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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda B9 Antiprogestins and the Treatment of Breast Cancer KATHRYN B. HORWITZ, Ph.D. Professor of Medicine and Pathology, and the Molecular Biology Program, University of Colorado Health Sciences Center, Denver INTRODUCTION Endocrine therapy used either prophylactically or therapeutically for the treatment of locally advanced or metastatic breast cancers offers many advantages to patients whose tumors contain functional estrogen receptors (ERs) and progesterone (PR) receptors. The range of treatments defined as endocrine includes surgical ablation of endocrine glands, administration of pharmacologic doses of steroid hormones, chemical blockade of steroid hormone biosynthesis, and inhibition of endogenous steroid hormone action at the tumor with synthetic antagonists. The last of these approaches is the most widely used, making the antiestrogen tamoxifen the preferred first-line therapeutic agent for treatment of hormone-dependent metastatic breast cancer. The widespread use of tamoxifen reflects its efficacy and low toxicity, and the fact that it makes good physiological sense to block the local proliferative effects of estrogens directly at the breast. But are estrogens the only hormones with a proliferative impact on the breast and on breast cancers? This review, abstracted from one recently published (Horwitz, 1992), focuses on evidence that progesterone also has proliferative actions in the breast; on the role of synthetic progestins in breast cancer treatment; and on the preliminary data showing that progesterone antagonists may be powerful new tools for the management of metastatic breast cancer because they block the local effects of endogenous progesterone on breast cell proliferation. The reader is also referred to the excellent general review on progestin regulation of cell proliferation by Clark and Sutherland (1990).
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda PROGESTERONE AND THE NORMAL BREAST Conventional wisdom holds that the mechanisms by which estradiol and progesterone regulate the proliferation and differentiation of uterine epithelial cells, apply equally to the breast. This is probably inaccurate (Anderson et al., 1987, 1989; Going et al., 1988). In the uterus, estrogens are clearly mitogenic, and the addition of progesterone to the estrogenized endometrium leads to the appearance of a secretory pattern characterized by cells engaged in protein synthesis rather than cell division (Berliner and Gerschenson, 1976). That is, in the uterus, estradiol is a proliferative hormone; progesterone is a differentiating hormone. For this reason the unopposed actions of estradiol are considered to be tumorigenic in the uterus, while the risk of endometrial hyperplasia and cancer is lowered when estrogens are combined with progestins. In fact, the combined regimen may even be protective since a decrease in endometrial cancers has been reported in women prescribed combined estrogens and progestins, compared to women receiving no treatment (Henderson et al., 1988). However, considerable evidence has now accrued to suggest that in the epithelium of the breast, progesterone has a different influence—that, like estradiol, progesterone in the breast has a strong proliferative effect. Studies in support of this come both from experimental models and from normally cycling women. The proliferation of normal mammary epithelium in virgin mice, and the lobular-alveolar development of mammary tissues in pregnant mice, both require progesterone (Imagawa et al., 1985; Haslam, 1988). A fundamental difference in the actions of estradiol and progesterone in the breast is that the latter stimulates DNA synthesis, not only in the epithelium of the terminal bud but also in the ductal epithelium (Bresciani, 1971). The stimulating effects of progesterone on the development of mammary gland buds can be inhibited by progesterone antagonists (Michna et al., 1991). Data from normal human mammary cells have been more difficult to obtain and are often equivocal. Compared to the increase caused by estradiol treatment (11.3-fold), progesterone treatment only marginally (2.0-fold) increases the mitotic index of normal human breast ductal epithelium maintained in intact athymic nude mice (McManus and Welsch, 1984). In fact, Mauvais-Jarvis and colleagues (Gompel et al., 1986; Mauvais-Jarvis et al., 1986) concluded, using primary cultures of epithelial cells from normal human mammary glands, that while estradiol treatment stimulates growth, progestins inhibit growth. Their data are difficult to interpret however, since the experiments using estradiol were done with cells growing in minimally supplemented medium, whereas the progestin treatment studies were done with cells in optimally supplemented medium, and any progestin growth-
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda stimulatory effect might have been masked. In contrast to the sparse and conflicting in vitro data are studies of the mitotic rate in breast epithelial cells during the normal menstrual cycle and in women taking oral contraceptives. These data show that the highest thymidine labeling indices occur during the progestin-dominated, secretory phase of the menstrual cycle. Both the estrogen and the progestin components of oral contraceptives increase the thymidine labeling index, with progestin-only formulations exhibiting high activity (Anderson et al., 1987, 1989; Going et al., 1988). The investigators conclude that it is difficult to sustain the idea that progestins are protective in the breast (Anderson et al., 1989). It would seem that more work must be done to understand the actions of progestins in the normal breast, but that clinical decisions based on an inappropriate uterine model system are unjustified (McCarty, 1989). PROGESTERONE AND BREAST CANCER A discussion of the role of progestins in breast cancer must distinguish between their effects on carcinogenesis and their role in regulating proliferation of established cancers. Progestin Agonists and Tumor Induction Progestin agonists have been shown to be carcinogenic or to increase the incidence of spontaneous mammary tumors in dogs and mice (Frank et al., 1979; Lanari et al., 1986, 1989; Nagasawa et al., 1988; Kordon et al., 1990). In mice, results vary with the strain tested, which suggests the contribution of a genetic component; however tumorigenic effects of progestins have been observed whether or not the strain harbors the mouse mammary tumor virus (MMTV). The importance of progesterone in carcinogen-induced rat mammary cancers is documented by the early reports of Huggins et al. (Huggins and Yang, 1962; Huggins et al., 1962; Huggins, 1965), who showed that pregnancy promotes the growth of dimethylbenzanthracene (DMBA)-induced mammary tumors, and that administration of progesterone together with the carcinogen to intact rats accelerates the appearance of tumors, increases the number of tumors, and augments the growth rate of established tumors. The relationship between progestins and carcinogenesis is temporally complex. In general, progesterone administered simultaneously with or after the carcinogen enhances tumorigenesis, whereas progesterone given prior to the carcinogen inhibits tumorigenesis (Welsch, 1985). Thus, the high progesterone level associated with pregnancy can be protective if it precedes administration of the carcinogen (Russo et al., 1989). Extrapolation of these experimental models to human disease is
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda unclear since the only data available for the latter are epidemiologic in nature and relate hormone use, particularly oral contraceptive use, to the risk of breast cancer. The trend toward increased risk with increased duration of hormone use appears repeatedly (Meirik et al., 1986; Hulka, 1990; Schlesselman, 1990), and a possible adverse effect of progestins seems likely (Ewertz, 1988; Bergkvist et al., 1989). This is discouraging when taken together with the likelihood that in the breast, unlike the uterus, progestins enhance proliferation during the menstrual cycle. Progestin Agonists and Growth of Established Tumors Carcinogen-induced rat mammary tumors are a major model for in vivo studies of progestin-regulated growth (Welsch, 1985). Following ovariectomy, progesterone alone is usually unsuccessful in preventing regression of established tumors. The rapid decrease of PR levels due to estrogen withdrawal is probably a critical factor (Horwitz and McGuire, 1977). In intact animals, which more closely mimic the clinical situation, progestin agonists at moderate doses have been reported to promote tumor growth and to reverse the antitumor effects of tamoxifen (Robinson and Jordan, 1987). Thus, there exists the possibility that endogenous circulating progesterone may enhance breast cancer growth. Enigmatically, progestins at higher pharmacologic doses appear to be growth inhibitory (Danguy et al., 1980). The molecular mechanisms responsible for the opposing actions of physiologic and high-dose progestins remain unclear and are discussed later in this paper. In vitro cell culture models designed to assess the role of progestin agonists in tumor cell proliferation have generated contradictory results. Experiments can be cited in support of any argument—that progestins stimulate (Dao et al., 1982; Hissom and Moore, 1987; Hissom et al., 1989), inhibit (Vignon et al., 1983; Chalbos and Rochefort, 1984; Horwitz and Freidenberg, 1985; Purohit et al., 1989; Poulin et al., 1990), or have no effect (Lippman et al., 1976) on growth. Explanations for the lack of a consensus are as varied as the results. Responses of cells in culture are critically dependent on the conditions in which they are grown. In a rich medium, where growth is optimized, further growth enhancement is difficult to demonstrate, while inhibitory stimuli may be exaggerated. In a deprived medium the reverse is true, although here, key co-factors may be lacking. There is no simple solution to these inherent problems. Couple this generic uncertainty with other variables including the use of different cell lines (Lippman et al., 1976; Vignon et al., 1983; Horwitz and Freidenberg, 1985), heterogeneity and genetic instability even within the same cell lines (Reddel et al., 1988; Graham et al., 1989) a burgeoning list of factors besides estradiol and progestins that directly or indirectly modulate progestin sensitivity through regulation of PR
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda levels (Eckert and Katzenellenbogen, 1983; Sarup et al., 1988; Katzenellenbogen, 1990; Aronica and Katzenellenbogen, 1991; Clarke et al., 1991) and the possibility that progestin-sensitive cells can generate resistant subpopulations (Graham et al., 1992) and it may be that no physiological consensus is likely to be forthcoming from using these in vitro models. These models remain invaluable, however, for the analysis of molecular mechanisms of progestin actions, as well as underscoring the complexities inherent in tumor cell biology. Where does this leave us on the critical issue of the use of progestin agonists in breast cancer treatment? Interestingly, here there is more agreement, but the data contradict the conclusion that physiological levels of progestins are growth stimulatory. Especially at high doses, progestins appear to be antiproliferative in breast cancers. A comprehensive review of the clinical literature (Sedlacek and Horwitz, 1984; Canobbio et al., 1987; Howell et al., 1987a; Lundgren et al., 1989; Gundersen et al., 1990; Pronzato et al., 1990; Parnes et al., 1991) shows that synthetic progestins, used at pharmacologic doses for first- or second-line therapy, are as effective as tamoxifen in the treatment of advanced breast cancer. That is, in patients whose tumors are not screened for steroid receptors, approximately 30 percent have an objective, positive response. Since in addition, progestins are well tolerated and have a relatively low toxicity (Henderson et al., 1989), their use in the treatment of advanced breast cancer is experiencing a resurgence (McGuire et al., 1985, 1989). However, the mechanisms underlying the actions of intermediate and high doses of progestin agonists in breast cancer regression remain unclear when compared to their proliferative actions at physiologic doses. Although some studies suggest that PR-negative tumors respond just as well as do PR-positive tumors (implying that progesterone receptors are not involved), others suggest that methodological problems produce false PR-negative values in responders (McGuire et al., 1989) and that progesterone receptors are indeed required to obtain a response to progestins. An interesting study comparing tamoxifen therapy to therapy in which tamoxifen was alternated with medroxyprogesterone acetate (MPA) in ER-positive patients, showed a 40 percent response to tamoxifen alone versus a 62 percent response to the alternating treatment (Gundersen et al., 1990). It is postulated that when tamoxifen is cycled, its agonist properties predominate, which increases PR levels, thereby enhancing the efficacy of MPA. The same argument is made for enhancing the therapeutic efficacy of antiprogestins (see below). In general, tamoxifen inducibility of the PR is considered to be a good indicator for a positive response to hormone therapy (Howell et al., 1987a). Thus, although definitive data are still lacking, it is likely that positive responses to therapy with progestins in breast cancer are mediated by the PR in the tumors.
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda PROGESTERONE ANTAGONISTS AND THE TREATMENT OF BREAST CANCER Because progestin antagonists are relatively new compounds, and because of the political controversy that surrounds them, their promise and use in the treatment of breast cancer are just beginning to be evaluated. What is the rationale for their use? What is the explanation for the paradox that both antiprogestins and high-dose progestin agonists inhibit the growth of breast cancers? Human Breast Cancer Cell Lines Two PR-positive human breast cancer cell lines that are phenotypically different have served as the major models for studies of growth regulation by antiprogestins. The MCF-7 cell line is classically estrogen-responsive cells; the cells are ER positive but have only low PR levels unless these are induced by estradiol (Horwitz and McGuire, 1978). The T47D cell line (Keydar et al., 1979) is more complex; it is genetically unstable (Reddel et al., 1988; Graham et al., 1989), and differs phenotypically among and within laboratories (Vignon et al., 1983; Horwitz and Freidenberg, 1985; Hissom et al., 1989), and this leads to reported differences in response to hormone treatment. One major T47D subline, clone 11, is ER positive and PR positive, and the cells respond to estradiol treatment by proliferating—a response that can be inhibited by tamoxifen (Vignon et al., 1983). Another subline, T47Dco, is estrogen resistant, and cell growth is neither accelerated by estradiol nor inhibited by tamoxifen (Horwitz et al., 1982). The response to antiprogestins is generally similar among these cell lines, but interpretation of the results differs. In general, RU 486 inhibits the growth of both T47D cells and MCF-7 cells (Bardon et al., 1985, 1987; Horwitz, 1985; Rochefort and Chalbos, 1985; Bakker et al., 1987; Gill et al., 1987; Sutherland et al., 1988). The antiproliferative effects are evident at low doses, and their magnitude correlates loosely with PR levels; T47D > estrogen-primed MCF-7 > unprimed MCF-7 (Bardon et al., 1985). That the antiproliferative effects are mediated by PR is also shown by the fact that in T47D cells, growth inhibition is confined to progestins; other steroid hormones are ineffective (Bardon et al., 1985; Sutherland et al., 1988). Moreover, the fact that RU 486 is not antiproliferative in PR-negative breast cancer cell lines also argues for a receptor-mediated mechanism of action (Bardon et al., 1985). The antiproliferative actions of RU 486 in these models of breast cancer would support a logical treatment strategy if it were not for one disturbing fact—progestin agonists, including R 5020, also inhibit their growth. This effect of R 5020 is seen even at low doses in the T47Dco cells
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda (Horwitz, 1985), but requires higher doses in the clone 11 cells in which, interestingly, low doses actually protect the cells from the antiproliferative effects of RU 486 (Bardon et al., 1985). Furthermore, in the estrogen-responsive clone 11 cells, R 5020 inhibits only the estradiol-stimulated growth fraction where it is cytostatic, whereas RU 486 reduces cell numbers below the estrogen-untreated baselines, suggesting that it has a more profound cytotoxic effect (Bardon et al., 1987; Gill et al., 1987). Thus the antiproliferative mechanisms for progestin agonists such as R 5020, and antagonists such as RU 486, may be fundamentally different. R 5020 appears to have dual proliferative/antiproliferative effects, depending on the dose that is tested, and the antiproliferative doses produce growth stasis. By contrast, RU 486 is more purely antiproliferative even at low doses, and it can produce growth regression. Indeed, ultrastructural studies show effects of RU 486 on cell and chromatin condensation and pyknosis that are consistent with the induction of cell death by apoptosis (Bardon et al., 1987). This cytotoxic effect is prevented by low doses of R 5020. To explain these results, Bardon et al. (1987) propose that there are three different mechanisms by which progestins inhibit the growth of breast tumor cells: (1) ''PR-mediated cytotoxic" mechanisms that apply to antagonists such as RU 486. These are observed only in PR-positive cells by using "physiological" hormone doses, and are preventable by receptor occupancy with an agonist. This cytotoxicity is characterized by ultrastructural evidence of cell death. (2) "PR-mediated cytostatic" effects are produced by physiological doses of antagonists or agonists, and are characterized by inhibition of the growth-stimulatory actions of unrelated (nonprogestin) growth factors including estradiol. (3) "Nonspecific cytotoxic" effects are not receptor mediated and are seen with most steroid hormones at high doses. The molecular explanations that underlie these three mechanisms are unknown, but they serve as an important departure for further research. For example, the prolonged DNA occupancy time of RU 486-bound PR compared to R 5020-bound PR may account for cytotoxic versus cytostatic effects (Sheridan et al., 1988). As described above, variables like the gene or cell being tested (Tora et al., 1988), and regulation by either the A or the B receptor (Meyer et al., 1990), may dictate an agonist or antagonist response. Alternatively, it has been suggested that R 5020 is inhibitory because it is "antiestrogenic" (Vignon et al., 1983) whereas RU 486 is inhibitory through a direct antiproliferative effect involving the PR (Bardon et al., 1985). Although these explanations may begin to address the paradox that allows both R 5020 and RU 486 to be growth inhibitory in the appropriate physiological setting, it is clear that a considerable amount of research remains to be done. Preliminary analyses of cell cycle parameters show no definitive differences between agonists and antag-
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda onists. Both appear to inhibit growth by significantly decreasing the proportion of cells in the S-phase of the cell cycle; cells accumulate in G0/G1 possibly due to increase in the G1 transit time (Sutherland et al., 1988; Michna et al, 1990). Although the majority of studies using cell culture models ascribe growth-inhibitory properties to progestins and to antiprogestins through direct effects involving PRs, contradictory results have also been reported. Given the fact that at physiological levels, progesterone is believed to be mitogenic in the normal breast (see above), it is not entirely surprising that Moore and colleagues (Hissom and Moore, 1987) consistently report proliferative effects of R 5020 at all doses in T47D cells. It is surprising, however, that RU 486 also stimulates growth in these cells (Bowden et al., 1989). The explanation for this discrepancy is unknown. However, it is now clear that T47D cells are exceptionally unstable; during prolonged time in culture, subpopulations can develop that are phenotypically different from the parental stocks, and some of these subpopulations may have responses to hormones that differ outright from the expected response. For example, some sublines or subpopulations of T47D cells respond to high doses of tamoxifen by growth stimulation (Graham et al., 1990, 1992). In the case of tamoxifen, these aberrant responses may be mediated by the presence of mutant or variant ER (Graham et al., 1990). By analogy, it is possible that the T47D cells of Moore and his colleagues have arisen from a subpopulation harboring a mutant PR. This scenario would be very interesting, but the PRs of these cells have not been analyzed in detail. Less interesting trivial explanations for discrepancies among laboratories studying growth regulation by progestins in cell culture are discussed above (see also Clark and Sutherland, 1990). Animal Models of Mammary Cancer The antiproliferative properties of progesterone antagonists are well documented in animal models of hormone-dependent mammary cancer. These include rats bearing DMBA-induced or nitroso-methyl urea (NMU)-induced tumors, and mice bearing the transplantable MXT tumor line. Growth of these tumors is inhibited by ovariectomy and maintained by physiological doses of estrogens (Bakker et al., 1989, 1990; Klijn et al., 1989; Michna et al., 1989a, b; Schneider et al., 1989). While treatment of rats with progestins at the time of DMBA administration accelerates tumor formation (Huggins and Yang, 1962; Huggins, 1965; Welsch, 1985), prophylactic treatment of rats with RU 486 at the time of DMBA administration delays the initial appearance of tumors from an average of 39 days to 81 days (Bakker et al., 1987). The reversal by progesterone of the inhibition of tumor induction produced by
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda tamoxifen can in turn be blocked by RU 486 (Robinson and Jordan, 1987). These implicate a PR-mediated mechanism. Treatment of established tumors with RU 486 for three weeks prevents their further enlargement, but tumor remission is not observed (Bakker et al., 1989). In contrast to the stasis seen with RU 486, ovariectomy leads to a decrease in tumor size (Schneider et al., 1989), which in MTX tumors is accompanied by necrosis and cytolysis of the tumor cells (Michna et al., 1989a). While ovariectomy-induced tumor regression is known to be accompanied by extensive loss of PR (Horwitz and McGuire, 1977), loss of tumor PRs was also seen with RU 486 treatment (Bakker et al., 1989). Since in the latter, the PR assay was not performed under exchange conditions or by immunologic methods, the validity of this decrease requires reexamination in light of other studies that show persistently high levels of PR in RU 486-treated human breast cancer cells (Sheridan et al., 1988). Since adrenal weights are unchanged by RU 486, participation of the antiglucocorticoid effects in the antitumor activity is considered to be unlikely (Schneider et al., 1989). This is supported by studies in human breast cancer cell lines, where the inhibitory effects of RU 486 cannot be rescued by dexamethasone (Bardon et al., 1985). Antigonadotropic effects have also been excluded as a mechanism (Schneider et al., 1989). In established DMBA tumors, inhibition of growth by tamoxifen resembles the inhibition seen with RU 486. The two hormones have equal growth-inhibitory effects when each is used alone (Bakker et al., 1989). When the two drugs are combined, however, the inhibitory effects are additive, and tumor remission similar to that induced by ovariectomy is observed (Bakker et al., 1990). This effect of combined treatment with an antiprogestin and an antiestrogen is extremely exciting and has considerable therapeutic promise. The mechanisms underlying these effects remain unclear, but several proposals have surfaced. First, tamoxifen can have agonist actions, among which is the induction of PR (Horwitz and McGuire, 1978). A tumor with increased, or restored, PR may have greater or more sustained sensitivity to RU 486. This hypothesis could be tested by the use of an antiestrogen having no agonist activity. Second, among the physiological effects seen in RU 486-treated intact female rats are increased plasma levels of luteinizing hormone (LH), prolactin, estradiol, and progesterone, as well as the persistence of numerous and actively secretory corpora lutea associated with hypertrophic pituitaries (Bakker et al., 1989, 1990; Michna et al., 1989a; Schneider et al., 1989). It has therefore been proposed that the efficacy of simultaneous tamoxifen results from its ability to counteract the proliferative effects of the high estrogen levels. Several newer antiprogestins, ORG 31710 and ORG 31806 (Bakker et al., 1990), and ZK 98299 and ZK 112993, have equal or greater antipro-
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda liferative actions than RU 486 (Michna et al., 1989a; Bakker et al., 1990). In the hormone-dependent MXT-transplantable tumor model, treatment with ZK 98299 or RU 486 starting one day after transplantation led to an almost complete inhibition of tumor growth. Their effect on established tumors was equivalent to that of ovariectomy (Michna et al., 1989a; Schneider et al., 1989). In this model, the potent antiproliferative actions of the antiprogestins completely counteracted the growth-stimulatory actions of estradiol or of approximately equimolar doses of MPA, but at higher MPA doses the agonist actions of the progestin prevailed (Michna et al., 1989b). It appears that antiprogestins inhibit growth by direct antagonism of progesterone action at the tumor, probably mediated by PR. This conclusion is bolstered by the fact that the hormone-independent MXT tumor is antiprogestin resistant (Michna et al., 1989a). In DMBA-induced tumors, ZK 98299 was more potent than an equal concentration of RU 486 (Michna et al., 1989a). It produced tumor regression analogous to that of ovariectomy, rather than the tumor stasis observed with RU 486. A similar trend was observed with NMU-induced rat mammary tumors (Michna et al., 1989a, b; Schneider et al., 1989). However, lack of comparative metabolic and pharmacokinetic data on the two antiprogestins in rats and mice makes these quantitative differences uninterpretable at present. Also of interest is the finding that strong antitumor activity was noted at 20 percent of the doses needed to obtain abortifacient actions in these rodent systems. This is important because by the use of lower doses of antiprogestins, their antiglucocorticoid effects may be mitigated. After treatment with the antiprogestins, the morphology of the hormone-dependent MXT and DMBA tumors showed signs of differentiation of the mitotically active polygonal epithelial tumor cells toward the nonproliferating glandular secretory pattern, with the formation of acini and evidence of secretory activity (Michna et al., 1989a). Based on this, it is suggested that the antiproliferative efficacy of the antiprogestins is related to their ability to induce terminal differentiation. Recall that they block tumor cells in G0/G1 (Sutherland et al., 1988; Michna et al., 1990). Note that an antiproliferative mechanism based on induction of terminal differentiation is fundamentally different from a mechanism involving tumor cell death. Tumor cell degradation and cytolysis were features of ovariectomy-induced regression (Michna et al., 1989a). The mechanisms underlying the antitumor effects of antiprogestational agents require further study, especially in human tissues and cells. Human Clinical Trials The enormous promise of progestin antagonists in treating breast cancer remains largely unexplored in clinical practice. Only two small
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda clinical trials using RU 486 have been reported, both from European laboratories. The first involved a series from France (Maudelonde et al., 1987) of 22 oophorectomized or postmenopausal patients in whom chemotherapy, radiotherapy, or tamoxifen and other hormonal therapy had already been used. RU 486 at 200 mg/day led to partial regression or stabilization of lesions in 12 of 22 (53 percent) women following four to six weeks of treatment. The response rate at three months had dropped to 18 percent. It is important to note that for ethical reasons, this untried therapy was used only in patients with advanced breast cancers in whom other treatment modalities had already failed. PR levels were not measured in all patients, but of the responders, 4/4 were PR positive, whereas of the nonresponders 0/4 were PR positive. In general, RU 486 was well tolerated in long-term treatment with few symptoms of adrenal dysfunction, but plasma cortisol levels were elevated. Of interest was the fact that a strong analgesic effect was observed in most of the patients with bone metastases. The second trial, from the Netherlands (Michna et al., 1989b; Bakker et al., 1990), involved 11 postmenopausal patients with metastatic breast cancer who were treated with 200 to 400 mg of RU 486 for 3 to 34 weeks as second-line therapy after first-line treatment with tamoxifen, irrespective of the response to tamoxifen. Six of eleven patients had a short-term (three to eight months) stabilization of disease, and one had an objective response lasting five months after RU 486 treatment. Again, response was associated with the presence of PR in the tumors. In this study, which involved prolonged use of RU 486, two patients had undesirable side effects associated with the antiglucocorticoid actions of the drug. Three days of treatment with dexamethasone reversed these symptoms after RU 486 was stopped. As in the animal studies, plasma estradiol levels increased despite the fact that these women were postmenopausal, and it is suggested that the simultaneous administration of tamoxifen might be beneficial because of its ability to blockade tumor ER. Alternatively, symptoms of adrenal hypersecretion and elevated plasma estradiol levels might be reduced with concurrent aminoglute-thimide or aromatase inhibitors. By modification of the dose and time of RU 486 administration, its antiglucocorticoid effects might be further minimized (Gaillard et al., 1984), although maintenance of high sustained blood levels of the drug is likely to be important. PROGESTIN RESISTANCE The emergence of hormone-resistant cells eventually reduces the effectiveness of all therapies in advanced breast cancer, and progestin agonists or antagonists are unlikely to be exceptions. Until recently, this has been an unexplored field. Murphy et al. (1991) generated a subline
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda of T47D cells that are resistant to the growth-inhibitory effects of progestins. This was done by sequential selection in medium containing 1 µM MPA. The cells remained PR positive, but receptor levels were halved. Transforming growth factor (TGF) and epidermal growth factor (EGF) receptor mRNA levels were both increased. The investigators suggest that increased growth factor expression and action, and decreased PR levels, may be involved in the development of progestin resistance. It is also likely that extensive heterogeneity in PR content exists within cell subpopulations of tumors that are PR positive based on analyses of solid tumors (Howell et al., 1987b) and of human breast cancer cell lines (Graham et al., 1992). Factors or treatments that lead to the selection and expansion of PR-poor or PR-negative populations would in the long run produce progestin resistance. Recent molecular analyses of human progesterone receptors (hPRs) are beginning to address mechanisms of resistance to progesterone antagonists. These studies suggest that the term "resistance" may be inappropriate. "Resistance" implies that the tumor stops responding to the drug, and ignores it instead. This may be an oversimplification, since under appropriate conditions, progesterone antagonists can behave like agonists. Rather than ignoring the drug the cell alters its transcriptional response to the drug. How is that possible? One explanation focuses on a mutant PR. Unlike the case for other members of the steroid-receptor family, no examples of natural PR mutants have yet been reported. The explanation for this may be, that unlike mutations in androgen receptors, systemic mutations in PRs are incompatible with life. However, theoretically, acquired mutations could develop in tumors as one mechanism for the development of resistance, and a systematic search might demonstrate them. In view of this, Vegeto et al. (1992) recently showed that a synthetic hPR mutant with a 42-amino acid truncation at the C-terminus of the 933-amino acid hPR B-receptors, loses its progesterone-binding ability but retains RU 486-binding ability. This receptor mutant, when occupied by RU 486, has agonist transcriptional activity. Additional models of resistance associated with functional reversion have emerged from our studies of progesterone antagonists as transcriptional inhibitors. These studies provide two scenarios in which antagonists can have inappropriate agonist-like effects on normal PR. We believe that the mechanisms underlying these functional switches may be analogous to mechanisms by which tumor cells become hormone resistant. The first case involves studies with the human breast cancer cell line T47D, which expresses high natural levels of PR and is stably transfected with the progestin-responsive MMTV promoter linked to the CAT reporter. In this model, PR-antagonist complexes are transcription-
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda ally silent, and the antagonists inhibit the effects of agonists. However, if cyclic adenosine 3',5'-monophosphate (cAMP) levels are elevated, the antagonists become strong transcriptional stimulators—they behave like agonists. This functional reversal occurs only if the receptors are bound to DNA, and it does not involve hPR phosphorylation by pathways dependent on cAMP. The model we propose involves transcriptional synergism, in which a promoter that is independently regulated by cAMP-responsive factors, and by hPR, is selected for positive or negative transcription, through cooperative interactions between the two, DNA-bound factors (Sartorius et al., 1993). The second case involves the functional difference between progesterone A- and B-receptors (Meyer et al., 1990). A-receptors occupied by progesterone antagonists are transcriptionally silent on a progesterone response element (PRE) thymidine kinase promoter-CAT reporter. By contrast, in the same cells and with the same promoter-reporter, antagonist-occupied B-receptors strongly stimulate transcription. Interestingly, this unusual property of B-receptors does not require the presence of the PRE (K.B.H., unpublished). Our working model is that transcription by antagonist-occupied B-receptors proceeds through a mechanism in which the receptors are tethered to a DNA-bound protein partner at the promoter, without being bound to DNA themselves. We have ruled out the possibility that the unknown protein is AP-1. In sum, each of these recent experimental models suggests that "resistance" can be a condition in which tumors respond inappropriately to hormone antagonists. These studies may also explain why, in some normal target cells, antagonists have tissue-specific, agonist-like activity. SUMMARY AND FUTURE PROSPECTS The foregoing suggests that progesterone antagonists could have an important place in the routine management of hormone-dependent breast cancers. Our knowledge of the actions of these compounds is rudimentary, however. The following points provide an outline for future directions: If endogenous progesterone has the mitogenic actions in normal breast epithelia that the current data would indicate, then it is likely that physiologic progesterone is also a mitogen in breast cancers. Blockade of endogenous progesterone with antiprogestins, especially in premenopausal women, would seem to be an important therapeutic goal. However, nothing is known about the pattern of mitosis in breast cancer cells during the normal menstrual cycle. Obtaining such data is important, and it should be possible to analyze the mitotic patterns of tumors taken from cycling patients. The problem, of course, is the difference in
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda mitotic indices among tumors. Ideally, each tumor should serve as its own control, with multiple samples analyzed for proliferative activity at different times of the cycle. This approach is fraught with ethical problems. However, it might be possible to obtain a fine-needle aspirate of a tumor for initial mitotic analysis, then to acutely treat the patient with RU 486 before the tumor is removed 24 hours later for reanalysis. Additionally, it is important to know whether RU 486 is cytostatic or cytolytic in human breast cancers. Electron microscopy of tumors taken from patients entered into trials may provide answers to this. However, while it is always preferable to ask biological questions using clinical tissues, much of the work on the mechanisms of the mitogenic actions of progestins and progestin antagonists will require well-controlled studies using organ-cultured human breast tumors, human breast cancer cell lines, and human tumors implanted into nude mice. There are sufficient theoretical and preclinical data to justify large-scale clinical trials. Trials must include accurate measurements of ER and PR in tumors, and analysis of pre- and posttreatment levels of endogenous hormones to monitor the status of the pituitary-adrenal-ovarian axis. Progestin antagonists may be useful both for adjuvant endocrine therapy, when used either alone or in combination with tamoxifen, and for therapy of locally advanced or metastatic cancers, again when used either alone or in combination with tamoxifen. The usual issues of drug doses, metabolism, and other pharmacokinetic parameters must be addressed. Schedules in which the antiprogestins and antiestrogens are combined or alternated must be tested. Is an antiestrogen with agonist properties preferable because it induces PR, or is a pure antiestrogen preferable because it blocks the actions of elevated circulating estrogens? Is the answer different in premenopausal versus postmenopausal women? The antiglucocorticoid side effects of RU 486 remain a major impediment to its long-term use. However, as the newer Schering and Organon antiprogestins show, it appears to be possible to design molecules with maximal antiprogestin activity and minimal antiglucocorticoid activity. Further, even RU 486 may be used for long-term treatment if it is combined with drugs that block adrenal steroidogenesis or prevent peripheral aromatization of adrenal steroids to estrogens. Additionally, since the antiglucocorticoid effects of RU 486 are apparently tolerable in the short term (i.e., two to three months), perhaps it is reasonable to ask if alternating RU 486 with tamoxifen maximizes its antiprogestin effects while minimizing its antiglucocorticoid ones. Basic tumor biological and molecular research must continue in order for us to understand the precise molecular targets and mechanisms of antagonist action. If different antagonists target different molecular sites, which antagonist would be best for clinical use? What
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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda are the genes in breast cells that are regulated by progestins, whose inhibition is associated with lowered cell proliferation? What is the underlying cause of the tissue-specific differences in progestin action at the breast and uterus? What is the molecular explanation for the dual agonist/antagonist effects seen on only some promoters, with only one or the other PR isoform, and in only some cells when using RU 486? Can pure progesterone antagonists devoid of antiglucocorticoid activity be synthesized? Are there mutant progesterone receptors in breast cancers? Where do we begin? Ensuring that scientists and clinicians have access to antiprogestins, unencumbered by the Byzantine bureaucratic obstacles and the "antagonistic" political climate currently encountered in the United States, is a good place to start. REFERENCES Anderson, T.J., Howell, A., and King, R.J.B. Comment on progesterone effects in breast tissue. Breast Cancer Research and Treatment 10:65, 1987. Anderson, T.J., Battersby, S., King, R.J.B., et al. Oral contraceptive use influences resting breast proliferation. Human Pathology 20:1139, 1989. Aronica, S.M., and Katzenellenbogen, B.S. Progesterone receptor regulation in uterine cells: Stimulation by estrogen, cyclic adenosine 3',5'-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. Endocrinology 128:2045, 1991. Bakker, G.H., Setyono-Han, B., Henkelman, M.S., et al. Comparison of the actions of the antiprogestin mifepristone (RU 486), the progestin megestrol acetate, the LHRH analog buserelin, and ovariectomy in treatment of rat mammary tumors. Cancer Treatment Reports 71:1021, 1987. Bakker, G.H., Setyono-Han, B., Portengen, H., et al. Endocrine and antitumor effects of combined treatment with an antiprogestin and antiestrogen or luteinizing hormone-releasing hormone agonist in female rats bearing mammary tumors. Endocrinology 125:1593–1598, 1989. Bakker, G.H., Setyono-Han, B., Portengen, H., et al. Treatment of breast cancer with different antiprogestins: Preclinical and clinical studies. Journal of Steroid Biochemistry and Molecular Biology 37:789–794, 1990. Bardon, S., Vignon, F., Chalbos, D., et al. RU 486, a progestin and glucocorticoid antagonist, inhibits the growth of breast cancer cells via the progesterone receptor. Journal of Clinical Endocrinology and Metabolism 60:692–697, 1985. Bardon, S., Vignon, F., Montcourrier, P., et al. Steroid receptor-mediated cytotoxicity of an antiestrogen and an antiprogestin in breast cancer cells. Cancer Research 47:1441, 1987. Bergkvist, L., Adami, H.O., Persson, I., et al. The risk of breast cancer after estrogen and estrogen-progestin replacement. New England Journal of Medicine 321:293, 1989. Berliner, J.A., and Gerschenson, L.E. Sex steroid induced morphological changes in primary uterine cell cultures. Journal of Steroid Biochemistry 7:153, 1976. Bowden, R.T., Hissom, J.R., and Moore, M.R. Growth stimulation of T47D human breast cancer cells by the anti-progestin RU 486. Endocrinology 124:2642–2644, 1989. Bresciani, F. Ovarian steroid control of cell proliferation in the mammary gland and cancer. P. 130 in Basic Actions of Sex Steroids on Target Organs. Basel: Karger Publishing Company, 1971.
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Representative terms from entire chapter: