphate, generating sufficient quantities of
`estradiol to stimulate growth in estrogen—withdrawn cells (0.1 nM estrone sulphate
`significantly increases S-phase fraction). Under these conditions, the nuclear steroids
`are unconjugated estrone and estradiol.29 This sulphatase activity is, however, lowered
`by the presence of ICI 1643 84,2931 with basal PR and p82 immunostaining abrogated,5
`an effect which appears not to be due to a direct inhibition of the sulphatase enzyme,
`but rather to be an indirect ER-mediated response.29
`Furthermore, interactions with other growth signalling pathways potentially am-
`plify the importance of low estradiol concentrations, with several studies demonstra—
`ting that the stimulatory actions of estrogens on tumor cell growth and gene expression
`can be potentiated by EGF/TGFOL, IGF—1 and the FGF family.22’32'34 In the case of
`EGF/TGth signalling, this may occur through the activation of EGF-receptor tyrosine
`kinase activity, which, in its turn, influences key intermediates that interact with ER
`in the nucleus.35 This concept is supported by the recent observations that an EGF-
`specific tyrosine kinase inhibitor36 can inhibit the expression of PR and p52 (FIG.
`2A,B) in estrogen-withdrawn MCF—7 cells in the presence of maintained amounts
`of the estrogen receptor and TGFoz (FIG. 2C,D), and that similar com ounds can
`inhibit the growth of MCF—7 cells.37 Importantly, EGF35 and dopamine3 have been
`shown to activate estrogen receptor signalling in the complete absence of its ligand
`and thus may operate to enhance the constitutive activity of the estrogen receptor in
`breast cancer cells in vitro. The existence of such mechanisms would normally act
`to protect cells against complete estrogen withdrawal. Importantly, pure antiestrogens
`have been shown to reduce the above interactions between estrogen receptor and
`growth factor signalling, generating cells which are desensitized to estrogens, partial
`antiestrogens, growth factors and dopamine.23'35'38 Pure antiestrogens diminish estro—
`gen receptor levels and thus reduce the impact of residual estrogens, and also secondary
`growth factor signalling. Additionally, ICI 182780 has been shown to reduce the
`intracellular levels of TGFa (FIG. 4B), thereby reducing its potential autocrine stimula-
`tion of breast cancer cells. Elevated TGFa immunostaining in estrogen receptor—
`positive breast cancer has been recently shown to be associated with a decreased
`sensitivity of the disease to tamoxifen therapy (39). Its reduction therefore in clinical
`samples might further improve the sensitivity of breast cancer to estrogen withdrawal.
`
`Biological Consequences of Exposure of Tamoxifen or Estrogen-Resistant Breast
`Cancer Cell Model Systems to Pure Antiestrogens
`
`Initial clinical responses in breast cancer patients to endocrine-type therapies are
`all too often overtaken by the development of tumor resistance. This is also the case
`in vitro, where cultured human breast cancer cells gradually gain a comparable
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`A
`PR—ICA HSOOI’O
`
`1
`
`B
`pSZ-ICA HScoro
`
`minus E2
`
`TKI (1 O'5M)
`
`O
`
`2
`
`4
`
`7
`Day: in culture
`
`1O
`
`1 4
`
`0
`
`2
`
`4
`
`7
`Days in Culture
`
`1 O
`
`1 4
`
`0.2
`0.5
`
`0‘9
`
`0.6
`
`0.4
`
`3
`
`2.5
`
`L5
`
`ERJCA H500"
`
`TKI (10‘5M)
`
`TGFa—ICA HScoro
`
`2
`
`1.5
`
`0.5
`
`minus E2
`
`-5
`TKI (1 o M)
`
`0
`
`2
`
`4
`
`7
`Days in Culture
`
`10
`
`14
`
`0
`
`2
`
`4
`
`7
`Day: in Culture
`
`10
`
`14
`
`FIGURE 2. Effect of 4-(3-methylanilino)quinazoline (ZM 163613) on MCF—7 cells. The cells
`were grown on TESPA-coated coverslips in medium A (minus E2) with and without the tyrosine
`kinase inhibitor (TKI). Assays for PR (A), p82 (B), ER (C) and TGFa (D) were performed
`according to the methods of Press & Greene19 (A), Char-pin et (11.50 (B), Walker et (11.51 (C)
`and Nicholson et (11.39 (D). The results are the mean values : SD of 5 replicates from a
`minimum of 2 coverslipsi
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`endocrine resistance following their prolonged exposure to either estrogen withdrawal
`or antiestrogens. This phenomenon tends to be associated with an increase in cell
`growth rate, while retaining the capacity to express estrogen receptors and thus
`potentially to demonstrate estrogen receptor-mediated responses. Studies with T47D
`and MCF-7 estrogen-responsive human breast cancer cells suggest that the mechanism
`underlying the development of tamoxifen resistancelz‘4043 is the outgrowth of subpop-
`ulations of cells whose growth is stimulated by tamoxifen,4°'4‘ rather than the selection
`of cells which are unaffected by tamoxifen.44
`Fortunately, it is likely that such tamoxifen—resistant cells retain a sensitivity to
`the growth—inhibitory actions of pure antiestrogens in vitro,12'13‘45 and thus it is feasible
`that these new compounds may be of clinical value in patients who relapse on
`tamoxifen therapy. In contrast, cross—resistance to several structurally diverse partial
`antiestrogens has been demonstrated in the antiestrogen—resistant LY2 cell line/‘6'47
`A similar resistance mechanism has been identified in vivo in animal model sys-
`tems,“47 with tamoxifen-resistant cells again exhibiting growth inhibition by pure
`aritiestrogens.48‘49 This is associated with a parallel decrease in the intracellular levels
`of estrogen receptors and an associated fall in estrogen-regulated gene products.49
`Furthermore, it is likely that estrogen—resistant breast cancer cells also retain a
`sensitivity to the inhibitory actions of pure antiestrogens.52'53 FIGURE 3A illustrates
`this phenomenon for a MCF—7 variant cell line, K353"55 This cell line was derived
`from estrogen—responsive MCF—7 cells by estrogen withdrawal and no longer shows
`a growth stimulation by estradiol (cmf. response of wild-type cells to oestradiol; FIG.
`3B). Despite this, it is growth inhibited by antiestrogens, with ICI 182780 (10'7 M)
`being more effective than 4—hydroxytamoxifen (10’7 M). Indeed, only two doublings
`of the initial cell number occurred throughout ICI 182780 treatment and contrasts
`with the 7 to 8 doublings recorded under estrogen—treated or -withdrawn conditions.
`Over several experiments the estimated tumor cell doubling time for ICI 182780-
`treated K3 cells is in excess of 150 hours. This compares favorably with the value
`that may be estimated following the treatment of wild—type MCF-7 cells with pure
`antioestrogens (FIG. 3B).
`The actions of ICI 164384 and ICI 182780 cells appear specific for ER signalling
`and may be reversed by the presence of estradiol.53 Indeed, K3 cells appear more
`sensitive to the growth—promoting effects of the steroid than do wild-type cells, with
`10‘9 M and 10’8 M estradiol reversing the effects of 10'7 M ICI 182780, respectively.
`As a possible contributor to the increased sensitivity of K3 cells to estradiol, the
`cells also show a higher basal expression of TGFor (FIG. 4A),53 which, in contrast to
`wild-type cells, is not substantially increased by the presence of estradiol.53 The
`intracellular concentration of TGFa is, however, lowered by the presence of ICI
`182780, but not 4-OHT. Importantly, the reduction in TGFOL levels in pure antiestro-
`gen-treated cells accompanies a substantial fall in their ER content (FIG. 4C,D).53’55
`This action would minimize the opportunity for cross—talk between ER and TGFa
`signalling pathways. Interestingly, K3 cells also show an elevated basal expression
`of p82 (FIG. 3C,D),52 a protein whose gene promoter contains a response element
`for both estrogens and TGFcL.56 Once again, the expression of this protein is efficiently
`reduced by the presence of the pure antiestrogen (FIG. 3C,D).53
`Although less is known about the emergence of breast cancer cells resistant to
`pure antiestrogens, breast cancer cell xenograft studies have indicated longer remission
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`A
`Calls/wolll1 000
`
`400
`
`B
`Cells/well“ 000
`
`400
`
`300
`
`200
`
`100
`
`300
`
`200
`
`100
`
`O
`O 1
`
`4
`
`6
`
`8
`
`10
`
`1 2
`
`1 4
`
`O
`O 1
`
`4
`
`6
`
`8
`
`10
`
`1 2
`
`14
`
`Days in Culture
`C
`psz-ICA HScoro
`2.5
`
`Buy: In Culture
`D
`pSZ-ICA HScoro
`
`1.5
`
`minus E2
`
`0.5
`
`O
`
`2
`
`4
`
`7
`Day: in Culture
`
`1 O
`
`1 4
`
`0
`
`2
`
`4
`
`7
`Days in Culture
`
`1 0
`
`‘l 4
`
`FIGURE 3. Growth and immunohistochemical characterisation of K3 and wild type (Wt)
`MCF—7 cells K3 (A) and Wt (B) cells were grown in multiwell dishes in medium A containing
`no additives (minus E2), estradiol (10'9 M; E2), 4 hydroxytamoxifen (10‘7 M; 4-OHT) and
`ICI 182780 (10‘7 M; 182). Cell numbers were asseSSCd using a Coulter Counter. K3 (C) and
`Wt (D) cells were grown on TESPA-coated coverslips in medium A containing no additives,
`4-OHT (10—7 M) and 182 (10—7 M) and assayed for p52.50 The results are the mean value x
`SD of 5 replicates from a minimum of 2 coverslips.
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`
`A
`TGFa HScoio
`
`2.5
`
`B
`TGFa-ICA HScoro
`
`2.5
`
`1.5
`
`1.5
`
`0.5
`
`0.5 o
`0.5 O
`
`O
`
`2
`
`4
`
`7
`
`1 O
`
`1 4
`
`O
`
`O
`
`2
`
`4
`
`7
`
`1O
`
`1 4
`
`Days in Culture
`
`Days in Culture
`
`ER-ICA HSoore
`
`3
`
`ER-ICA HScom
`
`1.5
`
`2
`
`4
`
`7
`
`1O
`
`14
`
`Day: in Culture
`
`Days in Culture
`
`FIGURE 4. Immunohistochemical characterization of K3 and Wt MCF-7 cells. K3 (A,C) and
`Wt (B,D) cells were grown on TESPA-coated coverslips under conditions as described in
`FIGURE 3C,D and assayed for TGFa (AB) and ER (CD). The results are the mean value :
`SD of 5 replicates from a minimum of 2 coverslips.
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`times with pure antiestrogens than with either tamoxifen or estrogen withdrawal.49
`Moreover, where ICI 182780-resistant tumors have been transplanted into castrated
`mice, their growth has been shown to be slightly impeded by tamoxifen alone or in
`combination with ICI 182780. Such data imply that true cross-resistance to these
`agents has not fully developed in this animal model, despite the fact that pure
`antiestrogens abrogate ER levels.49 Interestingly, this may not be the case in vitro,
`since cultured MCF—7/LCC9 ICI 182780 resistant cells are also tamoxifen resistant.45
`
`Properties of Pure Antiestmgens in Clinical Breast Cancer
`
`Antitumor Activity
`
`Although clinical trials with pure antiestrogens are in their infancy and conse-
`quently little is known about their clinical properties, in late 1991 a Phase I study
`of ICI 182780 (6 or 18 mg/day in a short acting propylene glycol-based formulation)
`was initiateds‘6 The purpose of this study was to assess the safety and pharmacokinetic
`properties of the drug and to begin to investigate its biological effects on tumor
`tissue. The latter was achieved by measuring a number of immunohistochemical end
`points on pretreatment needle core biopsies from newly diagnosed primary breast
`cancer patients, comparing results with identical measurements performed on the
`posttreatment specimen removed at the time of primary surgery seven days later.
`Analysis of these data has shown that, as observed in vitro and within animal model
`systems, the pure antiestrogen is capable of reducing both tumor cell proliferation
`and estrogen-regulated gene expression.
`In the Phase I study, ICI 182780 produced a highly significant decrease in ER
`expression in patients with tumors initially classified as ER positive, mirroring those
`effects observed in experimental systems. Comparison of these data with similar
`measurements derived from short-term tamoxifen-treated patients showed that al-
`though tamoxifen also produced a suppression of ER levels, the effects were not as
`great as those induced by the highest dose level of ICI 182780 (18 mg/day). Further—
`more, examination of PR levels in these samples revealed a divergence of response to
`the antiestrogens, with the pure antiestrogen inhibiting PR expression while tamoxifen
`promoted some increase in its tissue concentration. Thus, in initially ER—positive and
`PR—positive tumors, ICI 182780 caused a fall in PR levels in the majority of patients.
`Indeed, in several patients the tumors became negative for both of these steroid
`receptors after the seven-day treatment period. The estrogen-regulated protein ps2
`showed similar responses to PR, although the inhibitory effects of ICI 182780 appeared
`blunted. No short-term effects of ICI 182780 or tamoxifen were recorded on the
`
`mammotrophic growth factor TGFa or the bcl—2 protein. However, ICI 182780 (18
`mg/day) produced a significant reduction in the expression of the tumor cell prolifera-
`tion marker Ki—67, with staining values decreasing in the majority of ER—positive
`tumors examined.
`
`A more recent second study directly attempted to address the issues of whether
`pure antiestrogens can promote clinical tumor remissions of worthwhile duration and
`also if these drugs remain effective in tamoxifen-resistant patients.7 Initial results
`using an oil-based monthly depot again appear promising. Thus, approximately two-
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`159
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`thirds of women who had either received adjuvant tamoxifen (minimum of two years)
`and subsequently recurred, or who had relapsed following an initial favorable response
`to the antiestrogenic drug, gained further benefit from ICI 182780 treatment (7/19
`partial responses, 6/19 no change at 6 months). Within responding patients, 10/13
`women were still in remission at 9 months. However, although these results are better
`than would have been expected following tamoxifen withdrawals”8 or second line
`endocrine therapy,59 the study numbers were small and no direct randomized compari-
`sons were made with other endocrine measures. It is noteworthy, however, that in
`clinical studies where tamoxifen-resistant tumors have been treated with another
`
`triphenylethylene antiestrogen, toremifene, cross—resistance was demonstrated.56 This
`result parallels experimental studies on antiestrogen-resistant human breast cancer
`cells.
`
`Side Efi‘ects
`
`To date, no serious drug-related side effects have been reported in either of the
`above clinical studies. In particular, there has been no evidence of altered coagulation
`or thrombogenicity after treatment with ICI 182780. On long-term therapy a rise in
`serum gonadotrophin levels has been recorded, suggesting that ICI 182780 has an
`antiestrogenic effect on the pituitary gland. There were no significant changes in
`serum levels of sex hormone binding globulin, implying no estrogenic effect of the
`drug on the liver.
`
`CONCLUSIONS
`
`It is highly encouraging that the majority of experimental studies to date have
`shown the recently developed pure antiestrogens to be effective antitumor agents,
`certainly with regard to inhibition of tumor cell growth and proliferation, and estrogen—
`regulated gene expression. Indeed, in many instances their effects substantially surpass
`those observed following estrogen withdrawal or tamoxifen therapy. Treatment of
`breast cancer cells with pure antiestrogens appears to promote an efficient growth
`arrest in vitro and in vivo by their induction of a state of strict estrogen deprivation.
`Such estrogen withdrawal is likely to be induced primarily by the compounds antago-
`nizing the cellular actions of estrogens and, possibly, reducing other growth signalling
`activities through the estrogen receptor. Importantly, these mechanisms appear rele-
`vant to the treatment of endocrine-resistant states.
`
`In clinical breast cancer it is too early to judge the final value of these compounds.
`They have, however, passed the first major hurdle by successfully promoting tumor
`remissions in previously treated patients generating minimal adverse side-effects.
`The compounds seem capable of reducing cell proliferation and expression of both
`the estrogen receptor and several estrogen-regulated genes. However, their ultimate
`success or failure will depend on many factors, most notably the importance of small
`amounts of estrogen to the tumor cell. If we have not, as yet, passed the threshold
`of response to estrogen withdrawal, a potential exists for pure antiestrogens to improve
`the outcome of endocrine therapy“62 in such important areas as the rate and duration
`of remission and the prevention and treatment of resistant states.2 Over the next few
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`ANNALS NEW YORK ACADEMY OF SCIENCES
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`years, analysis of the clinical actions of pure antiestrogens will establish many
`unknowns in breast cancer. Let us hope that one of them is that the inhibition of all
`ER-mediated signalling is a worthwhile goal.2
`
`REFERENCES
`
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