The study reported here is to our knowledge the first chemoprevention study to compare the efficacy of two SERMs, using a transgenic mouse transplant model that fulfills the operational definition of premalignancy. We demonstrate that the transplanted premalignant lesions (MIN-Os) are ER-positive and their growth is ovarian-hormone-sensitive. Two related SERMs, ospemifene and tamoxifen, had an inhibitory effect on this mouse model for DCIS on the growth of premalignant mammary lesions and on the tumor incidence. The primary effect of these two agents was on proliferation, while apoptosis was less affected. In comparing the two SERMs, no significant differences were found in the growth of the premalignant lesion, tumor incidence, and markers for proliferation or apoptosis. In contrast, there was a significant difference in the ER status between the two SERM-treated groups. The ER score in the MIN-O lesions was reduced after treatment with tamoxifen but increased by treatment with ospemifene. Overall, these studies provide a foundation for studying and comparing the effects of hormonal manipulation in a chemoprevention setting in an immunocompetent transplantable mouse model for DCIS.
The MIN-O line, 8w-B, originally isolated from dysplastic lesions in the Tg(PyV-mT
) mammary glands, was found to be ER-positive and ovarian-hormone-sensitive in this study. Recent studies with Tg(PyV-mT
) mice have demonstrated that the distribution and number of ER-positive cells change with progression to malignancy [5
]. In our studies, we observed a specific distribution of ER-positive cells in the MIN-Os, showing a higher ER score near the leading edge of the growing outgrowth. Tumors, on the contrary, typically had cells with weak ER positivity randomly scattered throughout the tumor. To functionally assess this observation that the MIN-O is estrogen-positive, the host animals were ovariectomized to study the effect on MIN-O growth and tumor incidence. Ovariectomy significantly reduced the growth of the MIN-O and it significantly increased the tumor latency. This data, coupled to the receptor levels, suggests that MIN-O growth and progression to invasive tumor is ovarian-hormone-dependent, albeit not exclusively. This is reminiscent of human breast cancer, in which oophorectomy decreases recurrence and the incidence of contralateral invasive breast cancer [22
Both SERMs exerted an inhibitory effect on the premalignant tissue growth. As each initial transplant contains 1 mm3 of tissue, the results at 3 weeks of treatment show that the transplants are growing, albeit at a much slower rate. In addition, this decreased growth effect is not due to decreased PyV-mT expression in the treatment groups, as the PyV-mT expression was not different between the SERM groups and the control group, based on immunohistochemistry.
The SERMs also inhibited progression to invasive carcinoma. Typically, a high proportion of the MIN-Os will have tumors by 10 weeks after transplantation. The SERM-treated animals showed diminished MIN-O growth with significantly fewer tumors than controls at this time point. The major effect of the SERMs was on proliferation as seen by Ki-67 staining, rather than on apoptosis. In human breast cancer, reduced Ki-67 staining is seen after treatment with SERMs and aromatase inhibitors and has been used as efficacy marker in antiestrogen therapy [24
]. No statistically significant differences in MIN-O growth, tumor incidence, and rates of proliferation and apoptosis were observed between the two SERM treatments. One exception was the effect on ER expression. The ospemifene treatment increased the ER score, whereas tamoxifen decreased it. In ER-positive mammary tumors from Wnt-1 transgenic mice, tamoxifen treatment resulted in significant reduction of ER expression [21
]. In a portion of human tumors, decreased ER levels in tumors after tamoxifen treatment has been shown to predict tamoxifen resistance [25
]. Therefore, these differences in ER modulation between the two related SERMs may suggest different mechanisms of action and may portend different long-term outcomes. This may be reflected in the slight differences in cell proliferation and apoptosis rate between the two treatment groups.
The study detailed here is the first to compare these two related SERMs in a mouse mammary premalignant transplant model and shows that ospemifene has equal effects to tamoxifen in mammary lesions. This is important, because ospemifene is currently in phase III clinical trials for urogenital sequelae but limited data demonstrate its effectiveness in breast cancer. As phase I and II studies have shown it was well tolerated in healthy postmenopausal women, it may offer alternative hormonal options to women with breast cancer or at high risk for breast cancer. In particular, ospemifene is not known to cause menopausal symptoms, such as hot flashes, insomnia, [26
], melancholy, nervousness, dizziness, while it has some proestrogenic effects on the bone [17
] and vaginal tissue [26
] and, unlike other SERMs, does not cause vaginal dryness. Recently, aromatase inhibitors have been shown to provide better chemoprevention to the breast than tamoxifen, but similar to tamoxifen, they have significant side effects including bone loss, muscle and joint sequelae, and cardiac events [15
These studies provide evidence that both tamoxifen and ospemifene have effects on decreasing growth and progression in our model of DCIS. Previously, the chemopreventive effects of ospemifene have been studied in a dimethylbenz [a]anthracene (DMBA)-induced rat and mouse tumor models, where it reduced the incidence of mammary tumors [28
]. Although the DMBA-induced models have been utilized by many investigators in chemoprevention studies, they have significant drawbacks in that they can only test the ability of an agent to affect the progression to invasive carcinoma, rather than examining the effects on the early preneoplastic disease. In addition, the tumors that do arise from DMBA treatment are commonly heterogeneous and involve many organs. Moreover, a large portion of breast carcinomas derived in DMBA-treated animals are adenocanthomas, which do not represent or model typical human invasive carcinoma [29
]. The chemopreventive effects of tamoxifen have been studied in various mouse models, including transgenic mice with activated neu
expression. In Tg(MMTV-neuN
) mice, which exhibit estrogen-sensitive tumor development [30
], tamoxifen treatment reduced the mammary tumor incidence and size when the treatment was initiated before subclinical tumors had developed [32
]. More recently, tamoxifen was shown to delay tumorigenesis in an ER-positive Tg(P53-/-
) mammary premalignant transplant model [34
The MIN-O model illustrated in these studies offers many advantages over other mouse mammary carcinoma models for chemoprevention studies. In typical transgenic mouse mammary models, tumorigenesis occurs in a multifocal manner, that is, multiple tumor foci develop in a mammary fat pad arising independently and at different starting times. Thus, in a given mammary fat pad, multiple lesions at different stages of tumorigenesis can be seen. Since no two fat pads are the same with respect to the development of the lesions, interpreting the results of chemopreventative interventions can be very complicated. Moreover it may require a significant amount of animals to distinguish the effect of an intervention. In contrast, in the MIN-O model, the proliferation of the 'premalignant' growth begins upon transplantation, and therefore the time to malignant transformation is easily measurable. Chemopreventative interventions can be applied before transplantation, at transplantation, or at a defined time after the time of transplantation. In particular, line 8w-B, a MIN-O line used in this study, has a defined tumor latency period [2
]. This relatively short latency affords the opportunity to perform chemoprevention experiments rapidly. Secondly, the premalignant MIN-O and the invasive tumor mimic the histopathology of, respectively, human DCIS and invasive tumor [2
]. Third, since each experimental subject receives tissue from the same MIN-O, the comparison of the experimental and control groups is less prone to error due to differences in the biological potential of the tissue. Fourth, the outgrowths continue to maintain the same biological characteristics, such as tumor latency, histopathological characteristics, and molecular profiles, over multiple serial transplant generations [2
]. This phenotypic stability affords the opportunity to compare experiments over time, regardless of the transplant generation.