`
`Molecular Mechanisms of Resistance
`to Tamoxifen Therapy in Breast Cancer
`
`Monica Morrow, MD, V. Craig jordan, PhD, DSc
`
`linical data suggest that the use of adjuvant tamoxifen citrate (Nolvadex) for a mini-
`mum of 5 years, and possibly indefinitely, will result in maximal antitumor benefit.
`There is concern that long—term tamoxifen maintenance therapy may result in the in—
`duction of drug resistance. This article reviews the potential molecular mechanisms of
`resistance to tamoxifen and explores the possibility of tamoxifen-stimulated tumor growth.
`(Arch Surg. 1993;128:1187-1191)
`
`There are more than 4.5 million women
`
`years of experience with tamoxifen (Nol-
`vadex) for the treatment of breast cancer.
`
`During the past two decades, the initial ap-
`plication of tamoxifen as a palliative therapy
`for the treatment of stage IV breast cancer
`has expanded to establish this antiestro-
`gen as the endocrine treatment of choice
`for all stages of breast cancer. Indeed, the
`fact that adjunct tamoxifen produces a sur—
`vival advantage in both node—positive and
`node-negative breast cancer and also re-
`duces the incidence of second primary breast
`cancers by up to 40%1 has increased en-
`thusiasm to test the worth of tamoxifen to
`
`prevent breast cancer in normal women.2
`Tamoxifen has a low incidence of side
`
`effects that have resulted in a tendency to
`administer therapy for more than 5 years.
`Tamoxifen also has some positive estrogen-
`like effects that maintain bone density3 and
`reduce the incidence of fatal myocardial in-
`farction.4 Tamoxifen maintenance therapy
`can clearly be advantageous to patients with
`node-negative breast cancer as a hormone
`replacement therapy, but indefinite treat-
`ment of patients with stage I and II cancer
`
`From the Department of Surgery, University of Chicago (Ill) (Dr Morrow), and the
`Departments of Human Oncology and Pharmacology, University of Wisconsin, Madison
`(Dr Jordan). Dr Morrow is now with the Department of Surgery, Northwestern
`University, Chicago.
`
`raises the specter ofrapidly progressing dis-
`ease when drug resistance develops.
`By the end of the 20th century, be-
`tween 400 000 and 500 000 women in the
`
`United States could be taking tamoxifen
`to treat or prevent breast cancer. On a world—
`wide basis, this could be millions of women.
`
`It is clearly time to review the potential
`mechanisms of drug failure so that women
`can be treated successfully on a longer treat-
`ment regimen. At present, we have no de—
`finitive data about the clinical expression
`of drug resistance to tamoxifen during in-
`definite therapy because the clinical trials
`have not been completed. It is therefore
`appropriate to focus attention on this as-
`pect of the actions of tamoxifen so that suit-
`able strategies can be developed to aid pa-
`tient care.
`This article will review the current theo-
`ries about the various molecular mecha—
`
`nisms by which a responsive tumor could
`_ become either refractory or stimulated by
`tamoxifen.
`
`POTENTIAL MECHANISM
`OF DRUG RESISTANCE
`
`The mechanisms to be considered are il-
`
`lustrated in Figure 'I . but only the mo-
`lecular mechanisms will be discussed in de-
`
`tail. Since tamoxifen is a competitive inhibitor
`of estrogen action by blocking estradiol bind-
`
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`ARCH SURGNOI. 128, NOV 1993
`1187
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`should prove to be adequate to treat premenopausal women
`and avoid premature drug failure.
`The pharrnacokinetics and metabolism of tamoxifen
`have been extensively studied in patients?10 There is no
`evidence that poor absorption or systemic metabolism to
`estrogens contributes to drug resistance. However, recent
`laboratory studies have focused on the metabolism and sta-
`bility of antiestrogenic metabolites within the tumor itself
`as a potential mechanism of tamoxifen-stimulated growth.
`
`LOCAL METABOLISM
`
`It is possible that the tumor cells, or the stromal compo-
`nent, could locally metabolize tamoxifen to potent estro-
`gens that would stimulate tumor growth. In the labora-
`tory, tamoxifen will stimulate the growth of human breast
`(MCF—T) or endometrial tumors transplanted into athy-
`mic mice.”12 The tumors are ER positive and grow in
`response to estradiol, tamoxifen, and a variety of nonste-
`roidal antiestrogens.13 Since steroidal antiestrogens that
`have none of the estrogenlike properties of tamoxifen will
`block tamoxifen-stimulated tumor growth,” it is rea-
`soned that tamoxifen must be converted to estrogens that
`stimulate growth through the ER.
`Tamoxifen is metabolized to 4-hydroxytamoxifen in
`the mouse.15 This metabolite is a potent antiestrogen that
`has been shown to have antitumor activity in the athyrnic
`mouse model.16 However, the potent antiestrogenic Z iso-
`mer is unstable and can convert to the weakly antiestro—
`genic E isomer.17 If the isomerization occurs locally, the
`net antiestrogenicity of tamoxifen will decrease, but this
`would not in itself account for increased tumor growth;
`an estrogenic stimulus is required. Minute amounts of me-
`tabolite E (tamoxifen without the dimethylaminoethane
`side chain) have been detected in human tumors during
`tamoxifen therapy.18 Fortunately, this metabolite of tamox—
`ifen is too weakly estrogenic to promote tumor growth
`alone. Nevertheless, the metabolite is unstable and can
`
`isomerize to a potent estrogen.17 It is possible that if large
`quantities of this estrogenic metabolite accumulated in
`the tumors, this could account for tamoxifen-stimulated
`tumor growth by preferential binding of estrogenic ligands
`at the ER. This hypothesis19 is summarized in Figure 2.
`We recently addressed the question of metabolite
`isomerization as the mechanism of tamoxifen-stimulated
`
`growth by determining the ability of tamoxifen deriva-
`tives that cannot isomerize to cause tumor growth. Since
`we have found that tumor growth is adequately sup-
`ported by nonisomerizable derivatives of tamoxifen,20 it
`is unlikely that local metabolite instability is responsible
`for tamoxifen-stimulated growth. It is perhaps more likely
`that clones of cells that are extremely sensitive to the in-
`trinsic activity of tamoxifen as an estrogen are selected
`and gain a dominant growth advantage, Clearly, the mecha-
`nism of signal transduction that converts an antagonist to
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`uttered Signal
`Transduction
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`Poor Absorption
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`Graven Factors
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`Tamoxifen
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`Meiahotiies
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`Eatmaenio
`Maiaeotites
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`increased
`Eaitatiitii
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`Figure 1. The potential mechanisms of drug resistance to tamoxifen in the
`breast cancer cell. Estrogen binds to the estrogen receptor (ER) to form a
`receptor complex that activates gene transcription through an estrogen
`response element (EFlE) on the DNA. Tamoxifen and its metabolites block
`the competitive inhibition of estrogen binding to ER.
`
`\
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`4—Hydroxytamoxiten
`Potent Aniiestrogen
`H
`0
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`Weak Antiestrogen
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`Isomerization OH
`00HO
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`Potent Estrogen
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`\ O
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`\T
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`amoxifen
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`oM
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`etabolite E
`Weak Estrogen
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`Figure 2. A proposed scheme for the metabolism of tamoxifen in breast
`tumors that could cause tamoxifen-stimulated growth. Tamoxifen could be
`converted to the potent antiestrogen 4-hydroxytamoxiten and the weak
`estrogen referred to as metabolite E. The key event in the hypothesis is the
`instability of the metabolites in the tumor cells to isomerize to a weak
`antiestrogen and a potent estrogen. Compounds that cannot isomerize
`have been shown to produce tumor-stimulated growth that makes this
`proposal unlikely to occur.
`
`ing to the human estrogen receptor (ER),5 an increase in
`circulating estradiol could potentially reverse the antitu-
`mor action of tamoxifen. The administration of adjuvant
`tamoxifen to premenopausal women6 causes an increase
`in circulating estrogen levels; however, there is evidence
`that tamoxifen is effective in node-negative premeno~
`pausal women.1
`Nevertheless, patients with stage IV disease who ini-
`tially respond to tamoxifen and subsequently experience
`drug failure can respond to oophorectomy.7 This sug-
`gests that ovarian steroids may eventually reverse the an-
`titumor actions of tamoxifen. Clearly, tamoxifen will be
`more effective in a low estrogen environment, but con-
`sistently maintained levels (>100 ng/ml.) of tamoxifen
`
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`ARCH SURGNOL 128, NOV 1993
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`Ptasmirfi Vector
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`smith ER Gene
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`tiesmgein
`Resistance
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`"transcriptase
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`Figure 3. A diagrammatic representation of the isolation of the estrogen
`receptor (ER) complementary DNA (cDNA). The messenger RNA {mHNA}
`for ER is transcribed from the ER gene in a breast cancer cell, but it is
`then processed to cutout intervening sequences {introns) of the transcript
`to retain the exons that can be translated into the El? protein. The
`processed mRNA can be used as a template to produce the cDNA for the
`ER gene with the enzyme—reverse transcriptase (an enzyme identified from
`RNA-based oncogenic viruses). The cDNA can be spliced into a vector that
`will continuously transcribe the ER message from a cytomegalovirai
`promoter. The vector produces a polycistronic RNA of both the ER and an
`enzyme that confers neomycin resistance to transfected cells. Growth of
`cells in a normally lethal environment of antibiotic will select resistant
`clones that will also contain ER.
`
`Ii]
`Constitutive
`Constitutive
`Vector
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`Vector Q
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`Mutant ER
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`Wild-Type EH
`i‘: a Clone10A 5
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`MBA—M8431
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`Mutant EH + Clones
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`ER + Clones
`
`Figure 4. The human estrogen receptor (ER) has been cloned and the
`complementary DNA (cDNA) is available for molecular biological studies of
`gene transfectian. The ER cDNA is divided into different areas indicated at
`the top of the figure. The C region is the DNA-binding domain that is
`essential to interact with the estrogen response element on the genome
`(Figure 1). The DNA-binding domain is exposed when estradiol binds in
`the steroid-binding domain E. Both the wild-type and a mutant cDNA for
`the ER (ie, with a point mutation that now produces a protein with a valine
`[VAL] rather than a glycine {GL Y] at position 400 in the steroid-binding
`domain) have been spliced into a vector that can be transfected into an
`Eli-negative breast cancer cell line (MBA-M3231) so that the effects of
`estrogen on the resulting cell lines can be compared and contrasted.
`
`antiestrogens to estrogens and explain tamoxifen-
`stimulated growth in tumors.
`Screening of clinical tumor material has resulted in
`the identification of several mutations of the ER,28 but the
`biological relevance of the findings is unclear. However,
`it is possible to examine the impact of point mutations of
`the ER on the pharmacologr of antiestrogens under labo-
`ratory conditions. If MDA—MB-231 cells are transfected
`with either a wild-type ER gene or an ER gene with a
`glycine to valine mutation at amino acid 400, the result-
`
`an agonist is an area of great interest within the molecular
`biology community.
`
`LOSS OF THE ER
`
`Estrogen responsiveness of tissues and tumors is corre-
`lated with the presence or absence of the ER. Breast can-
`cer requires estrogen to promote the process of carcino-
`genesis, and it is generally accepted that tumors are initially
`ER positive but eventually loose the receptor, and growth
`becomes hormone independent.
`It is an important goal of laboratory research to de-
`velop models of human breast cancer progression. The
`objective is to study the biological processes involved in
`the evolution of hormone dependency to find a strategy
`to prevent, or at least delay, hormone-independent growth.
`Regrettably, there are only a few hormone-dependent hu-
`man breast cancer cell lines. Both ZR—75 and MCF-7 cell
`
`lines have been used to develop antiestrogen—resistant or
`estrogen-independent sublines, but invariably the tumor
`cells retain the ER. In contrast, T47D breast cancer cells
`
`that are ER positive and estrogen responsive for growth
`do lose the ER if the cells are maintained in an estrogen-
`free environment for many months,21 The cloned cells are
`insensitive to both estrogens and antiestrogens. We are
`currently using this new model system to devise ways to
`reactivate the ER gene to produce a functional receptor.
`During the 19805, the gene for the ER was isolated
`(Figure 3) and the resulting complementary DNA (cDNA)
`studied extensively to determine the important domains
`on the protein.
`Estrogen receptor genes have been transfected into
`receptor—negative animal and human cell lines with vary-
`ing degrees of successuv23 High levels of receptor result
`in a cidal effect from estrogen treatment.24 In related ex-
`periments, we have transfected the ER gene into the ER—
`negative breast cancer cell line MDA-MB-231.25 We chose
`to develop cell lines that contain levels comparable with
`those observed in hormone-responsive cells, ie, approxi-
`mately 150 to 300 fmoL/mg of cytosol protein. Estradiol
`decreases the growth rate of transfected breast cancer cells,
`an effect that is blocked by pure antiestrogens. It is pos-
`sible that the selective reactivation or transfection of
`
`cancer cells with steroid receptor could prove to be a
`novel therapeutic strategy to control previously refrac-
`tory disease.
`
`MUTATED ER
`
`There is much interest in determining the biological rel-
`evance of mutated steroid hormone receptors. Laboratory
`models have demonstrated that specific mutations of the
`androgen26 and progesterone receptors27 can change the
`biological properties of antiandrogens and antiprogestins
`to full agonist molecules. It is therefore possible that mu-
`tations in the ER could change the pharmacology from
`
`
`ARCH SURGNOL 128, NOV 1993
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`
`
`COMMENT
`
`The ubiquitous use of tamoxifen for the treatment of breast
`cancer has not only provided the clinical community with
`a safe and effective therapy but also has provided an in-
`sight into the molecular mechanisms of hormone-
`dependent tumor growth.
`However, a fundamental piece of information is miss-
`ing that might be obtained by the research strategies cur-
`rently being investigated in the laboratory. We do not know
`about the precise and specific control mechanisms that
`regulate the activation of the ER gene. The current ex-
`periments on the drift of hormone-dependent growth to
`independent growth through the controlled loss of the
`ER are an important start to find critical steps in the bio-
`chemistry that might respond to therapeutic modulation.
`Clearly, it must be a goal of laboratory research to
`elucidate the cascade of events that subverts effective tran-
`
`scriptional control through the ER. Conversely, it may be
`equally productive to discover precise ways to maintain
`receptor control. Cell-specific receptor reactivation could
`become a powerful tool for the molecular biologist to ap-
`ply to therapeutic research. The clues obtained from un-
`derstanding receptor mechanisms in breast cancer could
`become an important first step in developing strategies to
`treat all cancers.
`
`Accepted for publication August 6, 1993.
`These studies werefunded by a grant from the Susan G.
`Komen Foundation, Dallas, Tex, and grants CA-56143, CA»
`32713, and CA 14520 from the National Cancer Institute,
`National Institutes of Health, Bethesda, Md.
`Reprint requests to the Department of Surgery, North-
`western University, 250 E Superior, Wesley 201, Chicago, IL
`60611 (Dr Morrow).
`
`w 1
`
`. Early Breast Cancer Trialists Collaborative Group. Systemic treatment of early
`breast cancer by hormonal, cytotoxic or immune therapy. Lancet. 1992;339:1-
`15, 71-85.
`2, Fisher B. The evolution of paradigms for the management of breast cancer: a
`personal perspective. Cancer Res. 1992;52:2371-2383.
`3. Love RR, Mazess RB, Barden H0, at al. Effects of tamoxifen bone mineral den-
`sity in postmenopausal women with breast cancer. N Eng! J Med. 1992;326:
`852-856.
`4. MacDonald CC, Stewart HJ. Fatal myocardial infarction in the Scottish adjuvant
`tamoxifen trial. BMJ. 1991;303:435-437.
`5. Jordan VC, Koerner S. Tamoxifen (ICI4B,474) and the human tumour BS oestro-
`gen receptor. Eur J Cancer. 1975;11:205-206.
`6. Jordan VB, Fritz NF, Langan Fahey S, Thompson M, Tormey DC. Alteration of
`endocrine parameters in premenopausal women with breast cancer during tong-
`term adiuvant tamoxiten monotherapy. J Natf Cancer Inst. 1939118321488-
`1491.
`7. Sawka CA. Pritchard KI, Paterson DJA. et al. Role and mechanism 01 action of
`tamoxifen in premenopausal women with metastatic breast cancer. Cancer Res.
`1986;46:3152-3156.
`8. Lien EA, Solheim E, Kvinnsland S. Veland PM. identification of 4-hydroxy-N-
`desmethyltamoxiten as a metabolite of tamoxifen in human bile. Cancer Res.
`1938;43:2304-2308.
`
`ing transfectants (Figure 4) will respond to estrogen by
`decreasing the growth rate.25 This then becomes a labo-
`ratory model to determine the degree of estrogenicity ex—
`pressed by a test molecule under controlled conditions.
`Pure antiestrogens prevent the inhibitory effect of estra-
`diol in both wild-type and mutant transfectants.25
`In contrast, the antiestrogens 4-hydroxytamoxifen29
`and RU39411,3° which are partial estrogens with anties-
`trogenic properties in the wild—type transfectants, only ex-
`press estrogenic activity in the mutant transfectants. Clearly,
`these data indicate that the pharmacology of antiestrogen
`can be changed to express fully estrogenic properties. Should
`mutations of the ER be found in clinical specimens that
`are suspected of playing a role in the drug resistance to
`tamoxifen, the cDNA could be transfected into receptor—
`negative cells in the laboratory to study the actions of the
`translated mutant receptor.
`
`ALTERED SIGNAL TRANSDUCTION
`
`It is possible that hormone—independent cells could still
`synthesize a normal ER, but either the local environment
`or additional subcellular factors have changed. This would
`prevent the hormone (or antihormone) receptor complex
`from either binding with other transcription factors or pre-
`venting the complex binding adequately to estrogen re-
`sponse elements
`
`ARLY STUDIES with drug resistance to the an-
`tiestrogen LY117018 demonstrated that an
`ER—positive clone of MCF—7 cells could con—
`tinue to grow in an antiestrogenic environ—
`ment.31 The receptor was shown to have
`the same sequence as the wild-type hormone-responsive
`MCF-7 cell line.32 Similarly, we have described33 an ER-
`positive clone of MCF-7 cells that does not respond to
`either estrogens or antiestrogens for growth. Estradiol does
`not stimulate progesterone receptor production, but the
`ER sequence is not mutated. Clearly, there is a funda-
`mental alteration in the signal transduction mechanism
`that controls replication, but a vestigial receptor still re—
`mains. An intervention that could resolve the aberrant con-
`
`trol mechanism might potentially become a valuable new
`treatment strategy.
`The local environment of growth factors can alter
`hormone and antihormone responsiveness. Epidermal
`growth factor can stimulate cell replication and poten-
`tially reverse the inhibitory effects of antiestrogen on estrogen-
`stimulated growth.“35 Indeed, the increased local con-
`centration of growth factors within a heterogeneous tumor
`may be the reason why some ER—positive tumors (that are
`progesterone receptor negative) do not respond to tamox—
`ifen or other antihormonal therapy.36
`
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`ARCH SURGNOL 128, NOV 1993
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`
`Jordan VC, Bain RR, Brown RR, Gosden B, Santos MA. Determination and
`pharmacology of a new hyroxylated metabolite of tamoxifen observed in pa-
`tient sera during therapy for advanced breast cancer. Cancer Res. 1983;43:
`1446-1450.
`Langan Fahey SM, Tormey DC, Jordan VC. Tamoxifen metabolites in patients
`on long-term adjuvant therapy for breast cancer. EurJ Cancer. 1990;26:883-
`888.
`Gottardis MM, Jordan VC. Development of tamoxifen-stimulated growth on
`MCF—7 tumors in athymic mice after long-term antiestrogen administration.
`Cancer Res. 1988;48:6183-6187.
`Satyaswaroop PG, Zaino RJ, Mortel R. Estrogen-like effects of tamoxifen on
`human endometrial carcinoma transplanted into nude mice. Cancer Res. 1984;
`44:4006-4010.
`,
`_
`Gottardis MM, Ricchio ME, Satyaswaroop PG, Jordan VC. Effect of steroidal
`and nonsteroidal antiestrogens on the growth of a tamoxifen-stimulated hu-
`man endometrial carcinoma (EnCa101) in athymic mice. Cancer Res. 1990;
`50:3189-3192.
`Gottardis MM, Jiang SY, Jeng MH, Jordan VC. Inhibition of tamoxifen-
`stimulated growth of an MCF-7 variant in athymic mice by novel steroidal an-
`tiestrogens. Cancer Res. 1989;49:4090-4093.
`Robinson SP, Langan Fahey SM, Johnson DA, Jordan VC. Metabolites, phar-
`macodynamics and pharmacokinetics of tamoxifen in rats and mice compared
`to the breast cancer patient. Drug Metab Dispos Biol Fate Chem. 1991;19:36-
`43.
`Gottardis MM, Robinson SP, Jordan VC. Estradiol stimulated growth of MCF-7
`tumors implanted in athymic mice: a model to study the tumoristatic action of
`tamoxifen. J Steroid Biochem Mol Biol. 1988;20:311-314.
`Murphy CS. Langan-Fahey SM, McCague R, Jordan VC. Structure-function re-
`lationships of hydroxylated metabolites of tamoxifen that control the prolif-
`eration of estrogen responsive T470 breast cancer cells in vitro. Mol Phar-
`macol. 1990;38:737-743.
`Wiebe VJ, Osborne CK, McGuire WL, DeGregorio M. identification of estro-
`genic tamoxifen metabolite(s) in tamoxifen-resistant human breast tumors.
`J Clin Oncol. 1992;10:990-994.
`Osborne CK, Coronado E, Allred DC, Weibe VJ, DeGregorio M. Acquired tamox-
`ifen (TAM) resistance: correlation with reduced breast tumor levels of tamox-
`ifen and isomerization of trans-4-hydroxytamoxiten. J Natl Cancer Inst. 1991;
`83:1477-1480.
`Wolf DM, Langan—Fahey SM, Parker CJ, McCague R, Jordan VC. investigation
`of the mechanisms of tamoxifen-stimulated breast tumor growth with non»
`isomerizable analogues of tamoxifen and metabolites. J Natl Cancer Inst. 1993;
`85:806-812.
`Murphy CS, Pink JJ, Jordan VC. Characterization of a receptor-negative, hor-
`mone non-responsive clone derived from T47D human breast cancer cell line
`kept under estrogen free conditions. Cancer Res. 1990;50:7285-7292.
`Watts CK, Parker MG, King RJ. Stable transfection of the oestrogen receptor
`gene into a human osteosarcoma cell line. J Steroid Biochem Mol Biol. 1989;
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`Touitou I, Mathieu M, Rochefort H. Stable translection of the estrogen receptor
`cDNA into HELA cells induces estrogen responsiveness of endogenous cathe-
`psin D-gene but not of growth. Biochem Biophys Res Commun. 1990;169:
`109-115.
`
`Kushner PJ, Hort E, Shine J, Baxter JD, Greene GL. Construction of cell lines
`that express high levels of the human estrogen receptor and are killed by es-
`trogens. Mol Endocrinol. 1990;4z1465-1473.
`Jiang SY, Jordan VG. Growth regulation of estrogen receptor negative breast
`cancer cells transfected with cDNAs lo'r estrogen receptor. J Natl Cancer Inst.
`1992;84:580-591.
`'
`'
`.
`Veldscholte J, His-Stalpers C, Kuiper GGJM, et al. A mutation in the ligand
`domain ofthe androgen receptor of human LNCaP cells affects steroid binding
`characteristics and response to antiandrogens. Biochem Biophys Res Com-
`mon. 1990;173:534-540.
`Vegeto E, Allan GF, Schrader WT, Tsai MJ, McDonnell DP, O’Malley SW. The
`mechanism of RU486 antagonism is dependent on the conformation of the
`carboxyterminal tail of the human progesterone receptor. Cell. 1992;69:703—
`713.
`Murphy LC. Estrogen receptor variants in human breast cancer. Moi Celt En—
`docrinol. 1990;74:083-086.
`Jiang SY, Langan-Fahey SM, Stella AL, McCague R, Jordan VC. Point mutation
`of the estrogen receptor (ER) in the ligand binding domain changes the phar—
`macology of antiestrogens in ER-negative breast cancer cells stably expressing
`cDNA’s for ER. Mol Endocrinol. 1992;6z21GT-2174.
`Jiang SY, Parker CJ, Jordan VC. A model to describe how a point mutation of
`the estrogen receptor alters the structure function relationship of antiestro-
`gens. Breast Cancer Res Treat. In press.
`Bronzert DA, Greene GL, Lippman ME. Selection and characterization ot a breast
`cancer cell line resistant to the antiestrogen LY11701B. Endocrinology. 1985;
`117:1409-1415.
`Mullick A, Chambon P. Characterization of the estrogen receptor in two an-
`tiestrogen resistant cell
`lines LY2 and T4TD. Cancer Res. 1990;50:333-
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`AstraZeneca Exhibit 2018 p. 5
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