Breast cancer in humans is complex involving different histopathologies, genetic changes and clinical outcomes. These observations indicate that a single animal model may not mimic all features of human breast carcinogenesis (1
). In the present study, we evaluated the inhibitory effects of Gemini vitamin D analogs on tumorigenesis in two different models: an ER-positive NMU-induced breast cancer model in rats and an ER-negative MCF10DCIS.com
xenograft model in immunodeficient mice. NMU-induced mammary tumors showed similar gene expression profiles as were found in luminal type ER-positive, low to intermediate grade human breast cancer (28
). Selective estrogen receptor modulators, such as tamoxifen, raloxifene or arzoxifene, have shown inhibitory effects in the NMU model (29
), suggesting that this model may be a good ER-positive animal model of breast cancer for testing cancer preventive drugs.
As a xenograft model of breast cancer, we selected the MCF10DCIS.com
cell line, which is known to be a basal-like ER-negative cell type (24
human breast cancer cells are derived from Ha-ras
oncogene transfection and further passages in mice (30
). In addition to genetic ras
cells significantly overexpressed the breast cancer associated marker, Her-2, compared to the parent cells, MCF10A (unpublished data), and mammary tumors generated from MCF10DCIS.com
cells were shown to dynamically interact with stroma during progression from in situ
to invasive cancer (31
). We used this cell line for both in vitro
and in vivo
studies. Although xenograft models may not be the best model for testing preventive effects of Gemini vitamin D analogs, MCF10DCIS.com
model represents DCIS progression to invasive ductal carcinoma, which can be useful for testing new compounds for inhibition of breast cancer progression.
In this report, we demonstrated that several Gemini vitamin D analogs had better efficacy for growth inhibition of MCF10DCIS.com
cells than 1α,25(OH)2
() and that certain Gemini vitamin D analogs significantly suppressed tumor growth in both an ER-positive NMU-induced breast cancer model and in an ER-negative MCF10DCIS.com
xenograft model without hypercalcemic toxicity (, ). Hypercalcemia is a well-established toxicity induced by 1α,25(OH)2
and for many classical vitamin D derivatives. We found that 1α,25(OH)2
significantly increased the serum calcium level at doses showing tumor growth inhibition (). In contrast, Gemini 0097 exerted potent efficacy in preventing mammary tumor growth without causing hypercalcemia (). These results are in agreement with previous reports using a colon cancer model (19
). The lack of hypercalcemic effects of Gemini vitamin D analogs at anticancer doses may be because of the flexibility of the ligand binding pocket in the vitamin D receptor (32
). Recently, it was reported that structural rearrangement of the ligand binding pocket provides space to accommodate the second side chain of Gemini vitamin D analogs, which leads to expansion of the ligand binding pocket volume (33
). This conformational flexibility of the ligand binding pocket to accommodate different ligands may result in different ligand-specific cofactor binding and/or selectivity of transcription of target genes (32
), thus suggesting that Gemini vitamin D analogs may show different regulation of vitamin D target genes when compared with 1α,25(OH)2
In our mechanistic study, the expression levels of cyclin dependent kinase (CDK) inhibitor p21 and insulin-like growth factor binding protein-3 (IGFBP-3) in tumor tissues from both models were enhanced by Gemini 0097 treatment ( and ). Interestingly, however, the markers for apoptosis including cleaved PARP and caspase-3 were regulated by Gemini 0072 and 0097 in ER-positive mammary tumors induced by carcinogen (), but not in ER-negative tumors from the xenograft model (data not shown). It has been shown in several studies that 1α,25(OH)2
and its analogs suppress the mRNA and protein synthesis of ER through the negative vitamin D response element (nVDRE) in the ER promoter, which eventually leads to inhibition of cell proliferation through cell cycle arrest and apoptosis (35
). The involvement of ER in inducing apoptosis by Gemini vitamin D analogs in NMU-induced mammary tumors needs to be further investigated.
Insulin-like growth factor binding proteins (IGFBPs), a group of six different proteins, sequester free insulin-like growth factors (IGFs) by binding with high affinity, which inhibits the IGF signaling pathway having mitogenic activity (38
). The expression of IGFBPs has been shown to be up-regulated by 1α,25(OH)2
and certain of its analogs in different cancer cells including prostate (39
), colon (41
), and breast cancer (21
). In breast cancer, several studies suggest that IGFBP-3 acts as a tumor suppressive factor (42
). Recently, the functional vitamin D response element (VDRE) has been identified in the promoter region of the IGFBPs (44
), indicating that IGFBPs may be primary target genes of 1α,25(OH)2
and its analogs. In the present study, we demonstrated that Gemini vitamin D analog treatment increased the expression of IGFBP-3 in breast tumors from both the carcinogen-induced model and the xenograft model, suggesting that IGFBP-3 induction may be mediated via direct transcriptional regulation. Interestingly, IGFBPs were shown to be involved in the regulation of the CDK inhibitor, p21 (46
). IGFBP-3 knockdown by siRNA reversed the induction of p21 and growth inhibition by synthetic androgen R1881 in LNCaP human prostate cancer cells, indicating that IGFBP-3 was upstream of p21 (46
). These studies support our hypothesis that the Gemini vitamin D analog 0097 inhibits tumor growth, at least in part, through the regulation of the link between IGFBP-3 and p21.
In several previous studies, vitamin D and its analogs have been involved in the regulation of transforming growth factor-β (TGF-β) signaling, which is extensively reviewed in Ref (6
). We have also reported that Gemini vitamin D analogs were shown to activate the bone morphogenetic protein (BMP)/Smad signaling pathway via Ras/PKCα, which mediates the proliferation of the MCF10 breast epithelial cells (48
). In this report, we demonstrate for the first time that the Gemini vitamin D analog 0097 activates Smad signaling in vivo
transplanted tumors (), suggesting that the regulation of Smad signaling by vitamin D analogs may contribute to the suppression of mammary tumorigenesis. In addition, we showed that Gemini 0097 suppressed the apolipoprotein A-I protein expression in tumors from both animal models that we tested ( and ). Apolipoprotein A-I is one of the high-density lipoproteins, and 1α,25(OH)2
and other VDR modulators were shown to down-regulate the expression of apolipoprotein A-I in human hepatocytes and intestinal cells (50
). Since a low serum level of high-density lipoproteins, such as apolipoprotein A-I, has been associated with breast cancer, it will be important to determine whether inhibition of apolipoprotein A-I by vitamin D analogs has an effect on the regulation of mammary tumorigenesis.
In conclusion, we investigated the in vivo effect of certain Gemini vitamin D analogs on mammary tumor growth in a chemically-induced breast cancer model and in a xenograft model. We report here that Gemini vitamin D analogs significantly inhibit both ER-positive and ER-negative mammary tumor growth without increasing serum calcium levels. Mechanistic studies showed that the inhibitory activity was associated with the induction of IGFBP-3 and the CDK inhibitor p21 in both animal models. Taken together, these results suggest that Gemini vitamin D analogs may be promising agents for the prevention and treatment of breast cancer without hypercalcemic toxicity.