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Data represent the relative mean CT values from two experiments standard deviation

Data represent the relative mean CT values from two experiments standard deviation. (VDR) is a nuclear hormone receptor that Ikarugamycin protects against mammary tumor formation, progression and metastasis in the presence of 1,25D3 through induction of cell-cycle arrest, promotion of apoptosis, regulation of differentiation, and reduction in angiogenesis (reviewed in [22-25]). Ligand-dependent VDR signaling has been shown to antagonize the -catenin signaling pathway through several mechanisms in human and murine colon cancer cells (reviewed in [26]). For example, induced VDR activation promotes transcriptional upregulation of E-cadherin which inhibits -catenin nuclear localization and induces translocation to adherins junctions at the plasma membrane [27-29]. Ligand-dependent VDR signaling also increases mRNA expression of Dickkopf-related Ikarugamycin protein 1 (DKK-1), an inhibitor of the canonical Wnt signaling pathway, that subsequently prevents the activation of -catenin by way of Wnt signaling [30]. TCF-4 is transcriptionally regulated by VDR in murine colon and breast cancer cells for growth inhibition [31]. In colon cancer, ligand-bound VDR also competes with transcription factors for -catenin binding between the activator function-2 (AF-2) domain of VDR and the C terminus of -catenin [32] thus physically preventing transcriptional activation of TCF/LEF target genes [27]. Given the negative regulation of -catenin signaling by VDR activation, we hypothesized that vitamin D3-dependent VDR signaling would impede the aggressive progression of Ron-mediated breast tumorigenesis. Herein we demonstrate that VDR limits CSH1 Ron-induced mammary tumor initiation and growth by decreasing active Ikarugamycin -catenin levels and through a reduction in -catenin target genes. Further, vitamin D3 treatment reduced breast cancer cell growth, migration, and invasion in Ron expressing breast cancer cells. Moreover, Ron knockdown (KD) further sensitized breast cancer cells to the growth inhibitory effects of vitamin D3, while constitutive activation of -catenin reverted the effects Ikarugamycin of vitamin D3. Mechanistically, 1,25D3 reduced active -catenin levels, decreased -catenin transcriptional activity, increased expression of DKK-1, and diminished the association of active -catenin with the cyclin D1 promoter. Thus, combinational therapies integrating Ron or receptor tyrosine kinase (RTK) antagonists with vitamin D3 or potent vitamin D3 analogs in breast cancers exhibiting Ron overexpression may provide a beneficial alternative to current standard of care chemotherapeutic agents. RESULTS Loss of VDR signaling accelerates epithelial hyperplasia in the mammary glands of MMTV-Ron mice To examine the role of vitamin D receptor (VDR) signaling in the development of Ron-mediated breast tumorigenesis, MMTV-Ron transgenic mice were crossed to mice deficient for VDR (VDR?/?) generating offspring with VDR haploinsufficiency. Littermates were intercrossed to generate VDR+/+, VDR?/? mice or mice heterozygous for a functional VDR with Ron overexpression specific to the mammary epithelium. Analysis of protein and mRNA from transgenic mouse mammary gland lysates shows Ron and VDR expression levels in the MMTV-Ron VDR?/? model (Figures ?(Figures1A1A and ?and1B1B). Open in a separate window Figure 1 VDR signaling delays Ron-mediated mammary Ikarugamycin gland hyperplasiaA. Western blot of mammary lysates from MMTV-Ron VDR+/+ and VDR?/? mice depicting Ron expression levels. B. qRT-PCR analysis of Ron and VDR mRNA expression in mammary glands (MG) and tumors from MMTV-Ron VDR+/+ and VDR?/? mice. Data represent mean values from three independent experiments SE. C. Representative mammary whole mounts (upper panels) and H&E-stained tissue sections (lower panels) from 10 week-old MMTV-Ron VDR+/+ and VDR?/? mammary glands (= 8-11). D. Incidence of mice with hyperplastic mammary glands at 2.5, 4 and 6 months of age from MMTV-Ron VDR+/+ and VDR?/? animals. * 0.05. Previous studies demonstrated that tissues with targeted overexpression of Ron in the mammary epithelium develop hyperplasia by 12 weeks of age [13]. Mammary gland hyperplasia was evident in the majority of MMTV-Ron VDR?/? mice at 10 weeks (Figures ?(Figures1C1C and ?and1D).1D). Contrastingly, most age-matched glands from MMTV-Ron VDR+/+ mice exhibited a normal phenotype at this early time point suggesting that VDR delays Ron-mediated mammary hyperplasia. However, by 4 months of age almost all MMTV-Ron VDR+/+ and VDR?/? mice exhibited severely dilated, cystic acini similar to the mammary epithelium previously reported in MMTV-Ron induced tumorigenesis [13]. VDR signaling delays mammary tumor onset and reduces metastasis to the lungs and liver Mammary tumor formation occurred in all study mice regardless of VDR status. However, MMTV-Ron mice homozygous or heterozygous for VDR exhibited significantly longer times to palpable tumor formation compared to VDR?/? mice (Figure ?(Figure2A2A and inset). The mean time-to-tumor onset was 178.5 days for VDR?/? mice compared to 227 days for VDR+/? and 241.