Vitamin D Regulates CXCL12/CXCR4 and Epithelial-to-Mesenchymal Transition in a Model of Breast Cancer Metastasis to Lung.

Vitamin D deficiency is associated with poor cancer outcome in humans, and administration of vitamin D or its analogs decreases tumor progression and metastasis in animal models. Using the mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) model of mammary cancer, we previously demonstrated a significant acceleration of carcinogenesis in animals on a low vitamin D diet and a reduction in spontaneous lung metastases when mice received vitamin D through perfusion. We investigate here the action mechanism for vitamin D in lung metastasis in the same non-immunodeficient model and demonstrate that it involves the control of epithelial to mesenchymal transition as well as interactions between chemokine C-X-C motif chemokine 12 (CXCL12) and its receptor C-X-C chemokine receptor type 4 (CXCR4). In vitro, 10-9M vitamin D treatment modifies the phenotype of MMTV-PyMT primary mammary tumor cells and significantly decreases their invasiveness and mammosphere formation capacity by 40% and 50%, respectively. Vitamin D treatment also inhibits phospho-signal transducer and activator of transcription 3 (p-STAT3), zinc finger E-box-binding homeobox 1 (Zeb1), and vimentin by 52%, 75%, and 77%, respectively, and increases E-cadherin by 87%. In vivo, dietary vitamin D deficiency maintains high levels of Zeb1 and p-STAT3 in cells from primary mammary tumors and increases CXCL12 expression in lung stroma by 64%. In lung metastases, vitamin D deficiency increases CXCL12/CXCR4 co-localization by a factor of 2.5. These findings indicate an involvement of vitamin D in mammary cancer "seed" (primary tumor cell) and "soil" (metastatic site) and link vitamin D deficiency to epithelial-to-mesenchymal transition (EMT), CXCL12/CXCR4 signaling, and accelerated metastasis, suggesting vitamin D repleteness in breast cancer patients could enhance the efficacy of co-administered therapies in preventing spread to distant organs.

Author Correction: An atlas of genetic influences on osteoporosis in humans and mice.

In the version of this article initially published, in Fig. 5a, the data in the right column of 'DAAM2 gRNA1' were incorrectly plotted as circles indicating 'untreated' rather than as squares indicating 'treated'. The error has been corrected in the HTML and PDF versions of the article.

An atlas of genetic influences on osteoporosis in humans and mice.

Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). We assessed genetic determinants of BMD as estimated by heel quantitative ultrasound in 426,824 individuals, identifying 518 genome-wide significant loci (301 novel), explaining 20% of its variance. We identified 13 bone fracture loci, all associated with estimated BMD (eBMD), in ~1.2 million individuals. We then identified target genes enriched for genes known to influence bone density and strength (maximum odds ratio (OR) = 58, P = 1 × 10-75) from cell-specific features, including chromatin conformation and accessible chromatin sites. We next performed rapid-throughput skeletal phenotyping of 126 knockout mice with disruptions in predicted target genes and found an increased abnormal skeletal phenotype frequency compared to 526 unselected lines (P < 0.0001). In-depth analysis of one gene, DAAM2, showed a disproportionate decrease in bone strength relative to mineralization. This genetic atlas provides evidence linking associated SNPs to causal genes, offers new insight into osteoporosis pathophysiology, and highlights opportunities for drug development.

Tumoral Vitamin D Synthesis by CYP27B1 1-α-Hydroxylase Delays Mammary Tumor Progression in the PyMT-MMTV Mouse Model and Its Action Involves NF-κB Modulation.

Biologically active vitamin D (1,25-dihydroxycholecalciferol or 1,25(OH)2D) is synthetized from inactive prohormone 25-hydroxycholecalciferol (25(OH)D) by the enzyme CYP27B1 1-α-hydroxylase in kidney and several extrarenal tissues including breast. Although the development of breast cancer has been linked to inadequate vitamin D status, the importance of bioactive vitamin D production within tumors themselves is not fully understood. To investigate the role of tumoral vitamin D production in mammary epithelial cell progression to breast cancer, we conducted a Cre-loxP-mediated Cyp27b1 gene ablation in the mammary epithelium of the polyoma middle T antigen-mouse mammary tumor virus (PyMT-MMTV) mouse breast cancer model. Targeted ablation of Cyp27b1 was accompanied by significant acceleration in initiation of spontaneous mammary tumorigenesis. In vivo, cell proliferation, angiogenesis, cell cycle progression, and survival markers were up-regulated in tumors by Cyp27b1 ablation, and apoptosis was decreased. AK thymoma (AKT) phosphorylation and expression of several components of nuclear factor κB (NF-κB), integrin, and signal transducer and activator of transcription 3 (STAT3) signaling pathways were increased in Cyp27b1-ablated tumors compared with nonablated controls. In vitro, 1,25(OH)2D treatment induced a strong antiproliferative action on tumor cells from both ablated and nonablated mice, accompanied by rapid disappearance of NF-κB p65 from the nucleus and segregation in the cytoplasm. In contrast, treatment with the metabolic precursor 25(OH)D was only effective against cells from nonablated mice. 25(OH)D did not inhibit growth of Cyp27b1-ablated cells, and their nuclear NF-κB p65 remained abundant. Our findings demonstrate that in-tumor CYP27B1 1-α-hydroxylase activity plays a crucial role in controlling early oncogene-mediated mammary carcinogenesis events, at least in part by modulating tumoral cell NF-κB p65 nuclear translocation.

Chemoprevention activity of 25-hydroxyvitamin D in the MMTV-PyMT mouse model of breast cancer.

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)2D), 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 (25(OH)D) can delay tumor progression in vivo without hypercalcemic effects. A low vitamin D diet (25 IU/kg) in the non-immunodeficient MMTV-PyMT mouse model of metastatic breast cancer revealed a significant acceleration of mammary neoplasia compared with normal diet (1,000 IU/kg). Systemic perfusion of MMTV-PyMT mice with 25(OH)D or 1,25(OH)2D delayed tumor appearance and significantly decreased lung metastasis, and both metabolites reduced Ki-67, cyclin D1, and ErbB2 levels in tumors. Perfusion with 25(OH)D caused a 50% raise in tumor 1,25(OH)2D levels, indicating good tumor penetration and effective activation. Importantly, in contrast with 1,25(OH)2D, perfusion with 25(OH)D did not cause hypercalcemia. In vitro treatment of cultured MMTV-PyMT mammary tumor cells with 25(OH)D inhibited proliferation, confirming local activation of the prohormone in this system. This study provides an in vivo demonstration in a non-immunodeficient model of spontaneous breast cancer that exogenous 25(OH)D delays neoplasia, tumor growth, and metastasis, and that its chemoprevention efficacy is not accompanied by hypercalcemia.

Enhancement of taxol, doxorubicin and zoledronate anti-proliferation action on triple-negative breast cancer cells by a PTHrP blocking monoclonal antibody.

Triple-negative breast cancers (TNBCs) are heterogeneous cancers that present tumors without the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Because of the absence of these receptors, there are currently no known specific molecular targets for treatment, and although TNBC tumors are chemosensitive, prognosis is poor because this type of cancer relapses more frequently and more aggressively than hormone receptor-positive cancers. The mechanisms by which TNBCs escape control by chemotherapy are not clear, and it is crucial to identify novel molecular drivers that can be targeted in order to develop more efficient therapeutic approaches. We recently highlighted a pleiotropic role for parathyroid hormone-related protein (PTHrP) in all stages of breast cancer, and used our neutralizing anti-PTHrP monoclonal antibody (mAb M158) to efficiently inhibit progression and metastasis of human breast cancer xenografts in athymic mice. In the present study, we present evidence for a strong in vitro anti-proliferative effect of our blocking anti-PTHrP mAb M158 as a single agent on TNBC lines of various subtypes that are known to express PTHrP (MDA-MB-231, BT-549, MDA-MB-435). The same mAb is inactive in a TNBC line without detectable PTHrP expression (MDA-MB-468). In in vitro combination studies, the mAb enhances the effect of the chemotherapeutic drugs taxol and doxorubicin in PTHrP-positive TNBC cells in an additive manner. When combined with the bisphosphonate zoledronate, M158 can act in additive or antagonistic fashion in vitro depending on the cell line. Our observations identify PTHrP as a novel target against TNBC cell proliferation, and suggest that combination therapies that include an anti-PTHrP approach might increase treatment efficacy in patients with PTHrP-positive TNBC.

Switchable-polarity solvents prepared with a single liquid component.

Known liquids that can reversibly switch their polarity at atmospheric pressure are all prepared as mixtures of two liquid components; we now report a series of switchable-polarity solvents that consist, in their low-polarity form, of only a single liquid component, a secondary amine. These solvents operate in a polarity range that is significantly lower than those of previously reported switchable solvents. Application to the separation and purification of a polymer and recovery of a catalyst is described.