ORIGINAL ARTICLE
`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 mayresult 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_raises the specter ofrapidly progressing dis-
`years of experience with tamoxifen (Nol-
`ease when drugresistance develops.
`vadex) for the treatment ofbreast cancer.
`By the end of the 20th century, be-
`During the past two decades, the initialap-
`tween 400.000 and 500 000 womenin the
`plication of tamoxifen asa palliative therapy
`United States could be taking tamoxifen
`for the treatmentof stage IV breast cancer_totreat or prevent breast cancer. On a world-
`has expandedto establish this antiestro-
`widebasis, this could be millions of women.
`gen as the endocrine treatment of choice
`_It is clearly time to review the potential
`for all stages of breast cancer. Indeed, the—mechanismsof drug failure so that women
`fact that adjunct tamoxifen produces asur-
`_can betreated successfully on a longertreat-
`vival advantage in both node-positive and—ment regimen.At present, we have no de-
`node-negative breast cancer and also re-
`_finitive data aboutthe clinical expression
`ducesthe incidence of second primary breast_of drugresistance to tamoxifen during in-
`cancers by up to 40%' has increased en-_—_definite therapy becausetheclinicaltrials
`thusiasm to test the worth of tamoxifento
`have not been completed. It is therefore
`prevent breast cancer in normal women.?_—
`appropriate to focus attention on this as-
`Tamoxifen has alow incidence ofside_pect of the actionsof tamoxifen so that suit-
`effects that have resulted ina tendency to
`_able strategies can be developed to aid pa-
`administer therapy for more than 5 years._tientcare.
`Tamoxifen also has somepositive estrogen-
`This article will review the current theo-
`like effects that maintain bone density? and
`ries about the various molecular mecha-
`reducethe incidence of fatal myocardialin-|nisms by which a responsive tumor could
`farction.* Tamoxifen maintenance therapy
`_ becomeeitherrefractory or stimulated by
`can clearly be advantageouste patients with
`tamoxifen.
`node-negative breast cancer as a hormone
`replacement therapy, but indefinite treat-
`mentofpatients with stage I and II cancer
`
`POTENTIAL MECHANISM
`OF DRUG RESISTANCE
`
`From the Departmentof Surgery, University of Chicago (1) (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.
`
`The mechanisms to be consideredare il-
`lustrated in Figure 1 , butonly the mo
`lecular mechanismswill be discussed in de-
`tail. Since tamoxifen is a competitive inhibitor
`of estrogen action by blockingestradiol bind-
`
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`ARCH SURG/VOL 128, NOV 1993
`1187
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`Figure 1. The potential mechanisms ofdrug 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 (ERE) on the DNA. Tamoxifen and its metabolites block
`the competitive inhibition of estrogen binding to ER.
`
`Tamoxifen
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`4-Hydroxytamoxifen
`
`Potenta
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`spiE
`
`WeakEstrogen
`
`Weak Antiestrogen
`
`Isovnenizaonot:
`PotentEstrogen
`
`and avoid premature drugfailure.
`The pharmacokinetics and metabolism oftamoxifen
`have been extensively studied in patients.?”° 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 andsta-
`bility of antiestrogenic metabolites within the tumoritself
`as a potential mechanism of tamoxifen-stimulated growth.
`
`LOCAL METABOLISM
`
`It is possible that the tumorcells, or the stromal compo-
`nent, could locally metabolize tamoxifen to potentestro-
`gens that would stimulate tumor growth. In the labora-
`tory, tamoxifen will stimulate the growth of humanbreast
`(MCF-7) or endometrial tumors transplanted into athy-
`mic mice.''!? The tumors are ER positive and grow in
`response to estradiol, tamoxifen, and a variety of nonste-
`roidal antiestrogens.’* Since steroidal antiestrogens that
`have noneofthe estrogenlike properties of tamoxifen will
`block tamoxifen-stimulated tumor growth,'* it is rea-
`soned that tamoxifen must be converted to estrogensthat
`stimulate growth through the ER.
`Tamoxifen is metabolized to 4-hydroxytamoxifen in
`the mouse.!° This metabolite is a potent antiestrogen that
`has been shown to have antitumoractivity in the athymic
`mouse model.!© However, the potent antiestrogenic Z iso-
`meris unstable and can convert to the weakly antiestro-
`genic E isomer.’’ If the isomerization occurslocally, the
`het antiestrogenicity of tamoxifen will decrease, but this
`would not in itself account for increased tumor growth;
`an estrogenic stimulus is required. Minute amountsof me-
`tabolite E (tamoxifen without the dimethylaminoethane
`side chain) have been detected in human tumors during
`tamoxifen therapy.!® 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 potentestrogen.”’ It is possible thatif large
`quantities of this estrogenic metabolite accumulated in
`the tumors, this could account for tamoxifen-stimulated
`tumor growthby preferential binding of estrogenic igands
`at the ER. This hypothesis’® is summarized in Figure 2.
`Werecently 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,”? it
`is unlikely that local metabolite instability is responsible
`for tamoxifen-stimulated growth.It is perhaps morelikely
`that clonesofcells 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
`
`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-hydroxytamoxifen and the weak
`estrogen referred to as metabolite E. The key eventin 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
`proposalunlikely to occur.
`
`ing to the humanestrogen receptor (ER),’ an increase in
`circulating estradiol could potentially reverse the antitu-
`moraction of tamoxifen. The administration of adjuvant
`tamoxifen to premenopausal women?causes an increase
`in circulating estrogen levels; however, there is evidence
`that tamoxifen is effective in node-negative premeno-
`pausal women.’
`Nevertheless, patients with stage IV disease who ini-
`tially respond to tamoxifen and subsequently experience
`drug failure can respond to oophorectomy.’ This sug-
`gests that ovarian steroids may eventually reverse the an-
`titumoractions of tamoxifen. Clearly, tamoxifen will be
`more effective in a low estrogen environment, but con-
`sistently maintained levels (>100 ng/mL) of tamoxifen
`
`
`ARCH SURG/VOL 128, NOV 1993
`1188
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`

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`
`
`Vector ()\/ () Vector
`ey,M8-231
`sor/
`i ‘© Clone10A 5
`
`Mutant ER
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`Wild-Type ER
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`
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`biology community.
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`LOSS OF THE ER
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`ae ER Gere
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`Revere
`Tapsornee
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`Reonyeln
`Resistance
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`Figure 3. A diagrammatic representation of the isalation of the estrogen
`receptor (ER) complementary DNA (cDNA). The messenger RNA (mRNA)
`for ER is transcribed from the ER gene in a breast cancer cell, butit fs
`ihen processed te cut out intervening sequences (introns) of the transcript
`to retain the exons that can be translated into the ER 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
`ANA-based oncogenic viruses). The cDNA can be spliced into 2 vector that
`will continuously transcribe the ER message from a cytomegaloviral
`promoter. The vector produces a polycistronic RNA ef 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.
`
`Estrogen responsiveness of tissues and tumorsis corre-
`lated with the presence or absence of the ER. Breast can-
`cer requires estrogen to promote the process of carcino-
`genesis,andit is generally accepted that tumorsareinitially
`ERpositive but eventually loose the receptor, and growth
`becomes hormone independent.
`It is an important goal oflaboratoryresearch to de-
`velop models of human breast cancer progression. The
`objective is to study the biological processes involved in
`the evolution of hormone dependencyto find a strategy
`to prevent, orat least delay, hormone-independent growth.
`Regrettably, there are only a few hormone-dependent hu-
`man breast cancercell 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 cancercells
`that are ER positive and estrogen responsive for growth
`do lose the ERif the cells are maintained in an estrogen-
`free environment for many months.”! Theclonedcells are
`insensitive to both estrogens and antiestrogens. We are
`Constitutive Aen/Constitutive
`currently using this new model system to devise ways to
`reactivate the ER gene to produce a functional receptor.
`During the 1980s, 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 humancell lines with vary-
`ing degrees of success.**? High levels of receptor result
`in a cidal effect from estrogen treatment.”* In related ex-
`periments, we have transfected the ER gene into the ER-
`negative breast cancer cell line MDA-MB-231.?? We chose
`to developcell lines that contain levels comparable with
`those observed in hormone-responsivecells, ie, approxi-
`mately 150 to 300 fmoL/mgof cytosol protein. Estradiol
`decreases the growthrate of transfected breast cancercells,
`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.
`
`Mutant ER + Clones
`
`ER + Clones
`
`Figure 4. The human estrogen receptor (ER) has been cloned and the
`complementary DNA (cDNA) is available for motecular biological stucies of
`gene transfection. 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 fo interact with the estrogen response element on the genome
`(Figure 1). The DNA-binding domain is exposed when estradial binds in
`the steroid-binding domain E. Both the wild-type and a mutant cDNA for
`the ER (ie, with a paint mutation that now produces a protein with a valine
`{VAL] rather than a glycine [GLY] at position 400 in the steraid-binding
`domain) have been spliced into a vector that can be transfected into an
`ER-negative breast cancer ceil tine (MDA-MB-231) so that the effects of
`estrogen on the resufting celf lines can be compared and contrasted.
`
`MUTATED ER
`
`There is much interest in determining the biological rel-
`evance of mutated steroid hormonereceptors. Laboratory
`models have demonstrated that specific mutations of the
`androgen”* and progesterone receptors” 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
`
`antiestrogens to estrogens and explain tamoxifen-
`stimulated growth in tumors.
`Screening ofclinical tumor material has resulted in
`the identification of several mutations of the ER,?* 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 pharmacology of antiestrogens underlabo-
`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-
`
`
`ARCH SURG/VOL 128, NOV 1993
`1189
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`

`decreasing the growth rate.”° This then becomesa labo-
`ratory model to determinethe degree of estrogenicity ex-
`pressed by a test molecule under controlled conditions.
`Pure antiestrogens preventthe inhibitory effect of estra-
`diol in both wild-type and mutanttransfectants.”
`In contrast, the antiestrogens 4-hydroxytamoxifen”
`and RU39411,° which are partial estrogens with anties-
`trogenic properties in the wild-type transfectants, only ex-
`press estrogenic activity in the mutanttransfectants.Clearly,
`these data indicate that the pharmacology of antiestrogen
`can be changedto express fully estrogenic properties. Should
`mutations of the ER be foundin 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-independentcells could still
`synthesize a normal ER,buteither the local environment
`or additional subcellular factors have changed. This would
`prevent the hormone(or antihormone) receptor complex
`from either binding with othertranscription factors or pre-
`venting the complex binding adequately to estrogen re-
`sponse elements.
`
`The ubiquitous use of tamoxifen for the treatment of breast
`cancer has notonly 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 importantstart 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 mechanismsin breast cancer could
`become an importantfirst 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).
`
`ARLY STUDIES with drug resistance to the an-
`tiestrogen LY117018 demonstratedthat an
`ER-positive clone of MCF-7cells could con-
`tinue to grow in an antiestrogenic environ-
`ment.*! The receptor was shown to have
`EEEad
`the same sequenceas the wild-type hormone-responsive
`MCF-7cell line.* Similarly, we have described*? an ER-
`positive clone of MCF-7 cells that does not respond to
`either estrogensor 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 receptorstill re-
`mains. An intervention that could resolve the aberrant con-
`trol mechanism might potentially become a valuable new
`treatmentstrategy.
`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.**”° Indeed, the increased local con-
`centration of growth factors within a heterogeneous tumor
`maybe the reason why some ER-positive tumors (that are
`progesterone receptornegative) do not respond to tamox-
`ifen or other antihormonal therapy.*°
`
`1. Early Breast Cancer Triatists Collaborative Group. Systemic treatment of early
`breast cancer by hormonal, cytetoxic or immune therapy. Lancet. 1992;339:1-
`15, 71-85.
`2. Fisher B. The evolutian of paradigms for the management of breast cancer: a
`personal perspective. Cancer Res. 1992:52:2371-2383.
`3. Leve RR, Mazess RB, Barden HC, et 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 myocardialinfarction in the Scottish adjuvant
`tamoxifen trial. BM. 1991;303:435-437.
`5. Jordan VC, Koerner S. Tamoxifen (IC146,474} and the human tumour 85 oestro-
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`6. Jordan VC, Fritz NF, Langan Fahey S, Thompson M, Tormey DC.Alteration of
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`—__nn
`ARCH SURG/VOL 128, NOV 1993
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`tumors implanted in athymic mice: a model to study the tumoristatic action of
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`. Murphy CS, Langan-Fahey SM, McCagueR, Jordan VC. Structure-function re-
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`AstraZeneca Exhibit 2018 p. 5
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