Estradiol-induced mammary cancers in ACI.COP-Ept2 rats respond to the antiestrogen tamoxifen and express hormone receptors, confirming prior reports suggesting that tumors in this model are hormone responsive. The ACI rat and the ACI.COP-Ept2 rat have similar susceptibility to mammary cancers induced by 30-mm E2 capsules, with reduced pituitary hyperplasia in the ACI.COP-Ept2 rat [21
]. We report lower circulating estradiol and prolonged tumor latency compared to previous reports, likely the result of using the smaller, 9-mm E2 capsules [21
]. Both the ACI.COP-Ept2 rat and the lower E2 dose were chosen to minimize pituitary hyperplasia reported in prior studies [17
]. Our observations are consistent with an earlier report which observed a delayed latency in ACI rats treated with 9 mg E2 capsules compared to 27 mg, and which also reported a reduction in pituitary weight and in circulating prolactin [27
]. In rats treated with 3-mm E2 capsules, we have observed a further prolonged latency to tumor (unpublished observation). Although our observed mean serum estradiol was within observed physiological parameters of untreated rats with functioning ovaries, tumors are not seen in intact rats not treated with supplemental E2. Continuous estradiol exposure in conjunction with progesterone seems to be key, and is likely more critical than E2 dose [12
It has previously been reported that estradiol-induced tumors in ACI rats regressed after the estradiol capsule was removed [14
], and that tamoxifen prevented induction of tumors by estradiol in ACI rats [28
]. We show here that tamoxifen treatment leads to tumor regression in a rat model of estrogen-induced mammary cancer. These findings demonstrate that both tumor initiation and progression in this model is estrogen dependent and is mediated through estrogen receptor-dependent mechanisms. We found that both ERα and ERβ were expressed in cancers, with ERα levels higher in cancer tissue than in surrounding non-cancer tissue. Whether ERα or ERβ, or both, are critical for mammary cancer development is unknown.
Recent advances in imaging technology including MRI make possible non-invasive, sensitive and accurate monitoring of tumor growth or regression in small animals [29
]. Tumors as small as 0.5 mm diameter can be detected by MRI, while palpable tumors are at least 5 mm diameter (unpublished observation). Furthermore, imaged tumors can be measured in three dimensions, while only two dimensions can easily be obtained manually. Thus, MRI is a highly sensitive, accurate, non-invasive method to monitor tumor regression.
In both the ACI and ACI.COP-Ept2 rats, PR is upregulated in mammary tumors compared to adjacent non-tumor tissue [17
]. PR is upregulated in early mammary lesions as well as cancers [14
]. Thus, PR may contribute to the development of mammary cancers in these models. Alternatively, since PR is an estrogen responsive gene, the cancer cells may have a heightened response to estrogen, evidenced by increased PR. The upregulation of ER in tumors compared to adjacent non-tumor tissue, and increased mitotic index of tumor cells [14
], suggest that the cancer cells are more responsive to estrogen. On the other hand, we and others have established that ovariectomized rats treated with estradiol do not develop mammary cancers [5
], while replacing progesterone restores tumor development in ovariectomized rats [12
]. These suggest that estrogen and PR are important factors in this model, as in human breast cancer.
In humans, lifetime estrogen exposure is a known risk factor for breast cancer, with early menarche, late menopause, and hormone replacement therapy (HRT) increasing risk [33
]. Combination HRT, containing both estrogen and progesterone, increases breast cancer risk more than estrogen alone [33
]. In the Women's Health Initiative study, the combination HRT treatment arm was halted after a median treatment of 5.2 years because of increased breast cancer risk, while the estrogen-alone arm ended after a median 6.9 years of treatment due to increased stroke, not breast cancer risk [3
]. The Million Women Study also found a greater increase in breast cancer risk for combination HRT users than in estrogen-only users [34
]. In the ACI rat model, removal of ovaries in the presence of exogenous estrogen does not lead to tumor formation, whereas adding back progesterone and estrogen led to mammary tumor formation [12
]. The similarity to human disease suggests that our model is well suited to study hormone responsive breast cancer.
ERBB2 overexpression is associated with tumor relapse and poor survival [35
]. While Erbb2 was expressed in both benign and malignant mammary tissue, we did not find upregulation in cancer tissue. Erbb2 is encoded on rat chromosome 10, which is duplicated in 15% of ACI mammary tumors [16
]. Thus, we cannot exclude the possibility that Erbb2 overexpression might have been observed if more tumors were analyzed, and/or if mRNA or protein expression were quantified, rather than assessing expression by measuring percent Erbb2-positive cells.
The anti-inflammatory drug celecoxib, a selective COX-2 inhibitor, reduces breast cancer risk [36
]. COX-2 breast cancer expression is associated with unfavorable outcome, and is more common in breast cancers with poor prognosis [24
]. We observed a low degree of Cox-2 expression in cancer and non-cancer mammary tissue, consistent with the idea that ours is a model of breast cancer with good prognosis.
In summary, we demonstrate by physical examination and by MRI imaging that a lower dose than generally used of E2 leads to mammary tumor formation in 100% of ACI.COP-Ept2 rats, although with a prolonged latency compared to the higher dose. Tamoxifen induces rapid regression of E2-induced mammary cancers in the rats. The tumors overexpress ERα and PR, but not Erbb2 nor Cox-2. Based on biochemical and morphological characteristics, this model is most representative of hormone responsive breast cancers.