Breast cancers with a basal-like gene signature are primarily triple-negative, frequently metastatic, and carry a poor prognosis. Basal-like breast cancers are enriched for markers of breast cancer stem cells as well as markers of epithelial-mesenchymal transition (EMT). While EMT is generally thought to be important in the process of metastasis, in vivo evidence of EMT in human disease remains rare. Here we report a novel model of human triple-negative breast cancer, the DKAT cell line, which was isolated from an aggressive, treatment-resistant triple-negative breast cancer that demonstrated morphological and biochemical evidence suggestive of phenotypic plasticity in the patient. The DKAT cell line displays a basal-like phenotype in vitro when cultured in serum-free media, and undergoes phenotypic changes consistent with EMT/MET in response to serum-containing media, a unique property among the breast cancer cell lines we tested. This EMT is marked by increased expression of the transcription factor Zeb1, and Zeb1 is required for the enhanced migratory ability of DKAT cells in the mesenchymal state. DKAT cells also express progenitor-cell markers, and single DKAT cells are able to generate tumorspheres containing both epithelial and mesenchymal cell types. In vivo, as few as ten DKAT cells are capable of forming xenograft tumors which display a range of epithelial and mesenchymal phenotypes. The DKAT model provides a novel model to study the molecular mechanisms regulating phenotypic plasticity and the aggressive biology of triple-negative breast cancers.

Objective Ospemifene, a new drug indicated for the treatment of vulvovaginal atrophy, has completed Phase III clinical trials. A condition affecting millions of women worldwide, vulvovaginal atrophy has long been treated with estrogen therapy. Estrogen treatment carries with it risks of thromboembolism, endometrial proliferative effects, and breast cancer promotion. In this study, we test the effects of three dosing levels of ospemifene in both the prevention and treatment of breast cancer in the MTag.Tg mouse model. Methods The polyomavirus middle-T transgenic mouse model (MTag.Tg), which produces synchronized, multifocal mammary tumors in the immunologically intact C57BL/6 background, was used to examine the impact of ospemifene treatment. First, a cell line derived from an MTag.Tg mouse tumor (Mtag 34) was treated in vitro with ospemifene and its major metabolite, 4-OH ospemifene. MTag.Tg mice were treated daily by gavage with three different doses of ospemifene (5, 25, and 50 mg/kg) before or after the development of mammary tumors. Survival and tumor development results were used to determine the effect of ospemifene treatment on mammary tumors in both the preventive and treatment settings. Results Tumors and the MTag 34 cell line were positive for estrogen receptor expression. The MTag 34 line was not stimulated by ospemifene or its major, active metabolite 4-OH ospemifene in vitro. Ospemifene increased survival time and exerted an antitumor effect on the development and growth of estrogen receptor positive mammary tumors in the MTag.Tg mouse model at the 50 mg/kg dose. Levels of ospemifene and 4-OH ospemifene in both the tumors and plasma of mice confirmed dosing. Ospemifene did not exert an estrogenic effect in the breast tissue at doses equivalent to human dosing. Conclusions Ospemifene prevents and treats estrogen receptor positive MTag.Tg mammary tumors in this immune intact mouse model in a dose-dependent fashion. Ospemifene drug levels in the plasma of treated mice were comparable to those found in humans. Combined with our previous data, ospemifene does not appear to pose a breast cancer risk in animals, and slows cancer development and progression in the MTag.Tg model.

Purpose: In this study, we examine the immunomodulatory effects and antitumor activity of tamoxifen and letrozole when combined with the human epithelial mucin (hMUC1)-specific vaccine, L-BLP25, in the hMUC1-expressing mammary tumor (MMT) mouse model.Experimental Design: Dose-finding studies were conducted for both tamoxifen and letrozole. Letrozole and L-BLP25 combination studies used 69 MMT female mice assigned to five groups: untreated, cyclophosphamide þ placebo, cyclophosphamide þ L-BLP25, letrozole 0.8 mg/kg, and cyclophosphamide þ L-BLP25 þ letrozole. Tamoxifen and L-BLP25 combination studies used 48 MMT female mice assigned to five treatment groups: untreated, cyclophosphamide þ placebo, cyclophosphamide þ L-BLP25, tamoxifen 50 mg/kg, and cyclophosphamide þ L-BLP25 þ tamoxifen 50 mg/kg group. Mice were injected subcutaneously with L-BLP25 (10 mg) weekly for 8 weeks. Serum cytokines were serially measured using a Luminex assay, whereas splenocytes at termination were analyzed by ELISpot to determine T-helper (T H )1/T H 2 polarization of immune response.Results: Daily oral doses of 50 and 0.8 mg/kg of tamoxifen and letrozole, respectively, resulted in a significant survival advantage over controls (P < 0.05). A predominant T H 1-polarized immune response in vaccinated mice was seen with or without tamoxifen or letrozole treatments. In the L-BLP25 plus letrozole treatment group, statistically significant (P < 0.05) additive antitumor activity was observed, whereas tamoxifen plus L-BLP25 was not significantly different (P > 0.05). Conclusion:The results of this study show that hormonal therapy does not interfere with L-BLP25-induced predominant T H 1 response, and the combination of L-BLP25 with letrozole has additive antitumor activity in the MMT mouse model.

887noticeable improvement in about two thirds of the remainder. About one sixth of the total cases showed slight or no improvement.4 These differences between their and our results were mainly due to an inadequate dose of the drug being given for an inadequate time. We found that nodular lesions responded quickly and depigmented macules very slowly. Our high dose and longer duration of treatment were based on the fact that sodium stibogluconate is quickly excreted in the urine and six hours after an intravenous injection blood concentrations have fallen to less than 1% of peak values. The danger of cumulative toxicity might be exaggerated.5We thank Dr R S Jha, department of pathology; Mr R J Sharma, technician;and Mr C P Singh, who typed the manuscript. We received financial support from the United Nations Development Programme/World Bank/World Health Organisation special programme for research and training in tropical disease.