`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—terrn 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?
`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-
`farctionf‘ Tamoxifen maintenance therapy
`can clearly be advantageous to patients with
`node-negative breast cancer as a honnone
`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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`
`
`should prove to be adequate to treat premenopausal women
`and avoid premature drug failure.
`The pharmacokinetics and metabolism of tamoxifen
`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 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—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 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.” This metabolite is a potent antiestrogen that
`has been shown to have antitumor activity in the athyrnic
`mouse model.“ However, the potent antiestrogenic 2 iso-
`mer is unstable and can convert to the weakly antiestro-
`genic E isomer.” 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.” 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.” 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 hypothesis” 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,” 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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`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 (EHE) on the DNA. Tamoxifen and its metabolites block
`the competitive inhibition of estrogen binding to ER.
`
`
`
`
`0 /\/N:
`
`\H0
`
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`Potent Antiestrogen
`H
`0
`
`Weak Antiestrogen
`
`Weak Estrogen
`
`lsomarization O
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`00HO
`
`Potent Estrogen
`
`N 0
`
`‘T
`
`amoxifen
`
`0M
`
`etabolite E
`
`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 antim-
`rnor action 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.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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`
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`
`
`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, honnone-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.“ 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 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 .15 We chose
`to develop cell lines that contain levels comparable with
`those observed in hormone-responsive cells, ie, approxi-
`mately ISO 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
`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
`
`' Pia-srniszi ‘rector
`
`rransertptase
`
`cistslti. ER Ease
`1---———-——————————————-t
`
`tieosrgcin
`Resistance
`
`Figure 3. A diagrammatic representation of the isolation of the estrogen
`receptor (ER) complementary DNA (cDNA). The messenger RNA (mliWA)
`for ER is transcribed from the ER gene in a breast cancer cell, but it is
`then processed to cut out intervening sequences {introns) of the transcript
`to retain the exons that can be translated into the El? protein. The
`processed mRlUA can be used as a template to produce the cDlllA 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 cytomegaloviral
`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.
`
`Al B
`—I§I
`Constitutive
`
`Vector QMutant EH
`V‘: a Clone1DA a
`
`ER 4» CIOHBS
`
`MD!-\—M8-231
`
`Mutant EH + CIOFIES
`
`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 El? cDNA is divided into different areas indicated at
`the lap of the figure. The C region is the DNA-binding domain that is
`essential to interact with the estrogen response element on the genome
`(l-'igure 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 (Gt. Y] at position 400 in the steroid-binding
`domain) have been spliced into a vector that can he transfected into an
`ER-negative breast cancer cell line (MDA—MB-231) 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,” 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 pharmacolog 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-
`
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`
`ing transfectants (Figure 4) will respond to estrogen by
`decreasing the growth rate.” 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.”
`In contrast, the antiestrogens 4-hydroxytamoxifenzg
`and RU39411,” 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 horrnone—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 antihorrnone) receptor complex
`from either binding with other transcription factors or pre-
`venting the complex binding adequately to estrogen re-
`sponse elements.
`
`ARLY sruon-:s 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.” The receptor was shown to have
`the same sequence as the wild-type horrnone-responsive
`MCF-7 cell line.” Similarly, we have described” 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 becorne a valuable new
`treatment strategy.
`The local environment of growth factors can alter
`hormone and antihorrnone 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.“
`
`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).
`
`gm;
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`23.
`
`27.
`
`30.
`
`33.
`
`34.
`
`35.
`
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