Parathyroid hormone-related protein (PTHrP) is a secreted factor expressed in almost all normal fetal and adult tissues. It is involved in a wide range of developmental and physiological processes, including serum calcium regulation. PTHrP is also associated with the progression of skeletal metastases, and its dysregulated expression in advanced cancers causes malignancy-associated hypercalcemia. Although PTHrP is frequently expressed by breast tumors and other solid cancers, its effects on tumor progression are unclear. Here, we demonstrate in mice pleiotropic involvement of PTHrP in key steps of breast cancer -it influences the initiation and progression of primary tumors and metastases. Pthrp ablation in the mammary epithelium of the PyMT-MMTV breast cancer mouse model caused a delay in primary tumor initiation, inhibited tumor progression, and reduced metastasis to distal sites. Mechanistically, it reduced expression of molecular markers of cell proliferation (Ki67) and angiogenesis (factor VIII), antiapoptotic factor Bcl-2, cell-cycle progression regulator cyclin D1, and survival factor AKT1. PTHrP also influenced expression of the adhesion factor CXCR4, and coexpression of PTHrP and CXCR4 was crucial for metastatic spread. Importantly, PTHrP-specific neutralizing antibodies slowed the progression and metastasis of human breast cancer xenografts. Our data identify what we believe to be new functions for PTHrP in several key steps of breast cancer and suggest that PTHrP may constitute a novel target for therapeutic intervention.

Interferon g (IFN-g) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN-g ablation in vivo in C57BL/6 mice expressing the IFN-g receptor knockout phenotype (IFNgR1 À/À ). Compared with their wild-type littermates (IFNgR1 þ/þ ), IFNgR1 À/À mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNgR1 À/À mice showed a low-boneturnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone-formation and bone-resorption markers. Furthermore, administration of IFN-g (2000 and 10,000 units) to wildtype C57BL/6 sham-operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN-g signaling as a potential new anabolic therapeutic target for osteoporosis. ß

Interferon (IFN)c is a strong inhibitor of osteoclast differentiation and activity. However, its role in osteoblastogenesis has not been carefully examined. Using microarray expression analysis, we found that several IFNc-inducible genes were upregulated during early phases of osteoblast differentiation of human mesenchymal stem cells (hMSCs). We therefore hypothesized that IFNc may play a role in this process. We first observed a strong and transient increase in IFNc production following hMSC induction to differentiate into osteoblasts. We next blocked this endogenous production using a knockdown approach with small interfering RNA and observed a strong inhibition of hMSC differentiation into osteoblasts with a concomitant decrease in Runx2, a factor indispensable for osteoblast development. Additionally, exogenous addition of IFNc accelerated hMSC differentiation into osteoblasts in a dose-dependent manner and induced higher levels of Runx2 expression during the early phase of differentiation. We next examined IFNc signaling in vivo in IFNc receptor 1 knockout (IFNcR1 2/2

The retinoid X receptor ␣ (RXR␣) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR⅐RXR␣ heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXR␣ via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXR␣, inhibits signaling through several heterodimeric partners of the RXR␣. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D 3 but also of retinoic acid receptor ␣ ligands and LG1069 (an RXR␣ ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXR␣ mutant at serine 260 (RXR␣ Ser-260 3 Ala). Importantly, phosphorylation of RXR␣ at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR⅐RXR␣ complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR⅐RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXR␣-mediated signaling likely through structural changes in the H 1 -H 3 omega loop-AF2 coactivator(s) interacting domain.Ras activation has been detected in numerous cancers, including hepatocellular carcinoma, colorectal carcinomas, breast tumors, leukemias, and squamous tumors of the head and neck (1). Activation of the Ras-Raf-mitogen-activated protein kinase (MAPK-ERK) 2 signaling cascade leads to phosphorylation of downstream targets, including some nuclear receptors, resulting in a mechanism for receptor control (2). Signaling through nuclear receptors is required in many aspects of cellular functions (3). Several nuclear receptors require heterodimerization with the retinoid X receptor (RXR) to fulfill their signaling functions (4). Upon dimerization, the receptors recognize and bind bipartite regions of promoters of target genes, known as response elements, involving a discrete DNA binding domain within the receptors (5). HPK1Aras, a ras-transformed keratinocyte cell line, is resistant to the growth-inhibitory effects of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) (6), as are several pancreatic (7) and breast carcinoma cell lines (8). The phosphorylation of RXR␣ at serine 260 caused the resistance of the HPK1Aras cell line to the antiproliferative effects of 1,25(OH) 2 D 3 (9) as well as resistance to the antiproliferative effect of all-trans-retinoic acid (ATRA) in hepatocellular carcinoma cells (10) . This serine lies within a PSSP MAPK re...

Development of oncologic conditions is often accompanied by inadequate vitamin D status. The chemoprevention ability of this molecule is of high interest for breast cancer, the most common malignancy in women worldwide. Because current effective vitamin D analogues, including the naturally occurring active metabolite 1,25-dihydroxycholecalciferol (1,25(OH) 2 D), frequently cause hypercalcemia at pharmacologic doses, the development of safer molecules for clinical chemopreventive use is essential. This study examines whether exogenously supplied prohormone 25-hydroxycholecalciferol (