I. Introduction

Top Previous Next References

Endocrine manipulations are among the most effective, and least toxic, of the systemic therapies currently available for the management of hormone-responsive breast cancers. Ovariectomy in premenopausal women is the oldest of these therapies (Beatson, 1896) and has long been known to produce benefit in approximately one-third of all patients (Boyd, 1900). Although ovariectomy is still an effective therapy, currently the administration of antiestrogenic drugs is the most widely applied endocrine manipulation. Antiestrogenic drugs are effective in both premenopausal and postmenopausal patients and in the metastatic, adjuvant, and chemopreventive settings. The drugs are well tolerated, the incidence of dose-limiting toxicities is low, and responses are seen in approximately 70% of patients selected on the basis of the steroid hormone receptor expression profile of their tumors (Clark and McGuire, 1988). Additional benefits associated with some antiestrogens likely include reductions in the risk and/or severity of osteoporosis. Evidence also supports a possible reduction in the risk of cardiovascular disease (McDonald et al., 1995), but this is not consistent across all studies (EBCTCG, 1998; Fisher et al., 1998). Whether the estrogenic effects of Tamoxifen (TAM²) are responsible for any reduction in coronary heart disease has also become somewhat controversial, since the preventive effects of estrogenic hormone replacement therapy (HRT) on coronary heart disease have been questioned (Hulley et al., 1998).

Currently, the most widely used antiestrogen is the triphenylethylene TAM (ICI 46,477), which is administered orally as the citrate salt. Cole et al. (1971) described the first clinical study demonstrating TAM's efficacy. TAM was approved for use in advanced disease several years later. Clinical experience with this drug likely now exceeds 10 million patient years. Unfortunately, in most patients, cancers that initially respond to TAM will recur and require alternative systemic therapies. Despite extensive experience with this drug, the precise mechanisms that confer resistance remain unknown. This review will discuss evidence from recent clinical trials and experimental models that identify several possible mechanisms of resistance. Because the activity of antiestrogens is intimately involved with the role of estrogens and their receptors, a brief discussion of the role of estrogens and estrogen receptors (ERs) is included. Additional ER-independent events, which also may be important, are discussed.

The utility of antiestrogens as treatments and/or chemopreventives for breast cancer is closely associated with antagonizing the activity of estrogens. Estrogens have been widely implicated in affecting breast cancer risk in the postmenopause. Evidence includes the association of increased serum estrogens, or estrogen excretion, with postmenopausal breast cancer (Table 1) (see Thomas et al., 1997 for review). Prolonged HRT, which also elevates serum estrogen levels, can significantly increase breast cancer risk (CGHFBC, 1997), and the tumors arising tend to be primarily ER-positive (Lower et al., 1999). HRT is often prescribed to naturally perimenopausal or postmenopausal women, but may also be given to younger women with primary ovarian failure, or who have had their ovaries removed/irradiated.

The estrogenicity of HRTs can vary significantly, and dose is important, at least in some studies. For example, low potency oral and transdermal estrogens may not increase risk, whereas more potent estrogens significantly increase breast cancer risk (Magnusson et al., 1999). Serum estradiol concentrations can exceed 0.77 nM with some HRT regimens (Garnett et al., 1990). This concentration is almost 10-fold higher than that seen in untreated postmenopausal women and is comparable with that seen in the luteal phase of the menstrual cycle (Table 1). Recent evidence suggests that the greatest increase in breast cancer risk is associated with replacement therapies that combine estrogens and progestins (Schairer et al., 2000). Most studies observe the greatest risk in current/recent users, perhaps reflecting a promotional rather than initiating action of the estrogens.

Whereas HRT increases the risk of developing breast cancer, the resulting biology of the tumors may be different from those arising in the absence of HRT. Patients using HRT at the time of diagnosis have a reduced mortality from breast cancer (Schairer et al., 1999), perhaps reflecting a less aggressive biology (CGHFBC, 1997; Holli et al., 1997). Thus, the estrogenicity of HRT may have allowed the survival of less aggressive tumors. This is consistent with the observation that estrogen-dependent breast cancer cells selected in vivo for growth in a low estrogen environment, rather than in the presence of an adequate estrogenic stimulus, can acquire a more aggressive phenotype (Thompson et al., 1993).

Indirect evidence for a role for estrogens in affecting lifetime breast cancer risk is provided by several known risk factors. For example, breast cancer risk is increased in women who either began menstruating at a young age (<12 years) and/or ceased menstruating (menopause) at a late age (55 years) (Hulka and Stark, 1995). This would tend to increase the number of cycles and total lifetime exposure to ovarian estrogens. Postmenopausal obesity is also associated with increased breast cancer risk (Hulka and Stark, 1995). Peripheral adipose tissue is the primary source for the production of circulating estrogens in postmenopausal women, and serum estrogen concentrations are generally higher in obese postmenopausal women (Ingram et al., 1990; Madigan et al., 1998). There are also data implicating estrogenic exposure and risk of premenopausal breast cancer. Perhaps the most compelling evidence is the efficacy of ovariectomy and luteinizing hormone releasing hormone analogs in inducing responses in premenopausal patients (Crump et al., 1997).

Estrogens may affect carcinogenesis by acting either as initiators (i.e., directly damage DNA) or as promoters (i.e., promoting the growth and/or survival of initiated cells). For example, administration of estrogens alone can produce tumors in some rodents (Lacassagne, 1932). This may reflect an effect mediated through mouse mammary tumor virus, and/or activities of the more chemically reactive metabolites of 17-estradiol. Reactive estrogen semiquinone/quinone intermediates are produced by the redox cycling of the hydroxylated estrogen metabolites. These can produce DNA adducts (initiation). This has been most closely associated with the 4-hydroxy (Liehr and Ricci, 1996) and 3,4-hydroxy metabolites, with a recent study strongly implicating the catechol estrogen-3,4-quinones as initiators (Cavalieri et al., 1997). The production of these metabolites is a function of several cytochrome P-450 isoforms that are expressed in the breast, liver, and other tissues (Zhu and Conney, 1998).

The potential role of estrogens as promoters of carcinogenesis is more firmly established. Ovariectomywhether chemical, surgical, or radiation-inducedremains a highly effective treatment (Crump et al., 1997). Indeed, surgical ovariectomy and the suppression of gonadotropin secretion by luteinizing hormone releasing hormone analogs are as effective as TAM in managing premenopausal breast cancer (Jonat, 1998). Chemically induced mammary adenocarcinomas in rats also require functional ovaries (Russo et al., 1990), probably reflecting promotion of the carcinogen-initiated cells. Several human breast cancer cell lines require estrogen for proliferation in vitro and in vivo (Clarke et al., 1996). This proliferation can be blocked by the administration of antiestrogens, consistent with the removal of a mitogenic effect. Although estrogens may function as both initiators and promoters of carcinogenesis, for the purposes of this review the promotional effects are most relevant.

TAM provides a good illustration of several of these points. TAM is a classical partial agonist and exhibits both species and tissues specificity for inducing either an agonist or antagonist response. In the mouse, TAM is an agonist. In rats and humans, it exhibits partial agonism (Jordan and Robinson, 1987) [e.g., producing antagonist effects in the breast, but agonist effects in the vagina and endometrium (Harper and Walpole, 1967; Ferrazzi et al., 1977)]. Long-term TAM use is generally associated with a reduced incidence of contralateral breast cancer (antagonist), a reduced incidence of primary breast cancer in high-risk women (antagonist), maintenance of bone density (agonist), and increased risk of endometrial carcinomas (agonist) (Fisher et al., 1998).

The ability to generate these tissue-specific effects has lead to the search for other selective ER modulators, which will have the beneficial effects seen with TAM but without the increased risk of endometrial carcinoma. Several triphenylethelene variations on TAM are already available, including Toremifene (chloro-TAM) and Droloxifene (3-OH-TAM). Both drugs seem to be approximately equivalent to TAM in terms of their antitumor activities and toxicities; both drugs are partial agonists (Roos et al., 1983; Pyrhonen et al., 1999).

The clinical utility of several of these newer antiestrogens has recently been reviewed by others (Lien and Lonning, 2000), and an exhaustive review is beyond the scope of this article. Nonetheless, several of the newer compounds are notable. Many are not triphenylethylenes [e.g., Raloxifene is a benzothiophene (previously called keoxifene; LY 156,758)]. It is now available in the U.S. as a treatment for the prevention of osteoporosis in postmenopausal women. Evidence suggests that Raloxifene may not have the same uterotropic effects as TAM (Delmas et al., 1997) and that it may regulate gene expression through novel pathways (Yang et al., 1996). In the multiple outcomes of Raloxifene randomized trial, Raloxifene significantly reduced the number of breast cancer cases, from 27/2576 to 13/5129 (Cummings et al., 1999), but did not increase the incidence of endometrial cancers (Delmas et al., 1997; Cummings et al., 1999). It also produces beneficial effects comparable with TAM on other endpoints, including lowering levels of both total and low-density lipoprotein cholesterol (Delmas et al., 1997; Walsh et al., 1998) and increasing bone mineral density (Delmas et al., 1997). However, Raloxifene increases the incidence of hot flashes (Davies et al., 1999).

Other antiestrogens that have received attention are the steroidal compounds ICI 164,384 and ICI 182,780. Both ICI 164,384 and ICI 182,780 have high affinities for ER (Wakeling and Bowler, 1988). There may also be some preference for ER, since ICI 164,780's relative binding affinity for ER = 166%, but for ER = 85% (Kuiper et al., 1997). Both ICI 164,384 and ICI 182,780 seem to be antagonists, being devoid of agonist activity in most experimental models. For example, ICI 164,384 does not exhibit agonist activity either in MCF-7 cells growing in the absence of estrogens (Clarke et al., 1989c;Thompson et al., 1989), or in the uterus or vagina of rats and mice (Wakeling and Bowler, 1988). ICI 164,384 can inhibit the agonist effects of both estrogen and TAM (Wakeling and Bowler, 1988). The estrogenic activities of TAM induce expression of a series of estrogen-regulated genes, including the progesterone receptor (PgR) and pS2. ICI 164,384 has no notable estrogenic effects on the regulation of these genes (Wiseman et al., 1989), other than a modest induction of PgR in endometrial cells (Jamil et al., 1991). However, there is evidence that ICI 182,780 can produce an estrogen-like effect in KPL-1 breast cancer cells (Kurebayashi et al., 1998). When ICI 182,780 is administered to pregnant rats, their female offspring exhibit changes in their mammary glands similar to those seen in offspring exposed to exogenous estradiol in utero (Hilakivi-Clarke et al., 1997). This could reflect primarily ER-mediated events, since ER is the predominant form at least in some normal human and rodent mammary tissues (Speirs et al., 1999b;Saji et al., 2000). Furthermore, ICI 182,7870 is an activator of transcription at AP-1 sites (Paech et al., 1997).

The steroidal antiestrogen ICI 182,780 retains its potency in vivo as determined by its ability to inhibit MCF-7 and Br10 tumors. This compound also exhibits substantial antiuterotrophic activity in the immature rat (de Launoit et al., 1991). ICI 182,780 (trade name: Faslodex) has already completed initial phase I clinical evaluation. The first study was performed on patients who had previously demonstrated a response to TAM, but recurred. The overall reported response rate of 69% (Howell et al., 1995) is substantially higher than the 5% objective response rate reported for crossover to another triphenylethylene (Toremiphene) following TAM failure (Vogel et al., 1993) and is more in line with responses to alternative second line endocrine therapies [e.g., aromatase inhibitors (Dowsett et al., 1995)]. This observation suggests that the steroidal antiestrogens affect breast cancer cells differently than the triphenylethylenes.

The partial agonist activities of TAM and Raloxifene are thought to be responsible for their beneficial effects on bone resorption. Pure antagonists like ICI 182,780 may further exacerbate bone loss, a concern that also applies to aromatase inhibitors (Dowsett, 1997). However, when combined with alternative therapies for osteoporosis, such as bisphosphonates, these drugs may have considerable potential as first-line endocrine therapies.

Patients with ER-positive tumors have a significantly higher response rate to antiestrogens than patients with ER-poor/ER-negative tumors. This relationship holds whether ER is measured by ligand binding or immunohistochemistry, reflecting the high concordance seen with these different techniques (Molino et al., 1997). It also holds despite the range of cut-off values used for assessing ER positivity versus ER-poor/ER negativity. TAM also seems most effective in the suppression of ER-positive tumors in the chemopreventive setting (Fisher et al., 1998).

Expression of PgR also has been implicated as a predictor of response to TAM. Several studies have reported responses in patients with ER-negative but PgR-positive tumors. However, the number of tumors is small and could reflect false negative estimations of ER expression. Concurrent expression of both ER and PgR is often associated with a higher response rate than in ER-positive, but PgR-negative, tumors. In general, approximately 70% of patients with ER-positive/PgR-positive tumors will respond to TAM, whereas response rates of 45% are seen in patients with ER-negative, but PgR-positive tumors. A 34% response rate is seen in ER-positive, but PgR-negative, tumors (Honig, 1996). The predictive power of PgR expression is likely related to the ability of estrogens to induce its expression. Thus, the presence of both ER and PgR may reflect the existence of an at least partially functional ER signaling pathway (Horwitz et al., 1975).

The Early Breast Cancer Trialists Group's initial meta-analysis in 1992 reported both a significant reduction in recurrence or death, and a reduction in death from any cause, in patients with ER-poor tumors (Table 2). Their more recent meta-analysis found no significant reduction in recurrence rates in patients with ER-poor tumors. Indeed, a 3% (nonsignificant) increase in the risk of death from any cause was reported in women, receiving TAM, with ER-poor tumors (Table 2). These latter data do not strongly implicate ER-independent events in beneficial responses to TAM and possibly indicate an adverse effect in some women. What those adverse effects may be, whether they are real, and the extent to which they may be restricted to an undefined subset of patients, remain to be determined. It also may reflect the more aggressive biology of ER-negative tumors (Aamdal et al., 1984; Clark and McGuire, 1988). Whereas longer term TAM use (e.g., 10 yr) is less beneficial than 5 yr, it still produces an overall benefit (EBCTCG, 1992, 1998). Why the benefit should be lower with longer use is not known, but may also reflect an adverse effect in some women.

Antiestrogens clearly produce several beneficial effects in some patients, including improved disease-free survival and overall survival from breast cancer. However, most patients with initially responsive tumors will experience a recurrence, indicating acquired antiestrogen-resistant disease. There are several possible mechanisms that could influence response to antiestrogens and, when altered, contribute to resistance. These include changes in host immunity, host endocrinology, or antiestrogen pharmacokinetics. Competition with endogenous ligands for binding to an antiestrogen's primary intracellular target(s), or altered function of its target(s), could also contribute to resistance (Fig. 1). The low rate of responses in ER-negative tumors is most consistent with antiestrogen action being primarily mediated through interactions with ER. However, antiestrogens, and TAM in particular, have been shown to bind intracellular proteins in addition to ER. It might be expected that, if these targets were critical for generating a response, many ER-negative tumors also would be responsive. Although such responses are not common, the ability of antiestrogens to influence the function of targets other than ER may still be important.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Overview of the likely targets of antiestrogen action and resistance. E2, estradiol.

It is apparent that the cellular context (i.e., the gene/protein expression pattern in a cell) can affect how a cell responds to a specific stimulus (Clarke and Brünner, 1996). For example, ER's transcriptional activities can be influenced by phosphorylation events regulated by signaling, which activates mitogen-activated kinase (MAPK) (Kato et al., 1995). Downstream signaling from the ER also is likely to be complex and may interact/intersect with other (ER-independent) signaling pathways. Antiestrogens could influence the activities of these other pathways (e.g., through binding to non-ER proteins) and alter cellular context (Clarke and Brünner, 1996). Whereas such events are probably not sufficient to induce an antiestrogenic effect in most ER-negative cells, they may be necessary/permissive for signaling to a fully antiestrogenic effect in responsive cells. Thus, perturbations in the activity of some ER-independent effects could contribute to an acquired antiestrogen resistance. Both ER-mediated and ER-independent targets for antiestrogens are considered in this review.

	II. Endogenous and Exogenous Estrogens in Antiestrogen Resistance

Top Previous Next References

In women, the biosynthesis of estrogens may arise from several sources. Ovarian production is the main source of circulating estrogens in premenopausal women, the primary estrogen being 17-estradiol. The efficacy of ovariectomy and luteinizing hormone releasing hormone analogs in premenopausal women (Crump et al., 1997) strongly support a role for ovarian estrogen production in the breast cancers that arise in these women. Conversion of adrenal androgens in peripheral tissues is the predominant source of circulating estrogens in postmenopausal women. The primary estrogen produced in the postmenopause by the action of aromatase is the relatively weak estrone, which is generally present in serum as the inactive estrone sulfate. Breast cancer cells can release the biologically active estrone through the action of the steroid sulfatase enzyme (Pasqualini et al., 1988) and can further convert estrone to 17-estradiol through the action of 17-hydroxysteroid dehydrogenase type 1 (Brodie et al., 1997).

Mammary tissues accumulate serum estrogens to concentrations significantly higher than those present in serum (Masamura et al., 1997; Miller, 1997). However, breast tissues also synthesize estrogens through a pathway similar to that in peripheral adipose tissues. This biosynthesis can occur within the epithelial cells (Brodie et al., 1997), the associated breast adipose tissue (Bulun and Simpson, 1994), and in some infiltrating lymphoreticular cells (Mor et al., 1998).

The importance of the aromatase enzyme in generating biologically active estrogens is evidenced by the efficacy of aromatase inhibitors in inducing clinical responses in postmenopausal breast cancer patients. These drugs already are established as second-line endocrine therapies (Dowsett, 1997). Because they inhibit both peripheral and breast aromatase activities, it is often difficult to assess which site of synthesis predominates. Both peripheral and intratumor/stromal aromatase activities are likely to be important, with the relative contribution varying among tumors. Studies in experimental models suggest that local production may be more important (Santen et al., 1999). Although peripheral aromatization is reduced to comparable levels by both aminoglutethimide and testololactone in women, testololactone produces a much lower clinical response rate (Lonning et al., 1989a). However, aminoglutethimide significantly increases estrone sulfate clearance in addition to its inhibition of aromatase activity (Lonning et al., 1989b; Lonning et al., 1990). These data suggest that both serum estrogens and intratumor/stromal biosynthesis may contribute to intratumor estrogen concentrations.

High intratumor estrogen concentrations could prevent antiestrogens from blocking ER action and produce a resistant phenotype. Data in Table 3 show that normal, benign, and malignant breast tissues in postmenopausal women contain concentrations of 17-estradiol up to 10-fold higher than those seen in serum. The range among tumors is considerable, from undetectable to over 5 µM 17-estradiol, with these levels being essentially equivalent regardless of patients' menopausal status. The mean concentration estimated from these studies is 1.28 nM (Table 3). If this reflects the concentration in epithelial cells, and it is fully available for ER binding, there would be sufficient intratumor estradiol to produce a maximal stimulation of ER signaling. In serum, <5% of estrogens are "free" [i.e., not bound to serum proteins]. Using this as an estimate of intracellular availability within tumors, and with a K_d of approximately 0.1 nM in breast cancer and other cells (Bei et al., 1996), only 25% of ER would be occupied.

View this table: [in this window] [in a new window]   TABLE 3 17-Estradiol concentrations in breast tumors, normal and benign breast tissues, and in sera

Generally, biological response is proportional to receptor occupancy. However, some cells up-regulate receptor expression, these "spare" receptors producing a left shift in the dose-response relationship (Ross, 1996). If this occurred in some breast tumors, they might exhibit a greater biological response than would be predicted by the proportion of occupied receptors. Consistent with the concept of spare receptors, MCF-7 cells respond to 17-estradiol at concentrations well below its K_d for ER. Some MCF-7 cells selected in vitro for growth in the absence of estradiol further up-regulate ER expression (Jeng et al., 1998). However, MCF-7 cells, which represent the most widely used endocrine responsive experimental model (Levinson and Jordan, 1997), have ER levels of ~400 fmol/mg protein (Martin et al., 1991). This is 40 times greater than the lower limit used to determine ER positivity in tumors. Relatively few breast tumors express these very high levels of ER, nor the levels seen in an estrogen supersensitive MCF-7 variant (Masamura et al., 1995).

In the absence of spare receptors, our estimate of 25% receptor occupancy would predict that many breast tumors exist in a weak estrogenic environment. Evidence of a suboptimal estrogenic environment being present in tumors is apparent from the associations of increased serum estrogens, HRT (Table 1), and oral contraceptive use (Hulka and Stark, 1995; CGHFBC, 1997) with increased breast cancer risk in some populations. Similarly, some metastatic tumors, which develop while a patient is taking HRT, regress upon withdrawal of HRT (Dhodapkar et al., 1995). Generally, the effects of HRT are not seen in heavier women (Magnusson et al., 1999; Schairer et al., 2000), probably reflecting the ability of higher serum estrogen levels, derived from peripheral adipose tissues, to compensate for a low intratumor estrogenic environment. In lean postmenopausal women, HRT could stimulate tumors with otherwise suboptimal intratumor estrogen concentrations.

Tumors arising in women exposed to HRT tend to be ER-positive (Lower et al., 1999). In one recent study, the mitogenic effects of HRT (high S-phase fraction) were seen only in ER-positive tumors (Cobleigh et al., 1999). ER-positive tumors often proliferate more slowly than ER-negative tumors (Wenger et al., 1993), which have no obvious need of estrogens for proliferation. This may reflect a suboptimal estrogenic/mitogenic environment, and could contribute to the different biologies apparent between ER-positive and ER-negative tumors.

Some tumors with no effective estrogenic stimulation could be driven by a ligand-independent activation of the ER signaling network (Tzukerman et al., 1990; Clarke and Brünner, 1996). Others with insufficient ligand may benefit from a concurrent ligand-independent activation of the remaining unoccupied ER. Generally, ligand independent activation is weaker than ligand activation. Both forms of activation can be blocked by antiestrogens (Clarke and Brünner, 1996; Tzukerman et al., 1990). Thus, tumors driven exclusively or partly by ligand-independent activation of ER should still exhibit responses to several endocrine therapies.

The potential contribution of serum estrogens to intratumor estrogen concentrations implies that factors influencing serum estrogen concentrations might affect response to antiestrogens. Some early studies suggested that TAM is of greater benefit when administered to postmenopausal rather than premenopausal women. However, these data are not supported in the recent Breast Cancer Trialists Cooperative Group meta- analysis, where it is clear that TAM is equally effective in both postmenopausal and premenopausal patients (EBCTCG, 1998). This does not exclude possible important mechanistic differences concerning how tumors respond in premenopausal versus postmenopausal women. For example, the presence of functional ovaries, particularly if these provide a major component of intratumor estrogenicity, could affect responsiveness.

The release of estrogens from the ovaries is regulated by the pituitary-ovarian axis. Estrogens can regulate the release of gonadotropins at two levels: the release of gonadotropin releasing hormone from the hypothalamus and the release of gonadotropins from the anterior pituitary. If TAM effectively blocks the ER in both the hypothalamus and anterior pituitary, this would disrupt the negative feedback on gonadotropin releasing hormone, ultimately producing a "hyperstimulation" of the ovaries. This might partly explain how TAM increases the circulating levels of estrogens in some premenopausal women (Ravdin et al., 1988; Szamel et al., 1994). Other studies have not reported an ability of TAM to affect circulating estrogen levels. However, ovariectomy and aromatase inhibitors can induce remissions in premenopausal women who initially responded to TAM but eventually recurred. This suggests that TAM produced an incomplete antiestrogen action, possibly as a result of increased circulating estrogens.

TAM can affect gonadotropin levels in premenopausal women, but its ability to do so in postmenopausal women is not so clear (Lien and Lonning, 2000). Small increases in serum dehydroepiandrosterone, estrone, and estradiol levels are also produced by antiestrogens in postmenopausal women (Szamel et al., 1994; Pommier et al., 1999). This probably reflects an effect mediated either through the release of adrenal androgens and/or increases in adrenal estrogen production in postmenopausal women (Pommier et al., 1999).

Where serum estrogens are increased, a consequent elevation in intratumor 17-estradiol concentrations would be predicted, reflecting the ability of tumors to accumulate serum estrogens. Such an effect might compromise response to TAM by increasing intratumor estrogen competition for binding to ER. Whether this occurs to an extent sufficient to affect the response to TAM is unclear. Response rates to TAM are comparable in premenopausal and postmenopausal women, but serum estrogen levels are higher in premenopausal women. A clearer understanding of the role of serum estrogens in influencing TAM response will probably await data from appropriately designed clinical trials. Nonetheless, it is evident that estrogens can readily reverse the inhibitory effects of antiestrogens in experimental models in vitro and in vivo. Since the primary estrogen produced in premenopausal women in response to TAM is also the most potent (17-estradiol), and tumors can significantly accumulate estrogens to levels in excess of that seen in serum (Masamura et al., 1997; Miller, 1997), changes in serum estrogens could affect TAM responsiveness in some individual tumors.

Antiestrogens can block both ligand-dependent and ligand-independent ER activation (Tzukerman et al., 1990; Clarke and Brünner, 1996). Thus, the precise origin of the ligand, and whether or not it is required for receptor activation, is less important than the potential of available intratumor estrogens to prevent antiestrogen action. Free intracellular estrogens could compete with antiestrogens for binding to ER, reducing their ability to block ligand dependent receptor activations.

The mean intratumor concentration (1.28 nM from Table 3) would probably not be sufficient to fully compete with TAM and its metabolites. This is consistent with evidence from experimental models suggesting that combinations of an antiestrogen and an aromatase inhibitor is no better than either drug alone (Lu et al., 1999). However, where reduced intratumor TAM accumulation also occurs (Johnston et al., 1993), the higher intratumor estradiol concentrations in some tumors might overcome TAM's antiestrogenic activities. Very high intratumor estrogen levels (up to 5 µM) are only occasionally observed, but would provide sufficient estrogenicity to compete with the mean intratumor concentrations of triphenylethylene antiestrogens (3.4 µM; see Section III.A.). Assuming that both estrogens and antiestrogens have equivalent intracellular availability for binding ER, it is theoretically possible for some tumors to acquire sufficient intratumor estrogen concentrations to either eliminate or reduce the inhibitory effects of TAM and its major metabolites.

Although this is a reasonable hypothesis, it has been inadequately addressed in clinical trials. It is evident that approximately 30% of tumors that acquire TAM resistance will respond to a second-line aromatase inhibitor. The proportion may be higher in selected populations (Dowsett et al., 1995). This response pattern is consistent with an important role for estrogen biosynthesis in acquired TAM resistance. It implies that the responding tumors have retained both a functional ER signaling network and a dependence upon that network's estrogenic activation/regulation for continued survival/proliferation. In some of these tumors, the levels of intratumor estrogens may reach sufficient levels to overcome any antiestrogenic activities of TAM and support an estrogen-dependent proliferation.

Currently, determining the possible contribution of HRT to antiestrogen resistance can also be done only indirectly. The National Surgical Adjuvant Breast and Bowel Project (NSABP)-P1 TAM chemoprevention trial precluded women who were receiving HRT, but found a significant reduction in the incidence of invasive breast cancers (Fisher et al., 1998). The apparent lack of a chemopreventive effect of TAM in the Italian (Veronesi et al., 1998) and United Kingdom studies (Powles et al., 1998) has been partly attributed to their inclusion of women receiving HRT. This explanation for the failure of these studies remains somewhat controversial. For example, it is not clear that many HRTs, particularly those using low-dose/potency estrogens, would produce an environment any more estrogenic than that occurring naturally in TAM-responsive premenopausal women. Tumors in premenopausal patients have a response rate comparable with those arising in postmenopausal women (EBCTCG, 1998). Other differences in the chemoprevention trials probably account for the lack of activity in the European studies. These may include differences in the patient populations and the greater statistical power of the NSABP study (Pritchard, 2000).

The timing of TAM treatment relative to any HRT may affect clinical outcome. Initiation of HRT during TAM may have a greater inhibitory effect on TAM's ability to affect serum lipid profiles than initiation of TAM in current HRT users (Decensi et al., 1998). Since these are agonist cardiovascular endpoints rather than antagonist cancer endpoints, extrapolation to the antiestrogenic effects of TAM in breast cancer is difficult. Nonetheless, data raise the possibility that the timing of HRT may affect TAM's antineoplastic activity in these patients. Additional studies are required to definitively answer the possible contribution of HRT to TAM resistance. The limited information available does not provide strong evidence for an effect of HRT on TAM responsiveness, which, if it occurs, may be restricted to specific HRT formulations and/or specific populations.

	III. Pharmacokinetics in Resistance to Tamoxifen

Top Previous Next References

There are several pharmacologic properties of TAM that directly influence its biological activity and that, when significantly altered, could contribute to the emergence of an antiestrogen resistant phenotype. These include the classical pharmacokinetic parameters of absorption, distribution, biotransformation, and elimination. The intracellular availability of TAM will determine the concentration free to interact with ER. This could be affected by changes in TAM accumulation in tumors. There are several likely major intracellular binding compartments for TAM that could limit intracellular availability. These include binding to antiestrogen binding sites (AEBSs) and other intracellular proteins, and partition into the lipophilic domains of cellular membranes. Such interactions could effectively sequester active TAM and its metabolites to produce the resistance phenotype. Since TAM is extensively metabolized in humans, and several metabolites are agonists, a resistance phenotype could also be conferred by a switch to the generation of predominantly estrogenic metabolites.

Steady-state serum concentrations of TAM are generally achieved after approximately 4 weeks with the conventional dosing regimen of 20 mg TAM daily (Buckely and Goa, 1989; Etienne et al., 1989). Following administration of 30 mg/day, the mean steady-state plasma concentrations of parent drug and major metabolites can be up to 1.1 µM (Etienne et al., 1989). High-dose TAM, 150 mg/m² twice daily following a loading dose of 400 mg/m², produces plasma concentrations of 4 µM TAM and 6 µM N-desmethyl TAM (Trump et al., 1992). In most studies, clinical response does not seem to correlate with TAM plasma levels (Bratherton et al., 1984; Clarke and Lippman, 1992).

Greater than 98% of TAM and its major metabolites are bound to serum proteins. Most of this appears to reflect binding to serum albumin, which can bind drugs in a ratio of 1:1 (Lien et al., 1989). The extensive degree of association with albumin (Lien et al., 1989), peripheral tissues (Daniel et al., 1981; Lien et al., 1989) and cellular membranes (Clarke et al., 1990), and its large volume of distribution (Herrlinger et al., 1992) may contribute to TAM's long terminal elimination phase. The relatively low affinity binding to serum albumin might facilitate transport to tissues, where dissociation may occur to allow for tissue accumulation. This role for albumin as a transporter has been described for estrogens, with albumin-bound estrogens often being considered within the available component (Moore et al., 1986; Jones et al., 1987).

Despite the low free concentrations in serum, TAM concentrations of 5 to 110 ng/mg protein (25 ± 27 ng/mg protein; mean ± S.D.) have been reported in the breast tumors of women receiving 40 mg TAM/day (Daniel et al., 1981). This would approximate 0.67 to 14 µM (3.36 ± 3.63 µM; mean ± S.D.) using the conversions in the legend to Table 3. Similar intratumor concentrations have been described for brain metastases, with mean concentrations of TAM  4 µM, 4-hydroxytamoxifen (4-hydroxyTAM)  0.13 µM, and N-desmethyl TAM  8 µM (approximate values derived from the published data) detected in a small study of patients receiving 30 to 50 mg TAM/day (Lien et al., 1991). Thus, as with estrogens, there is clear evidence of intratumor accumulation of TAM and its major metabolites to concentrations significantly in excess of that seen in serum (MacCallum et al., 2000).

When compared with the mean intratumor 17-estradiol concentration (1.28 nM; Table 3), and assuming approximately equivalent intratissue availability, it is apparent that there should be sufficient TAM present to effectively compete with most concentrations of intratumor estrogens. This would be the case even if all the drug was present as either the relatively weak parent or the N-desmethyl TAM metabolite. The latter is present at concentrations of approximately 7 ± 8 µM (estimated from the values of Daniel et al., 1981). However, a significant proportion of the antiestrogenic activity will be provided by the 4-hydroxyTAM metabolite (77 ± 64 nM estimated from the values of Daniel et al., 1981), which has an affinity for ER  17-estradiol (Kuiper et al., 1997). Although these estimates were obtained several years ago, a more recent study by MacCallum et al. (2000) obtained mean intratumor concentrations of TAM and its major metabolites (4-hydroxyTAM = 0.18 µM; N-desmethyl TAM = 0.61 µM; TAM = 0.32 µM) within the range of these prior studies.

The potentially significant intratumor excess of antiestrogenicity over estrogenicity (>10-fold for 4-hydroxyTAM) explains, in part, why TAM is an effective therapy in many patients with ER-positive tumors. This likely also contributes significantly to the apparent lack of a strong dose-related response rate in clinical trials. Many of the lower doses studied could still produce antiestrogen concentrations in excess of any intratumor estrogens.

Several intracellular binding proteins have been identified for estradiol (Anderson et al., 1986; Takahashi and Breitman, 1989; Masamura et al., 1997), and it would be remarkable if none of these also bound TAM. Indeed, it is likely that there are several such proteins that can sequester TAM and reduce its intracellular availability. One intracellular binding component, at least for the triphenylethylenes, is the AEBS protein. AEBS seems to be predominately microsomal (Katzenellenbogen et al., 1985) and may represent a novel histamine receptor (Clemmons et al., 1990). More recent data imply a protein complex containing the microsomal epoxide hydrolase as one of the subunits (Mésange et al., 1998). This is a type II detoxification enzyme involved in the hydrolysis of aliphatic and aromatic electrophilic epoxides. TAM-AEBS interactions could contribute to the putative mutagenicity of TAM in some species (Greaves et al., 1993; Mésange et al., 1998). Whereas TAM induces expression of the epoxide hydrolase mRNA (Nuwaysir et al., 1995), it is an inhibitor of the enzyme's catalytic activity (Mésange et al., 1998). Such an inhibition could leave reactive epoxide metabolites of TAM, or other electrophilic epoxides, available to induce DNA damage (Mésange et al., 1998). TAM-induced hepatocellular carcinomas have been reported in rats (Greaves et al., 1993), but the incidence of these tumors is not increased in humans (Muhlemann et al., 1994). Any role for the epoxide hydrolase-TAM interactions may be tissue- and species-specific.

A basic alkylether side chain, as occurs in many of the nonsteroidal antiestrogens, seems important for recognition of AEBSs by triphenylethylenes (Murphy and Sutherland, 1985). AEBSs do not bind either the natural estrogens or the steroidal antiestrogens with high affinity (Pavlik et al., 1992) and will not interfere with intratumor estrogen activation of ER. Thus, overexpression of AEBSs could contribute to TAM resistance in the presence of continued ER expression. The antiestrogen-resistant LY2 cells (Bronzert et al., 1985; Clarke et al., 1989c) overexpress AEBSs relative to ER, as do a significant proportion of human breast (Pavlik et al., 1992) and ovarian carcinomas (Batra and Iosif, 1996). The affinity of TAM for AEBSs in ovarian cells is estimated <1 nM (Batra and Iosif, 1996) significantly greater than its affinity for ER. This implies a preferential binding of TAM to AEBSs relative to ER. Where TAM inhibits the epoxide hydrolase activity of AEBSs allowing reactive metabolites to persist, this could increase the genetic instability of some tumors. One consequence could be an increased potential to induce mutations in genes required for TAM function, with a subsequent increased risk of producing mutations that produce antiestrogen resistance.

The biological potency of antiestrogens does not correlate with their affinity for AEBSs (Katzenellenbogen et al., 1985). Although it has generally been assumed that the primary function of AEBSs has been to sequester drugs, several studies imply otherwise. Lymphoid cells that express AEBSs, but not ERs, are growth inhibited by antiestrogens (Tang et al., 1989; Hoh et al., 1990; Teo et al., 1992). The compound N,N-diethyl-2-(4 phenyl-methyl)-phenoxy ethamine HCl binds AEBSs, but not ERs, and is growth inhibitory in MCF-7 cells (Brandes, 1984). A TAM-resistant MCF-7 variant (RTx₆) does not express AEBSs (Faye et al., 1983) and is not inhibited by either benzylphenoxy ethanamine derivatives (Poirot et al., 1990) or other selective ligands for AEBSs (Fargin et al., 1988; Teo et al., 1992). Parental MCF-7 cells are growth inhibited by these compounds.

Polyunsaturated fatty acids can block TAM binding to AEBSs (Hoh et al., 1990). Cholesterol and lipoproteins can reverse the inhibitory effects of antiestrogens in an ER-negative lymphoid cell line (Tang et al., 1989). The antiproliferative activities of oxygenated sterols may be mediated by AEBSs. Ligand binding to AEBSs also affects cholesterol metabolism. Benzofurans can inhibit de novo cholesterol metabolism in ER-negative cells that express AEBSs (Teo et al., 1992). This raises the possibility that the hypocholesterolemic effects of some antiestrogens may be related to effects mediated by binding AEBSs.

Whereas AEBSs can sequester TAM, the extent to which antiestrogen-mediated activation of any AEBS function contributes to the antiproliferative effects of antiestrogens is unclear. If sufficient alone to confer responsiveness, the response rate to antiestrogens would be expected to be high in ER-negative tumors. However, responses in ER-negative tumors are infrequent (EBCTCG, 1998). The relationship between AEBS affinity and the IC₅₀ for antiproliferative effects is also of concern. The affinities of the antiestrogens TAM and clomiphene for AEBSs are two to three orders of magnitude greater than their respective antiproliferative IC₅₀s (Lin and Hwang, 1991). Whatever the role of AEBSs, these sites cannot affect the activities of the steroidal antiestrogens because steroids do not bind AEBSs (Pavlik et al., 1992).

Many lipophilic compounds are sequestered within plasma membranes and other intracellular bilipid membranes. This is probably a relatively nonspecific phenomenon, reflecting their physicochemical properties. Compounds with a high degree of lipophilicity would be expected to preferentially partition into lipophilic domains in cellular membranes. This has been widely reported for steroids (Duval et al., 1983). We have previously shown that both TAM and estradiol can affect membrane structure in breast cancer cells in vitro (Clarke et al., 1990). Sequestration of TAM in a cell's plasma membrane, and potentially within other intracellular bilipid membranes, could significantly reduce intracellular availability for binding to ERs. Some breast tumors exhibit a marked desmoplastic response, associated with the presence of fibroblastic and myofibroblastic cells, and/or significant infiltration of lymphoreticular cells (Clarke et al., 1992b). Thus, TAM could be further sequestered within the membranes of infiltrating cells and adjacent adipose tissue.

The precise mechanism for intracellular uptake of TAM is not known. Passive diffusion, as probably occurs for steroids, seems most likely. Although tumors can concentrate TAM relative to its levels in serum (Fromson and Sharp, 1974; Daniel et al., 1981; Lien et al., 1989), intracellular sequestration could produce a relatively low concentration of unbound TAM, favoring its diffusion from extracellular sources. Some tumors may appear to have high TAM concentrations, but respond poorly because of low intracellular drug availability.

Reduced uptake of TAM from extracellular sources could confer resistance, provided the intracellular levels of available drug/metabolites fell below those required to effectively compete with any intratumor estrogens. Lower intratumor levels of TAM have been reported in some resistant versus sensitive tumors (Osborne et al., 1991, 1992; Johnston et al., 1993) and in some cell lines (Kellen et al., 1986). However, data are inconsistent. In a recent study, tumor concentrations of TAM, 4-hydroxyTAM, and N-desmethyl TAM did not correlate with responsiveness or resistance. Indeed, the serum concentrations of 4-hydroxyTAM and N-desmethyl TAM were significantly higher among nonresponding patients (MacCallum et al., 2000). The sources of inconsistency require further study but one source may be related to the ER content of the tumors in the study population. For example, the subgroup of patients with ER-poor tumors seem to have lower serum levels of antiestrogens, and their tumors have a low response rate to TAM (MacCallum et al., 2000). Future studies may need to carefully control for the ER content of tumors in their study populations.

TAM is antiangiogenic (Haran et al., 1994; Lindner and Borden, 1997) and reduces tumor vascularization, leading to decreased tumor perfusion and TAM delivery. However, this could not explain the reduced accumulation of TAM in some cells growing in vitro (Kellen et al., 1986). If accumulation is dependent on the expression of intracellular binding proteins, altered expression of these could affect accumulation. Altered TAM levels are not seen in one TAM-stimulated MCF-7 xenograft model (Maenpaa et al., 1994). We also have not found any significant difference in accumulation of [³H]TAM among TAM-resistant and TAM-responsive breast cancer cells growing in vitro (unpublished results).

TAM's ability to diffuse into cells could be related to specific plasma membrane domains into which it initially partitions (Clarke et al., 1990). The structure of these domains might depend on critical membrane-associated proteins or lipids, the altered expression of which could contribute to reduced diffusion/uptake. A simple reduction in the number of such putative domains also could reduce accumulation. These comments are speculative; further studies are required to determine the extent to which TAM's association with, and diffusion through, the plasma membrane is dependent upon definable membrane domains and/or functions.

The mechanism for TAM efflux also is not known, although a passive diffusion again seems most likely. We and others (Ramu et al., 1984; Leonessa et al., 1994) have described the ability of TAM to interact with the P-glycoprotein (also known as MDR1, gp170, and PGP) efflux pump, the product of the mdr1 (multidrug resistance 1) gene. P-glycoprotein is widely expressed in human breast tumors and is associated with a worse than partial response to cytotoxic chemotherapy (Trock et al., 1997). To determine the ability of P-glycoprotein to alter response to TAM, the MDR1 gene was overexpressed in MCF-7 cells. TAM can compete with azidopine for binding to P-glycoprotein and reverse the multidrug resistance phenotype in the transfectants (Leonessa et al., 1994). However, the transfectants' response to TAM is unaffected (Clarke et al., 1992a), and TAM accumulation is equivalent to wild-type cells (Clarke and Lippman, 1996). Thus, TAM is an inhibitor but not a substrate for this efflux pump, and expression of P-glycoprotein is probably not a contributor to TAM resistance.

TAM is subject to extensive hepatic metabolism. Not surprisingly, several of the metabolites are predominately estrogenic, rather than antiestrogenic. Differences in TAM metabolism among mice, rats, and humans probably contribute to its species-specific agonist versus partial agonist properties (Jordan and Robinson, 1987).

The most relevant metabolites will be discussed only briefly, since the metabolism of TAM has been extensively reviewed elsewhere (Buckely and Goa, 1989; Lonning et al., 1992b). Demethylation of the aminoethoxy side chain produces N-desmethyl TAM, with further N-demethylation producing the primary amine (N-didesmethyl TAM). Deamination of the primary amine produces the primary alcohol (Kemp et al., 1983). Metabolite E is generated when the aminoethane side chain is removed. Hydroxylation of the parent drug produces the two more polar metabolites 4-hydroxyTAM and 3,4-dihydroxyTAM. Loss of the aminoethane side chain and hydroxylation at position 4 produces the bisphenol. Metabolite E and the bisphenol are estrogens and exhibit a lower affinity for ER than TAM (Jordan and Robinson, 1987). The other metabolites (B, D, X, Y, and Z) are partial agonists. The relative affinities for ERs are 4-hydroxyTAM  17-estradiol > TAM > N-desmethyl TAM > metabolite Y (Jordan et al., 1983; Katzenellenbogen et al., 1984).

Increased isomerization of TAM to estrogenic metabolites is observed in some TAM-resistant breast tumors (Osborne et al., 1991, 1992). A preferential generation of estrogenic metabolites could compete with the antiestrogenic metabolites for binding to ERs, perhaps interacting additively with existing intratumor estrogens to block antiestrogen action. It also would reduce the concentrations of antiestrogenic metabolites, potentially shifting the ratio of estrogenic:antiestrogenic metabolites in an unfavorable direction.

Evidence firmly establishing altered metabolism as a clinically relevant event remains elusive. Data from one animal model of TAM-stimulated growth, a phenotype that could reflect the preferential intracellular generation of estrogenic metabolites, clearly excluded the generation of such metabolites in this phenotype (Wolf et al., 1993). A series of elegant studies were performed using nonisomerizable TAM. These could not be metabolized to estrogenic metabolites, but the tumors still exhibited a mitogenic response to these derivatives (Wolf et al., 1993). Subsequent studies implicated a mutant ER protein in conferring the phenotype (Jiang et al., 1992). In a similar model from Dr. Osborne's laboratory (Baylor College of Medicine, Houston, TX), nonisomerizable TAM analogs also produced a stimulation of tumorigenesis. These data imply that the TAM-stimulated phenotype, at least in these models, is unlikely to be explained by the significant conversion of parent drug to estrogenic metabolites (Osborne et al., 1994).

The importance of reduced TAM accumulation also requires further study. It is unlikely that P-glycoprotein contributes to lower intratumor TAM levels. However, we have preliminary data suggesting that P-glycoprotein may confer resistance to steroidal antiestrogens (Leonessa et al., 1998). The role of other membrane transporters has not been well defined.

The extent to which metabolism of TAM to estrogenic metabolites confers resistance remains to be clearly established. TAM-stimulated growth, the predicted response to this mechanism, can arise from mutations in ER and may not require estrogenic metabolites (Jiang et al., 1992). Nonetheless, it may be premature to entirely exclude the generation of estrogenic metabolites as a possible contributing resistance mechanism in some breast tumors.

	IV. Cell Culture Models of Antiestrogen Responsiveness and Resistance

Top Previous Next References

The study of acquired resistance has been greatly facilitated by the generation of several series of resistant variants. Most have been obtained by in vitro selection of the MCF-7 human breast cancer cell line. Almost all of these variants retain ER expression and show various patterns of resistance and cross-resistance. Resistant variants of other estrogen-responsive cell lines also have been reported. Although not a full listing, Table 4 describes several antiestrogen-resistant models. This section will focus primarily on those models of apparent pharmacological resistance (i.e., cells that do not exhibit a growth response to specific antiestrogens). Models that are growth stimulated by TAM are discussed in Section V. The models presented are selected to reflect the most widely used models and the diversity of phenotypes.

These were among the first stable antiestrogen-resistant variants reported. R27 cells were obtained following anchorage-independent cloning of MCF-7 cells in the presence of TAM. The cells retain an attenuated response to estradiol and are resistant to the growth inhibitory activities of TAM (Nawata et al., 1981). The LY2 cells were generated by a stepwise selection against the benzothiophene antiestrogen LY 117,018 (Bronzert et al., 1985). While retaining some responsiveness to estrogens, LY2 cells are cross-resistant to 4-hydroxyTAM (Bronzert et al., 1985; Clarke et al., 1989c) and ICI 164,384 (Clarke et al., 1989c). Unfortunately, LY2 cells are nontumorigenic, restricting their use to in vitro studies (Clarke et al., 1989c). The tumorigenicity of R27 cells is not reported.

The MCF-7RR subline was obtained by selecting MCF-7 cells for their ability to grow in medium supplemented with 2% calf serum and 1 µM TAM (Butler et al., 1986). The cells exhibit an altered chromatin structure and chromatin acceptor sites for the antiestrogen 4-(N,N-diethylaminoethoxy)-4'methoxy-)-(p-hydroxyphenyl)-ethylstilbene (Singh et al., 1986). Of interest is MCF-7RR cells' retinoic acid cross-resistance (Butler and Fontana, 1992), which has not been fully studied in many other antiestrogen-resistant variants. Whereas the cross-resistance pattern among other antiestrogens is not reported for MCF-7RR, these cells provide a novel model for studying the relationships among responsiveness and resistance to both antiestrogens and retinoids. Another MCF-7 variant selected against 4-hydroxyTAM (MCF/TOT) has also been shown to exhibit cross-resistance to retinoic acid (Herman and Katzenellenbogen, 1996).

This series was established to facilitate a further evaluation of cross-resistance phenotypes and to identify underlying molecular mechanisms. LCC variants were established from an estrogen-independent variant of MCF-7 cells (MCF7/MIII), initially selected for growth in vivo in ovariectomized nude mice (Clarke et al., 1989b). Circulating estrogen concentrations in these mice are similar to those found in postmenopausal women (Seibert et al., 1983), and the parent MCF-7 cells were derived from a postmenopausal patient (Soule et al., 1973). MCF7/MIII cells form proliferating tumors in these mice, but their growth is further increased upon estrogen supplementation. The cells retain ER expression and are growth inhibited by antiestrogens (Clarke et al., 1989b). A further in vivo selection produced the MCF7/LCC1 variant (Brünner et al., 1993a). These cells are similar to the MCF7/MIII, but tend to produce tumors more rapidly in ovariectomized nude mice. MCF7/LCC1 cells also retain ER expression, are estrogen-independent for growth, and are inhibited by triphenylethylene and steroidal antiestrogens (Brünner et al., 1993a; Brünner et al., 1997).

To generate antiestrogen-resistant variants, MCF7/LCC1 cells were stepwise selected against increasing concentrations of either 4-hydroxyTAM or ICI 182,780. Cells selected against the TAM metabolite produced stable, TAM-resistant cells (MCF7/LCC2), which also retain estrogen-independent growth in vitro and in vivo (Brünner et al., 1993b; Coopman et al., 1994). However, the MCF7/LCC2 cells are not cross-resistant to ICI 182,780. This predicts that tumors that responded and then failed TAM might show a strong response to a steroidal antiestrogen (Brünner et al., 1997). This prediction has now been confirmed in the clinic. The first trial of ICI 182,780 was performed in TAM responders who subsequently recurred. Consistent with the MCF7/LCC2 phenotype, the overall response rate to ICI 182,780 (69%) was substantially higher than would be predicted if the patients had been treated with another triphenylethylene (Howell et al., 1995). Using similar approaches, others have reported a MCF-7 variant (MCF-7/TAM^R-1) expressing a phenotype similar to MCF7/LCC2 (Lykkesfeldt et al., 1994).

Cells resistant to ICI 182,780 (MCF7/LCC9) were generated by selecting the MCF7/LCC1 variant against ICI 182,780. The resulting phenotype is clearly ER-positive, ICI 182,780-resistant, estrogen-independent, and TAM-crossresistant. Indeed, TAM cross-resistance emerges at early passages during the selection, arising before stable ICI 182,780 resistance is apparent (Brünner et al., 1997). The cross-resistance pattern may reflect the greater potency of ICI 182,780 relative to TAM and/or the differences in its interactions with ER (Fawell et al., 1990; Dauvois et al., 1992), which may have more substantial effects on ER functioning/signaling. Others have selected MCF-7 cells against ICI 182,780, but have not seen TAM cross-resistance (Jensen et al., 1999). The clinical relevance of these diverse phenotypes remains to be established.

ZR-75-1 cells are another of the relatively few, well established, estrogen-responsive human breast cancer cell lines. They were established from an ascites that developed in a 63-yr-old woman with an infiltrating ductal breast carcinoma (Engel et al., 1978). The patient had been receiving TAM for 3 months before the time when cells were removed to establish the ZR-75-1 cell line (Engel et al., 1978). ZR-75-1 cells are ER-positive and PgR-positive (Engel et al., 1978<