Numerous preclinical, epidemiologic, and clinical studies have suggested the benefits of vitamin D and its analogues for the prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1α,25(OH) 2 D 3 , the hormonally active form of vitamin D. To identify vitamin D analogues with better efficacy and low toxicity, we have tested >60 novel Gemini vitamin D analogues with a unique structure of two side chains for growth inhibition of breast cancer cells. Our initial studies found that some Gemini analogues are 5-15 times more active than 1α,25(OH) 2 D 3 in growth inhibition assay. In vivo experiments were designed to study the inhibitory effect of selected Gemini vitamin D analogues against mammary carcinogenesis by using -hexafluoro-19-nor-cholecalciferol] administration inhibited by 60% the NMUinduced mammary tumor burden compared with the NMU-treated control group, but these compounds were devoid of hypercalcemia toxicity. In an ER-negative xenograft model, Gemini 0097 significantly suppressed tumor growth without hypercalcemia toxicity. We found that the inhibitory effect of Gemini 0097 was associated with an increased level of cyclin-dependent kinase inhibitor p21 and the insulin-like growth factor binding protein 3 in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogues may be potent agents for the prevention and treatment of both ER-positive and ER-negative breast cancer without hypercalcemia toxicity.In the United States, breast cancer remains the most frequently diagnosed cancer and the second leading cause of cancer death in women according to Cancer Statistics from the American Cancer Society. Because of the complexity and heterogeneity of mammary carcinogenesis (1), many pharmacologic agents have been studied for their effects on the prevention of breast cancer. For example, selective estrogen receptor (ER) modulators such as tamoxifen and raloxifene have achieved significant reduction of breast cancer incidence in women at high risk (2, 3). However, selective ER modulators are not effective in preventing ER-negative breast cancer, which corresponds to at least one third of the breast cancer cases (4). The vitamin D receptor (VDR), a member of the nuclear receptor superfamily, has been suggested as a target for both ER-positive and ER-negative breast cancer prevention (5, 6) because it is present in most breast tumors (4), and VDR ablation in mice was reported to enhance carcinogeninduced formation of mammary tumors (7). These results suggest a role of vitamin D signaling in the regulation of mammary tumorigenesis.The ligand for VDR, 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ; the key hormone in calcium/phosphate homeostasis) is a hormonally active metabolite synthesized from vitamin D 3 predominantly through hydroxylation by a 25-hydroxylase in the

Numerous preclinical, epidemiological and clinical studies with vitamin D and analogs have suggested the benefits of vitamin D and analogs for prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1α,25(OH)2D3, the hormonally active form of vitamin D, and many of its classical synthetic analogs as potent agents for cancer prevention and treatment. To identify vitamin D analogs with better efficacy and low toxicity, we have tested more than 60 novel Gemini vitamin D analogs, which have a unique structure of two six-carbon chains with a C-20-normal and a C-20-epi side chain. Our initial studies found that some Gemini analogs are many-fold more active than 1α,25(OH)2D3 in growth inhibition assay. We further examined the inhibitory effect of selected Gemini vitamin D analogs against mammary carcinogenesis in vivo by using N-methyl-N-nitrosourea (NMU)-induced mammary tumor model which is ER-positive. Among Gemini vitamin D analogs we tested, Gemini 0072 and Gemini 0097 treated groups showed approximately 60% suppressive activity in NMU-induced mammary tumor growth compared to the control group. In a dose dependent experiment, Gemini 0097 significantly reduced the average tumor burden per rat without affecting the body weight. At all doses tested, Gemini 0097 did not exert any calcemic toxicity. In addition, we determined the inhibitory effect of Gemini 0097 in MCF10DCIS xenograft model of ER-negative mammary tumors. Gemini 0097 significantly inhibited MCF10DCIS xenograft tumor growth without calcemic toxicity. We analyzed the tumor samples and found that the inhibitory effect of Gemini 0097 in vivo was through (a) increasing cyclin dependent kinase inhibitor, p21 and (b) inducing insulin-like growth factor binding protein 3 (IGFBP3) in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogs may be potent agents for the prevention and treatment of both ER positive and ER negative breast cancer without calcemic toxicity.