Preclinical Studies Using the Intratumoral Aromatase Model for Postmenopausal Breast Cancer

ABSTRACT: To determine the most effective strategies for the treatment of postmenopausal hormone dependent breast cancer, we recently developed a model system in nude mice. In this model, estrogen receptor-positive human breast cancer cells (MCF-7) stably transfected with the aromatase gene are inoculated into ovariectomized, immunosuppressed (nude) mice. These cells synthesize sufficient estrogen from androgen substrate to stimulate their proliferation and the development of tumors. Moreover, estrogen secreted by the tumor cells maintains uterine weight comparable to that of the intact mouse. In the present study, we employed this model to investigate the effects of the aromatase inhibitor, letrozole (CGS 20267 [Femara]) on mammary tumor growth and on the uterus. We also used this model to predict the effects of combining two aromatase inhibitors, letrozole and anastrozole (Arimidex), with the antiestrogen tamoxifen (Nolvadex). Letrozole was found to be a highly potent inhibitor of tumor proliferation and more effective than tamoxifen. No stimulation of uterine growth was observed with the aromatase inhibitors. However, the combination of letrozole or anastrozole and tamoxifen was no more effective than either aromatase inhibitor alone. The agonistic effect of tamoxifen on the uterus was observed when it was given alone and when combined with the aromatase inhibitors. Furthermore, letrozole had the most potent antitumor activity when compared to other aromatase inhibitors and antiestrogens. No additional benefit was observed by combining these agents with tamoxifen over treatment with aromatase inhibitors alone.[ONCOLOGY(Suppl 5):36-40, 1998]

When we began the development of aromatase inhibitors for breast cancer treatment a number of years ago, our objective was to block effectively the synthesis of estrogen in all tissues with compounds that had no estrogenic or agonistic effects, in contrast to the antiestrogens used at that time. Although substantial beneficial effects of tamoxifen (Nolvadex) are now apparent,[1,2] this antiestrogen has the disadvantage of acting as a weak agonist on the endometrium. This has led to endometrial hyperplasia and cancer in some breast cancer patients.[3,4] It also seemed possible that tamoxifen may not optimally suppress the effects of estrogen on tumor proliferation. Thus, complete blockade of estrogen synthesis may be more effective.[5]

We first reported on estrogen synthetase (aromatase) inhibitors in 1973,[6] and subsequently 4-hydroxyandrostenedione was developed as the first selective aromatase inhibitor for breast cancer treatment.[7] In the interim, aminoglutethimide (Cytadren), first used as an anticonvulsant but later found to inhibit steroidal hydroxylations by adrenal P450 enzymes, was employed to reduce estrogen production in breast cancer patients.[8] Numerous steroidal and nonsteroidal compounds have now been reported to inhibit aromatase.[5] The steroidal inhibitors are substrate analogs, whereas the nonsteroidal compounds tend to be less specific for aromatase.

Recently, two triazole compounds with good specificity for aromatase have been approved for the treatment of breast cancer in the United States.[9] Letrozole (CGS 20267 [Femara]), 4-[1-(cyanophenyl)-1-(1,2,4-triazolyl) methyl] benzonitril, is a highly potent inhibitor of aromatase that reduces plasma estrogen levels to the limit of detection in postmenopausal patients.[10] Similar effects are observed with anastrozole (ZD1033 [Arimidex]), 2,2¢[5-(1H-1,2,4-triazol-1-yl methyl)-1,3 phenylene]bis(2-methylpropiononitrile).

Clinical trials of these new agents have been carried out in patients with advanced breast cancer who had relapsed after treatment with other agents.[11] Results from these trials have provided the important observation that aromatase inhibitors are effective in some patients who have relapsed and become resistant to tamoxifen and other treatments. However, evaluating the inhibitors as first-line agents has been difficult since almost all patients receive tamoxifen initially. We have, therefore, developed a model that is relevant to postmenopausal patients with hormone-dependent disease in order to study the effects of aromatase inhibitors and antiestrogens.[12]

After menopause, when the ovary ceases production of estrogen, synthesis in nongonadal tissues increases. The main sources of circulating estrogens in postmenopausal women are adipose and muscle tissues, which make up the largest proportion of body mass. The breast also synthesizes estrogens. The concentrations of estrogen in the breast tissue of postmenopausal patients have been found to be higher than levels in plasma and similar to levels in the breast tissue of premenopausal women.

Aromatase activity has been detected in approximately 60% of tumors in breast cancer patients. Aromatase is expressed in epithelial and stromal cells of the normal breast and breast tumors.[13] Intratumoral estrogen synthesis can result in high local concentrations of estrogen. We recently demonstrated that this has functional significance and results in enhanced tumor proliferation.[13]

In our model, we use ovariectomized nude mice to simulate the postmenopausal situation.[12,14,15] Since the rodent has no significant production of estrogen from nonovarian tissue, we inoculate the mice with human breast cancer cells (MCF-7) transfected with the aromatase gene to provide the intratumoral source of estrogen. The tumors produced from the MCF-7 cells transfected with the aromatase gene proliferate faster than those in the same animal without aromatase, which depends on circulating estrogen from adjacent tumors.[16]

In the present study aimed at optimizing treatment strategies, we used this model to investigate the effects of letrozole and anastrozole on tumor growth, as well as on the uterus. We compared these effects with those of tamoxifen alone or in combination with the aromatase inhibitors.[17]

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