The successful treatment of childhood leukemia during the 1960s became the evidence-based catalyst to develop a lexicon of chemotherapies to cure all cancers, including breast cancer. The treatment of breast cancer evolved from radical surgery and radiation to incorporate combination cytotoxic chemotherapy. Furthermore, the new science of bone marrow transplantation, perfected in the treatment of childhood leukemia, was evaluated unsuccessfully in breast cancer.

To put the war against cancer into the words of General George Patton “if everyone in the room is thinking the same way, then somebody is not thinking”. Nevertheless, a body of evidence, using the failed contraceptive tamoxifen targeted to estrogen receptor (ER)-positive breast cancer, would serve as a foundation for all future progress in precision or targeted therapy.

The origins and initial development of tamoxifen will not be described here as the story has been described previously.1 Tamoxifen is an active agent in the treatment of metastatic breast cancer,2 but the key to the success of tamoxifen, as a pioneering anticancer agent, was the translational research strategy proven by subsequent clinical trials. During the 1970s, initially at the Worcester Foundation for Experimental Biology, and continued at the University of Leeds, the principles for the treatment and prevention of breast cancer with tamoxifen were established: (1) targeting only patients with ER-positive breast cancers;3 (2) deploying long-term (> 5 years) adjuvant tamoxifen therapy4,5; and (3) the discovery6 of the potential of tamoxifen as the first chemopreventive for breast cancer, in high-risk women. Following rigorous randomized clinical trials,7 each of these translational treatment principles is now US FDA approved. This path to clinical progress in the treatment and prevention of breast cancer illustrates it is not the availability of a therapeutic agent alone that creates an environment for success, but it is how the new agent is applied strategically for clinical care.

A move to the University of Wisconsin, Madison, USA, to establish a tamoxifen team (1980–1995), proved to be an ideal opportunity for research and clinical translation. The University of Wisconsin was unique as it had two National Cancer Institute (NCI)-funded cancer centers—the world famous McArdle laboratory, and the newly built Clinical Cancer Center. Additionally, Jack Gorski of ER fame,8 was working in the Department of Biochemistry. This university environment, along with the support of the founding director of the Wisconsin Comprehensive Cancer Center (WCCC), Harold Rusch, the new director Paul P. Carbone, and the head of the Breast Cancer Research and Treatment Program, Doug Tormey (Fig. 1) would be the place that changed healthcare dramatically with the discovery and development of SERMs.

Fig. 1
figure 1

University of Wisconsin Comprehensive Cancer Center faculty involved in hormone action and breast cancer therapy. Back row, left to right: Dr. Gerald C. Mueller, Dr. Jack Gorski, Dr. V Craig Jordan, and Dr. Douglas C. Tormey. Seated, left to right: Dr. Harold Rusch and Dr. Paul P. Carbone. This photograph was taken in 1984 just prior to the satellite symposium entitled “Estrogen and Antiestrogen Action: Basic and Clinical Aspects” hosted by the Cancer Center in Madison as part of the events surrounding the 7th International Congress of Endocrinology held in Montreal, Canada. The proceedings were published by the University of Wisconsin Press as a book titled “Estrogren/Anti-estrogen Action and Breast Cancer Therapy”, edited by V. Craig Jordan

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Translational Research in Breast Cancer at the University of Wisconsin Comprehensive Cancer Center (1980–1993)

The expanding clinical applications of tamoxifen demanded parallel laboratory studies to examine pharmacology and toxicology. Little information was available regarding (1) the mechanism of acquired resistance to tamoxifen; (2) how antiestrogens function to block estrogen action at a target gene, or regulate breast cancer cell replication; and (3) the long-term metabolic stability of tamoxifen.

(1) Acquired Resistance to Antihormone Therapy Multiple approaches were employed to study acquired resistance to tamoxifen and estrogen deprivation in breast cancer once aromatase inhibitors were developed. This knowledge was critical for safety and to determine how to develop second-line treatments.

The role of growth factor cell signaling was a ‘hot topic’ during the 1980s. It was possible that tumor cell growth factors could play a role in undermining the effectiveness of tamoxifen to control tumor cell growth. Two ideas were popular. Tamoxifen could increase levels of transforming growth factor (TGF)-β, which prevented the growth of adjacent ER-negative tumor cells.9 However, testing the theory in vivo in athymic mice did not support this hypothesis.10 An alternate proposition was the role of growth factors to subvert the action of tamoxifen through ‘growth factor cross talk’ at the tumor cell membrane. Studies demonstrated11,12,13 that paracrine mechanisms for growth factors subverted the antitumor actions of tamoxifen and reduced progesterone receptor synthesis. This latter observation was significant as the breast tumor progesterone receptor is a marker for good prognosis.

One interesting and important result of a study of growth factor regulation was the unanticipated finding that synthetic progestins in oral contraceptives, which were 19 nortestosterone derivatives, were also estrogenic for the growth of breast cancer.14,15,16,17

Tamoxifen treatment of athymic mice implanted with MCF7 breast tumor cells, to replicate long-term adjuvant therapy, resulted in a surprise. Marco Gottardis (a Ph.D. student) discovered that the growth of the ER-positive tumors occurred despite tamoxifen treatment. Subsequent studies retransplanting the tamoxifen-resistant tumors into a new generation of athymic mice or athymic rats, demonstrated that the tumors grew only with estrogen or tamoxifen.18,19 This was not autonomous tumor growth. These data were important for the development of the pure antiestrogen fulvestrant. The WCCC research program successfully tested the first pure antiestrogen in this animal model.20 Subsequent clinical studies demonstrated that either an aromatase inhibitor or fulvestrant should be used as second-line treatment for tamoxifen failure in metastatic breast cancer.21

In the 1980s/1990s, tamoxifen-stimulated tumors could not be grown in cell culture. As a result, tumor retransplantation into successive generations of tamoxifen-treated athymic mice was the only way to preserve the tamoxifen-resistant phenotype. This methodology resulted in another discovery. The plan was to document growth factor tumor cell regulation for estrogen- or tamoxifen-stimulated growth. Were the same growth factors being activated by estrogen or tamoxifen? Unexpectedly, Doug Wolf, another Ph.D. student, was the first to discover22 that physiological estrogen would cause rapid tumor regression of MCF7 tumors exposed to 5 years of tamoxifen in vivo. These data were confirmed23 and resulted in the new biology of estrogen-induced apoptosis24 that has been used to explain the reason why (1) estrogen replacement therapy administered to women over the age of 60 years results in a decreased incidence of breast cancer25; and (2) why women treated with tamoxifen for 5 years continue to have a decreasing recurrence rate after tamoxifen is stopped. It is proposed that a woman’s own estrogen is killing micrometastasis with acquired resistance to tamoxifen.26

With the advent of aromatase inhibitors for the treatment of breast cancer, long-term estrogen deprivation studies were conducted on T47D and MCF7 ER-positive breast cancer cell lines.27,28 Two mechanisms of ER regulations were discovered.29 The T47D cells lost the ER during long-term estrogen deprivation,30,31 and MCF7 cells increased ER levels and became autonomous for growth.28 Clonal selection resulted in MCF7:5C cells,32 where estradiol triggers apoptosis within 7 days,33 and MCF7:2A cells, where the process takes 14 days.34 These cells were used extensively to study the mechanism of estrogen-induced apoptosis. Clinical confirmation of the value of estrogen treatment following exhaustive endocrine therapy35 validated the translational research model at WCCC.

(2) An ER Model of Antiestrogen Action The discovery that tamoxifen was hydroxylated in the 4-position to an antiestrogen with high affinity for ER36,37 not only provided important knowledge in medicinal chemistry38,39 to synthesize future nonsteroidal antiestrogens, but also created a valuable reagent to be made radiolabeled for studies in vivo40 and in vitro41,42,43,44 with interactions at the ER. The first model cell culture system in vitro of estradiol-stimulated prolactin synthesis in isolated cells from the mouse pituitary gland45 was an important innovation to address the mechanics of estrogen and antiestrogen action at the ER. The model in vitro avoided concerns about metabolism in vivo. Extensive structure function relationship studies classified ligands as antagonist, partial agonist, and agonist,46,47,48,49,50 based on structure. This investigation at an estrogen-responsive gene was subsequently expanded to map the structure function relationships of breast cancer cell replication.51,52,53 The model developed was referred to as the ‘crocodile model’, as the antiestrogenic side chain was predicted to prevent ‘the jaws of the crocodile’ from closing.54 The antiestrogenic side chain was predicted to interact with a region referred to as the ‘antiestrogenic region’.55 This region was subsequently identified as asp351, which was found to be mutated to asp351tyr in a tamoxifen-stimulated tumor line.56,57 Most importantly, it was demonstrated that this specific natural mutation could convert raloxifene and antiestrogen to an estrogen. This scientific detective work was accomplished by isolation of the asp351tyr complementary DNA (cDNA),57 creation of the first stable transfectants of ER in ER-negative breast cancer cells,58,59 and, finally, the demonstration using a TGFα target that asp351tyr can convert raloxifene into an estrogen.60,61 These pharmacologic data complimented the subsequent X-ray crystallography of both the raloxifene and tamoxifen ER complexes.62,63 The antiestrogenic side chain interacts with asp351. Today, it is known that asp351 is an essential amino acid necessary to close the unoccupied mutant ERs noted in aromatase resistant breast cancer.64 This story illustrates how basic science deciphers the mechanics of how the ER complex functions in clinical situations. The validity of knowledge comes from multiple investigations, discovery, and then ‘rediscovery’.

(3) The Long-Term Metabolic Stability of Tamoxifen and Patient Endocrinology The major advantage of the WCCC was a mandatory serum collection process for all patients attending a cancer center clinic. These serial samples allowed not only monitoring of tamoxifen and metabolites65,66 but also circulating hormone levels in pre- and postmenopausal patients.67,68 Tamoxifen and metabolites were monitored over 5 years of adjuvant tamoxifen therapy69 and, subsequently, over 10 years.70 Little was known of the metabolism of tamoxifen, but almost immediately a new metabolite of tamoxifen metabolite Y was discovered.66,71 Other investigators studying the metabolism of the related antiestrogen toremifene discovered an equivalent metabolite. Today, this SERM is marketed for treating dyspareunia in postmenopausal women.72

Most importantly comparative studies of circulating tamoxifen and metabolites were completed in laboratory animals and athymic mice,73 and were compared with circulating patient levels.74,75

The Discovery and Proposed Applications of Selective Estrogen Receptor Modulators (SERMs)

A series of simultaneous investigations at WCCC created a database that resulted in the new science of selective estrogen receptor modulators (SERMs). It was known that tamoxifen could prevent the growth of human breast cancer cells in athymic mice, but the mouse uterus stimulated growth.76 The same metabolites of tamoxifen accumulated in the human tumor and the mouse uterus, therefore it was proposed that the drug receptor complex is perceived as either “a stimulatory or inhibitory signal in the different target tissues of different species”.76 Simultaneously, it was discovered that tamoxifen and raloxifene preserved bone density in ovariectomized rats similar to that observed with estradiol.77 This was a completely counterintuitive result as ‘antiestrogens’ were expected to decrease bone density. Tamoxifen and raloxifene were both found to inhibit rat mammary carcinogenesis.78 Subsequent studies by others confirmed the bone-sparing properties of tamoxifen and raloxifene.79,80

The use of long-term adjuvant tamoxifen raised a safety question about the effects of tamoxifen in the human uterus and in endometrial cancer, however nothing was known. To address this issue, Marco Gottardis conducted a pivotal experiment using athymic mice bi-transplanted with ER-positive breast cancer cells or an ER-positive human endometrial carcinoma.81 Tamoxifen blocked estrogen-stimulated breast cancer growth, but endometrial cancers grew dramatically. The presentation of these data at a symposium in Italy attracted considerable interest from the clinical community.82,83 It was subsequently found that long-term tamoxifen therapy produced a small but significant increase in the incidence of endometrial cancer in tamoxifen-treated patients.84 These data not only caused changes in clinical care but also restricted the use of tamoxifen to patients with breast cancer or at high risk for developing breast cancer.

Based on these laboratory data, it was concluded, “we have obtained valuable clinical information about this group of drugs that can be applied to other disease states. Research does not travel in straight lines and observations in one field of science often become major discoveries in another. Important clues have been garnered about the effects of tamoxifen on bone and lipids so it is possible that derivatives could find targeted applications to retard osteoporosis or atherosclerosis. The ubiquitous applications of novel compounds to prevent disease associated with the progressive changes after menopause may, as a side effect, significantly retard the development of breast cancer. The targeted population would be post-menopausal women in general, thereby avoiding the requirement to select a high risk group to prevent breast cancer”.85

Eli Lilly abandoned work on keoxifene (raloxifene), but clinical studies with adjuvant tamoxifen progressed at WCCC. Tamoxifen maintained bone density in postmenopausal patients,86 and decreased low-density lipoprotein cholesterol.87,88 However, the emerging toxicology of tamoxifen with endometrial cancer89 and the induction of rat liver carcinogenesis90,91,92,93 required a safer compound to implement the aforementioned strategy85 to treat multiple diseases in women with a single medicine.

Eli Lilly confirmed the WCCC bone data,80 and advanced the stated blueprint for women’s health85 with clinical trials to prevent osteoporosis. Their study demonstrated that raloxifene reduces fractures in postmenopausal women and produces a dramatic decrease in the incidence of ER-positive breast cancer.94 A subsequent clinical trial, referred to as the Study of Tamoxifen And Raloxifene (STAR), demonstrated that both SERMs produced a decrease in breast cancer in high-risk women, but raloxifene had fewer adverse effects than tamoxifen.95,96

There are now five FDA-approved SERMs on the market with discovery origins back to studies at WCCC: toremifene,97,98 raloxifene,77,78 bazedoxifene,99 ospemifene,66 and tamoxifen.5 One additional compound, lasofoxifene, is a miracle of medicinal chemistry.100 Unlike any of the other SERMs, lasofoxifene is extremely potent; it is used at 0.5 mg daily, compared with 60 mg daily for raloxifene, to prevent osteoporosis. Unlike the other SERMs, lasofoxifene decreases coronary heart disease and strokes. In addition, a decrease in breast and endometrial cancer has been observed. However, like other SERMs, there is a small but significant increase in thromboembolic events.

The SERM saga originating at the University of Leeds and the University of Wisconsin is an example of the value of comprehensive cancer centers to take ideas from the laboratory to aid patients with multiple diseases. Selective estrogen receptor modulation is a unique mechanism that has not been anticipated. Only by examining a broad range of estrogen target tissues, cell culture models, and animal models focused on a comparison with human disease in one laboratory, could the principles be deciphered correctly. Today, there is a clear understanding of the molecular mechanisms of SERM action in different target tissues (Fig. 2). It is possible that in future new selective modulators of other members of the nuclear receptor super family can be modulated to treat diseases previously believed to be impossible.

Fig. 2
figure 2

The molecular mechanism of action of SERMs. Molecular networks potentially influence the expression of SERM action in a target tissue. The shape of the ligands that bind to the ERs α and β programs the complex to become an estrogenic or antiestrogenic signal. The context of the ERC can influence the expression of the response through the number of CoRs or CoAs. In simple terms, a site with few CoAs or high levels of CoRs might be a dominant antiestrogenic site. However, the expression of estrogenic action is not simply the binding of the receptor complex to the promoter of the estrogen-responsive gene, but a dynamic process of CoA complex assembly and destruction. A core CoA, for example steroid receptor coactivator protein 39 (SRC3), and the ERC are influenced by phosphorylation cascades that phosphorylate target sites on both complexes. The core CoA then assembles an activated multiprotein complex containing specific CoCos that might include p300, each of which has a specific enzymatic activity to be activated later. The CoAc binds to the ERC at the estrogen-responsive gene promoter to switch on transcription. The CoCo proteins then perform Me or Ac to activate dissociation of the complex, simultaneously ubiquitylated and destroyed in the 26S proteasome. Therefore, a regimented cycle of assembly, activation, and destruction occurs on the basis of the preprogrammed ER complex. However, the coactivator, specifically SRC3, has ubiquitous action and can further modulate or amplify the ligand-activated trigger through many modulating genes that can consolidate and increase the stimulatory response of the ERC in a tissue. Therefore, the target tissue is programmed to express a spectrum of responses between full estrogen action and antiestrogen action on the basis of the shape of the ligand and the sophistication of the tissue-modulating network. Reproduced with permission from Nature Publishing Group; Jordan VC, Chemoprevention of Breast Cancer with Selective Estrogen Receptor Modulators. Nature Reviews Cancer. 2007;7:46-53. NFκB nuclear factor κB, SERMs selective estrogen receptor modulators, ERs estrogen receptors, ERC estrogen receptor complex, CoRs co-repressors, CoAs co-activators, CoCos co–co-activators, CoAc CoA complex, Me methylation, Ac acetylation

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Fig. 3
figure 3

The ladies of the UWCCC Tamoxifen Team in 1983. Far left, Ethel Cormier, and far right, Anna T. Riegel (née Tate), both Ph.D. students. In the center is Barbara Gosden (deceased) who spent 2 years in the laboratory following her MSc degree at the University of Leeds in 1979 with Anna Riegel. Mara E. Lieberman (deceased), between Barbara and Anna, joined my Team in 1981 from Jack Gorski’s laboratory. This article is dedicated to their memory. Both were outstanding practical scientists. Mara recruited Rich Koch, who performed hundreds of experiments using the isolated pituitary gland cells assay from immature mice to perform structure function relationships of SERMs that bound to the ER. UWCCC University of Wisconsin Comprehensive Cancer Center, SERMs selective estrogen receptor modulators, ER estrogen receptor

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Fig. 4
figure 4

The Wisconsin Tamoxifen Team in the mid 1980s. Front row, left to right: Simon Robinson (post doc from the University of Leeds), Wade Welshons (Assistant Scientist from Jack Gorski’s laboratory), V. Craig Jordan (estrogen receptor laboratory), Peter Ravdin (Medical Oncology Fellow), and Rich Koch (Technician). Center, back row: Marco Gottardis, Ph.D. student; right, Mike Wolf (estrogen receptor laboratory), Richard Bains (technician metabolite analysis), Eric Phelps (summer student), and Pat Mortons (Medical Illustrator for the hospital)

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Fig. 5
figure 5

The Wisconsin Tamoxifen Team at the Swan Hotel, Orlando, Florida, 1991. Front row, left to right: Professor Y. Iino, who visited for 1 year in the 1980s and subsequently ensured that the other Japanese physicians would join my later tamoxifen teams), M-Wei Jeng (Ph.D. student), and S-Y Jeng. Back row, near American Flag: Doug Wolf (Ph.D. Student), John Pink (Ph.D. Student), Chris Parker (estrogen receptor laboratory), Delinda Mauel (radioimmuno assay), Sue Langan-Fahy (tamoxifen assays), and Simon Robinson (post doc). The meeting on Long-Term Tamoxifen Treatment for Breast Cancer lasted for 3 days over 4 July. The University of Wisconsin published the book titled “Long Term Tamoxifen Treatment for Breast Cancer”, edited by V. Craig Jordan, in 1994

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