c91
Molecular and Cellular Endocrinology, 14 (1990) C91LC95 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
02436
At the Cutting Edge
“The only true antiestrogen is no estrogen” V. Craig Jordan Department
Key wora!s: Antiestrogen;
of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison,
Tamoxifen;
ICI-164,384;
Receptor
dimerization;
“The only true antiestrogen is no estrogen”. This was the statement made by Terry Robinson during discussion of a presentation entitled “Antiestrogens” by Cliff Emmens in 1961 (Emmens et al., 1962). Professor Emmens had reviewed his work with dimethylstilbestrol, which has local antiestrogenic activity when administered intravaginally but no antiestrogenic properties when administered systemically. Albert Segaloff was particularly concerned that such compounds should be given a more precise nomenclature; some agents blocked vaginal cornification, whilst others were antiuterotropic. He concluded with the statement “I think that we ought to start talking more specifically until we have a real agent which will duplicate the effects of castration, which to my mind will deserve to be called an antiestrogen”. Elwood Jensen noted that MER25, a compound first described by Lemer and coworkers (1958), inhibited the incorporation and retention of administered [3H]estradiol in the rat uterus. Thus an antiestrogen could be defined as a compound that blocks estrogen action in the target tissue by preventing the uptake of estradiol. Lemer and coworkers (1958) had demonstrated that MER25 has weak, but effective and clear-cut, antiestrogenic activity in the uterus and vagina of a variety of species; most importantly, however, MER25 had no other hormonal or antihormonal
Address for correspondence: V. Craig Jordan, Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, WI 53792, U.S.A.
0303-7207/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
MCF-7;
T47D;
Hormone
WI 53792, U.S.A.
antagonist
actions. In particular, MER25 has very little intrinsic estrogenic activity and in many biological tests it appears to be like “no estrogen”. Clinical trials were organized to evaluate the pharmacological activity of MER25 in patients but were terminated due to toxic side effects (Lemer, 1981). The drug discovery research teams focussed upon increasing potency rather than retaining pure antiestrogenicity. During the 1960s and early 1970s a whole range of antiestrogens were used as laboratory tools, as antifertility agents in rodents and profertility agents in women (Emmens, 1970; Lunan and Klopper, 1975). All of the compounds developed during this period were partial estrogen agonists, but their antiestrogenic potency was much higher than that of MER25. The subsequent successful development of tamoxifen (ICI-46,474) as a treatment for breast cancer (reviewed in Jordan, 1988) has caused an explosion of interest in antiestrogens as antitumor agents; indeed there are now plans to evaluate antiestrogens to prevent breast cancer (Cuzick et al., 1986; Fentiman and Powles, 1987; Powles et al., 1989). How could a drug like tamoxifen gain such wide-spread use in the treatment of breast cancer? The answer reflects a combination of proven efficacy and a low incidence of side effects (Furr and Jordan, 1984). Successful testing in advanced disease, and the changing practice of treating early disease after mastectomy (adjuvant therapy), have now resulted in the general acceptance of tamoxifen treatment for all stages of breast cancer. The current strategy for treatment is to employ longLtd.
C92
term adjuvant tamoxifen therapy (Jordan, 1990) a concept that has successfully been transferred to the clinic from the laboratory (Jordan, 1983). The safety of tamoxifen has facilitated its use in long-term adjuvant therapy to treat patients with estrogen receptor positive, node negative disease (Fisher et al., 1989) and encouraged the proposed use of tamoxifen in normal women who are at risk for breast cancer (Powles et al., 1989, 1990). The key, however, to the successful use of tamoxifen for long-term therapy is the fact that the molecule does have some estrogen-like qualities (Furr and Jordan, 1984). The concern that an “antiestrogen” would place women at risk for premature atherosclerosis and osteoporosis seems to be unfounded; for example, the estrogenic activity of tamoxifen causes a decrease in low density lipoprotein cholesterol (Bruning et al., 1988; Baglade et al., 1990; Love et al., 1990) and longterm adjuvant tamoxifen therapy does not appear to cause a decrease in bone density (Fornander et al., 1990). A balance of estrogenic and antiestrogenie properties would thus seem to be best for the patient. The fact that tamoxifen exhibits estrogen-like activity to support physiological functions in postmenopausal women may be a two-edged sword at some target sites. Satyaswaroop and coworkers (1984) have demonstrated that a human estrogen and progesterone receptor positive endometrial carcinoma will grow in athymic mice in response to either estrogen or tamoxifen treatment. Furthermore, tamoxifen exhibits target site specificity: if athymic animals are transplanted with both human endometrial carcinoma (EnCalOl) and breast carcinoma (MCF-7) and then treated with estradiol and tamoxifen, the growth of the breast carcinoma is controlled by tamoxifen but the endometrial carcinoma grows more rapidly (Gottardis et al., 1988). This kind of data has raised concerns about the possibility of an increased incidence of endometrial carcinoma in women receiving long-term adjuvant therapy with tamoxifen. A recent report by Fornander and colleagues (1989) has very clearly illustrated the target site specificity of tamoxifen in a randomized clinical trial. Patients (almost a thousand in either tamoxifen or placebo arms) had an increase in endometrial carcinoma (control 2; tamoxifen
treated 13), but a significant decrease in the occurrence of second primary breast tumors (control 32; tamoxifen treated 18). The observation that human endometrial carcinoma can become responsive to the estrogenie activity of tamoxifen by growth raises the question of whether breast tumors may similarly become dependent upon tamoxifen during longterm therapy. In this regard, the long-term treatment of MCF-7 tumor bearing athymic mice with tamoxifen is eventually followed by tamoxifen stimulated tumor growth (Gottardis and Jordan, 1988); the tumors are estrogen receptor positive and also respond to estradiol by growth (Gottardis et al., 1989). A withdrawal response to tamoxifen occurs with tamoxifen-stimulated MCF-7 tumor (Gottardis et al., 1989), and a similar phenomenon has been noted clinically (Canney et al., 1987); in this case, however, an additional agent needs to be given to prevent the tumor from using endogenous estrogens to grow. Aromatase inhibitors have been used with some success after tamoxifen (Smith et al., 1981) on the principle that “the only true antiestrogen is no estrogen”. Unfortunately increased side effects, and the fact that endogenous estrogenic steroids are not all controlled by inhibitors of aromatization, rather limit their usefulness. A true antiestrogen equivalent to “no estrogen” would be a valuable addition indeed to our therapeutic armamentarium. Research does not proceed in straight lines, and a series of apparently unrelated observations contributed to the discovery of pure antiestrogens. Scientists at ICI were investigating the structureactivity relationships of C6 and C7 substituted estrogenic steroids to act as “irreversible antiestrogens”, or as compounds that might alkylate the estrogen response elements (EREs) (Jordan et al., 1981). The chemists at ICI had noted that derivatives of estradiol with a long alkyl chain ((CH,),,) at the C7 position attached to resins can be used to purify the estrogen receptor (Bucort et al., 1978). The team at ICI (Wakeling and Bowler, 1987) then put all of the information together and tested long chain derivatives of estradiol as antiestrogens. A number of compounds are under investigation but only ICI164,384 (estradiol with a 7a substituted (CH2),0 side chain) is available for evaluation.
c93
In the laboratory ICI-164,384 will completely inhibit estrogen action and appears to exhibit no agonist action in rats or mice (Wakeling and Bowler, 1988); in addition, it inhibits the estrogen-stimulated growth and invasive properties of breast cancer cell lines in culture (Thompson et al., 1989). Indeed ICI-164,384 will inhibit the uterotrophic effects of tamoxifen and blunt the tamoxifen-stimulated growth of MCF-7 (Gottardis et al., 1989) and EnCalOl tumors (Gottardis et al., 1990) in athymic mice. Radiolabelled ICI-164,384 is a competitive inhibitor of estradiol binding to the estrogen receptor (Weatherall et al., 1988), an effect that is probably responsible for its reversible blockade of the G, phase of the cell cycle (Musgrove et al., 1989). Pierre Chambon’s group has demonstrated that both estradiol- and 4-hydroxytamoxifen-estrogen receptor complexes can compete at EREs to affect gene transcription (Metzer et al., 1988). Since 4hydroxytamoxifen is a partial agonist (Jordan et al., 1977), clearly it must interact at EREs; in contrast, however, the pure antiestrogens may have a unique mechanism that is responsible for an effect like “no estrogen”. Dimerization of the estrogen receptor is necessary to bind to EREs; receptors with a mutation at the dimerization site will not bind to DNA (Fawell et al., 1990a). Recent studies by,Malcolm Parker and colleagues (Fawell et al., 1990b) have demonstrated that the pure antiestrogen ICI-164,384 prevents receptor dimerization and binding to DNA. Interestingly, a bivalent monoclonal antibody to the estrogen receptor will permit receptor binding to DNA, presumably by creating “dimers”; the monovalent antibody is ineffective. It is possible that antiestrogens will eventually fall into two categories, (1) triphenylethylenes that interact at the ligand binding site of the estrogen receptor, and that prevent the tertiary changes necessary for appropriate activation of receptor (Lieberman et al., 1983); such complexes can bind to EREs but cannot initiate full agonist actions; and (2) compounds with a long alkyl side chain which prevents receptor dimerization (Fawell et al., 1990b). What is the therapeutic potential of a pure antiestrogen? Currently, the use of long-term adjuvant tamoxifen may “educate” breast cancer
cells to become tamoxifen dependent, and pure antiestrogens could be an effective second line therapy. A second question, however, is what will happen to the growth of an estrogen responsive breast cancer if a pure antiestrogen is a first-line therapy? It has only recently been possible to evaluate the effects of complete estrogen deprivation on hormone responsive breast cancer cells in the laboratory, since estrogen receptor positive breast cancer cell lines have been cultured routinely (for up to 20 years!) in apparently estrogen-free media containing phenol red, an estrogenic contaminant (Berthois et al., 1986; Bindall and Katzenellenbogen, 1988). Long-term culture of MCF-7 breast cancer cells in media without phenol red initially results in the slowing of growth, but after about 6 months the growth rate increases (Katzenellenbogen et al., 1987; Welshons and Jordan, 1987). Cells still retain estrogen receptors (and are progesterone receptor negative); estrogen administration increases progesterone receptor level but not growth rate; and antiestrogens slow the growth of these adapted cells. Variants of the cell line T47D (Keydar et al., 1979) have been reported to be hormone responsive (Chalbos et al., 1982) and nonresponsive (Horowitz et al., 1982) depending upon the culture conditions used to derive the subline. We recently reported a hormone responsive subline (A line) derived from ATCC stock cells that we have used to study the effects of tamoxifen derivatives on breast cancer cell replication (Murphy and Jordan, 1989; Murphy et al., 1990a). Long-term (12 months) growth of T47D (line A) in medium without phenol red results in a dramatic decrease in the high levels of progesterone receptor, and eventually estrogen receptors become undetectable (Murphy et al., 1989); the derived cell line (line C) neither grows in response to estrogen nor produces progesterone receptor. Not surprisingly, the antiestrogens 4-hydroxytamoxifen and ICI-164,384 do not affect the growth rate of T47D line C. It should be pointed out, however, that the growth rate of line C (and an estrogen and progesterone receptor negative clonal line C,) is low compared to the growth rate of estrogen treated cells from line A, and of an estrogen and progesterone receptor positive clonal
c94
line Al8 (Murphy et al., 1990b). This property contrasts with the MCF-7 breast cancer cell line, which grows rapidly under estrogen deprived conditions and is sensitive to the effect of antiestrogens (Welshons and Jordan, 1987). Levels of mRNA for transforming growth factor (Yand pi,2 in C, cells are not altered compared with hormone responsive cell lines and contrast dramatically with the high levels of mRNAs observed in hormone independent cell lines like MDA-MB-231 (Murphy et al., 1990b), though the mRNA for both estrogen and progesterone receptor is undetectable. This raises an important therapeutic consideration. Westley and May (1988) have demonstrated that estrogen receptor mRNA is regulated by estrogen, and have cautioned about the therapeutic use of pure antiestrogens: if a pure antiestrogen completely prevents the synthesis of estrogen receptor, there is the potential for genetic instability to produce hormone-independent daughter cells. Although estrogen-deprived cells might then grow more slowly it is possible to envision the influence of paracrine growth factors causing tumor cell replication even in the presence of effective antiestrogenic concentrations of ICI164,384 (Cormier and Jordan, 1989; Robinson and Jordan, 1989). Despite this theoretical possibility, the question of whether pure antiestrogens will be significantly more effective given as front line therapy or as second line therapy after tamoxifen can be evaluated only in the clinic. Indeed, the position can be taken that a pure antiestrogen might be used to best advantage at an early stage of the disease, i.e. when hormone-dependent growth is more likely and there is a low tumor burden. Obviously, if pure antiestrogens are used in node negative women following mastectomy special attention must be paid to the potential problems of osteoporosis and atherosclerosis. Only 30% of such women will have a recurrence of disease 5-10 years after initial diagnosis and treatment, and those women who would not have had a recurrence will have to undergo the physiological consequences of absolute estrogen deprivation. Clinical strategies must be devised to find a duration of pure antiestrogen treatment which provides optimal control of tumor recurrence in those at risk, whilst not jeopardizing the health of the majority of women who are cured.
In summary, the past three decades have seen the introduction of a new class of drugs which have aided the survival of breast cancer patients and facilitated our understanding of the normal processes controlled by estrogen (Lerner and Jordan, 1990). From their humble beginnings as laboratory tools, the clinical use of antiestrogens by physicians has become commonplace. All stages of breast cancer are being treated with long-term and in some cases indefinite adjuvant tamoxifen therapy. The key to success has undoubtedly been the high degree of patient acceptability, but it is clear that some level of estrogenic activity is essential if tamoxifen is to be used as a preventive measure in normal women (Jordan, 1990). Whether the use of tamoxifen in postmenopausal women ever becomes ubiquitous, its estrogen-like qualities are clearly an advantage to node negative postmenopausal women with breast cancer who are denied normal hormone replacement therapy. Pure antiestrogens should soon be available for clinical evaluation. Whether they prove to be significantly superior to tamoxifen as therapeutic agents or not, they constitute a valuable new tool for studying the molecular endocrinology of estrogen action. Pure antiestrogens are like “no estrogen”; we may, however, be able to take Terry Robinson’s dictum one step further. Some estrogen receptor positive breast cancer cells appear to be able to grow rapidly without an estrogenic stimulus (Welshons and Jordan, 1987) but an antiestrogen can prevent growth, presumably by preventing dimerization of the unoccupied estrogen recptor. A pure antiestrogen may thus be even better than no estrogen. Acknowledgements Some of the studies described were supported by Cancer Center Support grant P30 CA14520, ROl CA32713, PO1 CA20432 and training grant 5T32-CA09471 in Human Cancer Biology. References Bagdade, J.D., Walter, J., Sabbaiah, P.V. and Ryan, W. (1990) J. Endocrinol. Metab. 70, 1132-1135. Berthois, Y., Katzenellenbogen, J.A. and Katzenellenbogen, B.S. (1986) Proc. Nat]. Acad. Sci. U.S.A. 83, 2496-2500.
c95 BindaIl, R.D. and Katzenellenbogen,
J.A. (1988) J. Med. Chem.
31,1978-1983. Bruning, P.F., Banfrer, J.M.G., Hart, A.A.M., DeJorg-Bakker, M., Linders, D., Van Loor, J. and Nooyen, W.J. (1988) Br. J. Cancer 58, 497-500. Bucourt, R., Vignau, M., Torelli, V., Richard-Roytt, S., Geynet, C., Secco-Millet, C., Redcailk, G. and Baulieu, E.E. (1978) J. Biol. Chem. 253, 8221-8228. Canney, P.A., Griffiths, T., Latief, T.N. and Priestman, T.J. (1987) Lancet i, 36. Chalbos, D., Vignon, F., Keydar, I. and Rochefort, H.I. (1982) J. Clin. Endocrinol. Metab. 55, 276-283. Cormier, E.M. and Jordan, V.C. (1989) Eur. J. Cancer Chn. Oncol. 25, 57-63. Cuzick, J., Wang, D.Y. and Bulbrook, R.D. (1986) Lancet i, 83-85. Emmens, C.W. (1970) Br. Med. Bull. 26, 45-51. Emmens, C.W., Cox, R.I. and Martin, L. (1962) Recent Prog. Horm. Res. 18,415-466. Fawell, S.E., Lees, J.A., White, R. and Parker, M.G. (1990a) Cell 60, 953-962. Fawell, SE., White, R., Hoare, S., Sydenham, M., Page, M. and Parker, M.G. (1990b) Proc. Natl. Acad. Sci. U.S.A. 87, 6883-6887. Fentiman, I.S. and Powles, T.J. (1987) Lancet ii, 1070-1072. Fisher, B., Constantine, J., Redmond, C. and other members of the NSABP (1989) New Engl. J. Med. 320, 479-484. Fomander, T., Rutqvist, L.E., Cedarmark, B., Glass, U., Mattson, A., Silversward, J.C., Skoog, L., Somell, A., Theve, T., Wilking, N., Askergren, J. and HjoImar, M.L. (1989) Lancet i, 117-120. Fornander, T., Rutqvist, L.E., Sjiiberg, H.E., Blomqvist, L., Mattsson, A. and Glas, U. (1990) J. Clin. Oncol. 8, 10191023. Furr, B.J.A. and Jordan, V.C. (1984) Pharmacol. Ther. 25, 127-205. Gottardis, M.M. and Jordan, V.C. (1988) Cancer Res. 48, 5183-5187. Gottardis, M.M., Robinson, S.P., Satyaswaroop, P.G. and Jordan, V.C. (1988) Cancer Res. 48, 812-815. Gottardis, M.M., Jiang, S.Y., Jeng, M.H. and Jordan, V.C. (1989) Cancer Res. 49, 4090-4093. Gottardis, M.M., Ricchio, M.E., Satyaswaroop, P.G. and Jordan, V.C. (1990) Cancer Res. 50, 3189-3192. Horwitz, K.B., Mockus, M.B. and Lessey, B.A. (1982) Cell 28, 633-642. Jordan, V.C. (1983) Beast Cancer Res. Treat. 3 (Suppl.), 73-86. Jordan, V.C. (1984) Pharmacol. Rev. 36, 245-276. Jordan, V.C. (1988) Breast Cancer Res. Treat. 11, 197-209. Jordan, V.C. (1990) Cancer Treat. Rev. (in press). Jordan, V.C., Collins, M.M., Rowsby, L. and Prestwich, G. (1977) J. Endocrinol. 75, 305-316. Jordan, V.C., Fenuick, L., Allen, K.E., Cotton, R.E., Richardson, D.N., Walpole, A.L. and Bowler, J. (1981) Eur. J. Cancer Clin. Oncol. 17, 193-200. Katzenellenbogen, B.S., Kendra, K.L., Norman, M.J. and Berthois, Y. (1987) Cancer Res. 47, 4355-4359.
Keydar, I., Chen, L., Karby, S., Weiss, F.R., Delarea, M., Rachu, M., Chaitcik, S. and Brenner, H.J. (1979) Eur. J. Cancer 15,659-670. Lemer, L.J. (1981) in Non-Steroidal Antiestrogens: Molecular Pharmacology and Antitumour Activity (R.L. Sutherland and V.C. Jordan, eds.), pp. l-16, Academic Press, Sydney. Lemer, L.J. and Jordan, V.C. (1990) Cancer Res. 50, 41774189. Lemer, L.J., Holthaus, Jr., F.J. and Thompson, C.R. (1958) Endocrinology 63, 295-318. Lieberman, M.E., Gorski, J. and Jordan, V.C. (1983) J. Biol. Chem. 258, 4741-4745. Love, R.R., Newcomb, P.A., Wiebe, D.A., Surawicz, T.S., Jordan, V.C., Carbone, P.P. and DeMets, D.L. (1990) J. Natl. Cancer Inst. 82, 1327-1331. Lunan, C.S. and KIopper, A. (1975) Chn. Endocrinol. 4, 557572. Metzer, D., White, J.H. and Chambon, P. (1988) Nature 334, 31-35. Murphy, C.S. and Jordan, V.C. (1989) J. Steroid Biochem. 34, 407-411. Murphy, C.S., Meisner, L.F., Wu, S.-Q. and Jordan, V.C. (1989) Eur. J. Cancer Clin. Oncol. 25, 1777-1788. Murphy, C.S., Langan-Fahey, S.M., McCague, R. and Jordan, V.C. (1990a) Mol. Pharmacol. (in press). Murphy, C.S., Pink, J.J. and Jordan, V.C. (1990b) Cancer Res. (in press). Musgrove, E.A., Wakeling, A.E. and Sutherland, R.A. (1989) Cancer Res. 49, 2398-2402. Powles, T.J., Hardy, J.R., Ashley, S.E., Farrington, G.H., Cosgrove, D., Davey, J.B., Dowsett, M., McKirma, J.A., Wash, A.G., Sinnett, H.D., Tillyer, C.R. and Treleaven, J.G. (1989) Br. J. Cancer 60, 126-131. Powles, T.J., Tillyer, CR., Jones, A.L., Ashley, S.E., Trebaven, J., Davey, J.B. and McKinna, J.A. (1990) Eur. J. Cancer 26, 680-684. Robinson, S.P. and Jordan, V.C. (1989) Eur. J. Cancer Chn. Oncol. 25, 493-497. Satyaswaroop, P.G., Zaino, R.J. and Mortel, R. (1984) Cancer Res. 44,4006-4010. Smith, I.E., Harris, A.L., Morgan, M., Ford, H.T., Gazet, J.C., Harmer, C.L., White, H., Parsons, C.A., Villardo, A., Walsh, G. and McKinna, J.A. (1981) Br. Med. J. 283, 1432-1434. Thompson, E.W., Katz, D., Shima, T.B., Wakehng, A.E., Lippman, M.E. and Dickson, R.B. (1989) Cancer Res. 49, 6929-6934. Wakehng, A.E. and Bowler, J. (1987) J. Endocrinol. 112, R7-RlO. Wakeling, A.E. and Bowler, J. (1988) J. Steroid B&hem. 30, 141-148. Weatherill, P.J., Wilson, A.P.M., Nicholson, R.I., Davis, P. and Wakehng, A.E. (1988) J. Steroid Biochem. 30, 263-268. Welshons, W.V. and Jordan, V.C. (1987) Eur. J. Cancer Clin. Oncol. 23, 1935-1939. Westley, B.R. and May, F.E.B. (1988) B&hem. Biophys. Res. Commun. 155. 1113-1118.
Molecular and Cellular Endocrinology, 14 (1990) C91LC95 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
02436
At the Cutting Edge
“The only true antiestrogen is no estrogen” V. Craig Jordan Department
Key wora!s: Antiestrogen;
of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison,
Tamoxifen;
ICI-164,384;
Receptor
dimerization;
“The only true antiestrogen is no estrogen”. This was the statement made by Terry Robinson during discussion of a presentation entitled “Antiestrogens” by Cliff Emmens in 1961 (Emmens et al., 1962). Professor Emmens had reviewed his work with dimethylstilbestrol, which has local antiestrogenic activity when administered intravaginally but no antiestrogenic properties when administered systemically. Albert Segaloff was particularly concerned that such compounds should be given a more precise nomenclature; some agents blocked vaginal cornification, whilst others were antiuterotropic. He concluded with the statement “I think that we ought to start talking more specifically until we have a real agent which will duplicate the effects of castration, which to my mind will deserve to be called an antiestrogen”. Elwood Jensen noted that MER25, a compound first described by Lemer and coworkers (1958), inhibited the incorporation and retention of administered [3H]estradiol in the rat uterus. Thus an antiestrogen could be defined as a compound that blocks estrogen action in the target tissue by preventing the uptake of estradiol. Lemer and coworkers (1958) had demonstrated that MER25 has weak, but effective and clear-cut, antiestrogenic activity in the uterus and vagina of a variety of species; most importantly, however, MER25 had no other hormonal or antihormonal
Address for correspondence: V. Craig Jordan, Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, WI 53792, U.S.A.
0303-7207/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
MCF-7;
T47D;
Hormone
WI 53792, U.S.A.
antagonist
actions. In particular, MER25 has very little intrinsic estrogenic activity and in many biological tests it appears to be like “no estrogen”. Clinical trials were organized to evaluate the pharmacological activity of MER25 in patients but were terminated due to toxic side effects (Lemer, 1981). The drug discovery research teams focussed upon increasing potency rather than retaining pure antiestrogenicity. During the 1960s and early 1970s a whole range of antiestrogens were used as laboratory tools, as antifertility agents in rodents and profertility agents in women (Emmens, 1970; Lunan and Klopper, 1975). All of the compounds developed during this period were partial estrogen agonists, but their antiestrogenic potency was much higher than that of MER25. The subsequent successful development of tamoxifen (ICI-46,474) as a treatment for breast cancer (reviewed in Jordan, 1988) has caused an explosion of interest in antiestrogens as antitumor agents; indeed there are now plans to evaluate antiestrogens to prevent breast cancer (Cuzick et al., 1986; Fentiman and Powles, 1987; Powles et al., 1989). How could a drug like tamoxifen gain such wide-spread use in the treatment of breast cancer? The answer reflects a combination of proven efficacy and a low incidence of side effects (Furr and Jordan, 1984). Successful testing in advanced disease, and the changing practice of treating early disease after mastectomy (adjuvant therapy), have now resulted in the general acceptance of tamoxifen treatment for all stages of breast cancer. The current strategy for treatment is to employ longLtd.
C92
term adjuvant tamoxifen therapy (Jordan, 1990) a concept that has successfully been transferred to the clinic from the laboratory (Jordan, 1983). The safety of tamoxifen has facilitated its use in long-term adjuvant therapy to treat patients with estrogen receptor positive, node negative disease (Fisher et al., 1989) and encouraged the proposed use of tamoxifen in normal women who are at risk for breast cancer (Powles et al., 1989, 1990). The key, however, to the successful use of tamoxifen for long-term therapy is the fact that the molecule does have some estrogen-like qualities (Furr and Jordan, 1984). The concern that an “antiestrogen” would place women at risk for premature atherosclerosis and osteoporosis seems to be unfounded; for example, the estrogenic activity of tamoxifen causes a decrease in low density lipoprotein cholesterol (Bruning et al., 1988; Baglade et al., 1990; Love et al., 1990) and longterm adjuvant tamoxifen therapy does not appear to cause a decrease in bone density (Fornander et al., 1990). A balance of estrogenic and antiestrogenie properties would thus seem to be best for the patient. The fact that tamoxifen exhibits estrogen-like activity to support physiological functions in postmenopausal women may be a two-edged sword at some target sites. Satyaswaroop and coworkers (1984) have demonstrated that a human estrogen and progesterone receptor positive endometrial carcinoma will grow in athymic mice in response to either estrogen or tamoxifen treatment. Furthermore, tamoxifen exhibits target site specificity: if athymic animals are transplanted with both human endometrial carcinoma (EnCalOl) and breast carcinoma (MCF-7) and then treated with estradiol and tamoxifen, the growth of the breast carcinoma is controlled by tamoxifen but the endometrial carcinoma grows more rapidly (Gottardis et al., 1988). This kind of data has raised concerns about the possibility of an increased incidence of endometrial carcinoma in women receiving long-term adjuvant therapy with tamoxifen. A recent report by Fornander and colleagues (1989) has very clearly illustrated the target site specificity of tamoxifen in a randomized clinical trial. Patients (almost a thousand in either tamoxifen or placebo arms) had an increase in endometrial carcinoma (control 2; tamoxifen
treated 13), but a significant decrease in the occurrence of second primary breast tumors (control 32; tamoxifen treated 18). The observation that human endometrial carcinoma can become responsive to the estrogenie activity of tamoxifen by growth raises the question of whether breast tumors may similarly become dependent upon tamoxifen during longterm therapy. In this regard, the long-term treatment of MCF-7 tumor bearing athymic mice with tamoxifen is eventually followed by tamoxifen stimulated tumor growth (Gottardis and Jordan, 1988); the tumors are estrogen receptor positive and also respond to estradiol by growth (Gottardis et al., 1989). A withdrawal response to tamoxifen occurs with tamoxifen-stimulated MCF-7 tumor (Gottardis et al., 1989), and a similar phenomenon has been noted clinically (Canney et al., 1987); in this case, however, an additional agent needs to be given to prevent the tumor from using endogenous estrogens to grow. Aromatase inhibitors have been used with some success after tamoxifen (Smith et al., 1981) on the principle that “the only true antiestrogen is no estrogen”. Unfortunately increased side effects, and the fact that endogenous estrogenic steroids are not all controlled by inhibitors of aromatization, rather limit their usefulness. A true antiestrogen equivalent to “no estrogen” would be a valuable addition indeed to our therapeutic armamentarium. Research does not proceed in straight lines, and a series of apparently unrelated observations contributed to the discovery of pure antiestrogens. Scientists at ICI were investigating the structureactivity relationships of C6 and C7 substituted estrogenic steroids to act as “irreversible antiestrogens”, or as compounds that might alkylate the estrogen response elements (EREs) (Jordan et al., 1981). The chemists at ICI had noted that derivatives of estradiol with a long alkyl chain ((CH,),,) at the C7 position attached to resins can be used to purify the estrogen receptor (Bucort et al., 1978). The team at ICI (Wakeling and Bowler, 1987) then put all of the information together and tested long chain derivatives of estradiol as antiestrogens. A number of compounds are under investigation but only ICI164,384 (estradiol with a 7a substituted (CH2),0 side chain) is available for evaluation.
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In the laboratory ICI-164,384 will completely inhibit estrogen action and appears to exhibit no agonist action in rats or mice (Wakeling and Bowler, 1988); in addition, it inhibits the estrogen-stimulated growth and invasive properties of breast cancer cell lines in culture (Thompson et al., 1989). Indeed ICI-164,384 will inhibit the uterotrophic effects of tamoxifen and blunt the tamoxifen-stimulated growth of MCF-7 (Gottardis et al., 1989) and EnCalOl tumors (Gottardis et al., 1990) in athymic mice. Radiolabelled ICI-164,384 is a competitive inhibitor of estradiol binding to the estrogen receptor (Weatherall et al., 1988), an effect that is probably responsible for its reversible blockade of the G, phase of the cell cycle (Musgrove et al., 1989). Pierre Chambon’s group has demonstrated that both estradiol- and 4-hydroxytamoxifen-estrogen receptor complexes can compete at EREs to affect gene transcription (Metzer et al., 1988). Since 4hydroxytamoxifen is a partial agonist (Jordan et al., 1977), clearly it must interact at EREs; in contrast, however, the pure antiestrogens may have a unique mechanism that is responsible for an effect like “no estrogen”. Dimerization of the estrogen receptor is necessary to bind to EREs; receptors with a mutation at the dimerization site will not bind to DNA (Fawell et al., 1990a). Recent studies by,Malcolm Parker and colleagues (Fawell et al., 1990b) have demonstrated that the pure antiestrogen ICI-164,384 prevents receptor dimerization and binding to DNA. Interestingly, a bivalent monoclonal antibody to the estrogen receptor will permit receptor binding to DNA, presumably by creating “dimers”; the monovalent antibody is ineffective. It is possible that antiestrogens will eventually fall into two categories, (1) triphenylethylenes that interact at the ligand binding site of the estrogen receptor, and that prevent the tertiary changes necessary for appropriate activation of receptor (Lieberman et al., 1983); such complexes can bind to EREs but cannot initiate full agonist actions; and (2) compounds with a long alkyl side chain which prevents receptor dimerization (Fawell et al., 1990b). What is the therapeutic potential of a pure antiestrogen? Currently, the use of long-term adjuvant tamoxifen may “educate” breast cancer
cells to become tamoxifen dependent, and pure antiestrogens could be an effective second line therapy. A second question, however, is what will happen to the growth of an estrogen responsive breast cancer if a pure antiestrogen is a first-line therapy? It has only recently been possible to evaluate the effects of complete estrogen deprivation on hormone responsive breast cancer cells in the laboratory, since estrogen receptor positive breast cancer cell lines have been cultured routinely (for up to 20 years!) in apparently estrogen-free media containing phenol red, an estrogenic contaminant (Berthois et al., 1986; Bindall and Katzenellenbogen, 1988). Long-term culture of MCF-7 breast cancer cells in media without phenol red initially results in the slowing of growth, but after about 6 months the growth rate increases (Katzenellenbogen et al., 1987; Welshons and Jordan, 1987). Cells still retain estrogen receptors (and are progesterone receptor negative); estrogen administration increases progesterone receptor level but not growth rate; and antiestrogens slow the growth of these adapted cells. Variants of the cell line T47D (Keydar et al., 1979) have been reported to be hormone responsive (Chalbos et al., 1982) and nonresponsive (Horowitz et al., 1982) depending upon the culture conditions used to derive the subline. We recently reported a hormone responsive subline (A line) derived from ATCC stock cells that we have used to study the effects of tamoxifen derivatives on breast cancer cell replication (Murphy and Jordan, 1989; Murphy et al., 1990a). Long-term (12 months) growth of T47D (line A) in medium without phenol red results in a dramatic decrease in the high levels of progesterone receptor, and eventually estrogen receptors become undetectable (Murphy et al., 1989); the derived cell line (line C) neither grows in response to estrogen nor produces progesterone receptor. Not surprisingly, the antiestrogens 4-hydroxytamoxifen and ICI-164,384 do not affect the growth rate of T47D line C. It should be pointed out, however, that the growth rate of line C (and an estrogen and progesterone receptor negative clonal line C,) is low compared to the growth rate of estrogen treated cells from line A, and of an estrogen and progesterone receptor positive clonal
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line Al8 (Murphy et al., 1990b). This property contrasts with the MCF-7 breast cancer cell line, which grows rapidly under estrogen deprived conditions and is sensitive to the effect of antiestrogens (Welshons and Jordan, 1987). Levels of mRNA for transforming growth factor (Yand pi,2 in C, cells are not altered compared with hormone responsive cell lines and contrast dramatically with the high levels of mRNAs observed in hormone independent cell lines like MDA-MB-231 (Murphy et al., 1990b), though the mRNA for both estrogen and progesterone receptor is undetectable. This raises an important therapeutic consideration. Westley and May (1988) have demonstrated that estrogen receptor mRNA is regulated by estrogen, and have cautioned about the therapeutic use of pure antiestrogens: if a pure antiestrogen completely prevents the synthesis of estrogen receptor, there is the potential for genetic instability to produce hormone-independent daughter cells. Although estrogen-deprived cells might then grow more slowly it is possible to envision the influence of paracrine growth factors causing tumor cell replication even in the presence of effective antiestrogenic concentrations of ICI164,384 (Cormier and Jordan, 1989; Robinson and Jordan, 1989). Despite this theoretical possibility, the question of whether pure antiestrogens will be significantly more effective given as front line therapy or as second line therapy after tamoxifen can be evaluated only in the clinic. Indeed, the position can be taken that a pure antiestrogen might be used to best advantage at an early stage of the disease, i.e. when hormone-dependent growth is more likely and there is a low tumor burden. Obviously, if pure antiestrogens are used in node negative women following mastectomy special attention must be paid to the potential problems of osteoporosis and atherosclerosis. Only 30% of such women will have a recurrence of disease 5-10 years after initial diagnosis and treatment, and those women who would not have had a recurrence will have to undergo the physiological consequences of absolute estrogen deprivation. Clinical strategies must be devised to find a duration of pure antiestrogen treatment which provides optimal control of tumor recurrence in those at risk, whilst not jeopardizing the health of the majority of women who are cured.
In summary, the past three decades have seen the introduction of a new class of drugs which have aided the survival of breast cancer patients and facilitated our understanding of the normal processes controlled by estrogen (Lerner and Jordan, 1990). From their humble beginnings as laboratory tools, the clinical use of antiestrogens by physicians has become commonplace. All stages of breast cancer are being treated with long-term and in some cases indefinite adjuvant tamoxifen therapy. The key to success has undoubtedly been the high degree of patient acceptability, but it is clear that some level of estrogenic activity is essential if tamoxifen is to be used as a preventive measure in normal women (Jordan, 1990). Whether the use of tamoxifen in postmenopausal women ever becomes ubiquitous, its estrogen-like qualities are clearly an advantage to node negative postmenopausal women with breast cancer who are denied normal hormone replacement therapy. Pure antiestrogens should soon be available for clinical evaluation. Whether they prove to be significantly superior to tamoxifen as therapeutic agents or not, they constitute a valuable new tool for studying the molecular endocrinology of estrogen action. Pure antiestrogens are like “no estrogen”; we may, however, be able to take Terry Robinson’s dictum one step further. Some estrogen receptor positive breast cancer cells appear to be able to grow rapidly without an estrogenic stimulus (Welshons and Jordan, 1987) but an antiestrogen can prevent growth, presumably by preventing dimerization of the unoccupied estrogen recptor. A pure antiestrogen may thus be even better than no estrogen. Acknowledgements Some of the studies described were supported by Cancer Center Support grant P30 CA14520, ROl CA32713, PO1 CA20432 and training grant 5T32-CA09471 in Human Cancer Biology. References Bagdade, J.D., Walter, J., Sabbaiah, P.V. and Ryan, W. (1990) J. Endocrinol. Metab. 70, 1132-1135. Berthois, Y., Katzenellenbogen, J.A. and Katzenellenbogen, B.S. (1986) Proc. Nat]. Acad. Sci. U.S.A. 83, 2496-2500.
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Keydar, I., Chen, L., Karby, S., Weiss, F.R., Delarea, M., Rachu, M., Chaitcik, S. and Brenner, H.J. (1979) Eur. J. Cancer 15,659-670. Lemer, L.J. (1981) in Non-Steroidal Antiestrogens: Molecular Pharmacology and Antitumour Activity (R.L. Sutherland and V.C. Jordan, eds.), pp. l-16, Academic Press, Sydney. Lemer, L.J. and Jordan, V.C. (1990) Cancer Res. 50, 41774189. Lemer, L.J., Holthaus, Jr., F.J. and Thompson, C.R. (1958) Endocrinology 63, 295-318. Lieberman, M.E., Gorski, J. and Jordan, V.C. (1983) J. Biol. Chem. 258, 4741-4745. Love, R.R., Newcomb, P.A., Wiebe, D.A., Surawicz, T.S., Jordan, V.C., Carbone, P.P. and DeMets, D.L. (1990) J. Natl. Cancer Inst. 82, 1327-1331. Lunan, C.S. and KIopper, A. (1975) Chn. Endocrinol. 4, 557572. Metzer, D., White, J.H. and Chambon, P. (1988) Nature 334, 31-35. Murphy, C.S. and Jordan, V.C. (1989) J. Steroid Biochem. 34, 407-411. Murphy, C.S., Meisner, L.F., Wu, S.-Q. and Jordan, V.C. (1989) Eur. J. Cancer Clin. Oncol. 25, 1777-1788. Murphy, C.S., Langan-Fahey, S.M., McCague, R. and Jordan, V.C. (1990a) Mol. Pharmacol. (in press). Murphy, C.S., Pink, J.J. and Jordan, V.C. (1990b) Cancer Res. (in press). Musgrove, E.A., Wakeling, A.E. and Sutherland, R.A. (1989) Cancer Res. 49, 2398-2402. Powles, T.J., Hardy, J.R., Ashley, S.E., Farrington, G.H., Cosgrove, D., Davey, J.B., Dowsett, M., McKirma, J.A., Wash, A.G., Sinnett, H.D., Tillyer, C.R. and Treleaven, J.G. (1989) Br. J. Cancer 60, 126-131. Powles, T.J., Tillyer, CR., Jones, A.L., Ashley, S.E., Trebaven, J., Davey, J.B. and McKinna, J.A. (1990) Eur. J. Cancer 26, 680-684. Robinson, S.P. and Jordan, V.C. (1989) Eur. J. Cancer Chn. Oncol. 25, 493-497. Satyaswaroop, P.G., Zaino, R.J. and Mortel, R. (1984) Cancer Res. 44,4006-4010. Smith, I.E., Harris, A.L., Morgan, M., Ford, H.T., Gazet, J.C., Harmer, C.L., White, H., Parsons, C.A., Villardo, A., Walsh, G. and McKinna, J.A. (1981) Br. Med. J. 283, 1432-1434. Thompson, E.W., Katz, D., Shima, T.B., Wakehng, A.E., Lippman, M.E. and Dickson, R.B. (1989) Cancer Res. 49, 6929-6934. Wakehng, A.E. and Bowler, J. (1987) J. Endocrinol. 112, R7-RlO. Wakeling, A.E. and Bowler, J. (1988) J. Steroid B&hem. 30, 141-148. Weatherill, P.J., Wilson, A.P.M., Nicholson, R.I., Davis, P. and Wakehng, A.E. (1988) J. Steroid Biochem. 30, 263-268. Welshons, W.V. and Jordan, V.C. (1987) Eur. J. Cancer Clin. Oncol. 23, 1935-1939. Westley, B.R. and May, F.E.B. (1988) B&hem. Biophys. Res. Commun. 155. 1113-1118.
