To Block Estrogen’s Synthesis or Action: That Is the Question

Richard J. Santen

Division of Endocrinology, Department of Medicine, University of Virginia Health System, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Richard J. Santen, M.D., Division of Endocrinology, Department of Medicine, University of Virginia Health System, P.O. Box 800379, Charlottesville, Virginia 22908. E-mail: . rjs5y{at}virginia.edu

Several disorders in patients require estradiol to produce clinical manifestations, and abrogation of the effects of this sex steroid ameliorates the related signs or symptoms. Included in this list are hyperplasia and neoplasia of the breast and endometrium as well as gynecomastia, premature thelarche, precocious and delayed puberty, mastodynia, oligo- and anovulation, leiomyomata uteri, and endometriosis. Two separate treatment strategies, available for at least three decades, can reduce the target organ effects of estrogen. One is designed to block estrogen action with antiestrogens that bind to the estrogen receptor and interfere with receptor-mediated transcriptional events. The other uses aromatase inhibitors to block the rate-limiting step in synthesis of estradiol involving the conversion of androgens to estrogens (1). Aromatase inhibitors lower the concentrations of estradiol in plasma and tissue and reduce the amount of estrogen available to stimulate estrogen receptor (ER)-mediated transcription. The major question is whether one strategy is superior to the other. Very recent evidence suggests the superiority of aromatase inhibitors over the anti-estrogens for certain indications. This article will review the background surrounding this issue, outline the evidence available, and discuss the potential mechanistic basis for the favorable effects of the aromatase inhibitors.

Clomiphene citrate, the first clinically available antiestrogen, was approved by the U.S. Food and Drug Administration (FDA) in 1967 and used for ovulation induction as well as for treatment of endocrine-dependent breast cancer. In the mid-1970s, tamoxifen gained approval for use in the United States as a treatment for breast cancer and later, toremiphene, a close cousin. Raloxifene is used for treatment of osteopenia and osteoporosis and is undergoing trial for breast cancer prevention. All four of these medications exert strong estrogen antagonist effects on certain tissues and partial estrogen agonistic activity on others and thus are considered selective ER modulators (SERMs). More recently, pure antiestrogens that lack agonistic properties have been developed and are undergoing extensive clinical testing (2).

Investigators first developed the concept of using aromatase inhibitors for treatment of a variety of estrogen- dependent processes in the 1960s. Later, clinical trials with the first-generation aromatase inhibitor, aminoglutethimide, provided practical proof that aromatase inhibitors could be used for treatment of hormone-dependent breast cancer. Direct clinical comparisons with the antiestrogen, tamoxifen, demonstrated equal efficacy for this indication (3). However, aminoglutethimide produced substantial side effects and toxicity, which diminished its usefulness. Over the last 30 yr, more potent and selective but less toxic aromatase inhibitors evolved (1). The third-generation agents, now approved for use worldwide, are nearly completely selective for the aromatase enzyme, 1,000- to 10,000-fold more potent than aminoglutethimide, and much better tolerated. These new inhibitors comprise two categories with respect to mechanism of action: 1) the competitive inhibitors that bind to the active site of the aromatase enzyme and block estradiol formation, and 2) the inactivators that are modified by the catalytic effects of aromatase to form reactive compounds that bind covalently to the active site of the enzyme and irreversibly destroy its enzymatic action. The latter are called suicide or mechanism-based inactivators. The FDA has now approved three third-generation aromatase inhibitors for use in the United States: the competitive inhibitors anastrozole and letrozole and the inactivator exemestane.

During the process of sequential development and testing in breast cancer patients, the second-generation (i.e. fadrozole, 4-hydroxy androstenedione) inhibitors demonstrated equal but not superior clinical efficacy when compared with the antiestrogens (1). Consequently, it came as a surprise to investigators in the field that third-generation aromatase inhibitors appear to be superior to tamoxifen for treatment of advanced hormone-dependent breast cancer (4, 5, 6, 7, 8, 9, 10, 11). Preliminary evidence also suggests superiority in the adjuvant and neoadjuvant setting for breast cancer prevention and perhaps also for ovulatory dysfunction in infertile women (12, 13, 14).

Direct head-to-head studies of tamoxifen and the third-generation aromatase inhibitors provided substantial power for assessing relative efficacy. Five recent trials compared tamoxifen with a third-generation aromatase inhibitor as the first hormonal treatment of recurrent or advanced breast cancer (Table 1Go; Refs. 6, 7, 8, 10). Each comprised a large multicenter study with randomization of patients and use of double-blind methodology. The largest trial, involving 907 women, compared the third-generation inhibitor, letrozole, with tamoxifen (7). Planned study end points included clinical benefit (defined as complete objective regression, partial objective regression, and stable disease for more than 6 months), time to progression of objectively measured tumors, and overall survival. This trial demonstrated the superiority of letrozole over tamoxifen with respect to clinical benefit (49% vs. 38%; P < 0.001) and time to disease progression (9.5 months vs. 6.0 months; P < 0.0001). A recent update also indicated that women receiving letrozole survived significantly longer than those given tamoxifen (4). A similar, multicenter North American trial with 353 participants compared 1 mg of anastrozole with 20 mg of tamoxifen daily (8). Eighty-nine percent of these women had ER- and/or progesterone receptor-positive tumors, whereas in the others the receptor status was unknown. Anastrozole proved statistically significantly superior to tamoxifen with respect to both clinical benefit and time to progression. A comparable European trial of similar design found equivalence between these two agents but may have been flawed in that only 45% of women had ER and/or progesterone receptor-positive tumors, whereas the receptor status in the others was unknown (6). Separate analysis of the subset of ER-positive patients in this trial and a combined analysis including only ER-positive patients in both anastrozole trials supported the superiority of the aromatase inhibitor over tamoxifen with respect to time to progression (8). A similarly conducted Spanish trial also showed superiority of anastrozole over tamoxifen with respect to time to progression and overall survival (10). At the time of data cut-off, only 61% of women had died in the anastrozole group and 92% in the tamoxifen group. Finally, a preliminary report comparing the aromatase inactivator, exemestane, with tamoxifen provided early evidence of superiority with respect to clinical benefit and time to progression (Ref. 15 ; Table 1Go).


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Table 1. Trials comparing the anti-estrogen tamoxifen (TAM) with aromatase inhibitors (AI) in patients with breast cancer

 
Two large, randomized clinical trials have also compared the aromatase inhibitor anastrozole with tamoxifen in the neoadjuvant setting (16, 17). The goal of this approach was to facilitate breast conservation surgery in patients whose large tumors would otherwise require mastectomy. Hormonal therapy for 4 months before surgery (neoadjuvant therapy) can shrink the size of the tumor sufficiently to allow lumpectomy. Comparison of letrozole with tamoxifen in this setting demonstrated an objective reduction of tumor size in 60% of patients receiving letrozole vs. 40% in those given tamoxifen (P < 0.004). Breast conservation was achieved in 40% of patients receiving letrozole and 25% given tamoxifen (P < 0.036; Ref. 16).

The relative clinical efficacy of each of the third-generation aromatase inhibitors under various clinical circumstances is not currently known. No head-to-head comparison of one inhibitor vs. the others has been published as yet, although such trials are ongoing. However, a recent study demonstrated that letrozole suppressed total body aromatase and plasma estrogen levels to a greater extent than did anastrozole (18). This would support the possibility, as suggested by nonhead-to-head clinical studies, that letrozole may be more potent than anastrozole.

A current research focus is to determine the relative efficacy of tamoxifen vs. aromatase inhibitors in the adjuvant setting and for prevention of contralateral breast cancer. Historically, the ability to prevent contralateral breast cancer when using adjuvant hormonal therapy provided the rationale for designing a breast cancer prevention trial in healthy women. For example, tamoxifen reduces the incidence of contralateral breast cancer by 50% in women receiving tamoxifen as adjuvant therapy (19). Presentation of data from a very large randomized trial provided evidence of the superiority of the aromatase inhibitors in this setting (13). The trial involved 9366 women with ER-positive tumors randomized to receive anastrozole alone, tamoxifen alone, or the two in combination (13, 20). The study is called the ATAC trial, which represents its acronym (anastrozole alone, tamoxifen alone, and in combination). Significantly (P < 0.05) fewer recurrences occurred in the anastrozole arm (223 patients) than in the tamoxifen arm (264 patients) after 33 months of follow-up. Even more striking was the fact that significantly fewer contralateral tumors developed in women in the anastrozole arm (14 patients) than in the tamoxifen alone arm (33 patients). Patients in the arms receiving the combination of tamoxifen plus anastrozole experienced recurrences and new contralateral tumors at the same rate as did patients given tamoxifen alone (28 patients). Several other ongoing studies are examining the sequential use of tamoxifen followed by aromatase inhibitors and the converse sequence vs. tamoxifen alone in the adjuvant setting (1).

The aromatase inhibitors, which lack estrogenic actions on liver and uterus, also appear to be associated with fewer adverse effects than tamoxifen. In the three trials involving anastrozole, the incidence of deep venous thromboses, pulmonary emboli, and vaginal bleeding were lower in the aromatase inhibitor than in the tamoxifen arms (5, 6, 8, 10). In the ATAC trial, endometrial cancer occurred significantly less commonly in women receiving anastrozole (2 patients) than in those given tamoxifen (11 patients; Ref. 13).

On the basis of known physiology, one might expect certain adverse effects of the aromatase inhibitors on bone and on lipid levels when compared with tamoxifen. The SERM properties of tamoxifen produce estrogen agonistic effects on bone and liver, whereas the aromatase inhibitors should abrogate estrogenic effects on these tissues (1, 21). The potentially harmful effects of estrogen reduction induced by the aromatase inhibitors are not problematic for women treated in the short term for metastatic disease. However, in the adjuvant treatment setting or for prevention of breast cancer, such effects could represent a major problem. Preliminary reports suggest no major effects of aromatase inhibitors on lipids (22). However, concomitant use of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) or bisphosphonates may be needed for selected patients. Ongoing substudies in the ATAC trial are examining comparative effects of tamoxifen and anastrozole on bone, but data are not yet forthcoming.

Antiestrogens have also been compared with aromatase inhibitors in benign disease, but data are limited. A recent study involved use of an aromatase inhibitor in women failing initial treatment with clomiphene citrate for infertility (14). Twenty-two women (12 with anovulatory polycystic ovary syndrome and 10 with ovulatory infertility) were selected for study on the basis of unsuccessful treatment with clomiphene citrate. Criteria for failure to clomiphene included lack of ovulation (55.6%) or endometrial thickness no more than 5 mm after a standard course of clomiphene citrate. Use of the third-generation inhibitor letrozole (2.5 mg on d 3–7 after menses) induced ovulation in 75% of women with PCOS vs. 44.4% ovulating in response to clomiphene. In the 10 patients with ovulatory infertility, letrozole treatment resulted in a mean number of 2.3 follicles and a mean endometrial thickness of 8 mm vs. no more than 5 mm in the clomiphene group. No untoward side effects of letrozole occurred. Overall, four pregnancies were achieved in response to letrozole. No data are yet available to determine the safety of letrozole with respect to fetal outcome.

A published study recently demonstrated the clinical efficacy of an aromatase inhibitor in the treatment of severe endometriosis in a postmenopausal patient (23). This therapeutic approach was based upon the finding that endometrial implant tissue contains high levels of aromatase, whereas normal endometrium does not. Further studies will be required to determine the precise role of aromatase inhibitors in this condition, particularly in premenopausal women. Another recent study demonstrated that aromatase inhibitors significantly improved predicted adult height in boys with delayed puberty treated with testosterone (24). This supported the concept that estradiol rather than testosterone mediates epiphyseal closure in boys during puberty. Ongoing studies are examining the efficacy of aromatase inhibitors as treatment for gynecomastia and premature puberty (25, 26). No data are available at present on the treatment of mastalgia or leiomyomata uteri.

The question posed in the title of this article (to block estrogen’s action or its synthesis) appears to be answered by the clinical trials. Blockade of synthesis is superior, at least for use in women with breast cancer and perhaps in selected women with infertility. However, the physiology underlying the differences in responses to aromatase inhibitors vs. antiestrogens requires extensive study to fully understand the mechanisms involved. The new third-generation aromatase inhibitors are so potent that they probably reduce the action of estrogens on tissue to a greater extent than tamoxifen. Substantial data also suggest the possibility that the estrogen agonistic properties of the commonly used antiestrogens might explain the differences in response rate in patients with established cancer. Tamoxifen is a SERM (21, 27, 28). It can exert estrogen-agonistic effects under conditions that depend on the cell or organ involved and on the context under study. The C-terminal region of ER {alpha} and ß contains an activation function 1 region that can exert agonistic effects on ER transcription, provided that an appropriate balance of coactivators, corepressors, integrator proteins, and receptor-binding proteins is present. In some patients with breast cancer, such factors may allow tamoxifen to exert agonistic effects. This phenomenon would not occur with aromatase inhibitors that merely lower the tissue levels of estradiol. Such estrogen agonistic effects of the antiestrogens might also explain why the combination of an aromatase inhibitor with tamoxifen appeared less effective than use of the aromatase inhibitor alone in the ATAC trial (13).

Antiestrogens without agonistic properties, such as faslodex, might be preferable to tamoxifen in women with breast cancer (2). Indeed, preliminary data from two studies involving a total of 851 women suggest that faslodex and anastrozole are equally effective in women relapsing after one prior endocrine therapy. From these observations, one could infer that faslodex may be superior to tamoxifen. Direct comparisons are necessary, however, to confirm this possibility. Taken together, these observations suggest that the agonistic properties of tamoxifen may be detrimental in women with hormone-dependent breast cancer. Clearly, further work is now necessary to fully explain the mechanisms for differential responses to the aromatase inhibitors and the SERMs in women with breast cancer.

The efficacy of an aromatase inhibitor in women failing to respond to clomiphene citrate probably results from different mechanisms. The rationale for use of clomiphene in infertile women is to interrupt estradiol-negative feedback and induce increments in LH, FSH, and estradiol which then stimulate ovulation. Clomiphene citrate (as well as tamoxifen) has a long half-life in the body. Consequently, after interruption of estradiol-negative feedback with clomiphene, residual antiestrogen effects may alter the ovulatory process or interfere with development of appropriate endometrial thickness. Aromatase inhibitors block the production of estradiol by the ovaries and thus interrupt negative feedback inhibition of LH and FSH. Additionally, the aromatase inhibitors may block brain aromatase as another mechanism to increase FSH (29). The blocking effects of aromatase inhibitors can be counteracted in premenopausal women by the large increase in androstenedione as substrate that is induced by the rise in LH (30). In addition, the FSH increments stimulate production of aromatase itself. The half-life of these agents is much shorter than for clomiphene and tamoxifen. Together, these effects allow the body to overcome the effects of the aromatase inhibitor. Consequently, ovulation and implantation can take place without hindrance. This hypothetical explanation for the superiority of the aromatase inhibitors over the antiestrogens for treatment of infertility will also require further experimental support. Further clinical studies are also required to fully validate the observations from the single study of letrozole in women failing to respond adequately to clomiphene citrate (14).

Estradiol derives from three sources: the ovary, the breast itself, and extraglandular tissues, predominantly fat. The ability of reflex increments of LH and FSH to overcome the estrogen blockade of the ovary in premenopausal women renders the aromatase inhibitors inactive when used alone in premenopausal women (30). With very high doses, such as those used in animal studies, ovarian blockade is possible (31). Because of insufficient ovarian blockade in premenopausal women with breast cancer, the aromatase inhibitors must be combined with a GnRH agonist to prevent the reflex LH and FSH increments. This approach is being tested as therapy for premenopausal women with recurrent breast cancer. In postmenopausal women, the aromatase inhibitors block estradiol production directly in the breast (32) as well as in fat tissue. LH and FSH do not regulate extraglandular aromatase or substrate levels, and thus suppression of estradiol is nearly complete.

The preliminary data suggesting a greater inhibition of new contralateral breast tumor formation in women receiving aromatase inhibitors is most intriguing (13). A large body of data suggests that estradiol can enhance the rate of development of new breast cancers (33, 34). The commonly accepted theory is that estradiol acts to stimulate cell proliferation with a concomitant increase in the number of cell divisions. As cells divide, errors in DNA replication occur, albeit uncommonly (34). As cells replicate more rapidly, the chances for errors increase mathematically (i.e. stochastically), and the time available for DNA repair during the cell cycle is reduced. In this way, estradiol is thought to initiate genetic mutations that can lead to neoplastic transformation. At the same time, estradiol causes propagation of cells bearing these mutations and promotes tumor growth. Most investigators believe that these initiating and promotional events explain the ability of estradiol to induce breast and endometrial cancer. However, if this theory were correct, one would expect a similar reduction of contralateral tumor development with the aromatase inhibitors than with tamoxifen.

An alternate theory of estrogen-induced carcinogenesis has gained substantial experimental support recently and was the subject of an international meeting and a monograph published by the Journal of the National Cancer Institute (35, 36). As (36) outlined in Fig. 1Go, estradiol can be metabolized to 4-hydroxy estradiol and then to the 3,4-estradiol quinone. This highly reactive quinone metabolite of estradiol can bind covalently to adenine and guanine on the DNA helix. Through activation of a glycosidase, these adducts are removed to produce a depurinated segment of DNA, a process called depurination. Error-prone DNA repair can then result in mutations that could provide the major mechanism for initiation of cancer. Catechol-O-methyl-transferase and glutathione are present as protective mechanisms to detoxify these potentially carcinogenic metabolites. An imbalance between the formation of reactive estradiol metabolites and their detoxification might then result in breast cancer.



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Figure 1. Diagrammatic representation of the two mechanisms whereby estrogens may cause breast cancer. The left side of the diagram demonstrates that estradiol may bind to the ER and stimulate transcription of genes involved in cellular proliferation. With a sufficient number of cell divisions, mutations occur that are promoted by the effects of estradiol on cell proliferation. The right side of the diagram illustrates the metabolic pathway whereby estradiol is converted into genotoxic metabolites. Estradiol is converted into 4-hydroxy-estradiol, which can then be converted into the 3,4-quinone. This molecule is highly reactive and binds covalently to guanine or adenine on DNA and causes depurination. With error-prone DNA repair, mutations occur. With a sufficient number of critical mutations, breast cancer may occur. The antiestrogens block the pathways shown on the left side of the diagram, and aromatase inhibitors block pathways on both sides.

 
Recent studies measured each of these metabolites in benign and malignant breast tissue, thus documenting the existence of this potentially carcinogenic pathway (37). The enzyme catalyzing the conversion of estradiol to 4-hydroxy estradiol, cytochrome p450 1B1, has been cloned and characterized (35, 36). In vitro studies have also now shown that estradiol can cause genetic mutations and transform cells that do not contain an ER. In addition, some but not all studies report an increase in the relative risk of breast cancer in women who express low levels of the protective enzyme, catechol-O-methyl-transferase (38, 39, 40).

An integrative hypothesis combines the two potential mechanisms of breast cancer etiology. This theory suggests that receptor-mediated proliferation and formation of genotoxic metabolites act in an additive or synergistic fashion to both initiate and promote breast cancer. This theory of estradiol-induced carcinogenesis would require that postmenopausal women have high levels of estradiol present in their breast tissue so that a sufficient number of metabolites would develop. Although not commonly appreciated, the amount of estradiol present in the breast tissue of pre- and postmenopausal women is similar, although blood levels of estradiol differ by a factor of nearly 50 (34). The local synthesis of estradiol in breast tissue appears to be the most likely explanation for this observation. Substantial amounts of aromatase are present in breast tissue, and direct isotopic studies demonstrate local synthesis from aromatase in breast tumors from women before surgical excision.

Analysis of the new theory about estrogen-induced carcinogenesis provides an interesting but speculative explanation for the superiority of aromatase inhibitors over tamoxifen for prevention of contralateral breast cancer. Tamoxifen would only block the effect of estradiol on breast proliferation, whereas the aromatase inhibitors would both inhibit proliferation and reduce breast tissue estradiol levels, and thus depurinating metabolite formation (Ref. 34 ; Fig. 1Go). If this hypothesis were correct, the aromatase inhibitors might prevent ER-positive as well as ER-negative breast tumors, whereas tamoxifen is known to prevent the formation of only ER-positive tumors. Data from the ATAC trial on this point should be forthcoming in the near future.

The above discussion has focused on the superiority of aromatase inhibitors for certain indications. However, antiestrogens such as tamoxifen, toremiphene, or raloxifene are SERMs and can exert beneficial effects on bone and lipids while exerting antiestrogenic actions on the breast (21). A large trial examining the effects of raloxifene demonstrated a significant reduction in both fracture and breast cancer risk (33). Thus, in this setting, the antiestrogens that act as SERMs might be preferable to the use of aromatase inhibitors, whereas a pure antiestrogen such as faslodex would not. If one then expands the question about estrogen synthesis inhibitors and antiestrogens to the pure antiestrogens, the question about relative efficacy remains open, and further studies are required.

In summary, recent data suggest that aromatase inhibitors may be superior to tamoxifen for treatment of breast cancer and perhaps also for its prevention. These unexpected observations emphasize the need to examine in great detail the physiological mechanisms responsible for these differences. Particularly important, in this observer’s view, is the need to determine precisely how estradiol causes breast cancer and whether the depurinating hypothesis is correct. If so, the use of aromatase inhibitors might provide an effective means of preventing breast cancer without the unwanted effects of increasing the risk of endometrial cancer at the same time.

Footnotes

Abbreviations: ATAC, Arimidex, tamoxifen, alone or in combination; ER, estrogen receptor; SERM, selective ER modulators.

Received February 1, 2002.

Accepted April 8, 2002.

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