Critical Reviews in Oncology/Hematology, 1993; 14: 173-188
`© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 1040-8428/93/$24.00
`
`173
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`ONCHEM 00068
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`Tamoxifen resistance in breast cancer
`
`Valerie J. Wiebe, C. Kent Osborne, Suzanne A.W. Fuqua and Michael W. DeGregorio
`Department of Medicine, Division of Oncology, The University of Texas Health Science Center at San Antonio, Texas, USA
`(Accepted 22 January 1993)
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`Contents
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`174
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`I
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`II.
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`III.
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`Introduction .
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`Mechanisms of anti-estrogen action .
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`Potential mechanisms of acquired tamoxifen resistance .
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`A. Altered levels of estrogen receptor .
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`1.
`Clinical clues .
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`B. Altered estrogen receptor .
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`1.
`ER variants .
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`2.
`Tissue specific transcription factors .
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`Enhanced biologic mechanisms for circumvention of tamoxifen cytotoxicity .
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`1. Growth factors
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`Antiestrogen binding sites (AEBS) .
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`D. Decreased intracellular drug . . . .
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`E.
`Tamoxifen metabolites and the development of resistance .
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`Estrogenic metabolites .
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`2,
`Tamoxifen isomers .
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`F. Other contributing factors to anti-estrogen failure ,
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`G. Circumvention
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`Summary .
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`185
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`References
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`Tamoxifen (TAM) resistance is the underlying cause
`of treatment failure in many breast cancer patients
`receiving TAM. The mechanism(s) involved in TAM
`resistance are poorly understood. A variety of mech-
`anisms have been proposed but only limited evidence
`exists to substantiate them. Studies have now shown
`
`that in many patients TAM resistance is not related to
`the down regulation or loss of estrogen receptors (ER).
`Variant ER have been identified, but their significance
`clinically remains to be proven. Since breast cancer cells
`secrete several estrogen-regulated growth factors and
`growth inhibitors that may have autocrine or paracrine
`
`Correspondence to: Dr. Valerie Wiebe, Department of Medicine,
`Division of Oncology, The University of Texas Health Science Center.
`7703 Floyd Curl Drive, San Antonio, Texas, 78284-7884, USA.
`
`activity, altered growth factor production is another
`possible mechanism for TAM resistance. Tissue-specific
`transcription activating factors that may alter how the
`signal induced by TAM binding to the receptor is inter-
`preted by the cell also require further investigation. An
`increase in antiestrogen binding sites (AEBS), which
`could effectively partition TAM and reduce its concen-
`tration at the ER has also been proposed as a potential
`mechanism. Pharmacologic mechanisms, such as a shift
`in metabolism toward the accumulation of estrogenic
`metabolites, are supported by recent data demonstrating
`metabolite E and bisphenol
`in tumors from TAM-
`resistant patients. Furthermore, a decrease in tumor
`TAM accumulation and an altered metabolite profile
`have been reported in TAM-resistant breast
`tumors
`
`Astrazeneca Ex. 2019 p. 1
`Mylan Pharms. Inc. V. Astrazeneca AB IPR2016-01326
`
`

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`174
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`grown in nude mice. These and other studies suggest
`that TAM resistance may be multifactorial in nature,
`but definitive identification of mechanisms that are op-
`erative in clinical TAM resistance requires further study.
`
`I. Introduction
`
`Tamoxifen (TAM) is a nonsteroidal antiestrogen that
`was originally synthesized in 1966 as an antifertility drug
`[1]. However, in the 1970s TAM was noted to have ac-
`tivity in the treatment of metastatic breast cancer, and
`clinical trials began in the United States in 1974. In 1978
`TAM was primarily used to treat postmenopausal
`women with estrogen receptor positive, metastatic
`breast cancer. However, its clinical role has expanded to
`include all stages of the disease (Stage I and II) and both
`pre- and postmenopausal patients [2]. Response rates
`and duration of response in studies comparing TAM
`and oopherectomy in premenopausal patients are
`similar. Response rates to TAM increase with higher
`tumor ER levels. Overall, TAM prolongs both the
`
`disease-free and overall survival of women following
`primary surgery [3], and it induces tumor regression in
`about half of women with advanced estrogen receptor
`positive, metastatic breast cancer [4]. TAM has demon-
`strated efficacy in the prevention of contralateral breast
`cancer and it is also currently being evaluated for use as
`a chemopreventative agent in healthy women at high
`risk of breast cancer.
`
`Although approximately 50% of estrogen receptor-
`positive (ER+) tumors will
`respond to TAM, only
`60-75% of patients with metastatic breast cancer have
`estrogen receptor-positive tumors. Therefore only 35%
`of metastatic breast cancer patients actually benefit
`from TAM therapy [5]. In addition, all patients who
`initially respond to therapy will eventually develop
`acquired TAM resistance following prolonged adminis-
`tration.
`
`The development of acquired TAM resistance, where
`cell populations initially sensitive to TAM become
`insensitive, differs from innate resistance where cell
`populations are insensitive to TAM from the onset of
`
`Potential Mechanisms of Tamoxifen Inhibitory Activity
`
`TAM
`
`Mammary Cell
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`Nucleus
`
`Cell Replication
`
`Autocrine
`
`Paracrine
`
`Shows the mechanism of estrogen (E2) binding to the estrogen receptor (ER) and the growth inhibitory effects of tamoxifen (TAM). TAM
`Fig. 1.
`competitively blocks the binding of E2 to the ER (1), it also binds to the antiestrogen binding sites (AEBS) (2). TAM blocks cells in GO/G1 (3)
`inhibiting cell replication. TAM may also decrease concentrations of TGF-oz, a growth factor that is stimulatory (4) and may increase levels of
`TGF-B, an inhibitory growth factor (5).
`
`Astrazeneca Ex. 2019 p. 2
`
`

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`drug exposure. Unfortunately, the cellular and molecu-
`lar mechanisms underlying the development of acquired
`resistance to antiestrogens remains unclear. However, a
`variety of potential mechanisms have been suggested,
`and several reports concerning potential mechanisms for
`acquired TAM resistance have recently been published.
`
`II. Mechanism of antiestrogen action
`
`The mechanism(s) by which TAM inhibits tumor cell
`growth are believed to be mediated is primarily through
`interaction with ER (Fig. 1). Competitive antagonism of
`estrogen at
`the ER by TAM slows the growth of
`estrogen-dependent cancer cells by blocking them in the
`G0/Gl phase of the cell cycle [6]. Binding of TAM to the
`receptor is believed to form a complex that, when bound
`to estrogen—response elements, fails to trigger transcrip-
`tion of target genes. The resulting blockade is believed
`to be predominantly cytostatic in nature and may be
`reversed by the addition of estradiol. Whether TAM in-
`duces apoptosis or cell death is not yet clear.
`The antiestrogenic activity of TAM has also been
`evaluated in several species, and the biological effects of
`the drug appear to be dependent both on the species
`studied and the target
`tissue examined. In rats and
`humans, TAM has similar biological activity. In both
`species TAM has partial estrogen agonist effects on
`uterine tissues, but it is primarily considered an estrogen
`antagonist [7]. TAM’s weak estrogenic like effects have
`also been noted in postmenopausal patients in whom
`estrogenic effects were noted on gonadotrophin levels.
`plasma proteins and vaginal epithelium [7—9]. Whether
`the difference in antiestrogenic action is related to spe-
`cies specific or tissue specific metabolism of TAM. or to
`the presence of specific transcription factors that alter
`signal interpretation by the cell following interaction of
`the antiestrogen with the estrogen receptor is unknown.
`However, many other factors may also play a role in the
`cellular response to TAM.
`Several studies have now shown that cellular inhibi-
`
`tion by TAM may involve a complex series of events.
`Modulation of breast cancer cell growth by the differen-
`tial stimulation or inhibition of growth factor produc-
`tion from cells may also be involved in antiestrogen
`action. Recent evidence now suggests that estrogens
`may stimulate cell growth in part by inducing cells to
`synthesize growth factors and/or receptors. TAM on the
`other hand may act by inhibiting the estrogen-induced
`production of growth factors, while at the same time
`stimulating the production of growth inhibitory factors.
`At least one pathway may involve the stimulation of
`transforming growth factor-beta (TGF-B) production
`
`175
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`by TAM. TGF-B has both growth inhibitory and stimu-
`latory effects. In stromal cells such as fibroblasts or
`endothelial cells, it stimulates cell growth. However, in
`most types of epithelial tumor cells,
`including breast
`tumors, it acts as a growth inhibitor [10]. Although the
`exact mechanism of TGF-B growth inhibitory effects are
`poorly understood, it does appear to inhibit tumor cell
`growth independent of the ER. A number of other
`growth factors are produced by breast cancer cells, and
`their expression is modified by estrogens and antiestro-
`gens. These include TGF-alpha, IGF-II, PDGF, and
`members of the EGF family [11]. However, the exact
`role that each of these growth factors plays in the induc-
`tion of cell growth by estrogen and the inhibition of cell
`growth by TAM remains to be elucidated.
`TAM has also been noted to bind to sites that are
`
`independent of the ER. These high affinity binding sites
`(Kd = 1 nM) are referred to as antiestrogen binding
`sites (AEBS) [12]. AEBS have been identified in several
`tissues with the highest concentrations noted in liver,
`uterus, ovaries, brain, and kidneys [l 3]. AEBS appear to
`be distinct from the ER and are only observed following
`prior treatment with estradiol [12].
`The affinity of antiestrogens for AEBS does not close-
`ly correlate with the biological potency of antiestrogens,
`suggesting that AEBS do not directly mediate antiestro-
`gen action [14,15]. Many studies have attempted to cor-
`relate binding of AEBS to other cellular events related
`to antiestrogen actions including protein kinase C in-
`hibition [16], calmodulin inhibition [17] and interac-
`tions with a variety of receptors, including histamine
`[18], dopamine [19] and muscarinic receptors [20]. In-
`deed much interest has been placed on the study of
`AEBS over the years; however, their true function and
`role in the antitumor efficacy of TAM remains to be
`established.
`
`III. Potential mechanisms
`resistance
`
`of
`
`acquired
`
`tamoxifen
`
`A variety of mechanisms has been implicated in the
`development of acquired resistance to TAM. However,
`little definitive data are available to support many of the
`proposed mechanisms. At one time it was assumed that
`drugs were responsible for inducing some biochemical
`modification in cells that resulted in acquired resistance
`to that drug. However, for many types of drug resistance
`the drug does not play a direct role in the development
`of resistance, but instead provides a strong selective
`pressure in favor of drug-resistant subclones. Drug-
`resistant subclones resulting from spontaneous muta-
`tions differ genetically from the original population. A
`
`Astrazeneca Ex. 2019 p. 3
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`176
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`deletion or modification of a specific enzyme, or altera-
`tion in some other cellular property in the genetically
`altered cell population may be responsible for the
`altered sensitivity of cells to the drug.
`Recent evidence derived from in vitro and in vivo
`
`studies suggests that TAM can stimulate cells to grow
`following prolonged exposure [2l—23]. Whether TAM
`is selecting a subclone of TAM-stimulated cells, whether
`the cells are altering TAM in such a way as to generate
`a stimulatory signal, or whether TAM is capable of
`inducing a genetic mutation that results in altered sensi-
`tivity to the drug remains to be established. One study
`has now shown that TAM can produce DNA adducts in
`the liver of rats suggesting that it may have genotoxic
`activity that could theoretically lead to mutations [24].
`Although the mechanisms underlying TAM resistance
`remain vague, a variety of recent studies suggest that
`multiple mechanisms may contribute to TAM resis-
`tance. Studies examining (A) altered level of ERs, (B) a
`decrease in ER affinity, (C) enhancement of cellular
`mechanisms
`for bypassing TAM cytotoxicity,
`(D)
`decreased cellular TAM concentration, (E) increased
`concentration of antagonizing metabolites and (F) a
`variety of other pathways, have contributed to our
`understanding of the possible mechanisms underlying
`TAM resistance.
`
`III-A. Altered levels of estrogen receptor
`
`III-A.1. Clinical clues
`
`ER expression is regulated to meet the demands of the
`cell. In the presence of high concentrations of estradiol,
`down regulation of receptors is believed to occur. The
`absence or loss of estrogen receptors could explain the
`development of hormonal independence or TAM resis-
`tance, particularly since ER-negative tumors rarely re-
`spond to TAM. However, clinical studies suggest that
`resistance to TAM is not always caused by selection of
`a hormone independent and/or ER-negative clone of
`tumor cells. Sequential biopsy studies have shown that
`apparent loss of ERs is common when the second biopsy
`is performed while the patient is taking TAM or within
`two months of stopping TAM presumably, due to recep-
`tor occupancy by the drug causing a false-negative
`ligand binding assay [25]. When the second biopsy is
`performed after two months, tumors frequently remain
`ER-positive, suggesting that there is no selection of a
`truly ER—negative clone. In a recent study of tumors
`from patients with TAM-resistance, an immunohisto-
`chemical technique was used to detect both bound and
`free receptors, ER was found in seven out of 13 tumors
`examined [26]. Maintenance of ER and/or PgR levels
`
`and responses to secondary hormonal therapies are not
`uncommon in patients with acquired TAM resistance
`[4,25]. Furthermore, in vitro studies suggest that follow-
`ing the selection of antiestrogen-resistance cells, many
`resistant cell
`lines may remain sensitive to estrogens
`[27,28].
`Thus, ER loss may play a role in acquired TAM resis-
`tance in some patients, but it cannot account for the
`resistance noted in the majority of patients. In addition,
`clinical evidence suggests that patients that
`initially
`respond to TAM, but who later develop resistance,
`frequently respond to secondary hormonal treatment,
`suggesting that the development of resistance to anti-
`estrogens does not confer resistance to other hormonal
`agents [4].
`
`III-B. Altered estrogen receptor
`
`Protein structure modifications leading to altered affi-
`nity of the ER for TAM is a plausible resistance mecha-
`nism. Site—specific mutations,
`including nonsense or
`frameshift mutations in the structural gene coding for
`the ER may potentially result in various types of func-
`tionally abnormal
`receptors. These mutations may
`render the ER entirely nonfunctional; thus, the tumor
`would appear clinically as if it were ER-negative. Alter-
`natively, if mutations result in amino acid substitutions
`in important domains of the receptor, then the result
`may be the generation of ER species which are func-
`tionally active, but which exhibit altered specilicities for
`estrogens and antiestrogens.
`
`III—B.1. ER variants
`Much is known about the structure and function of
`
`the ER [29]. The ER contains discrete domains involved
`in hormone binding, DNA binding, and subsequent
`activation of estrogen-responsive genes. Human ERs
`have now been shown to contain five distinct functional
`
`domains A/B, C, D, E and F [30]. Although there is
`some overlap between domains, regions E and D appear
`to primarily involve the horrnone-binding and dimeriza-
`tion domains [30,31]. Region C is the DNA binding do-
`main, and the A/B and E regions contain the two
`transcription activating functions.
`The presence of these discrete functional domains has
`led investigators
`to examine alterations in TAM-
`resistant, or hormone-independent model
`systems.
`Many earlier studies failed to show differences in the ER
`in in vitro systems. For example, Mullick and Chambon
`[32] used two independently isolated TAM-resistant,
`ER-positive breast cancer cell lines, LY2 and T47D, to
`demonstrate that the ER was still functional in these
`
`Astrazeneca Ex. 2019 p. 4
`
`

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`cells in spite of their hormone—insensitive growth. The
`ER was shown to be wild-type using RNase protection
`assays and by its ability to stimulate estrogen—responsive
`reporter constructs in these cells. However, neither of
`these assays rule out the possibility that mutated ER
`species may be present along with wild—type ER, and
`that
`these mutated forms may contribute to the
`tamoxifen-resistant phenotype. Direct sequence analysis
`of the ER from these cells may be required to definitive-
`ly answer this question.
`[33] have examined the
`In fact, Graham et al.
`presence of mutated ERs by cloning and sequence
`analysis in T47D cells that have been maintained in their
`laboratory. Several different ER mutations were
`detected in complementary DNAs prepared from these
`cells, including frameshift mutations within the DNA
`and the hormone binding domains of the receptor. If
`expressed, these mutated ER species could be defective
`in activity, and could contribute to the hormone-
`independent phenotype of this T47D subline. Of impor-
`tance from this study is
`that highly sophisticated
`technologies were required to identify ER mutations
`present
`in cells which were heterogenous
`for ER
`expression.
`Raam et al. [34] have utilized immunohistochemical
`procedures to demonstrate the presence of ERs deficient
`in nuclear binding in ER-positive tumors. Interestingly,
`patients with either constitutive nuclear binding, or
`those with ER which could not bind nuclei, were refrac-
`tory to hormone therapy. This encouraging result, that
`tumors may contain defective ERs, has been recently
`substantiated by studies using larger series of human
`breast tumor specimens [35,36]. Even though endocrine
`response data was not available on these later studies,
`both studies suggest that truncated forms of the ER,
`which fail to bind DNA in gel-retardation assays, may
`be present in tumors. Of note,
`is that DNA binding-
`deficient ER was most prevalent in tumors with minimal
`PgR expression, agreeing with the commonly-held doc-
`trine that PgR expression closely correlates with an in-
`tact ER response pathway. It will be interesting to apply
`the gel-retardation methodology to tumors with clinical
`response data to determine whether ERs defective in
`DNA binding may contribute to TAM failure.
`Murphy et al. [37] have identified abnormal sized ER
`mRNAs by Northern hybridization, and they have
`recently cloned these altered ERs from human breast
`tumors. Three different ER mRNAs have been iden-
`
`tified, all of which diverge from the known ER sequence
`at exon/intron borders. At the point of divergence, non-
`ER sequences have been inserted. These insertions are
`either unknown or are homologous to long interspersed
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`177
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`repetitive LINE-1 sequences. These three altered ERs
`are all missing the hormone binding domain of the
`receptor
`in addition to containing unique non-ER
`segments. One of the mutated ERs, designated clone 4,
`is widely expressed in breast tumor samples.(Dozlaw,
`unpublished results). Although clone 4 ER was devoid
`of transcriptional activity in in vitro assays, its presence
`may be functionally significant in tumors. Experiments
`are currently underway to examine its distribution in
`breast tumors where clinical information is available
`
`(Murphy and Fuqua, unpublished observations).
`The progression of steroid sensitive cells to steroid in-
`sensitivity was evaluated by Darbe and King [38]. In
`their study they demonstrate that clones of steroid-
`responsive cells can give rise to a population of
`unresponsive cells in a series of phenotypic changes
`which are brought about solely by long-term withdraw]
`of the hormone. In a further study they report that
`transfection of a steroid inducible gene into unrespon-
`sive cells (S1 15) results in that gene being fully inducible
`by steroids. Therefore, the machinery for steroid respon-
`siveness, including receptors, appears to be intact. They
`suggest that the process appears to be independent of
`the loss of steroid receptor function [39]. This was fur-
`ther substantiated by Clarke et al. who examined the
`progression of human breast cancer cells from hormone-
`dependent to hormone-independent growth in vitro and
`in vivo. They report that an ovarian—independent, but
`honnone-responsive phenotype may occur early in the
`natural progression to hormone—independence, but that
`altered hormone receptor expression may be a late event
`in the acquisition of this phenotype [40].
`ER variants have also been isolated from a variety of
`ER-positive,
`and
`supposedly ER-negative
`breast
`tumors, using sensitive RNA-directed polymerase chain
`reaction (PCR) methodologies [41,42]. Tumors which
`are ER-negative, but PgR-positive often express high
`levels of a variant ER lacking exon 5 of the hormone
`binding domain of the receptor. This deletion results in
`the production of a variant ER truncated within the hor-
`mone binding domain, and as such, is unable to bind es-
`trogen. However, the receptor appears to bind DNA
`and is constitutive for activation of estrogen-responsive
`genes. When the exon 5 ER variant is coexpressed with
`wild-type receptor
`in MCF—7 cells, TAM-resistant
`growth is conferred to these cells. Thus, overexpression
`of the variant, even in the presence of wild-type recep-
`tor, may contribute to TAM resistance. Furthermore,
`ER-positive tumors which express wild-type ER often
`coexpress the exon 5 ER deletion variant. Tumors with
`this variant may escape the normal growth dependence
`of estrogens, and subclones may be selected for under
`
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`
`

`
`[78
`
`conditions where TAM is present which would inhibit
`only the wild-type receptor present in these cells. The
`heterogenous expression of variant ERs within a tumor
`may contribute to the emergence of TAM resistance
`during treatment. Thus, the selection of TAM-resistant
`ER variants may be clinically significant with TAM ac-
`tually providing the selective pressure for their eventual
`outgrowth. These are testable hypotheses, and we anx-
`iously await the answers.
`
`III-B.2. Tissue specific transcription factors
`TAM resistance may also involve alterations in tissue-
`specific transcription activating factors that might not
`alter TAM or estrogen binding, but could alter how the
`signal induced by TAM binding to the receptor is inter-
`preted by the cell (antiestrogenic versus estrogertic).
`Transcriptional activation functions (TAF) have been
`associated with certain regions of the estrogen receptor
`(regions E and A/B). At least two TAFS (TAF-1 and
`TAF-2) have been associated with antiestrogen action.
`TAF-2, from region E,
`is
`located in the hormone-
`binding domain and is reported to be stimulated by es-
`trogens and inhibited by antiestrogens.
`In contrast,
`TAF-1 is believed to have constitutive activity in the ab-
`sence of estrogen binding in some cells [43]. TAF—1’s
`role in stimulating transcription may therefore be de-
`pendent on the cell type, promoter and the presence of
`other TAFS. Mutations in either TAF-1 or TAF-2 do-
`
`mains could potentially modify the cellular response to
`TAM leading to an estrogenic rather
`than an an-
`tiestrogenic effect. To date there is little evidence in sup-
`port of this mechanism. In preliminary sequence studies
`performed in our laboratory, no mutations in either of
`the two transcription—activating domains or
`in the
`hormone-binding domain of the ER in a TAM-resistant
`MCF-7 in vivo tumor model were identified (unpublish-
`ed observations).
`
`III-C. Enhanced biologic mechanisms for circumvention
`of tamoxifen cytotoxicity
`
`The adaptation of estrogen dependent cells to survive
`in the presence of estrogen antagonists may also be re-
`lated
`to
`several
`physiologic
`and
`biochemical
`mechanisms that help the cell
`to circumvent
`the in-
`hibitory effects of TAM.
`
`III—C.I. Growth factors
`Recent research has demonstrated that breast cancer
`
`cells and stromal cells can produce and secrete a variety
`of polypeptide growth factors and growth inhibitors,
`that by autocrine and paracrine mechanisms, may con-
`
`tribute to tumor growth regulation [1l,44,45]. Expres-
`sion of several of these factors is regulated by estrogens
`and antiestrogens, suggesting the hypothesis that they
`may mediate, at least in part, the growth effects of these
`agents. As discussed earlier, one of these factors is TGF-
`6, a family of growth inhibitors that are produced by
`breast cancer cells [46,47]. A consequence of estrogen
`treatment is a reduction in the expression of TGF-6
`which might then result in enhanced growth. TAM, by
`binding to ER, blocks the effect of estrogen and induces
`the production of the growth inhibitor. Interestingly,
`TAM has also been reported to stimulate TGF-/3 pro-
`duction by breast
`tumor
`fibroblasts by unknown
`mechanisms [48]. If confirmed, these data could explain
`the infrequent response of an ER—negative tumor with
`TAM treatment.
`
`However, the role of TGF-3 in mediating the effects
`of estrogens and antiestrogens is not yet clear. High-
`passage MCF—7 breast cancer cells, that have very low
`levels of TGF-B receptors and are not
`inhibited by
`exogenous TGF-[3, are still
`inhibited by TAM [47].
`Exogenous TGF-[3 injected into nude mice in sufficient
`concentrations to cause significant
`toxicity had little
`effect on tumor growth [44,45]. Furthermore, recent
`data have shown that when a mixture of MCF—7 (ER-
`positive) and MDA MB-231
`(ER-negative) breast
`cancer cells are injected into nude mice at a ratio of
`100021, tumor growth is not inhibited by TAM, and ER-
`negative tumors result [49]. Therefore, while TAM may
`be able to stimulate the production of TGF18 from ER-
`positive cells. it does not appear to do so in sufficient
`quantities to inhibit proliferation of the ER-negative cell
`population, at least in this model.
`Thus, the effects of TGF-B on breast cancer cells in
`vivo remain speculative. If TGF-B is eventually shown
`to be important in estrogen/antiestrogen action, then it
`could play a role in TAM resistance, which could
`theoretically result from the failure of the tumor or
`stromal cells to secrete active TGF-B in response to
`TAM.
`
`Other stimulatory growth factors could also poten-
`tially play a role in TAM resistance. TGF-oz, IGF-II,
`PDGF, cathepsin D, and other growth factors have
`been shown to be produced and secreted by breast
`cancer cells [1l,44,45]. Secretion by ER-negative cells is
`constitutive, while that by ER-positive cells is regulated
`by estrogen. TAM treatment inhibits the production of
`these autocrine growth factors, perhaps contributing to
`its growth suppressive effects. The acquired ability of
`the cells to express these growth factors constitutively,
`despite TAM treatment, could theoretically result
`in
`TAM resistance. In vitro studies have, in fact, shown
`
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`
`

`
`that certain growth factors can reverse the inhibitory ef-
`fects of TAM [50,51]. At this time, however, there is no
`supportive evidence from experimental models or from
`patients to suggest that altered tumor production of
`autocrine growth factors or growth inhibitors is a cause
`of TAM resistance. This possibility, nevertheless,
`deserves additional investigation.
`
`III-C.2. Antiestrogen binding sites (AEBS)
`One theoretical mechanism by which breast cancer
`cells could potentially become resistant
`to the an-
`tiestrogenic effects of TAM is increased binding of drug
`to intracellular sites that do not participate in its anti-
`tumor actions, thereby reducing TAM concentrations at
`the ER. The fact that antiestrogen resistance can occur
`in the presence of functional ER has lead to the hypo-
`thesis that antiestrogen action is mediated independent-
`ly of ER by specific AEBS [52]. In the presence of
`antiestrogen, but not estrogens, AEBS are competitively
`reduced [l3,53—56]. However, the affinity of antiestro-
`gens for AEBS does not appear to closely correlate to
`the biopotency of specific antiestrogens [l5,57,58], and
`AEBS specific ligands with no affinity for the ER do not
`demonstrate functional antagonism [59]. Furthermore,
`AEBS remain expressed in antiestrogen-resistant tumor
`cell variants [60]. Thus,
`the role of AEBS in TAM-
`
`induced tumor growth inhibition is questionable.
`Although in theory increased binding of TAM to
`AEBS by either an increase in AEBS concentration or
`increased affinity for TAM could reduce available
`TAM, only limited evidence has been found supporting
`this theory. Pavlik et al. examined the possibility that
`AEBS may prevent the antiestrogen from interacting
`with ERs [52]. In their study, antiestrogen binding was
`compared in uterine preparations, where ER activity ex-
`ceeded AEBS, and in liver preparations, where AEBS
`binding predominated. Their results suggest that when
`AEBS activity predominates, TAM was virtually all
`bound to AEBS with little remaining available to
`associate with the ER. Furthermore, they also noted
`that the ratio of AEBS: ER was three times greater in
`antiestrogen-resistant MCF-7 cells
`(LY-2)
`than in
`antiestrogen—sensitive wild type MCF-7 cells. In addi-
`tion, examination of 128 human breast carcinomas
`
`showed that AEBS activity exceeded ER activity, sug-
`gesting that AEBS could partition TAM from ER in
`human breast tumors. They conclude that AEBS may
`provide a mechanism of antiestrogen-resistance whereby
`cells may lose their sensitivity to antiestrogens but retain
`their sensitivity to estrogens.
`However, other data do not support this theory. Two
`TAM-resistant MCF-7 clones (R3 and R27) have
`
`179
`
`unaltered levels of AEBS [61,62], and one resistant clone
`has been reported in which no AEBS could be identified
`[63].
`
`III-D. Decreased intracellular drug
`
`A decrease in intracellular drug concentration is one
`mechanism by which cells become resistant to drugs.
`Multidrug resistance is one form of resistance whereby
`a decreased intracellular drug concentration has been
`associated with an active efflux of drug from the cell.
`Drug resistance due to overexpression of P-glycoprotein
`is a well characterized form of acquired multidrug resis-
`tance (MDR) to natural product antineoplastics such as
`doxorubicin.
`In this form of resistance tumor cells
`
`develop resistance to a broad range of structurally
`unrelated drugs [64]. The mechanism underlying MDR
`is believed to involve a 170 000-Da cell membrane pro-
`tein, termed P-glycoprotein, which is overexpressed in
`MDR cells and appears to function as a drug efflux-
`pump [65]. Classical MDR is associated with the
`overexpression of the MDR-l gene that codes for the
`plasma membrane P-glycoprotein (p170). Overexpres-
`sion of the MDR-1 gene is associated with decreased
`cellular accumulation of drug due to the active energy-
`dependent efflux mechanism.
`Efforts focused on the pharmacologic modulation of
`P-glycoprotein have identified several drugs that are ef-
`fective in reverting MDR in vitro. These include calcium
`channel blockers, chlorpromazine, cyclosporin, steroids
`and calmodulin antagonists [66,67]. Interestingly. anti-
`estrogens, including both TAM and toremifene, have
`also been shown to reverse MDR in vitro. and
`toremifene is now undergoing clinical evaluation for this
`indication [68,69].
`Although no similar efflux pump has been identified
`in TAM resistance, several recent studies