0163-769X/90/1104-0578$02.00/0 Endocrine Reviews Copyright © 1990 by The Endocrine Society
Vol. 11, No. 4 Printed in U.S.A.
Endocrine Pharmacology of Antiestrogens as Antitumor Agents* V. CRAIG JORDAN AND CATHERINE S. MURPHYf Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792
I. Introduction II. Cell Culture Systems to Test Antiestrogens A. Structure activity relationships 1. The side chain 2. Positioning of the side chain 3. Planarity of the binding ligand III. New Antiestrogens Used in the Laboratory and Clinics A. Trioxifene and keoxifene B. Zindoxifene C. Toremifene D. Droloxifene E. Pure antiestrogens IV. Subcellular Actions of Estrogens and Antiestrogens A. Physicochemical differences of the ER bound to agonists or antagonists B. Indirect actions of antiestrogens C. Non-ER mediated effects of antiestrogens D. Summary V. Animal Tumor Models A. Athymic mouse models B. Spontaneous mouse mammary tumorigenesis C. Carcinogen-induced models D. Summary VI. Adjuvant Tamoxifen Therapy VII. Long Term Tamoxifen Therapy A. Chemotherapy plus tamoxifen B. Tamoxifen alone C. Future strategies VIII. Resistance to Tamoxifen Therapy A. Antiestrogen-resistant cells B. Paracrine growth factor mediated mechanisms C. Tamoxifen-stimulated growth D. Metabolism to estrogens E. Summary XI. Concerns about Long Term Tamoxifen Therapy A. Reproductive side effects 1. Ovarian stimulation 2. Teratogenesis
B. Ophthalmic C. Antiestrogenic effects 1. Osteoporosis 2. Atherosclerosis D. Estrogenic effects 1. Thromboembolic disorders 2. Uterine stimulation E. Liver carcinogenesis F. Summary X. Prevention of Breast Cancer A. Chemosuppression of occult disease B. Tamoxifen maintenance XI. Summary and General Conclusions
I. Introduction
I
N 1958, Lerner and co-workers (1) described the biological properties of the first nonsteroidal antiestrogen MER25 (see Fig. 1). The discovery of this new class of drugs opened up a variety of clinical possibilities (2, 3), but early studies were terminated because of toxic side effects. Another member of this new class of drugs, clomiphene (then known as chloramiphene or MRL41), held the promise of being a potential antifertility agent (4); however, clinical studies demonstrated that ovulation was induced (5). The drug is now available as a profertility agent in subfertile women (6). During the 1960s a range of antiestrogens was synthesized by the pharmaceutical industry for potential applications in gynecology (7, 8). In the main though, these agents were used only to study reproductive endocrinology in the laboratory. However the focus of clinical application slowly changed from the broad market of contraception to the relatively small market of advanced breast cancer therapy. It was known that about one in three premenopausal women with advanced breast cancer will respond to ovarian ablation (9). Similarly about one in three postmenopausal women will respond to high dose estrogen (usually diethylstilbestrol) therapy (10, 11). The identification of the estrogen receptor (ER) system in some hormone-sensitive breast tumors (12,13) and the knowl-
Address requests for reprints to: Dr. V. Craig Jordan, Department of Human Oncology, University of Wisconsin, Clinical Cancer Center, Madison, Wisconsin 53792. * Supported by grants P30-CA-14520 awarded to the Wisconsin Clinical Cancer Center and R01-CA-32713. t Current address, Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232.
578
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November, 1990
ANTIESTROGENS AS ANTITUMOR AGENTS
OCH3
Ethamoxytriphetol (MER 25)
Clomiphene (cis and trans isomers)
Tamoxifen (trans isomer)
FIG. 1. Structures of nonsteroidal antiestrogens.
edge that antiestrogens could inhibit the binding of estradiol to the ER from either the estrogen target tissues of laboratory animals or human breast tumors (14, 15) naturally led to clinical studies to determine whether antiestrogens could control the growth of at least some advanced breast cancers. Several antiestrogens were tested in clinical trials (16), but only tamoxifen (Fig. 1) was developed further because of demonstrated efficacy and a low reported incidence of side effects (17-21). In 1978 tamoxifen was made available to physicians in the United States for the treatment of advanced breast cancer in postmenopausal patients. Evidence has now been accrued to demonstrate that tamoxifen is equivalent to oophorectomy in the treatment of premenopausal patients with advanced disease (22, 23). Although these studies (22, 23) are small, and do not have the statistical power to detect significant differences between the efficacy of tamoxifen and oophorectomy, the Food and Drug Administration has approved the use of tamoxifen to treat ER-positive advanced disease in premenopausal women. However, the changing fashions for the treatment of breast cancer that have occurred over the past two decades prompted the testing of tamoxifen as an adjuvant therapy after mastectomy in women with stage II disease. A recent overview of all randomized clinical trials around the world has demonstrated that at least 2 yr of adjuvant tamoxifen therapy provides a survival advantage for women treated with this antiestrogen (24). The finding has now established tamoxifen as the antihormonal treatment of choice in postmenopausal women with stage II disease. Indeed the drug now appears to be accepted in the treatment of all stages of breast cancer following the recommendation by the National Cancer Institute that women with ER-positive stage I disease
579
should be offered the opportunity of adjuvant therapy with an antiestrogen. Tamoxifen has now become an essential part of any therapeutic strategy for the control of breast cancer. The early evaluation of the general pharmacology, endocrinology, and clinical use of antiestrogens has been adequately reviewed (25-34). However, the number of articles about the mechanism of action and clinical usefulness of tamoxifen is increasing exponentially. This article will focus upon recent reports concerning the mechanisms of action of antiestrogens, new agents, long term adjuvant therapy for the treatment of breast cancer, and the problems to be addressed with the development of therapeutic resistance. Indeed, there are now plans to use tamoxifen to prevent breast cancer; therefore it is appropriate to consider this potential application. II. Cell Culture Systems to Test Antiestrogens Standard antiestrogens such as tamoxifen are often used in cell culture as control substances to demonstrate that a particular event is estrogen regulated through the ER. The alternative strategy is to describe an estrogenregulated cellular event to study the structure activity relationships (SAR) of antiestrogens. The last half-decade has seen a consolidation of our understanding of estrogen action in breast cancer cells and a shift of emphasis from animal models of hormone-dependent events to focus upon breast cancer cells. Estrogen stimulates the induction of PRL by immature rat pituitary gland cells in primary culture (35). The system has proved to be valuable to describe the SAR of tamoxifen and a variety of novel agents (36-43). The assay was developed, indirectly, to understand the action of antiestrogens as antitumor agents in the dimethylbenzanthracene (DMBA)-induced rat mammary carcinoma model (44). These hormone-dependent tumors are primarily dependent upon estrogen-stimulated PRL release for growth although progesterone and the direct effects of estrogen on the tumor are probably necessary for an optimal growth rate (45). Indeed there is probably not one single factor that can be described as the most important so the actual mechanism of action of antiestrogens in the model system in vivo is multifaceted. The value of the PRL assay in vitro is that the direct effects of antiestrogens can be described upon one facet of the control mechanism for DMBA-tumor growth. The value of the assay for studying SAR is that there is only a remote chance of metabolic intervention and the complicating effects of pharmacokinetics, with rapidly excreted compounds, are avoided. Nevertheless compound stability is still an important consideration that will be addressed later (Section II.A2). The most important factor that facilitated the devel-
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JORDAN AND MURPHY
580
opment of a primary culture system from rat pituitary glands to study the SAR of antiestrogens was that there was no equivalent system for breast cancer. A number of hormone-responsive breast cancer cell lines were available to study estrogen-regulated events (46, 47), but estrogen-stimulated breast cancer cell growth was hard to demonstrate in the laboratory. Nevertheless some investigators (48, 49) were able to describe the direct effects of estrogen on growth, although these conclusions were not universally accepted. Indeed the ease with which estrogen-stimulated growth can be demonstrated in vivo, but infrequently in vitro, naturally led to the suggestion (50) that estrogen caused cell replication by an indirect mechanism. With regard to antiestrogens the model was complicated further because the drugs could inhibit basal cell growth although this inhibition could be reversed by estrogen ("estrogen rescue") (51). Perhaps antiestrogens had an effect as antagonists of estrogen action other than through the ER? The discovery (52) that the pH indicator in culture medium, phenol red (Fig. 2), exhibited estrogen-like properties changed the situation completely. Breast can-
Bisphenolic Compounds
Diethylstilbesterol
OH
o
cer cell lines only grow very slowly in "phenol red free" media, but the addition of estradiol causes increased replication. Under these short term estrogen-deprived conditions antiestrogens inhibit estradiol-stimulated growth. The refinement of the culture conditions has illustrated how sensitive replication is as an estrogenregulated event. The breast cancer cells that have been maintained in the estrogenic medium for nearly 20 yr have adapted optimally to the environment; therefore further growth from added estrogen might only be expected to produce a modest effect. In contrast, protein synthetic events such as progesterone receptor induction have a different dose-response curve to estrogen (53). This also explains why PRL synthesis in primary cultures of pituitary cells is sensitive to estrogen in standard culture medium. It is also fair to point out that pituitary cells are grown under higher serum conditions (12.5%) compared with breast cancer cells (5%). Less "phenol red" is available for biological action as it is absorbed nonspecifically on the protein (54). As an aside, the chemical responsible for the estrogenlike effects of phenol red indicator is in fact a contaminant present in difference batches of material (54, 55). The similarity of the structure of phenol red to the bisphenolic compounds (Fig. 2), with known estrogenic activity (56), naturally drew the conclusion that the indicator was an estrogen. Nevertheless the potent estrogenic contaminant has been identified (57) (Fig. 2). In fact this discovery has a parallel that occurred 50 yr earlier. Dodds and Lawson (58) described the "minimum" structure, anol, necessary to produce estrogen action in vivo. Unfortunately the report could not be confirmed (59, 60). A potent contaminant of anol, dianol (Fig. 2), was found to be responsible for the estrogenic actions (61, 62). Dianol bears a structural resemblance to diethylstilbestrol (Fig. 2) (63, 64) whose biological properties were described during the same period. The structures of AREA OF ANTIESTROGENIC INFLUENCE TO PREVENT PROTEIN FOLDING
o
Phenol Red
Vol. 11, No. 4
Bis(4-hydroxyphenyl)[2-(phenoxysulfonyl)phenyl]methane
STILBENE-LIKE STRUCTURE FOR OPTIMAL FIT AND CORRECT FOLDING
Anol Dianol FlG. 2. The formulae of a variety of nonsteroidal estrogens with either actual or presumed estrogenic activity. The references that describe the biological activity of the compounds are in the text.
PHENOLIC HYDROXYL FOR HIGH AFFINITY INTERACTION
FIG. 3. The important aspects of the 4-hydroxytamoxifen molecule to produce high affinity for the estrogen receptor and antiestrogenic activity.
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November, 1990
ANTIESTROGENS AS ANTITUMOR AGENTS
581
the relevant compounds are shown in Fig. 2 for comparison. Breast cancer cell lines are now used routinely to determine the structural features essential for a ligand to control cell replication.
-CH3 OCH 2 CH 2 N
A.SAR
fixed ring "trans" 4-hydroxytamoxifen
The antiestrogenic potency of tamoxifen is increased by metabolic conversion to 4-hydroxytamoxifen (65). Since the dimethylaminoethane side chain is required for antiestrogenic activity, recent studies using breast cancer cell lines in culture have focused upon both the composition and positioning of the side chain. The salient features of 4-hydroxytamoxifen are shown in Fig. 3. 1. The side chain. Triphenylethylene derivatives of tamoxifen that lack the side chain are full estrogen agonists (40, 66). The composition of the side chain is also critical for antagonist activity (67-69). Several triphenylethylene compounds with alterations in the side chain region have been tested for their effects on the growth of a hormone-responsive line of T47D breast cancer cells in vitro (70). Substitution of the aminoethoxy side chain with an allyl side chain produces a compound with partial agonist activity. Compounds containing bulky aryl groups in place of the side chain are also partial agonists in this system. Therefore, the antiestrogenic activity of -CH OCH 2 CH 2 N,
,CH3
fixed ring "cis" 4-hydroxytamoxifen
B
compound a b c
O S
FlG. 5. Fixed ring derivatives of 4-hydroxytamoxifen to prevent isomerization (A) or to change the planarity of the molecule (B).
4 - hydroxytamoxifen ("trans" isomer)
4 - hydroxytamoxifen ("cis" isomer)
high affinity for ER antiestrogen
low affinity for ER antiestrogen
tamoxifen is not solely due to the length or bulk of the constituents in the side chain region. Interestingly, compounds that have oxygen-containing side chains are antiestrogenic in this system. Carbonylcontaining side chains of particular length are antiestrogenic. The addition of a single hydroxyl group onto an alkyl chain reverses the biological activity of the compound, making it antiestrogenic (70). These studies support the conclusion that side chain constituents containing a lone pair of electrons (such as oxygen or nitrogen) are necessary for antiestrogenic activity (68).
isomerization
Metabolite E ("cis" isomer)
Metabolite E ("trans" isomer)
potent estrogen
weak partial agonist
FlG. 4. The instability of hydroxylated triphenylethylenes results in the rapid isomerization, in vitro, to their alternate geometric isomer. The isomers on the left of the diagram are stable in solution.
2. Positioning of the side chain. The effect of triphenylethylene isomerization, and subsequent repositioning of the side chain region, has been studied extensively (71-76). Isomerization of the trans form of tamoxifen around the central double bond repositions the side chain to form its cis isomer. This results in a change in activity of the compound converting it from an antiestrogen to a partial agonist. Addition of a hydroxyl group to tamoxifen produces
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JORDAN AND MURPHY
582
TABLE 1. Biological activity of a fixed ring derivative of triphenylethylene substituted in various positions with hydroxyl or a dimethylaminoethoxy side chain FORMULA
NAME
EC 5 0 » OR I C 5 0 '
a.) ESTROGENS ESTRADIOL-17|i
3x10° 2 M
f.r. els met E
6x10" 1 1 M
,OCH 2 CHjN(CH 3 ) 2
COMPOUND"B"
PARTIAL AGONIST
COMPOUND"D"
PARTIAL AGONIST
PARTIAL AGONIST
l.r. trans met E
OCH 2 CH 2 N(CH 3 )2
b.) ANTIESTROGENS
l.r. trans 4-OHT
3x10" 1 1 M
COMPOUND"A"
8x10" 9 M
(CH 0 ) 2 NCH 2 CHjO
3x10' 8 M
COMPOUND"C"
(CHJJJNCHJCHJO
RU 39411
3x10° 1 M
Vol. 11, No. 4
the potent tamoxifen metabolite 4-hydroxytamoxifen (65). The trans form of 4-hydroxytamoxifen is a potent antagonist (IC50 = 3 X 10~n to 1 X 10~10 M). However, the true activity of the cis form of 4-hydroxytamoxifen was not known because it readily isomerizes (Fig. 4). Previous studies (37, 72) had shown that cis-4-hydroxytamoxifen was an antiestrogen in vitro and in vivo. However, based on studies done in the early 1980s, investigators suggested that the antiestrogenic activity of cis-4-hydroxytamoxifen could be due to its isomerization to the potent trans form (73, 74). To prevent isomerization a series of fixed-ring triphenylethylenes (Fig. 5A) were synthesized to test the activities of individual isomers (77-79). For these studies (42, 80), the growth response of hormone-responsive T47D human breast cancer cells and PRL synthesis in pituitary cell cultures were both used. The fixed-ring form of cis-4-hydroxytamoxifen (Fig. 5A) is antiestrogenic (IC50 = 4 X 10"8 to 2 X 10~7 M) (42, 80) and is not estrogenic. These findings support earlier predictions of the pharmacological actions of the cis isomer of 4-hydroxytamoxifen (37). The cis fixed-ring form of metabolite E (an estrogenic metabolite of tamoxifen) is a potent estrogen in this system (EC50 = 4 x 10~12 to 1 x 10" u M). However, the true biological activity of the trans form of metabolite E could not be determined because this compound also readily isomerizes to its opposite isomeric form (74) (Fig. 4). It was presumed that trans metabolite E, like cis metabolite E, was a full estrogen agonist because it lacked an aminoethoxy side chain in the relevant position (39). Surprisingly, it was found (80) that trans fixed ring metabolite E was only a weak partial agonist. These studies utilizing fixed-ring compounds have provided information regarding the true intrinsic activities of metabolites of tamoxifen. The fixed-ring compounds could also be useful to study the effects of various substituents on biological activity. Clearly isomerization of hydroxylated triphenylethylenes can produce changes in both pharmacological properties and potency so this should be considered during the evaluation of SAR studies (81). We have recently determined the relationship between the biological activity and the relative positioning of the hydroxyl and aminoethoxy side chain groups of 4-hydroxytamoxifen (Murphy, C. S., C. J. Parker, R. McCague, and V. C. Jordan, submitted). We have found that retention of the aminoethoxy side chain groups in a The activity of the compounds was determined by dose-response studies with T47D breast cancer cells in culture (82). The effective concentration 50% (EC50) to stimulate full cell replication was calculated for estrogens, and the inhibitory concentration 50% (IC50) against 10"10 M estradiol was calculated for antiestrogens. 0 EC50. fc IC60.
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November, 1990
ANTIESTROGENS AS ANTITUMOR AGENTS
position analogous to that found in 4-hydroxytamoxifen (Fig. 3) is necessary for antiestrogenic activity (Table 1). Repositioning of the aminoethoxy side chain para to the other available ring of 4-hydroxytamoxifen greatly decreased both the potency and the activity of the compound. It has been previously proposed that this area might be important for the correct folding of the ER protein around the molecule (30). Substitution of groups in this area reduces the potency of the ligand (Murphy, C. S., C. J. Parker, R. McCague, and V. C. Jordan, submitted). Clearly, there is an area of interference that prevents tight binding if large substitutions are made in the area of the D-ring of estradiol. This fact is well illustrated with the fixed-ring compounds with different substitutions (Fig. 6). The compound D (Table 1) has about 1% of the estrogenic potency of fixed-ring cis metabolite E. The aminoethoxy side chain is clearly preventing binding to the ligand binding site (Fig. 6). It is clear from these SAR that the positioning and rela-
583
tionship of both the hydroxyl and aminoethoxy side chain groups on tryphenylethylene-like ligands are essential to program cell replication. 3. Planarity of the binding ligand. The degree of planarity of the ligand is important for binding to the ER. Increased planarity of the triphenyl rings decreases the binding affinity of the molecules for the ER and abolishes any biological activity of these compounds in vivo (83). Compounds that contain linker groups of various size between the aryl rings of 4-hydroxytamoxifen (Fig. 5B) were tested for their effects on estradiol-stimulated growth of T47D cells in culture (Fig. 7). A small linker group, such as an oxygen at the X position (Fig. 5B), increases the planarity of the compound significantly, whereas a large linker group, such as a CH2-CH2 group at position X, increases the angle between aryl rings, thereby decreasing the planarity of the compound (83). As predicted, the most planar compound is a weak estrogen antagonist, whereas the nonplanar molecule is a much more potent inhibitor of estradiol-stimulated growth (Fig. 7). In summary, it appears that the composition and positioning of the side chain and hydroxyl groups of tamoxifen and its derivatives determine the
LU 0)
O)
Vol. 11, No. 4 Printed in U.S.A.
Endocrine Pharmacology of Antiestrogens as Antitumor Agents* V. CRAIG JORDAN AND CATHERINE S. MURPHYf Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792
I. Introduction II. Cell Culture Systems to Test Antiestrogens A. Structure activity relationships 1. The side chain 2. Positioning of the side chain 3. Planarity of the binding ligand III. New Antiestrogens Used in the Laboratory and Clinics A. Trioxifene and keoxifene B. Zindoxifene C. Toremifene D. Droloxifene E. Pure antiestrogens IV. Subcellular Actions of Estrogens and Antiestrogens A. Physicochemical differences of the ER bound to agonists or antagonists B. Indirect actions of antiestrogens C. Non-ER mediated effects of antiestrogens D. Summary V. Animal Tumor Models A. Athymic mouse models B. Spontaneous mouse mammary tumorigenesis C. Carcinogen-induced models D. Summary VI. Adjuvant Tamoxifen Therapy VII. Long Term Tamoxifen Therapy A. Chemotherapy plus tamoxifen B. Tamoxifen alone C. Future strategies VIII. Resistance to Tamoxifen Therapy A. Antiestrogen-resistant cells B. Paracrine growth factor mediated mechanisms C. Tamoxifen-stimulated growth D. Metabolism to estrogens E. Summary XI. Concerns about Long Term Tamoxifen Therapy A. Reproductive side effects 1. Ovarian stimulation 2. Teratogenesis
B. Ophthalmic C. Antiestrogenic effects 1. Osteoporosis 2. Atherosclerosis D. Estrogenic effects 1. Thromboembolic disorders 2. Uterine stimulation E. Liver carcinogenesis F. Summary X. Prevention of Breast Cancer A. Chemosuppression of occult disease B. Tamoxifen maintenance XI. Summary and General Conclusions
I. Introduction
I
N 1958, Lerner and co-workers (1) described the biological properties of the first nonsteroidal antiestrogen MER25 (see Fig. 1). The discovery of this new class of drugs opened up a variety of clinical possibilities (2, 3), but early studies were terminated because of toxic side effects. Another member of this new class of drugs, clomiphene (then known as chloramiphene or MRL41), held the promise of being a potential antifertility agent (4); however, clinical studies demonstrated that ovulation was induced (5). The drug is now available as a profertility agent in subfertile women (6). During the 1960s a range of antiestrogens was synthesized by the pharmaceutical industry for potential applications in gynecology (7, 8). In the main though, these agents were used only to study reproductive endocrinology in the laboratory. However the focus of clinical application slowly changed from the broad market of contraception to the relatively small market of advanced breast cancer therapy. It was known that about one in three premenopausal women with advanced breast cancer will respond to ovarian ablation (9). Similarly about one in three postmenopausal women will respond to high dose estrogen (usually diethylstilbestrol) therapy (10, 11). The identification of the estrogen receptor (ER) system in some hormone-sensitive breast tumors (12,13) and the knowl-
Address requests for reprints to: Dr. V. Craig Jordan, Department of Human Oncology, University of Wisconsin, Clinical Cancer Center, Madison, Wisconsin 53792. * Supported by grants P30-CA-14520 awarded to the Wisconsin Clinical Cancer Center and R01-CA-32713. t Current address, Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232.
578
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November, 1990
ANTIESTROGENS AS ANTITUMOR AGENTS
OCH3
Ethamoxytriphetol (MER 25)
Clomiphene (cis and trans isomers)
Tamoxifen (trans isomer)
FIG. 1. Structures of nonsteroidal antiestrogens.
edge that antiestrogens could inhibit the binding of estradiol to the ER from either the estrogen target tissues of laboratory animals or human breast tumors (14, 15) naturally led to clinical studies to determine whether antiestrogens could control the growth of at least some advanced breast cancers. Several antiestrogens were tested in clinical trials (16), but only tamoxifen (Fig. 1) was developed further because of demonstrated efficacy and a low reported incidence of side effects (17-21). In 1978 tamoxifen was made available to physicians in the United States for the treatment of advanced breast cancer in postmenopausal patients. Evidence has now been accrued to demonstrate that tamoxifen is equivalent to oophorectomy in the treatment of premenopausal patients with advanced disease (22, 23). Although these studies (22, 23) are small, and do not have the statistical power to detect significant differences between the efficacy of tamoxifen and oophorectomy, the Food and Drug Administration has approved the use of tamoxifen to treat ER-positive advanced disease in premenopausal women. However, the changing fashions for the treatment of breast cancer that have occurred over the past two decades prompted the testing of tamoxifen as an adjuvant therapy after mastectomy in women with stage II disease. A recent overview of all randomized clinical trials around the world has demonstrated that at least 2 yr of adjuvant tamoxifen therapy provides a survival advantage for women treated with this antiestrogen (24). The finding has now established tamoxifen as the antihormonal treatment of choice in postmenopausal women with stage II disease. Indeed the drug now appears to be accepted in the treatment of all stages of breast cancer following the recommendation by the National Cancer Institute that women with ER-positive stage I disease
579
should be offered the opportunity of adjuvant therapy with an antiestrogen. Tamoxifen has now become an essential part of any therapeutic strategy for the control of breast cancer. The early evaluation of the general pharmacology, endocrinology, and clinical use of antiestrogens has been adequately reviewed (25-34). However, the number of articles about the mechanism of action and clinical usefulness of tamoxifen is increasing exponentially. This article will focus upon recent reports concerning the mechanisms of action of antiestrogens, new agents, long term adjuvant therapy for the treatment of breast cancer, and the problems to be addressed with the development of therapeutic resistance. Indeed, there are now plans to use tamoxifen to prevent breast cancer; therefore it is appropriate to consider this potential application. II. Cell Culture Systems to Test Antiestrogens Standard antiestrogens such as tamoxifen are often used in cell culture as control substances to demonstrate that a particular event is estrogen regulated through the ER. The alternative strategy is to describe an estrogenregulated cellular event to study the structure activity relationships (SAR) of antiestrogens. The last half-decade has seen a consolidation of our understanding of estrogen action in breast cancer cells and a shift of emphasis from animal models of hormone-dependent events to focus upon breast cancer cells. Estrogen stimulates the induction of PRL by immature rat pituitary gland cells in primary culture (35). The system has proved to be valuable to describe the SAR of tamoxifen and a variety of novel agents (36-43). The assay was developed, indirectly, to understand the action of antiestrogens as antitumor agents in the dimethylbenzanthracene (DMBA)-induced rat mammary carcinoma model (44). These hormone-dependent tumors are primarily dependent upon estrogen-stimulated PRL release for growth although progesterone and the direct effects of estrogen on the tumor are probably necessary for an optimal growth rate (45). Indeed there is probably not one single factor that can be described as the most important so the actual mechanism of action of antiestrogens in the model system in vivo is multifaceted. The value of the PRL assay in vitro is that the direct effects of antiestrogens can be described upon one facet of the control mechanism for DMBA-tumor growth. The value of the assay for studying SAR is that there is only a remote chance of metabolic intervention and the complicating effects of pharmacokinetics, with rapidly excreted compounds, are avoided. Nevertheless compound stability is still an important consideration that will be addressed later (Section II.A2). The most important factor that facilitated the devel-
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JORDAN AND MURPHY
580
opment of a primary culture system from rat pituitary glands to study the SAR of antiestrogens was that there was no equivalent system for breast cancer. A number of hormone-responsive breast cancer cell lines were available to study estrogen-regulated events (46, 47), but estrogen-stimulated breast cancer cell growth was hard to demonstrate in the laboratory. Nevertheless some investigators (48, 49) were able to describe the direct effects of estrogen on growth, although these conclusions were not universally accepted. Indeed the ease with which estrogen-stimulated growth can be demonstrated in vivo, but infrequently in vitro, naturally led to the suggestion (50) that estrogen caused cell replication by an indirect mechanism. With regard to antiestrogens the model was complicated further because the drugs could inhibit basal cell growth although this inhibition could be reversed by estrogen ("estrogen rescue") (51). Perhaps antiestrogens had an effect as antagonists of estrogen action other than through the ER? The discovery (52) that the pH indicator in culture medium, phenol red (Fig. 2), exhibited estrogen-like properties changed the situation completely. Breast can-
Bisphenolic Compounds
Diethylstilbesterol
OH
o
cer cell lines only grow very slowly in "phenol red free" media, but the addition of estradiol causes increased replication. Under these short term estrogen-deprived conditions antiestrogens inhibit estradiol-stimulated growth. The refinement of the culture conditions has illustrated how sensitive replication is as an estrogenregulated event. The breast cancer cells that have been maintained in the estrogenic medium for nearly 20 yr have adapted optimally to the environment; therefore further growth from added estrogen might only be expected to produce a modest effect. In contrast, protein synthetic events such as progesterone receptor induction have a different dose-response curve to estrogen (53). This also explains why PRL synthesis in primary cultures of pituitary cells is sensitive to estrogen in standard culture medium. It is also fair to point out that pituitary cells are grown under higher serum conditions (12.5%) compared with breast cancer cells (5%). Less "phenol red" is available for biological action as it is absorbed nonspecifically on the protein (54). As an aside, the chemical responsible for the estrogenlike effects of phenol red indicator is in fact a contaminant present in difference batches of material (54, 55). The similarity of the structure of phenol red to the bisphenolic compounds (Fig. 2), with known estrogenic activity (56), naturally drew the conclusion that the indicator was an estrogen. Nevertheless the potent estrogenic contaminant has been identified (57) (Fig. 2). In fact this discovery has a parallel that occurred 50 yr earlier. Dodds and Lawson (58) described the "minimum" structure, anol, necessary to produce estrogen action in vivo. Unfortunately the report could not be confirmed (59, 60). A potent contaminant of anol, dianol (Fig. 2), was found to be responsible for the estrogenic actions (61, 62). Dianol bears a structural resemblance to diethylstilbestrol (Fig. 2) (63, 64) whose biological properties were described during the same period. The structures of AREA OF ANTIESTROGENIC INFLUENCE TO PREVENT PROTEIN FOLDING
o
Phenol Red
Vol. 11, No. 4
Bis(4-hydroxyphenyl)[2-(phenoxysulfonyl)phenyl]methane
STILBENE-LIKE STRUCTURE FOR OPTIMAL FIT AND CORRECT FOLDING
Anol Dianol FlG. 2. The formulae of a variety of nonsteroidal estrogens with either actual or presumed estrogenic activity. The references that describe the biological activity of the compounds are in the text.
PHENOLIC HYDROXYL FOR HIGH AFFINITY INTERACTION
FIG. 3. The important aspects of the 4-hydroxytamoxifen molecule to produce high affinity for the estrogen receptor and antiestrogenic activity.
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November, 1990
ANTIESTROGENS AS ANTITUMOR AGENTS
581
the relevant compounds are shown in Fig. 2 for comparison. Breast cancer cell lines are now used routinely to determine the structural features essential for a ligand to control cell replication.
-CH3 OCH 2 CH 2 N
A.SAR
fixed ring "trans" 4-hydroxytamoxifen
The antiestrogenic potency of tamoxifen is increased by metabolic conversion to 4-hydroxytamoxifen (65). Since the dimethylaminoethane side chain is required for antiestrogenic activity, recent studies using breast cancer cell lines in culture have focused upon both the composition and positioning of the side chain. The salient features of 4-hydroxytamoxifen are shown in Fig. 3. 1. The side chain. Triphenylethylene derivatives of tamoxifen that lack the side chain are full estrogen agonists (40, 66). The composition of the side chain is also critical for antagonist activity (67-69). Several triphenylethylene compounds with alterations in the side chain region have been tested for their effects on the growth of a hormone-responsive line of T47D breast cancer cells in vitro (70). Substitution of the aminoethoxy side chain with an allyl side chain produces a compound with partial agonist activity. Compounds containing bulky aryl groups in place of the side chain are also partial agonists in this system. Therefore, the antiestrogenic activity of -CH OCH 2 CH 2 N,
,CH3
fixed ring "cis" 4-hydroxytamoxifen
B
compound a b c
O S
FlG. 5. Fixed ring derivatives of 4-hydroxytamoxifen to prevent isomerization (A) or to change the planarity of the molecule (B).
4 - hydroxytamoxifen ("trans" isomer)
4 - hydroxytamoxifen ("cis" isomer)
high affinity for ER antiestrogen
low affinity for ER antiestrogen
tamoxifen is not solely due to the length or bulk of the constituents in the side chain region. Interestingly, compounds that have oxygen-containing side chains are antiestrogenic in this system. Carbonylcontaining side chains of particular length are antiestrogenic. The addition of a single hydroxyl group onto an alkyl chain reverses the biological activity of the compound, making it antiestrogenic (70). These studies support the conclusion that side chain constituents containing a lone pair of electrons (such as oxygen or nitrogen) are necessary for antiestrogenic activity (68).
isomerization
Metabolite E ("cis" isomer)
Metabolite E ("trans" isomer)
potent estrogen
weak partial agonist
FlG. 4. The instability of hydroxylated triphenylethylenes results in the rapid isomerization, in vitro, to their alternate geometric isomer. The isomers on the left of the diagram are stable in solution.
2. Positioning of the side chain. The effect of triphenylethylene isomerization, and subsequent repositioning of the side chain region, has been studied extensively (71-76). Isomerization of the trans form of tamoxifen around the central double bond repositions the side chain to form its cis isomer. This results in a change in activity of the compound converting it from an antiestrogen to a partial agonist. Addition of a hydroxyl group to tamoxifen produces
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JORDAN AND MURPHY
582
TABLE 1. Biological activity of a fixed ring derivative of triphenylethylene substituted in various positions with hydroxyl or a dimethylaminoethoxy side chain FORMULA
NAME
EC 5 0 » OR I C 5 0 '
a.) ESTROGENS ESTRADIOL-17|i
3x10° 2 M
f.r. els met E
6x10" 1 1 M
,OCH 2 CHjN(CH 3 ) 2
COMPOUND"B"
PARTIAL AGONIST
COMPOUND"D"
PARTIAL AGONIST
PARTIAL AGONIST
l.r. trans met E
OCH 2 CH 2 N(CH 3 )2
b.) ANTIESTROGENS
l.r. trans 4-OHT
3x10" 1 1 M
COMPOUND"A"
8x10" 9 M
(CH 0 ) 2 NCH 2 CHjO
3x10' 8 M
COMPOUND"C"
(CHJJJNCHJCHJO
RU 39411
3x10° 1 M
Vol. 11, No. 4
the potent tamoxifen metabolite 4-hydroxytamoxifen (65). The trans form of 4-hydroxytamoxifen is a potent antagonist (IC50 = 3 X 10~n to 1 X 10~10 M). However, the true activity of the cis form of 4-hydroxytamoxifen was not known because it readily isomerizes (Fig. 4). Previous studies (37, 72) had shown that cis-4-hydroxytamoxifen was an antiestrogen in vitro and in vivo. However, based on studies done in the early 1980s, investigators suggested that the antiestrogenic activity of cis-4-hydroxytamoxifen could be due to its isomerization to the potent trans form (73, 74). To prevent isomerization a series of fixed-ring triphenylethylenes (Fig. 5A) were synthesized to test the activities of individual isomers (77-79). For these studies (42, 80), the growth response of hormone-responsive T47D human breast cancer cells and PRL synthesis in pituitary cell cultures were both used. The fixed-ring form of cis-4-hydroxytamoxifen (Fig. 5A) is antiestrogenic (IC50 = 4 X 10"8 to 2 X 10~7 M) (42, 80) and is not estrogenic. These findings support earlier predictions of the pharmacological actions of the cis isomer of 4-hydroxytamoxifen (37). The cis fixed-ring form of metabolite E (an estrogenic metabolite of tamoxifen) is a potent estrogen in this system (EC50 = 4 x 10~12 to 1 x 10" u M). However, the true biological activity of the trans form of metabolite E could not be determined because this compound also readily isomerizes to its opposite isomeric form (74) (Fig. 4). It was presumed that trans metabolite E, like cis metabolite E, was a full estrogen agonist because it lacked an aminoethoxy side chain in the relevant position (39). Surprisingly, it was found (80) that trans fixed ring metabolite E was only a weak partial agonist. These studies utilizing fixed-ring compounds have provided information regarding the true intrinsic activities of metabolites of tamoxifen. The fixed-ring compounds could also be useful to study the effects of various substituents on biological activity. Clearly isomerization of hydroxylated triphenylethylenes can produce changes in both pharmacological properties and potency so this should be considered during the evaluation of SAR studies (81). We have recently determined the relationship between the biological activity and the relative positioning of the hydroxyl and aminoethoxy side chain groups of 4-hydroxytamoxifen (Murphy, C. S., C. J. Parker, R. McCague, and V. C. Jordan, submitted). We have found that retention of the aminoethoxy side chain groups in a The activity of the compounds was determined by dose-response studies with T47D breast cancer cells in culture (82). The effective concentration 50% (EC50) to stimulate full cell replication was calculated for estrogens, and the inhibitory concentration 50% (IC50) against 10"10 M estradiol was calculated for antiestrogens. 0 EC50. fc IC60.
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November, 1990
ANTIESTROGENS AS ANTITUMOR AGENTS
position analogous to that found in 4-hydroxytamoxifen (Fig. 3) is necessary for antiestrogenic activity (Table 1). Repositioning of the aminoethoxy side chain para to the other available ring of 4-hydroxytamoxifen greatly decreased both the potency and the activity of the compound. It has been previously proposed that this area might be important for the correct folding of the ER protein around the molecule (30). Substitution of groups in this area reduces the potency of the ligand (Murphy, C. S., C. J. Parker, R. McCague, and V. C. Jordan, submitted). Clearly, there is an area of interference that prevents tight binding if large substitutions are made in the area of the D-ring of estradiol. This fact is well illustrated with the fixed-ring compounds with different substitutions (Fig. 6). The compound D (Table 1) has about 1% of the estrogenic potency of fixed-ring cis metabolite E. The aminoethoxy side chain is clearly preventing binding to the ligand binding site (Fig. 6). It is clear from these SAR that the positioning and rela-
583
tionship of both the hydroxyl and aminoethoxy side chain groups on tryphenylethylene-like ligands are essential to program cell replication. 3. Planarity of the binding ligand. The degree of planarity of the ligand is important for binding to the ER. Increased planarity of the triphenyl rings decreases the binding affinity of the molecules for the ER and abolishes any biological activity of these compounds in vivo (83). Compounds that contain linker groups of various size between the aryl rings of 4-hydroxytamoxifen (Fig. 5B) were tested for their effects on estradiol-stimulated growth of T47D cells in culture (Fig. 7). A small linker group, such as an oxygen at the X position (Fig. 5B), increases the planarity of the compound significantly, whereas a large linker group, such as a CH2-CH2 group at position X, increases the angle between aryl rings, thereby decreasing the planarity of the compound (83). As predicted, the most planar compound is a weak estrogen antagonist, whereas the nonplanar molecule is a much more potent inhibitor of estradiol-stimulated growth (Fig. 7). In summary, it appears that the composition and positioning of the side chain and hydroxyl groups of tamoxifen and its derivatives determine the
LU 0)
O)
