Abstract
Triple negative breast cancer (TNBC) represents a subgroup of mammary cancers associated with particularly poor prognosis as they are refractory to currently available targeted therapy used to treat other breast cancers. Opposed In contrast to the normal 60 mg/day dose of raloxifene used to reduce the risk of estrogen receptor-α (ER)-positive breast cancer, low dose raloxifene was previously shown to be effective at reducing tumor growth in xenografts of TNBC that lack the ER expression. This study aimed to verify the TNBC growthinhibition induced by low dose raloxifene and to investigate molecular changes within the tumor to gain insights into the mechanism of raloxifene action. For this female CD1 athymic nude mice (5-6 weeks old) were implanted with MDA-MB-468 cells (8 × 106) and once a palpable tumor of approximately 200 mm3 had formed, the mice were randomly assigned into three treatment groups: 0.25% DMSO (vehicle; n=9), 0.5 mg/kg raloxifene (n=11) and 0.85 mg/kg raloxifene (n=12). Mice were treated daily for 10 weeks via oral gavage and the tumor volume was measured weekly. Upon necropsy, tumors were weighed, sliced in half and each half was appropriately prepared for immunohistochemistry (IHC) and Western blotting. Treatment with 0.85 mg/kg raloxifene treatment, resulted in a significant reduction in tumor volume of 32% compared to control, whereas the 0.5 mg/kg raloxifene dose decreased tumor size by 23%. These results were further supported by a reduction of up to 40% in tumor weight. Immunohistochemical analysis of tumor tissue showed that expression of Ki67 was significantly increased by at least 50% in tumors from both raloxifene groups. The results from the Western blot analysis showed that NF-κB expression was reduced by 37% in tumors treated with 0.85 mg/kg raloxifene. Furthermore, p38 activation was reduced by 80% in tumors treated with raloxifene compared to control. Expression of the ER was confirmed to be absent in the tumors tested. Surprisingly expression of EGFR was increased by 85% in tumors from the 0.5 mg/kg raloxifene group and by 115% in tumors from the 0.85 mg/kg raloxifene group. These results indicated that raloxifene reduced cell proliferation, mediated through a mechanism involving the inhibition of proliferative mediators NF-κB and p38 rather than acting as an ER antagonist. The possibility remains however that other proteomic or genomic alterations induced by raloxifene are responsible for the reduction in tumor growth. Overall, this study showed that raloxifene effectively reduced tumor growth in a xenograft model of TNBC at a dose 15-fold lower than the currently used dose in the clinic for ER-positive breast cancer.