Effects of Exemestane and Tamoxifen in a Postmenopausal Breast Cancer Model

Estrogen plays an important role in development and progression of breast cancers. Although the incidence of breast cancer increases with age, circulating levels of estrogen are low in postmenopausal women as the ovaries are no longer the major source of the hormone. Production of estrogens in peripheral tissue, such as adipose tissue is increased and accounts for the circulating and tissue concentrations. Despite low circulating levels of estrogens, levels in the breast are the same in postmenopausal as in premenopausal women because of hormone uptake as well as local production (1).

Current treatment for postmenopausal hormone-dependent breast cancers includes two strategies to reduce the effects of estrogen on tumor growth. One strategy is inhibition of estrogen action with antiestrogens, and the other is inhibition of estrogen synthesis with aromatase inhibitors. The antiestrogen tamoxifen has been a primary treatment for breast cancer since the 1970s (2) and has been shown to delay recurrences and contra-lateral breast cancer. The agent is well tolerated and can be taken for 5 years (3). However, tamoxifen is a partial agonist as well as an antagonist and can cause endometrial hyperplasia and occasionally endometrial cancer as well as increased risk of stroke because of its estrogenic effects (4). In addition, the compound may not be optimally effective in terms of its antagonistic actions on the tumor. For these reasons, we have investigated a different approach using inhibition of estrogen production with aromatase inhibitors (5, 6, 7). The two approaches have been compared in recent clinical trials and the results suggest that aromatase inhibitors are more effective than tamoxifen as first-line therapy for postmenopausal patients with hormone-responsive breast cancer (8, 9, 10) and also for patients with early breast cancer (11, 12). Two classes of aromatase inhibitors, steroidal (formestane, exemestane), and nonsteroidal (anastrozole, letrozole) compounds have been developed. Steroidal, irreversible inhibitors (also known as suicide inhibitors) of aromatase interact with the substrate-binding site of the enzyme, binding irreversibly and causing enzyme inactivation (13). Thus, no estrogen can be synthesized until new enzyme is produced. In contrast, nonsteroidal compounds interact with the heme-iron group of the cytochrome P450 aromatase in a reversible reaction (13).

To investigate the effectiveness of aromatase inhibitors, our laboratory developed a xenograft tumor model using human hormone responsive (estrogen-receptor positive) breast cancer cells stably transfected with the human aromatase gene (MCF-7Ca). In this model, MCF-7Ca cells (14) are grown as tumors in the ovariectomized, immune suppressed mice (15, 16). The tumor serves as an autocrine source of estrogen that stimulates its growth, and it is sensitive to both the antiproliferative effects of antiestrogens and of aromatase inhibitors (17, 18, 19). The model simulates the postmenopausal breast cancer patient as a major source of the hormone after menopause is breast tissue and where the aromatase enzyme is not under gonadotropin regulation. As the production of adrenal androgen precursors for aromatization is deficient in these mice (20), we supplement them with androstenedione. We have shown previously that androstenedione does not affect the growth of tumors directly. Thus, growth of tumors of MCF-7 cells without aromatase transfection are not stimulated by androstenedione (15).

Because both antiestrogens and aromatase inhibitors are effective in controlling tumor growth, blocking both estrogen receptors and estrogen synthesis might have an additive effect and result in better control of tumor growth. In this study, we have investigated whether the steroidal inhibitor exemestane combined with tamoxifen might be more effective than each alone. In our previous studies, we have compared the effects of combining tamoxifen with nonsteroidal aromatase inhibitors anastrozole and letrozole on tumor growth (18, 19). Although there was no benefit in combining treatment compared with the aromatase inhibitors alone, the effect of treatment with the combination was similar to that of tamoxifen alone. These data predicted the outcome of the clinical Anastrozole, Tamoxifen, and the Combination trial (11), which found that anastrozole was more effective than tamoxifen and the combination of the two agents in reducing recurrences in patients with early stage breast cancer. To date, it is not known whether combining a steroidal aromatase inhibitor with the antiestrogen tamoxifen will be more effective in slowing tumor progression compared with treatment with either of these drugs alone. We were particularly interested in examining the value of combining exemestane plus tamoxifen in this model as it had been reported previously that the combination is superior to either agent alone in the rat model with carcinogen [7,12-dimethylbenz(a)anthracene]-induced mammary tumors (21).

In the current study, we have also investigated several strategies with the goal of defining regimens that will be the basis of clinical trials to optimize treatment for postmenopausal breast cancer patients with these agents. Thus, we have investigated the effect of sequential treatment with tamoxifen and exemestane as second-line therapy and the effect of letrozole alone and in sequential therapy after tumors progressed on exemestane and tamoxifen.

MCF-7 human breast cancer cells stably transfected with the human aromatase gene (MCF-7Ca; ref. 14). Dulbecco’s PBS, DMEM, penicillin/streptomycin solution, trypsin-EDTA solution, and geneticin (G418) were from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Hyclone (Logan, UT). Androstenedione (Δ4A), tamoxifen, and hydroxypropyl cellulose were obtained from Sigma Chemical Company (St. Louis, MO). Matrigel was obtained from BD Biosciences (Bedford, MA). Letrozole was kindly provided by Dr. Dean Evans (Novartis Pharma, Basel, Switzerland). Exemestane was kindly provided by Dr. Lori Hollis (Pharmacia/Pfizer, Inc.).

MCF-7 human breast cancer cells stably transfected with the human aromatase gene (MCF-7Ca; ref. 14) were cultured in DMEM with 5% fetal bovine serum, 1% penicillin/streptomycin solution, and 750 μg/mL G418. The culture medium was changed twice weekly.

Female BALB/c athymic ovariectomized mice 4 to 6 weeks old were obtained from NCI (Frederick, MD). The animals were housed in a pathogen-free environment under controlled conditions of light and humidity and received food and water ad libitum. Animals were allowed to acclimatize for 48 hours after shipment and before tumor inoculation was done.

MCF-7Ca cells were inoculated into the mice as described previously (15, 18). Subconfluent (80%) MCF-7Ca cells were washed with Dulbecco’s PBS and scraped into Dulbecco’s PBS. Cells were collected by centrifugation and resuspended in Matrigel (10 mg/mL). Each mouse received subcutaneous injections at two sites on each flank with 0.1 mL of cell suspension (2.5 × 10⁷ cells/mL). Because athymic mice are deficient in adrenal androgens, they were supplemented with daily injections of the aromatase substrate androstenedione (Δ4A) 100 μg/day for the duration of the experiment. Treatment started when tumors reached a measurable size (∼300 mm³) 4 weeks after inoculation. Tumors were measured weekly with calipers, and volumes were calculated with the formula 4/3π × r₁² x r₂ (r₁ < r₂), where r¹ is the smaller radius. Mice were grouped for treatment (five mice per group) so that total tumor volume was similar in each group, and mice received subcutaneous injection with vehicle, nonsteroidal aromatase inhibitor letrozole (10 μg/day), antiestrogen tamoxifen (500 μg/day), and 5 different doses of steroidal aromatase inhibitor exemestane (50, 100, 250, 500, and 1,000 μg/day) prepared as suspensions in 0.3% hydroxypropyl cellulose. In the second experiment, mice were treated as above with vehicle, tamoxifen (100 μg/day), exemestane (100 and 250 μg/day), the combination of tamoxifen and exemestane at the same doses, and letrozole (10 μg/day). When tumors had doubled in volume during treatment, mice were crossed over to tamoxifen or exemestane for second-line treatment and then subsequently to letrozole. Mice treated with the combination were switched to letrozole treatment when their tumors doubled in volume. Animals were treated for the indicated times as shown in Scheme 1, after which they were sacrificed by decapitation, and tumors and uteri were excised, cleaned, and weighed.

Linear mixed-effects models were used to estimate average tumor weight and volume across the treatment groups (22). Each experiment was analyzed separately. The mixed effect of treatment in each group and random effects for each animal within a group were estimated. The simple covariance structure for the repeated measures within a subject was selected. The set of appropriate contrasts was used to compare treatment means across the groups. Data on tumor volume were grouped longitudinally and unbalanced with various treatment duration and number of measurable tumors per animal. For tumor growth, diagnostic plots suggested that models of exponential growth were appropriate to the data. Therefore, linear mixed-effects models were fitted to the natural logarithm of tumor volume over time. This approach allows the estimation of an exponential variable controlling the rate of tumor growth for each of these treatment groups. The multilevel model was more efficient and was preferred. Model with two random effects for the intercept and the slope at the mouse level and a single random effect for the intercept at tumor level (within a mouse) was chosen. The variance covariance matrix for the random effects was block diagonal. The first-order autoregressive structure was selected for these diagonal blocks.

The general linear models technique was used to analyze the uterine weight data. The treatment groups were compared with one another at 0.05 level of statistical significance. Either Tukey’s or Dunnett’s procedure (23, 24) was used to make the adjustments for multiple comparisons. The results of the comparisons across treatment groups are presented as the difference in growth rate, the mean difference in tumor weight, or uterine weight with a corresponding 95% confidence interval.

To identify the optimal dose of exemestane on tumor growth in vivo, several doses of the inhibitor were evaluated in comparison to treatment with letrozole, tamoxifen, and vehicle (control). All of the treatments were effective at suppressing tumor growth compared with the vehicle-treated tumors (Fig. 1,A). The tumor volumes in the letrozole-treated animals regressed by 15% over the 4 weeks of treatment, whereas the tamoxifen-treated tumors increased slightly by 35%. Nevertheless, although the tamoxifen-treated tumors did not regress, their growth was significantly reduced compared with the control tumors that increased by 2.8-fold during the 4 weeks of treatment. The estimated difference in the weekly growth rate was 0.15 mm³ with the 95% confidence interval from 0.08 to 0.21 mm³ (Fig. 1 A). The inhibition of tumor growth by exemestane was shown to be dose-dependent. At doses of 50 and 100 μg/day, tumor volumes increased by 1.1 and 0.9-fold, respectively, over 4 weeks. In the animals treated with exemestane at doses of 250, 500, and 1,000 μg/day, tumor volumes remained close to pretreatment levels indicating that these doses of exemestane effectively inhibited tumor growth to a maximum extent. Exemestane at doses of 250 μg/day was superior to tamoxifen (500 μg/day; P = 0.037). When exemestane was compared with letrozole, there was no statistically significant difference between the tumor growth rates of the mice treated with higher dose (250 μg/day) of exemestane compared with those treated with letrozole (10 μg/day).

At the end of treatment, animals were sacrificed and tumors and uteri were dissected and weighed. The results for tumor volumes correlated well with tumor weights. Thus, tumor weights (Fig. 1 B) in the control group (344 ± 56 mg) were significantly greater than in all of the treatment groups (P = 0.05). The mean weight of letrozole-treated tumors (48 ± 7 mg) was significantly lower than that of tamoxifen-treated tumors (P < 0.001). There was a statistically significant difference in tumor volumes between letrozole and exemestane-treated tumors at doses of 250 μg/day (P = 0.03). Also, the tumor weights of the letrozole-treated group were significantly less. The difference in treatment means is 29.1 mg with the 95% confidence interval (3.5–64.7 mg). The weight of tumors treated with the lowest dosage, 50 mg of exemestane, was significantly lower than in the control group (P = 0.05) but was not significantly different from tamoxifen.

The mouse uterus is very sensitive to the effects of estrogen and can be used as a bioassay of the estrogenic effects of compounds. In the tumor model, sufficient estrogen is produced by the tumors to maintain uterine weight comparable with that of the mouse in diestrus. Treatment with aromatase inhibitors letrozole and exemestane reduced uterine weights significantly compared with those of the control and tamoxifen-treated mice (Fig. 1 C). For instance, the estimated difference in treatment means between the control and the group treated with 50 mg of exemestane was 17.4 mg with the 95% confidence interval (6.7–27.8 mg). This result suggests that the two aromatase inhibitors cause marked reduction in estrogen levels. In contrast, tamoxifen did not cause any significant difference in uterine weight compared with control. This finding is consistent with previous reports that this antiestrogen acts as an estrogen on the uterus (25).

In the previous experiment, we identified submaximal (100 μg/day) and maximally (250 μg/day) effective doses of exemestane (Fig. 1,A). These doses and a submaximal dose of tamoxifen (100 μg/day) were evaluated as single agents and in combination. After tumors reached a measurable size, animals were assigned to seven groups of five mice each for first-line treatment with vehicle, letrozole (10 μg/day), tamoxifen (100 μg/day), exemestane (100 μg/day), exemestane (250 μg/day), tamoxifen (100 μg/day) plus exemestane (100 μg/day), and tamoxifen (100 μg/day) plus exemestane (250 μg/day; Fig. 2).

After 9 weeks, the control tumors had increased by 4.4-fold. Tamoxifen-treated tumors grew slowly over the period of 9 weeks, and increased in size by 2.3-fold. Exemestane at 250 μg/day was more effective than at 100 μg/day, and tumors increased in size by 1.8- and 3.5-fold, respectively, by 9 weeks. However, treatment with tamoxifen (100 μg/day) combined with exemestane in doses of 100 and 250 μg/day was more effective in suppressing the growth rate than either tamoxifen (P = 0.02) or exemestane (P = 0.04) alone and was not significantly different from letrozole. Letrozole treatment initially caused tumors to regress by 20% in the first 4 weeks. Tumors then slowly resumed growth and reached the pretreatment volume by 9 weeks.

To determine the effect of these agents in sequential therapy, mice were changed to second-line treatment when tumor volume had increased by 2- to 3-fold. Control tumors doubled their initial volume after <4 weeks (Table 1). Tamoxifen-treated tumors had doubled after 8 weeks of treatment. Tumors treated with exemestane at the dose of 250 μg/day doubled between 9 and 10 weeks whereas those treated with the combination of tamoxifen and exemestane (250 μg/day) doubled in 16 weeks. Thus, the time of tumor progression was significantly delayed compared with either drug alone. Letrozole-treated tumors did not double until 24 weeks of treatment.

After 9 weeks of treatment with tamoxifen when tumor volumes had increased 2.3-fold increased compared with their initial size, animals were switched to treatment with exemestane (250 μg/day; Fig. 2). Tumors became static for a brief period with exemestane and then continued to grow. After 3 weeks of treatment with exemestane, tumor volume had increased up to 3.4-fold. In a further effort to control tumor growth, mice were switched to letrozole treatment. However, tumors regressed for 3 weeks but then tumor growth resumed. When mice initially treated with exemestane (250 μg/day) were switched to treatment with tamoxifen at week 12, tumor growth continued unabated until week 16. The mice were then switched to letrozole treatment, which slowed tumor growth for 3 weeks before growth resumed. By week 21, the two groups started on exemestane or tamoxifen and receiving three sequential treatments had similar tumor volumes. Also, a second group of animals treated with exemestane (100 μg/day) for 9 weeks (data not shown) followed by second-line treatment with tamoxifen and third-line treatment with letrozole showed a similar growth pattern as tumors treated initially with the higher dose of exemestane. Combining tamoxifen plus exemestane prolonged the time of tumor doubling to 16 weeks. However, these tumors were unresponsive to second-line treatment with letrozole after 1 week. Overall, letrozole treatment alone produced the best results in delaying tumor progression (tumors did not double for 24 weeks). The weekly growth rate was less with continuous treatment with letrozole only compared with the tumor growth rate of mice treated first line with exemestane (250 μg/day) plus tamoxifen and then switched to letrozole (P = 0.046; Fig. 2).

The effect of exemestane on tumor growth in the MCF-7Ca xenograft model suggests that this aromatase inhibitor at optimal doses (250, 500, and 1,000 μg/day) is similar to or slightly better than the maximal dose of tamoxifen (500 μg/day) in reducing tumor weight after 4 weeks of treatment. Furthermore, aromatase inhibitors did not have estrogenic effects on the uterus unlike the antiestrogen tamoxifen. In the second experiment, a submaximal dose of tamoxifen (100 μg/day) was used in combination with 100 and 250 μg/day of exemestane to determine whether the two agents acting by different mechanisms have additive effects. There was a significantly greater reduction in tumor growth with both doses of exemestane (100 and 250 μg/day) used in the combined treatment than with either doses of exemestane alone or tamoxifen alone. A similar result was reported previously by Zaccheo et al., (21) who found that growth of carcinogen [7,12-dimethylbenz(a)anthracene]-induced tumors of the rat were inhibited to a greater extent by the combination of exemestane and tamoxifen than with either alone. The combined treatment, which inhibited estrogen action and estrogen synthesis, delayed tumor doubling for nearly 16 weeks, twice as long as with tamoxifen alone. This suggests that combining tamoxifen with this steroidal aromatase inhibitor may be more effective in breast cancer patients than the drugs administered separately. The results in the mouse model are in contrast to our previous findings with combinations of nonsteroidal aromatase inhibitors and antiestrogens (18, 19). However, whereas the exemestane-tamoxifen combination was more effective than either agent alone, it was less effective than the aromatase inhibitor letrozole only. Thus, tumors were significantly larger after 28 weeks of the combined treatment than those treated with letrozole.

The xenograft model bearing tumors of MCF-7Ca human breast cancer cells has predicted the results of clinical trials evaluating tamoxifen and aromatase inhibitors (17, 19). For example, letrozole was superior to tamoxifen in the mouse model (17, 19) and in time to progression and time to treatment failure in breast cancer patients (8). Anastrozole also significantly increased time to progression compared with tamoxifen in patients (P = 0.005; refs. 8 and 9). In addition, results of the Anastrozole, Tamoxifen, and the Combination trial (11) confirmed our findings of combining nonsteroidal aromatase inhibitors with tamoxifen in the mouse model (17, 19). Letrozole plus tamoxifen in the xenograft model was less effective than letrozole alone, and the combination was not significantly different in delaying tumor doubling than tamoxifen alone. One possible explanation for the difference in results between the two types of aromatase inhibitors is that letrozole is more potent than exemestane (Fig. 1 A). The slight reductions reported in serum levels of nonsteroidal aromatase inhibitors (27 and 38%) when combined with tamoxifen seems unlikely to influence their activity because estrogen concentrations remain maximally suppressed (19, 26). Clearance rates of exemestane and tamoxifen in combination compared with those of the drugs alone are reported not to be altered in breast cancer patients (27, 28). Additional studies are therefore needed to explore whether these or other mechanisms explain the difference between the two classes of aromatase inhibitors.

When tamoxifen was used in sequence followed by exemestane, tumor growth was arrested briefly. This finding in our model of advanced breast cancer is consistent with reports that patients who progressed on tamoxifen responded to subsequent exemestane treatment (10). Although the period of response was relatively brief in the mouse, recent studies indicate better results are achieved in early breast cancer. Thus, women who switched from tamoxifen to exemestane after 2 to 3 years had 32% fewer recurrences than those on tamoxifen (12).

Tumors that were initially treated with exemestane and crossed over to tamoxifen grew continuously without interruption. Although this finding is not consistent with response to these drugs in patients, tumor growth was arrested for a short time (3 weeks) when mice were switched to letrozole treatment. However, when tumors were initially exposed to tamoxifen plus exemestane in combination, their response time was even shorter (1 week) to subsequent treatment with letrozole as a second-line treatment. Nevertheless, growth of tumors treated with the combination initially was reduced, and tumors did not reach the same volume as those of groups treated with three sequential therapies, for 7 additional weeks. In conclusion, exemestane was more effective in controlling tumor growth than tamoxifen. In addition, the combination of exemestane plus tamoxifen was clearly more effective than sequential use of these agents in the tumor model. However, the nonsteroidal aromatase inhibitor letrozole as first-line therapy was overall the most effective treatment in controlling tumor growth.