Breast Cancer Research and Treatment 32: 49-55, 1994. © 1994 Kluwer Academic' Publishers. Printed in the Netherlands.

Mechanisms of tamoxifen resistance C. Kent Osborne and Suzanne A.W. Fuqua

Department of Medicine, Division of Medical Oncology, University of Texas Health Science Center, San Antonio, Texas, USA

Key words: animal models, antiestrogens, estrogen receptor variants, ICI 182,780, pure steroidal antiestrogens, tamoxifen resistance Summary Though the antiestrogen tamoxifen prolongs disease-fiee and overall survival in the adjuvant setting, and induces remissions in over half of the patients with estrogen receptor positive metastatic disease, all patients eventually acquire tamoxifen resistance. Furthermore, many of the resistant tumors actually appear to be stimulated by tamoxifen just as they are by estrogens. In both animal models and clinical specimens, we have found lower tamoxifen uptake and somewhat altered tamoxifen metabolism in resistant tumors, but neither appears to explain tamoxifen stimulation of the resistant tumors. Nor do estrogen receptor losses or mutations appear to explain this phenomenon, although altered expression of transcriptional variant forms of the receptor may well contribute. Pure steroidal antiestrogens such as ICI 182,780 are capable of reversing tamoxifen-stimulated as well as estrogen-stimulated growth of these resistant tumors, and are now in clinical trials for this purpose.

Introduction Breast cancer development and progression are influenced by steroid hormones, particularly estrogen, via their interaction with specific target cell receptors. Tamoxifen is a non-steroidal antiestrogen which is now the most frequently used drug in breast cancer treatment. Tamoxifen is thought to inhibit breast cancer growth by competitively blocking estrogen receptor (ER), thereby inhibiting estrogen-induced growth. In the adjuvant setting after primary surgery for breast cancer, tamoxifen has been shown to prolong disease-free and overall survival, and it has

also been shown to induce remissions in more than half of patients with metastatic disease who have ER-positive tumors [1,2]. Although tamoxifen is initially effective in many patients, 50% of patients fail to respond to the drug despite the presence of ER. Furthermore, even patients who initially respond eventually acquire tamoxifen resistance, leading to tumor progression and death. The mechanisms for either intrinsic or acquired tamoxifen resistance are unknown, but they are probably multifactorial. Our group has been interested in the mechanisms by which tumors develop resistance to tamoxifen, with the ultimate goal of developing

Address for correspondence and off'prints. C. Kent Osborne, M.D., Department of Medicine/Medical Oncology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7884, USA; Tel. 210-567-4777; Fax; 210567-6687

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CK Osborne & SAW Fuqua

Tamoxifen .._ E "

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Figure 1. Mechanism of tamoxifen action

new strategies for preventing or reversing the emergence of resistant cells. There are several possible mechanisms by which tamoxifen resistance could develop in breast cancer cells. Clues to these mechanisms can be gleaned from an understanding of the myriad of effects that tamoxifen has at the cellular level, as outlined in Figure 1. Tamoxifen binds to the ER and competitively blocks estrogen-induced transcription of specific genes encoding proteins involved with regulation of cell proliferation. Some of these proteins are in fact polypeptide growth factors, such as transforming growth factor c~, insulin-like growth factor II, and members of the fibroblast growth factor family, which by autocrine and paracrine mechanisms may enhance tumor growth. Downregulation of the expression of these growth factors by tamoxifen may result in suppression of tumor growth. Oddly enough, breast cancer cells, as well as other tumor cells, may also synthesize and secrete growth inhibitors, such as transforming growth factor-[3. Expression of TGF[3 is reduced by estrogen, but enhanced by tamoxifen treatment. Thus, increased expression of growth inhibitors by tamoxifen may also contribute to tumor growth suppression. Clearly, alterations in the expression of these growth factors or growth inhibitors, or their specific cell membrane

receptors, could provide the tumor cell with sufficient growth stimulation to overcome the tamoxifen block, resulting in tamoxifen resistance. Cross-talk between polypeptide growth factor pathways and ER-mediated events could also theoretically result in tamoxifen resistance. It has been shown, for instance, that increasing the level of cellular cyclic AMP pharmacologically alters the cellular response to tamoxifen, converting it from an antiestrogen to a weak estrogen agonist [3]. The mechanism for this phenomenon is not yet understood, but it could be related to changes in the phosphorylation state of the ER itself. Other potential mechanisms for the development of tamoxifen resistance include loss of or mutations in the ER, or altered expression of accessory proteins that could modify the transcriptional signal generated by ligands binding to estrogen receptor. Also, since certain metabolites of tamoxifen are known to be less antiestrogenic, or even to be full estrogen agonists, changed systemic metabolism of tamoxifen or altered uptake or metabolism of tamoxifen in the tumor itself could also result in tamoxifen resistance. Finally, high levels of the so-called antiestrogen binding sites, cytoplasmic binding sites whose function is not yet well understood, could theoretically serve as a sump, soaking up tamoxifen

Tamoxifen resistance in breast cancer

molecules and preventing their binding to ER. We have initiated studies in laboratory models investigating several of these possibilities.

Clinical studies in tamoxifen-resistant patients Clinical studies with tamoxifen provide several clues for mechanisms by which acquired resistance may develop. Patients whose tumors initially lack ER have a very low response rate to the drug, and thus, selection of an ER-negative clone of tumor cells could result in an estrogen-independent tumor refractory to tamoxifen. Some patients with tamoxifen resistance do develop resistance to all forms of endocrine therapy via selection of an ER-negative tumor cell clone. However, we have recently reported a series of patients with acquired tamoxifen resistance in whom tumor estrogen and progesterone receptors were measured by both ligand binding and immunohistochemical assays (to circumvent the problem of receptor occupancy by the drug) [4]. More than 60% of tumors continued to express ER and/or PgR even while progressing in the face of tamoxifen. These data indicate that while ER negativity may account for some cases of resistance, mechanisms of resistance other than receptor loss must be common. If patients' tumors remain ER-positive after development of tamoxifen resistance, one might expect that some of these tumors have retained estrogen sensitivity and will respond to other endocrine treatments. In fact, clinical experience demonstrates that patients who have initially responded to tamoxifen but who later develop tumor progression, frequently respond to second or third-line endocrine therapies. Thus, acquired tamoxifen resistance in these patients does not necessarily indicate global hormonal unresponsiveness, but rather selective resistance to tamoxifen itself. Although it has not been studied systematically, anecdotal experience suggests that some patients with tamoxifen resistance will respond to a rechallenge with the drug after an

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interval in which they receive other treatments. Furthermore, clinical reports suggest that patients who receive tamoxifen adjuvant therapy and then later recur, not infrequently will respond to a rechallenge with the drug. This suggests that tamoxifen resistance in some cases may not be a permanent phenotype, but rather may be reversible when the drug is stopped. Patients may also respond to an increase in the tamoxifen dose after developing progression with a lower dose schedule. Finally, similar to reports of patients treated with high dose estrogen therapy, some patients who have responded to tamoxifen will have a withdrawal response when the drug is stopped at the time of tumor progression. The prolonged half-life of tamoxifen makes it difficult for clinicians to withhold alternative therapy while waiting for a withdrawal response to the drug. Nevertheless, these data strongly suggest that in some patients with acquired resistance, tamoxifen may actually be stimulating tumor growth. Two previously published clinical trials also suggest that tamoxifen-stimulated tumor growth may be a cause of tamoxifen resistance in some patients [5,6]. In these studies, premenopausal women with advanced breast cancer were treated with second-line ovarian ablation after they first responded and then progressed on tamoxifen. In one of these studies, the secondary response to ovarian ablation was common in patients who had previously responded to tamoxifen, suggesting that tamoxifen treatment served as an in vivo tumor estrogen sensitivity assay. However, in the other study, opposite results were obtained and no patients responded to second-line ovarian ablation. In this latter study tamoxifen therapy was continued after the surgery, while in the first study tamoxifen treatment was stopped. Secondary response to ovarian ablation would not be expected in the latter study if tamoxifen itself was behaving as an estrogen agonist and stimulating tumor growth. Tamoxifen-stimulated tumor growth as a mechanism for acquired resistance is further supported by data from our own laboratory as well as others using experimental models. A major focus of our group is to better understand

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mechanisms by which tamoxifen-stimulated tumor growth occurs.

In vivo laboratory model of tamoxifen

resistance We have developed an in vivo experimental model of tamoxifen resistance in which MCF-7 human breast cancer cells are inoculated subcutaneously into athymic nude mice [7,8]. Tamoxifen treatment of these mice suppresses tumor growth for several months, but then tumor growth resumes despite continued treatment with the drug. Transplantation of fragments from these tamoxifen-resistant tumors demonstrates that their growth has not become estrogen-independent, but in fact is now stimulated by tamoxifen as well as by estrogen: These data, which have also been reported by others [9], again suggest that one form of acquired tamoxifen resistance may be due to the acquired ability of the tumor cells to be stimulated rather than inhibited by tamoxifen. Since certain metabolites of tamoxifen have estrogenic properties, we first investigated pharmacologic explanations for this tamoxifen-stimulated growth.

Tamoxifen pharmacology and metabolism in tamoxifen resistance To investigate potential mechanisms for the tamoxifen-stimulated tumor growth seen in our experimental model, we first compared levels of tamoxifen and several of its metabolites in serum and in tumor extracts [8]. We found no differences in levels of tamoxifen or its major metabolites in serum from tamoxifen-resistant mice compared to mice with tamoxifen-sensitive tumors. However, when we examined the tumors themselves, we found that extracts from resistant tumors had on average a 10-fold lower tamoxifen concentration than did extracts from sensitive tumors. We also found a reduced concentration of other metabolites of tamoxifen in tamoxifen-

stimulated tumors, but there was a relative increase in the cis isomer of 4-hydroxy tamoxifen, a much less potent antiestrogen, relative to the trans isomer. We do not yet have an explanation for the reduced tamoxifen concentration in tamoxifen-stimulated tumors. The fact that serum levels of tamoxifen from mice with these tumors remain normal while tumor levels are markedly reduced suggests the possibility of an effiux mechanism that reduces net uptake of tamoxifen by the tumor. We do know that P-glycoprotein, an effiux pump that is known to bind to tamoxifen, is not overexpressed in these tumors. A reduction in tamoxifen concentration in the tumor and a relative increase in the cis isomer of 4-hydroxy tamoxifen alone do not fully explain tamoxifen-stimulated growth. Therefore, we next investigated the possibility that accumulation of estrogenic metabolites such as metabolite E and bisphenol might account for the development of tamoxifen-stimulated growth in this model. Using HPLC and mass spectrometry, both metabolites were identified in tumors from our experimental model, as well as in tumor specimens from patients on tamoxifen, lending further support to the estrogenic metabolite hypothesis [10]. If isomerization of tamoxifen and/or conversion to estrogenic metabolites are important for this form of resistance, then tamoxifen analogs resistant to these metabolic conversions would not be expected to stimulate tumor growth. Two nonisomerizable 7-membered ring analogs fixed in the trans position were used to determine the importance of cis/trans isomerization to the development of resistance. The importance of conversion to metabolite E or bisphenol was evaluated using a deoxytamoxifen analog resistant to cleavage of the dimethylaminoethoxy side chain - - this side chain is necessary for antiestrogenic activity. Finally, a pure steroidal antiestrogen, ICI 182780, was also studied since it has a different mechanism of action than tamoxifen, and does not undergo a similar metabolic fate [11,12]. Using mice with transplanted fragments of a tamoxifen-resistant tumor, we first found that tumor growth was stimulated by both estrogen

Tamoxifen resistance in breast cancer

and tamoxifen as well as by another related non-steroidal antiestrogen, toremifene, and that the fixed-ring analogs that could not undergo isomerization had a similar stimulatory effect on tumor growth [13]. Furthermore, tumor growth stimulation was also observed with the deoxytamoxifen analog which was resistant to metabolism to metabolite E. In contrast, the pure steroidal antiestrogen ICI 182780 not only failed to stimulate tumor growth when used alone, but also inhibited the stimulatory effects of estrogen and tamoxifen. These data suggest that tamoxifen-stimulated tumor growth is mediated through the ER, and that by virtue of a different mechanism of action, pure steroidal antiestrogens may be useful in reversing this form of tamoxifen resistance. ICI 182780 is therefore now in clinical trial in tamoxifen-resistant patients. If tamoxifen-stimulated tumor growth were due to conversion of tamoxifen to a pure estrogen agonist, then combinations of tamoxifen with estrogen might be expected to have an additive effect when used in suboptimal concentrations. In transplant experiments, we again found that both tamoxifen and estrogen are capable of stimulating growth of these tumors. Interestingly, however, when tamoxifen was combined with estrogen, antagonistic rather than additive properties were observed. Thus, tamoxifen in this setting has a dual effect capable of stimulating tumor growth when used alone (agonist activity), but still able to antagonize estrogen when the drugs are used together. The results of the tamoxifen analog experiments suggest that tamoxifen metabolism is unlikely to be responsible for the phenomenon of tamoxifen-stimulated growth. Whether the reduced concentrations of tamoxifen in tamoxifenstimulated tumors are somehow related to the mechanism of tamoxifen resistance remains unknown, but the results of two clinical trials studying tamoxifen concentrations in tumor tissues from tamoxifen-resistant patients support this possibility [14,15]. In our own study, we measured tumor concentrations of tamoxifen and several of its metabolites in a small series of

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patients who were on tamoxifen at the time of tissue biopsy [14]. Although tumor tamoxifen levels varied over a wide range, in general low concentrations were observed in tumors from patients with acquired tamoxifen resistance, while moderate to high concentrations were found in patients responding to tamoxifen. As in our experimental model, an increased ratio of cis to trans 4-hydroxy-tamoxifen was also observed in these patients. Reduced tumor tamoxifen concentrations in patients with acquired tamoxifen resistance, relative to those with de novo resistance, have recently been confirmed in another larger study [15]. Whether reduced tumor tamoxifen concentrations are simply a marker of resistance, or are related mechanistically to the development of resistance, requires further study.

Altered E R as a m e c h a n i s m for t a m o x i f e n resistance

Our group has also been interested in the possibility that altered structure and/or function of ER could lead to tamoxifen resistance. ER contains at least five functional domains. Transcriptionactivating functions are located in the A/B region and in the E region, which also serves as the hormone binding domain. The C region is involved in DNA binding, and the D region is involved with receptor dimerization. Mutations in the receptor might render it nonfunctional, or might even alter the transcriptional activating activity, thus leading to tamoxifen resistance. The transcriptional activating activity in the A/B region of ER (TAF-1) is constitutive and is active even when the receptor is bound by tamoxifen. Mutations in this region, or the presence of other ancillary proteins that modify transcription, could conceivably lead to increased activity of TAF-1 resulting in tamoxifen-stimulated growth. By cloning and sequence analysis we have not yet been able to identify mutations either in the A/B region or in the E region of ER extracted from our tamoxifen-stimulated MCF-7 nude mouse tumors. These results, together with our

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observation that these tumors remain responsive to estrogen, suggest that the ER is normal in these tumors. We have, however, isolated several ER variants from a variety of ER-positive, and also supposedly ER-negative, human breast tumors using sensitive RNA-based polymerase chain reaction methodologies. Tumors which are ERnegative but PgR-positive often express high levels of a variant ER lacking exon 5 of the hormone binding domain [16]. This deletion resuits in the production of a variant ER truncated within the hormone binding domain which is unable to bind estrogen. The receptor, however, is still able to bind DNA and constitutively activates estrogen-responsive genes. When co-expressed with wild-type ER in MCF-7 cells, the exon 5 ER variant confers tamoxifen-resistant growth. Thus, a relative overexpression of this variant, even in the presence of wild-type receptor, could conceivably contribute to tamoxifen resistance in patients. We have also isolated an exon 7 deletion variant which acts as a dominant-negative receptor and could also lead to hormone-independent growth [17]. Therefore, the selection of tamoxifen-resistant ER variants could conceivably be clinically significant, with the drug actually providing the selective pressure for the eventual outgrowth of cells containing these or other ER variants. This hypothesis remains an important question currently under investigation by our group.

Current and future directions

The mechanisms by which tamoxifen acquires the ability to stimulate tumor growth after a period of growth inhibition in some tumor models remains a mystery. The studies described above suggest that this phenomenon is not due to tamoxifen metabolism, or to the presence of mutated ER. It has been known for a long time that tamoxifen has dominant, estrogen agonist properties in some tissues or in some animal species, while antagonist properties dominate in other tissues such as the breast. In our MCF-7 tumor model, estrogen antagonist properties dominate initially and tumor

growth is inhibited. Thereafter tumor progression occurs as the agonist activity of the drug increases, although estrogen antagonist properties are still preserved. It is interesting to speculate that changes in accessory proteins that affect transcriptional activation through ER might contribute to tamoxifen-stimulated growth. We are currently investigating the hypothesis that tamoxifen-stimulated tumor growth is due to increased transcriptional activity of TAF-I, the weakly constitutive transcriptional activating function located in the A/B region, perhaps due to loss of a transcription repressor, or to increased levels of other transcription factors that lead to increased transcriptional activity. The presence of estrogen receptor variants remains an important possible mechanism for tamoxifen resistance in some breast cancer patients, and we are continuing our studies of the function and clinical relevance of these mutants using tumor tissue from patients with documented tamoxifen resistance obtained from the San Antonio Tumor Bank. In addition, by employing PCR amplification, cloning, and sequence analysis we are searching for mutations in the two transcription-activating functional domains of ER. Once identified, the function of these variant ERs will be studied using estrogen-responsive yeast and mammalian expression systems. These studies should help to provide insight into whether altered ER contributes to tamoxifen resistance, and whether tamoxifen resistance in some patients can be predicted by the presence of these variant receptors. It is apparent that the problem of clinical tamoxifen resistance is complex and multifactorial; many different cellular pathways could contribute to a cell's ability to circumvent the inhibitory effects of tamoxifen. The estrogen response pathway is still poorly understood, and it is entirely possible that previously unknown genes may play a crucial role. With the help of our experimental in vivo model system of tamoxifen-stimulated growth and a large number of tumor samples from patients with tamoxifen resistance, our group is searching for new genes

Tamoxifen resistance in breast cancer p o t e n t i a l l y i n v o l v e d in t a m o x i f e n r e s i s t a n c e u s i n g a powerful molecular differential expression technique. Identification of such genes would yield important new information about breast c a n c e r h o r m o n a l r e s p o n s i v e n e s s , and w o u l d also provide potential markers of tamoxifen resistance to b e t e s t e d in future d i a g n o s t i c studies.

Acknowledgements This work was supported by Cancer Center Support Grant NIH P30 CA 54174 and SPORE Grant N I H P 5 0 C A 58183.

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7. Osborne CK, Coronado EB, Robinson JP: Human breast cancer in the athymic nude mouse: Cytostatic effects of long-term antiestrogen therapy. Eur J Cancer Clin Oncol 23:1189-1196, 1987 8. Osborne CK, Coronado E, Allred DC, Wiebe V, DeGregorio M: Acquired tamoxifen resistance: Correlation with reduced tumor levels of tamoxifen and isomerization of trans-4-hydroxytamoxifen. J Nat1 Cancer Inst 83:1477-1482, 1991 9. Gottardis MM, Jordan VC: Development of tamoxifenstimulated growth of MCF-7 tumors in athymic mice after long-term antiestrogen administration. Cancer Res 48:5183-5187, 1988 10. Wiebe V J, Osborne CK, McGuire WL, DeGregorio MW: Identification of estrogenic tamoxifen metabolite(s) in tamoxifen-resistant human breast tumors. J Clin Oncol 10:990-994, 1992 I I. Wakeling AE: Steroidal pure antiestrogens. In: Lippman M, Dickson R (eds) Regulatory Mechanisms in Breast Cancer. Kluwer, Boston, 1991, pp 239-257 12. Parker MG: Action of "pure" antiestrogens in inhibiting estrogen receptor action. Breast Cancer Res Treat 26:131-137, 1993 13. Osborne CK, Jarman M, McCague R, Coronado EB, DeGregorio MW, Hilsenbeck SG, Wakeling AE: The importance of tamoxifen metabolism in tamoxifenstimulated breast tumor growth. Cancer Chemother Pharmacol, in press 14. Osborne CK, Wiebe VJ, McGuire WL, Ciocca DR, DeGregorio MW: Tamoxifen and the isomers of 4hydroxytamoxifen in tamoxifen-resistant tumors from breast cancer patients. J Clin Oncol 10:304-310, 1992 15. Dowsett M, Johnston SRD, Haynes BP, Sacks NPM, Caum MB, Ebbs SR, Smith IE: Impaired intra-tumoral uptake as a mechanism for resistance in human breast cancer. Proc Am Assoc Cancer Res 34:1463 (Abstract), 1993 16. Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O'Malley BW, McGuire WL: Variant human breast tumor estrogen receptor with constitutive transcriptional activity. Cancer Res 51:104-109, 1991 17. Fuqua SAW, Fitzgerald SD, Allred DC, Elledge RM, Nawaz Z, McDonnell DP, O'Malley BW, Greene GL, McGuire WL: Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors. Cancer Res 52:483-486, 1992