The First Hormone-Responsivebreast Cancer Cell Line'

[CANCERRESEARCH57. 3071-3078, August 1. 1997] Perspectives in Cancer Research

MCF-7: The First Hormone-responsiveBreast Cancer Cell Line'

Anait S. Levenson and V. Craig Jordan2 Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611

!ntroduction

On Thursday,January2, 1997, Dr. HerbertSoule, the scientist who Marc Lippman (3) demonstrated that the antiestrogen tamoxifen in developed the MCF-7 breast cancer cell line, died. At the time, we hibited the growth of MCF-7 cells, but the inhibition could be re were in the processof writingthis tributeto markthe 25th anniversary versed by estrogen. The drug was classified as a competitive inhibitor of Dr. Soule's remarkableaccomplishment.The cells, derivedfrom a of estrogen action, but at high concentrationstamoxifen killed cells. breast cancer patient in the Detroit area and developed at the Michigan Because these results appeared to parallel the emerging clinical oh CancerFoundation,Detroit,became a standardmodel in hundredsof servations (4), Lippman (3) went on to predict a future path for laboratories around the world. In retrospect, the story of the diverse endocrine research: â€œThepotential value of a hormone-dependent uses of these cells is really the history of our developing knowledge human breast cancer in long term tissue culture for the study of of hormone-regulatedcell replication, and they provided a unique mechanism(s) by which steroid hormones exert their trophic effects is insight into the endocrine therapyof breastcancer. significant,particularlyin view of the likelihood of obtainingregula Our article is offered as a tributeand memorial to Dr. Soule. We tory variantsor mutantswhich are hormone independent.â€•Atabout will trace researchwith MCF-7 cells to illustratethe change in our the same time, KathrynHorwitz,who was completingherPh.D. in the ideas about cell replication and to highlight the advances in our late Dr. Bill McGuire's laboratory, identified the receptors for glu understanding of the signal transduction pathway of estrogen and the cocorticoids, progestins, and androgens, as well as the ER (5). She molecular biology of estrogen action. All of these advances depended concluded, â€œMCF-7maybe an excellent in vitro model for studying on the uniquepropertiesof MCF-7 cells. Additionally,it is important the mechanismof tumorresponseto endocrinetherapyas well as the to appreciate that the cell system has now found applications in complex relationshipsbetweenbindingandbiological actionsof these experimental therapeutics, and the results from these studies are being hormonesâ€•(5).The predictionsfrom both researchprogramswere to translated to the clinic for the treatment of patients. None of this be proved correctover the next 2 decades. would have been possible withoutDr. Soule's skill as a cell biologist. Horwitz and McGuire focused initially on the regulation of PgR synthesis in MCF-7 cells by estrogens and antiestrogens. They devel Characterization oped a largebody of evidence to associate processing(destruction)of nuclearreceptorcomplexes with the initiationof PgR synthesis (6, 7). The MCF-7 breast cancer cell line was derived from a pleural This work paralleled their suggestion of using PgR assays as a effusion taken from a patient with metastatic breast cancer (1). The predictive test for hormone-dependent breast cancer (8). Turning to 69-year-old patient had undergone a mastectomy of her right breast the effects of antiestrogens,Horwitzet a!. (9) found thattamoxifen is for a benign tumorand a radicalmastectomyof her left breastfor a sufficiently estrogenic to initiate PgR synthesis by itself. The estro malignant adenocarcinoma 7 and 3 years, respectively, before primary genic propertiesof tamoxifen would subsequently be importantto cultureof cells was started.Interestingly,the Soule et aL article (1) explain the additionalbenefits of tamoxifen on bones and lipids and notes that local recurrences were controlled for 3 years with radio the possible development of drug resistance (10). However, at that therapy and hormone therapy. In the days before tamoxifen, the time in the late l970s, work on the antiproliferative actions of antics patientwas probablytreatedwith high doses of the syntheticestrogen trogens was of paramountimportance. Tamoxifen and other corn diethylstilbestrol.Clearly, the disease was very hormoneresponsive, pounds were shown to inhibit replication(11). Drs. Rob Sutherland because it was controlled for three times longer than the 1-year (12, 13) and Kent Osborne (14, 15) would demonstrate subsequently, average to be expected. Two months after widespread nodular recur using MCF-7 cells, that tamoxifen produces a reversible block in the rences occurred, in June of 1970, samples were taken from a pleural G1 phase of the cell cycle. effusion for laboratory studies. The cells were proven to be of human Despite the interesting findings with antiestrogens, the central focus origin, and cytogenetic studies indicated a distinct stem line of 88 of laboratoryresearchin the 1970s andearly 1980s was to prove that chromosomes. Dr. Sam Brooks, working with Dr. Soule (2), first estrogen actually stimulated tumor growth directly. Dogma predicted described the ER3 in MCF-7 cells by both Scatchard and sucrose thatthe MCF-7 ER-positive cell line should grow faster with exoge density gradient analysis. This was a pivotal discovery. nous estrogen. The experiment could be accomplished routinely if From this point onward,researchacceleratedrapidly.In 1975, Dr. cells first were treated with antiestrogens for a few days. Estrogen could â€œrescueâ€•antiestrogen-blockedcells (3), but the effects of es Received 4/10/97; accepted 5/30/97. trogen alone were less dramatic.Here was a paradox. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section1734solelyto indicatethisfact. Cells Grown in Estrogen 1 These studies were supported by NIH Grant CA-56143, Breast Cancer Program Development Grant P11 CA-657634, and the Lynn Sage Breast Cancer Foundation of Although Lipprnan'sgroup consistently demonstratedstimulatory Northwestern Memorial Hospital, Chicago. 2 To whom requests for reprints should be addressed, at the Robert H. Lutie Cancer effects with estrogen using [3H]thymidineincorporationas a method Center, Northwestern University Medical School, 303 East Chicago Avenue, Olson of monitoringDNA replication(3, 11, 16), otherswere unableto show Pavilion8258,Chicago,IL60611.Phone:(312)908-4148;Fax:(312)908-1372. profound effects on cell proliferation (17â€”19).This led to intense 3 The abbreviations used are: ER, estrogen receptor, PGR, progesterone receptor; TGF, transforming growth factor; [OF, insulin-like growth factor. debate (20) and also to the suggestion that estrogen really produced 3071

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1997 American Association for Cancer Research. MCF-7 BREAST CANCER CELLS growth by an indirect method (21). This latter conclusion was based pivotal to all of the subsequent progress in the understanding and on the observation that estrogen did not stimulate MCF-7 cells to interpretation of data obtained with MCF-7 cells in culture. replicate in vitro, but if the same cells were grown in athymic mice, then estrogen stimulated growth. It was reasoned that estrogen must be triggeringa second messengerin vivo. At the time (1980), this was Monoclonal Antibodies to the ER not an unreasonable conclusion, because it was accepted that carcin ogen-induced rat mammary tumors require both estrogen and estro The discovery and detection of the ER protein (30â€”33)in the rat gen-stimulated prolactin for growth (22). Subsequently, Huseby et a!. uterus was of fundamental importance to progress in breast cancer (23), at the Michigan Cancer Foundation, provided evidence, in athy therapy. However, studies on the biochemistry of the protein mid mice, to demonstrate that estrogen had a direct growth-stimulat involved expensive experiments using rats or the collection of calf ing effect on MCF-7 tumor cells in vivo. Indirect growth stimulation uteri from slaughterhouses. Clearly, the development of antibodies through a second messenger was improbable. to the human receptor would be extremely important in research The breakthrough in our understanding of direct estrogen action and for the convenient detection of ER by clinical laboratories. came with the discovery that MCF-7 cells had been grown in estro Progress in the detection of the ER using antibodies primarily gen-containing media from the start. Indeed, if the occult estrogen had centered on Jensen's group at the Ben May Laboratory in Chicago. not been there, the MCF-7 cell model would not have been possible. Polyclonal antibodies against ER protein were obtained initially The story unfolded through a series of fortuitous accidents. Dr. using highly purified ER as an antigen to immunize rabbits and a John Katzenellenbogen was particularlyinterested in developing a goat (34, 35). To avoid the heterogeneity in the antibodies, the fluorescent-tagged estrogen so that ER could be detected easily in hybridoma techniques of Kohler and Milstein (36) were used breast cancer specimens under the fluorescence microscope. Natu subsequently to obtain monoclonal antibodies to the calf uterine rally, he was using MCF-7 cells as his laboratory model, but the nuclear ER (37). Unfortunately, there was no absolute proof that finding that unwashed cells fluoresced led to an examination of the monoclonal antibodies to animal ER would be of value for human chemical constituents in media. Phenol red (Fig. 1) is the indicator studies. Almost immediately, but working in collaboration with used routinely in commercial media to monitor the oxidative state of scientists at Abbott Laboratories, Dr. Geof Greene, who played the the cells. The indicator is, however, present not as a few drops but in leading experimental role in the antibody research at the Ben May @.LMconcentrations,and its structure is reminiscent of nonsteroidal Laboratories, described the preparation of monoclonal antibodies estrogens first synthesized by Sir Charles Dodds in the l930s (24) to the human ER from MCF-7 breast cancer cells (38). The prior to his landmark discovery of diethylstilbestrol (25). Drs. John property of the cells to replicate indefinitely under carefully con and Benita Katzenellenbogen established, in a seminal publication, trolled conditions provided a constant supply of receptor to make that removal of phenol red from media resulted in the exquisite antibodies. sensitivity of MCF-7 cells to the growth-promoting effects of estrogen Although the development of monoclonal antibodies to the ER (26).Aspredictedfromdata,invivoantiestrogenswerepartialago would ultimately revolutionize the detection of ER in clinical breast nists and were antagonists of estradiol action. cancer samples, the new laboratory tool would also change the sub As an aside, it was found that different preparations of phenol red cellular model of estrogen action. The original model for estrogen had different potencies as estrogens (27), and it was a dimerization action placed the ER in the cytoplasmic compartment. Estrogen bound product, which occurred during manufacture, that was responsible for to the receptorandcausedtranslocationof the complex to the nucleus, estrogenicity (28, 29). The similarity of the structure of the contam where the events associated with hormone action would be triggered. inant with the most potent synthetic estrogen, diethylstilbestrol, is This model had been challenged in 1977 by Zava and McGuire, who illustrated in Fig. 1. used careful fractionation methods and found unoccupied ER in the The discovery of an occult estrogen that is ubiquitously present in nuclear compartment of MCF-7 cells (39). However, the ability to see the culture media is an important lesson, obvious in hindsight, but receptor without cell disruption would change the textbook model that had stood for nearly 2 decades. King and Greene (40) used monoclonal antibodies to the MCF-7 OH ER to demonstrate that ER was in the nucleus of cells. Although the results were published before the discovery that MCF-7 cells were actually growing in the estrogenic phenol red-containing media, these data (Fig. 2) altered the scientific concepts. Parallel studies by Weishons et a!. (41), who used cytochalasin B to prepare nucleoplasts from the rat pituitary cell line GH3, found that the majority of ER was 4..@ 10\@ I HO contained in the nucleoplasts and not the cytoplasts. Subsequently, 0 Welshons et al. (42) showed that nucleoplasts from MCF-7 cells PhenolRed Estrogenic Contaminant bis(4-hydroxyphenyl)[2-(phenoxy. grown in phenol red-freemedia containedER. Thus, it would not be sulfonyl)phenyljmethane unreasonable to state that the MCF-7 cell line was essential for the understanding of the subcellular organization of not only the ER but also all of the other steroid hormone systems. The importance of the development of monoclonal antibodies to ER extends to mechanistic studies of receptor dynamics during estrogen and antiestrogen action (43, 44). Additionally, antibodies can be used as probes to study the direct effects of antiestrogens on the ER protein (45, 46). Indeed, an Diethylstilbestrol additional epitope has been discovered to occur when ER is liganded Fig. 1. Structures of estrogenic components identified in tissue culture media and the with 4-hydroxytamoxifen (47), and this may be important to under most potent synthetic nonsteroidal estrogen, diethyistilbestrol. stand antiestrogen action. 3072

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Fig. 2. Immunocytochemistry-specific nuclear staining for ER in MCF-7 cells grown on glass slides and incubated with H226 after fixation in picric acid/paraformaldehyde. Controlpanel (left), cellsthatwereincubatedwithnonspecificratIgCi. 7 Specific staining was visualized with diaminoben S zidine following incubation with rabbit antirat IgG and rat peroxidase-antiperoxidase complex.

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Cloning and Sequencing of the ER Growth Factor Regulation Without the preparationof monoclonal antibodies to the MCF-7 The MCF-7cell model has been examinedextensively to determine ER, it would not have been possible to make progress in cloning and the mechanism(s) of estrogen-stimulatedgrowth. The early work of sequencing the ER from MCF-7 cells (48). The development of Dr. Henri Rochefort identified a secreted glycoprotein from MCF-7 probes that hybridized in the cDNA library preparedfrom MCF-7 cells that was believed originally to be a growth accelerator (64, 65). cells resulted in two simultaneous reports of the sequencing and However, identification of the protein with monoclonal antibodies expression of the full-lengthcDNA for ER (49, 50). However, it was (66)andthecloningandsequencingofthegeneshowedtheproteinto noted initially that there was an alteration in the cloned MCF-7 ER be the enzyme cathepsin D (67). The clinical community has exten cDNA [Gly-400 (GGG) -+ Va1-400(GTG)]. Otherspecies (chicken, sively studied the protein as a potential marker of prognosis in mouse, rat,andXenopus)all have a glycine at position400, butinitial node-positive and node-negative breast cancer. In contrast, a protein speculation discounted the relevance of the finding for the human ER, of unknown function, pS2, was identified in MCF-7 cells by Cham because the binding affinity for estradiol was unaffected at 4Â°C(50, bon's group (68â€”70).The protein has, like the PgR, been linked to 51). However, it was concluded eventually that the original cDNA good prognosis in breast cancer. The estrogen response element for designated HEO produced a protein that has decreased affinity for pS2 is used routinely as an analytical system for reporter genes to estradiol at 25Â°C(52), and it is, because of the mutation, unstable and study the molecular biology of ER activation (e.g., see Ref. 71). rapidly turned over when expressed in cells. Subsequently, it was also The majorconceptualbreakthroughinourunderstandingofcellular found that the mutation increases the estrogen-like activity of tamox communication occurred in the late 1970s with the identification of ifen (53, 54). The wild-type ER from MCF-7 cells is now referredto positive and negative growth factors. It was reasoned that a link must as HEGO, with G denoting the presence of glycine at position 400. exist between estrogen action and growth factor secretion. Lippman's The human ER gene is >140 kb in length and is split into eight groupused MCF-7 cells extensively throughoutthe l980s to describe exons with the position of the introns highly conserved with other the hormonalregulationof the TGF-a/epidermalgrowthfactorrecep receptors (51). The MCF-7 ER mRNA is 6322 nucleotides in length tor system (72â€”76).Thishas been particularlydifficultto accomplish, and encodes a 595-amino acid protein (50). The functional organiza because, unlike hormone-independent cells that overexpress growth tion of the ER has been demonstrated by site-directed mutagenesis factors and their receptors, the MCF-7 system has only low levels of (55). The human ER comprises six regions (regions Aâ€”F),with a the relevant components. Studies showed initially that factors secreted small, centrally located DNA-binding domain and a large, COOH terminal ligand-binding domain (Fig. 3). The development of reverse transcription-PCR (56) allowed a close examination of the mRNAs NLS HSP-BINDING from MCF-7 cells to establish whether variantsof the ER exist and DNA- I @ whether these could have biological relevance. The research strategy BINDING LIGAND-BINDINGDOMAIN is controversial, because there is only clear evidence that the variants DOMAIN I AF.2 and I NLS actually produce a protein in one sublime of MCF-7 cells called 2A DIMERIZAT1ONI HSP-BINDING (57). The protein is a high molecular weight ER variant (Mr 77,000 AF.1 I @l I DIMERIZAT1ON I @I rather than Mr 65,000) that does not bind estradiol or antiestrogens @ (58). This is because the protein has a duplication of exons 6 and 7 in H2NIAIB C IDI E 1F1c00H the ligand-bindingdomain (58, 59). Otherinvestigatorsusing reverse 138 180 263302 553 595 transcription-PCR have produced variant cDNA from MCF-7 cells Fig. 3. Schematic representation of the strucwre and functional organization of ER. AF. (60);however,thebiologicalrelevanceofanyof the variantshas transcriptional activation function; NIS. nuclear localization signal, HSP-binding. heat recently been questioned (61â€”63). shock protein binding. 3073

from MCF-7 cells could support the growth of the same cells in cause the antiestrogen completely saturated receptors. In contrast, a athymic mice (77); however, the transfection of TGF-a cDNA into study of the direct effects of estrogen withdrawal on hormone-respon MCF-7 was not, by itself, able to provoke estrogen-independent sive breast cancer cells was not possible until the discovery of phenol growth in animals (78). Nevertheless, transfection of v-ras1' does red in the media (26). cause an elevation of stimulatory growth factors that can support cell The first two published reportsof the effects of phenol red-free growth in animals (79, 80) and the interaction of MCF-7 cells with media on the growth of MCF-7 cells came to the same conclusions basement membranes (81). Lippman's group also described antiestro (103, 104). The cells increase their growth rate as adapted clones grow gen-induced production of the inhibitory growth factor TGF-@(82). In out. There is an increase in the levels of ER, but PgR synthesis ceases. fact, these studies were performed before the knowledge that MCF-7 Estrogen can reinduce the PgR; however, there is little effect on cells were grown in phenol red and the realization that the estrogenic cellular growth rate. The cells now appear to grow maximally in the contaminant was blocking TGF-@3synthesis. Despite this, the princi absence of ligand but remain sensitive to the inhibitory actions of plc was clear. Antiestrogenscould maintainthe secretion of TGF-@ antiestrogen. In fact, Masamura et a!. (105) have demonstrated sub from ER-positive cells, and the growth factor could have a paracrine sequently that estrogen-deprived MCF-7 cells become hypersensitive role in inhibiting the growth of adjacent ER-negative cells (82). to estradiol. The MCF-7 cells providedan experimentalsystem for defining the Several cell lines have subsequently been cloned from the surviving role of insulin and IGFs in the estrogen-induced proliferation of breast MCF-7 cells in an estrogen-free medium. The line MCF-7.5C is ER cancer cells. Although IGF-I and IGF-ll mRNAs are detected easily in positive (wild type), but PgR cannot be induced by estrogen (106). If the majorityof breasttumors,conflicting results have been reported the original MCF-7 could be described as making up the ER-positive for the expression of IGFs in breast cancer cells. Initially, Huff et a!. PgR-positive tumor that has a functioning receptorwith good prog (83, 84) reported that IGF-I is synthesized under estrogen control in nosis, then the MCF-7.5C cells make up the ER-positive PgR-nega MCF-7 cells. However, Karey and Sirbasku (85), using an immuno tive tumorthathas a poorerprognosis (107). Dr. Benita Katzenellen assay to measure IGF-I in conditioned medium, reported that MCF-7 bogen has also developed numerous new cell lines from MCF-7 (108), cells do not produce biologically significant concentrations of IGF-I, and their growth characteristicshave been described in athymic ani and subsequently,Yee et a!. (86), using a sensitive RNase protection mals by Dr. Robert Clark at Georgetown University in Washington, assay, could not detect IGF-I mRNA in breast cancer cell lines, D.C. (109). Additionally, Dr. Katzenellenbogen has recently de including MCF-7 cells. scribed a tamoxifen (resistant)-stimulated breast cancer cell line de The majormitogenic effects of both IGF-I and IGF-IIare thought rived from MCF-7 cells in vitro (110). The cells are extremely to be mediated via the type I IGF receptor identified in MCF-7 cells valuable to compare and contrast with wild-type lines by differential as well as in other breast cancer cells (87â€”89).The action of IGF-I at display. Identificationandelucidationof the new growthpathwaysin its receptor may be modified by IGF-bindingproteins, which have the tamoxifen-resistantER-positive cells will have importantimpli been identified in conditioned media from breast cancer cells, includ cations for the design of new therapeutic agents. ing MCF-7cells (90, 91). It has been shown thattamoxifenresistance andestrogen independencein humanbreastcancercells is associated Tamoxifen-stimulated Tumor Growth with complex changes in IGF-binding protein secretion patterns that accompany changes in IGF receptor expression (92). The developmentof immunodeficientathymicmice (111) provided Despite the appeal of the autocrine model of growth regulation in a new dimension for the study of breast cancer growth in vivo. breast cancer by different growth factors, the data are not consistent Regrettably, early studies with primary breast tumors were, in the with an autocrine model of estrogen-regulated growth involving IGF-I main, unsuccessful in producing hormone-responsive disease, because and IGF-H. In the nude mouse model, antibody-induced blockade of tumor takes were poor, and hormone-independentdisease usually the type I IGF receptordoes not affect growthof the MCF-7 tumors, grew preferentially despite estrogen administration (1 12â€”114). whereas the antibody inhibits growth of estrogen-independent MDA The breakthroughcamewiththe transplantationofthe breastcancer MB-23l tumors (93). However, a stimulatory effect of exogenous cell line MCF-7 (1 15). Estrogen is required for the growth of MCF-7 IGF-I on MCF-7 tumor growth in nude mice has been reported (94), breast cancer cells in ovariectomized athymic mice. It should be suggesting that these cells do not lose the type I IGF receptors in vivo. noted, however, that MCF-7 cells do not grow vigorously in intact Experiments in vitro with phenol red-free medium demonstrate that athymic mice, because the animals have endocrine deficiencies and do IGF-I alone has very little effect on cell proliferationand only in the not have estrouscycles. Estrogensupplementationis obligatory(116). presence of estradiol can these agents act synergistically to stimulate However, there are considerablevariationsin MCF-7 cell lines, and growth (95â€”97).The observation that estradiol regulates type I IGF this produces different abilities to grow in athymic mice (117). receptor in MCF-7 cells at the transcriptional level (95) suggests a It is interesting to note that, although tamoxifen is described as direct sensitizationof breastcancer cells to IGFs by estradiol. estrogen in the mouse (118), tamoxifen does not initially support the growth of MCF-7 cells. Tamoxifen inhibits estrogen-stimulated Hormone and Antiestrogen Resistance in Vitro MCF-7 tumor growth in vivo (119, 120). This observationparallels the observationsin vitro. Tamoxifen does, however, maintainoccult It has been a century since the first observation that some human disease for long periods after inoculation into mice, and tumors can be breast cancers will regress if estrogen is withdrawn(98). Naturally, reactivated by estrogen treatment when the drug is stopped (120). the developmentof antiestrogensas a successful breastcancertherapy By the mid-l980s, prolonged(2â€”5years)adjuvanttamoxifentreat (99) resulted in the quest to understand antiestrogen resistance and the ment was being used routinely in clinical practice, and it was clear that slip to hormone-independent growth. In the laboratories, MCF-7 cells this strategy produced a survival advantage (121, 122). However, it were used to develop several antiestrogen-resistantcell lines (100), was unreasonable to expect that drug resistance would not occur one of which, LY2 (101), was found subsequentlyto have a wild-type eventually. An endometrial carcinoma, EnCa 101, had been shown to ER (102). The finding of wild-type receptors is a consistent finding in grow partiallyduring tamoxifen treatment(123), so it was possible antiestrogen-resistant disease. Early laboratory studies to develop thattamoxifen-stimulatedbreastcancer would also occur. Osborneet antiestrogen-resistant breast cancer cell growth were successful be a!. (124) first showed that MCF-7 cells could become tamoxifen 3074

stimulated for growth in vivo but, paradoxically, in vitro, the tumor vances in our knowledge of hormone action and the standardization of cells revert to their original phenotype; i.e., tamoxifen blocks estro therapy with antiestrogenic drugs. Dr. Herbert Soule had the vision gen-stimulated growth. In parallel studies, we showed (125) that and good fortune to establish the first hormone-responsive breast tamoxifen-stimulated breast tumors grown from MCF-7 cells could be cancer cell line that played a fundamental role in advancing knowl passaged routinely and the tumors would grow in vivo in response to edge. He was involved in the three key studies: cell characterization either estradiol or tamoxifen. Furthermore, tamoxifen-stimulated fti (1), receptor identification (2), and estrogen-stimulated growth in mom grow in athymic rats and naturalkiller cell-deficient (beige) athymic mice (115). Together, the results of his labors revolutionized mice (126), so the growth effect is neither species nor immunology the opportunities for breast cancer research. In retrospect, it is remark specific. In fact, target site-specific actions of tamoxifen were noted in able that this single cell line has contributed so much to our basic the animals. The uteri in mice treated with tamoxifen become quies understandingof hormone action and the treatmentpossibilities in cent and refractoryto estrogen, despite the fact that a tamoxifen breastcancer. However, the story does not end here. Dr. Soule left a stimulated MCF-7 tumor is growing in the host (125). second legacy, MCF-lO (149, 150). The model of tamoxifen resistance in breast cancer appears to be The research challenge for the 21st century is to prevent breast cancer. uniqueto MCF-7 cells, andthereis intense interestin discoveringthe MCF-l0 and its variantcells are, surprisingly,naturallyimmortalized, mechanismof tamoxifen-stimulatedtumorgrowth.Earlytheoriesthat normal epitheial cells of mammary origin that do not produce tumors in the receptormay be mutatedhave provedto be incorrect.Despite one athymic animals (151). The key for a future generation of research @ exception, with a 35l@ mutant ER (127) that is observed in one workers is to dissect the molecular events involved in the change from transplant line, the majority of tumors have wild-type ER (128). normalto malignantbreastepitheium (152â€”155).Itis ourbelief thatby Similarly, ideas that tamoxifen might be metabolized peripherally to diligent research endeavors, and the example of MCF-7 cells before, the estrogens (129, 130) or locally (131â€”133)in the MCF-7 tumor have MCF-lO cells can be used effectively to formalate a rational strategy for not been supportedby experimentation.The growth(134) and devel the preventionof breastcancer. opment of tamoxifen-stimulated MCF-7 tumors (135) occurs with analogues that are incapable of being converted to estrogenic metab Acknowledgments olites. Clearly, the cells are using the intrinsic efficacy of the antics trogen-ER complex to drive cell replication by an alternate pathway. This history is offered as a tribute and memorial to Dr. Herbert Soule and Most importantly,compoundssuch as IC! 164,384 andIC! 182,780 the staff of the MichiganCancerFoundation,who made possible the devel (pure antiestrogens), which have no intrinsic estrogenic properties in opment of the remarkable MCF-7 breast cancer cell line. It is also dedicated to estrogen target tissues (136), inhibit the growth of tamoxifen-stimu Sister Catherine Frances, the patient with breast cancer whose cells ultimately lated MCF-7 tumorsin vivo (137, 138). extended the lives of thousands of women. We offer this history as a prime example of the value of laboratory research that translates, over decades, to the aid of patientswithbreastcancer. Clinical Advances and the Future Possibilities We thankDr. GeoffreyGreenefor generouslyprovidingFig. 2 and Henry Muenzner for preparing other figures. It would be inappropriate to finish this story of the laboratory discoveries made possible by MCF-7 cells withouta brief mentionof some of the clinical advances that have occurred. The systematic References dissection of hormone action has led to significant progress in the 1. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan, M. A human cell line treatment of breast cancer. The following list of examples is itself from a pleural effusion derived from a breast carcinoma. J. NatI. Cancer Inst., 51: 1409â€”1416,1973. impressive but does not claim to be exhaustive. 2. Brooks, S. C., Locke, E. R., and Soule, H. D. Estrogen receptors in a human cell line Abbott Laboratories developed kits for the quantitation of ER (MCF-7) from breast carcinoma. J. Blot. Chem., 248: 6251â€”6253, 1973. (ER-EIA) and PgR (PgR-EIA) in breast tumor (139â€”141).Steroid 3. Lippman, M. E., and Bolan, G. Oestrogen-responsive human breast cancer in long-term tissue culture. Nature (Lond), 256: 592â€”593,1975. receptors from MCF-7 cells were key to this innovation. Specific 4. Cole M., Jones C. T., and Todd I. D. H. A new antioestrogenic agent in late breast monoclonal antibodies for ER and PgR are now used routinely to cancer an early clinical appraisal of IC! 46,474. Br. 3. Cancer, 25: 270â€”275, 1971. determine receptors in tumor cells by flow cytometry and to determine 5. Horwitz, K. B., Costlow, M. E., and McGuire, W. L. MCF-7: a human breast cancer cell line with estrogen, androgen, progesterone and glucocorticoid receptors. Ste the heterogeneityof cell populationsin small biopsy samples (142â€” roids, 26: 785â€”795,1975. 144). 6. Horwitz, K. B., and McGuire, W. L Estrogencontrol of progesteronereceptorin human breast cancer: correlation with nuclear processing of estrogen receptor. The ubiquitoususe of long-termadjuvanttamoxifen therapy(145) J. Biol. Chem., 253: 2223â€”2228,1978. originally presented a problem for the determination of receptor status 7. Horwitz, K. B., and McGuire W. L Nuclear mechanism of estrogen action: effects of recurrencesusing conventional ligand-bindingassays. Tamoxifen of estradiol and antiestrogens on estrogen receptors and nuclear receptor processing. J.Biol.Chem.,253:8185â€”8191,1978. and metabolites saturate all of the tissues and produce false-negative 8. Horwitz, K. B., McGuire, W. L., Pearson, 0. H., and Segaloff, A. Predicting results in breast cancer biopsies because of receptor occupancy. response to endocrine therapy in human breast cancer a hypothesis. Science (Wash ington DC), 189: 726â€”727,1975. Monoclonal antibodies changed all of that, and it is now known that 9. Horwitz, K. B., Koseki, Y., and McGuire, W. L. Estrogen control of progesterone many recurrences remain receptor positive (146). Additionally, there receptor in human breast cancer: role of estradiol and antiestrogen. Endocrinology, is evidence that tamoxifen-stimulated disease occurs in patients (147), 103:1742â€”1751,1978. 10. Jordan, V. C., and Murphy, C. S. Endocrine pharmacology of antiestrogens as which is analogous to the tamoxifen-stimulated MCF-7 tumor in the antitumor agents. Endocr. Rev., II: 578â€”610, 1990. laboratories that remains receptor positive (128). 11. Lippman, M. E., Bolan, G., and Huff, K. The effects ofestrogens and antiestrogens The ability of pure antiestrogens to control the growth of tamox on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res.,36:4595â€”4601,1976. ifen-stimulated MCF-7 tumors (137, 138) was strong evidence to 12. Sutherland, R. L, Green, M. D., Hall, R. E., Redell, R. R., and Taylor, I. W. progress toward clinical trials. Currently, IC! 182,780, the first of Tamoxifen induces accumulation of MCF-7 human mammary carcinoma cells in the 00G1 phase of the cell cycle. Eur. i. Cancer Clin. Oncol., 19: 615â€”621,1983. several pure antiestrogens, is performing as predicted. Preliminary 13. Sutherland, R. L., Hall, R. E., and Taylor, I. W. Cell proliferation kinetics of MCF-7 trials (148) indicate good patient acceptability, a response rate of human mammary carcinoma cells in culture and effects of tamoxifen on exponen about 30%, and a duration of response that extends for several years. tially growing and plateau phase cells. Cancer Res., 43: 3998â€”4006,1983. 14. Osborne, C. K., Boldt, D. H., Clark, 0. M., and Trent. J. M. Effect of tamoxifen on During the past 25 years, the symbiotic relationshipbetween lab human breast cancer cell cycle kinetics: accumulation of cells in early G@phase. oratoryresearchand clinical investigations has resulted in clear ad Cancer Res., 43: 3583â€”3585,1983. 3075

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Anait S. Levenson and V. Craig Jordan

Cancer Res 1997;57:3071-3078.

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