Modeling vitamin D insufficiency and moderate deficiency in adult mice via dietary cholecalciferol restriction.

PURPOSE: We sought to develop and characterize a model of human vitamin D nutritional insufficiency/deficiency in the adult mouse, which could have broad utility in examining health consequences of this common condition. METHODS: Adult mice were fed diets containing cholecalciferol contents of 0.05 IU/g, 0.25 IU/g, 0.5 IU/g or 1.5 IU/g for four months. We studied induction of steady-state vitamin D insufficiency, and its consequences on primary cholecalciferol metabolite levels, calcium homeostasis, parathyroid physiology, and bone morphology. RESULTS: All diets were well tolerated, without adverse effects on body weight. Diets containing 0.05 IU/g and 0.25 IU/g cholecalciferol significantly lowered serum 25-hydroxyvitamin D levels (median 25OHD, 10.5 ng/ml, and 21.6 ng/ml, respectively), starting as early as one month following initiation of the diets, maintained through the four-month experimental period. The 0.05 IU/g diet significantly decreased 1,25-dihydroxyvitamin D (1,25OH2D) levels (median, 78 pg/ml). Despite these decreased 25OHD and 1,25OH2D levels, the diets did not alter parathyroid gland morphology or parathyroid cell proliferation. There were no statistical differences in the serum total calcium and serum PTH levels among the various dietary groups. Furthermore, the 0.05 IU/g diet did not cause any alterations in the cortical and trabecular bone morphology, as determined by microCT. CONCLUSIONS: The dietary manipulations yielded states of vitamin D insufficiency or modest deficiency in adult mice, with no overtly detectable impact on parathyroid and bone physiology, and calcium homeostasis. This model system may be of value to study health effects of vitamin D insufficiency/deficiency especially on extraskeletal phenotypes such as cancer susceptibility or immune function.

Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis.

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

Regulation of Osteoblast Migration Involving Receptor Activator of Nuclear Factor-kappa B (RANK) Signaling.

Bone remodeling requires osteoclast activation, resorption, and reversal, prior to osteoblast migration into the bone pit. The Receptor Activator of NF-κB (RANK) signaling pathway plays an important role in bone remodeling. Two components of the RANK signaling pathway, RANK Ligand (RANKL) and the decoy receptor Osteoprotegerin (OPG), are expressed predominantly on the surface of osteoblasts, while RANK is principally expressed on the surface of osteoclasts. However, RANK has also been reported to be expressed on the surface of osteoblasts and osteosarcoma tumor cells. Treatment with soluble RANKL (sRANKL) of both normal osteoblasts and osteosarcoma tumor cells activated phosphorylation of ERK, p38(MAPK) , Akt, and p65(NF-κB). However, modified Boyden chamber assays and wound repair assays showed differential response to sRANKL-induced chemotactic migration in normal osteoblasts and osteosarcoma tumor cells. In contrast to previously published results, both normal osteoblasts and osteosarcoma tumor cells responded to sRANKL-induced chemotactic migration but the normal osteoblasts did so only in the presence of an ERK pathway inhibitor. For both normal and tumor cells, the chemotactic response could be blocked by inhibiting the PI3K/Akt or p65(NF-κB) pathway. Response to sRANKL in normal and tumor cells suggests a role for RANK/ERK-mediated signaling in normal osteoblasts chemotactic migration during bone remodeling that is altered or lost during osteosarcoma tumorigenesis.

ChIP sequencing of cyclin D1 reveals a transcriptional role in chromosomal instability in mice.

Chromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1(-/-) mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNA-bound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1-rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland-targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability.

Tissue-specific regulatory regions of the PTH gene localized by novel chromosome 11 rearrangement breakpoints in a parathyroid adenoma.

Parathyroid adenomas can contain clonal rearrangements of chromosome 11 that activate the cyclin D1 oncogene through juxtaposition with the PTH gene. Here we describe such a chromosomal rearrangement whose novel features provide clues to locating elusive cis-regulatory elements in the PTH gene and also expand the physical spectrum of pathogenetic breakpoints in the cyclin D1 gene region. Southern blot analyses of the parathyroid adenoma revealed rearrangement in the PTH gene locus. Analysis of rearranged DNA clones that contained the breakpoint, obtained by screening a tumor genomic library, pinpointed the breakpoint in the PTH locus 3.3 kb upstream of the first exon. Accordingly, highly conserved distal elements of the PTH 5' regulatory region were rearranged at the breakpoint approximately 450 kb upstream of the cyclin D1 oncogene, resulting in overexpression of cyclin D1 mRNA. Thus, PTH-cyclin D1 gene rearrangement breakpoints in parathyroid tumors can be located far from those previously recognized. In addition to expanding the molecular spectrum of pathogenetic chromosomal lesions in this disease, features of this specific rearrangement reinforce the existence of one or more novel cis-enhancer/regulatory elements for PTH gene expression and narrow their location to a 1.7-kb DNA segment in the distal PTH promoter.

Comprehensive mutational analysis and mRNA isoform quantification of TP63 in normal and neoplastic human prostate cells.

BACKGROUND: The role of TP63 in cancer remains controversial since both oncogenic and tumor suppressive actions have been reported. p63 protein is found in the nuclei of basal cells of the normal prostate, yet it is absent in the vast majority of prostate cancer nuclei. Since a complex array of TP63 mRNA transcripts encode polypeptides with distinct functional properties, it is important to determine which forms are expressed in normal and prostate cancer tissue. METHODS: We used real-time RT-PCR to distinguish TP63 mRNA isoforms in prostate cancer cell lines (n = 7), and samples from prostate cancer patients. We sequenced all TP63 exons from 20 primary tumors, 20 metastases, 28 tumor xenografts, and 7 prostate cancer cell lines. RESULTS: TP63 mRNA isoforms were present in all tumors, albeit at levels lower than in normal prostate. The most abundant N-terminal variant was DeltaN; the most abundant C-terminal variant was the alpha form. The prostate tumor cell line CWR22Rv1 contained a single G to T substitution in exon 8 that is identical to a dominant-negative DNA binding inactivation mutation occurring in patients with a congenital TP63 deficiency syndrome. One patient tumor contained a somatic mutation in exon 11. CONCLUSIONS: The pattern of TP63 mRNA expression in normal prostate tissue is retained in reduced amounts in prostate cancer, and a potentially functional TP63 mutation was identified in one prostate tumor. Thus, if TP63 is a prostate cancer gene it likely functions as a tumor suppressor. Further study of the role of TP63 isoforms in regulating stem cell functions of normal and neoplastic prostate epithelial cells is needed. Prostate 69:559-569, 2009. (c) 2009 Wiley-Liss, Inc.

Somatic mutations in SQSTM1 detected in affected tissues from patients with sporadic Paget's disease of bone.

Paget's disease of bone (PDB) is a focal disorder of bone remodeling that leads to overgrowth of affected bone, with rare progression to osteosarcoma. Extensive studies of familial PDB showed that a majority of cases harbor germline mutations in the Sequestosome1 gene (SQSTM1). In contrast, little is known about the mutational status of SQSTM1 in sporadic PDB. We hypothesized that somatic SQSTM1 mutations might occur in the affected tissues of sporadic PDB and pagetic osteosarcoma. We used laser capture microdissection to capture homogeneous populations of cells from the affected bone or tumor of patients with sporadic PDB or pagetic osteosarcoma, respectively. DNA from these samples and appropriate controls was used for sequence analysis and allelic discrimination analysis. Two of five patients with sporadic PDB had SQSTM1(C1215T) mutations detected in their affected bone but not in their blood samples, indicating a somatic origin of the mutations. Samples from three of five sporadic pagetic osteosarcoma patients had the SQSTM1(C1215T) mutation, whereas the normal adjacent tissue from two of these tumors clearly lacked the mutation, again indicating an occurrence of somatic events. No SQSTM1 mutations were found in primary adolescent osteosarcomas. The discovery of somatic SQSTM1 mutations in sporadic PDB and pagetic osteosarcoma shows a role for SQSTM1 in both sporadic and inherited PDB. The discovery of somatically acquired mutations in both the diseased bone and tumor samples suggests a paradigm shift in our understanding of this disease.

Expression of secretory phospholipase A2 in colon tumor cells potentiates tumor growth.

Secretory phospholipase A2 (sPLA2-IIA) has been shown to attenuate intestinal tumorigenesis in Apc(Min) mice, demonstrating that it is a tumor modifier. To further explore the actions of sPLA2-IIA in tumorigenesis, sPLA2-IIA was overexpressed in two cell lines where it is normally absent, the murine colon tumor cell line AJ02nm0, and human colon carcinoma cell line HCT-116. Two allelic variants of sPLA2-IIA were tested in this study; sPLA2-IIA(AKR) and sPLA2-IIA(SWR), which are derived from AKR/J and SWR/J mice, respectively, and differ by a single amino acid at position 63 in the calcium- and receptor-binding domain. There was no change in cell-doubling time for either allele when compared to vector controls. Furthermore, sodium butyrate and arachidonic acid (AA)-induced cell death were unchanged in control and transfected cells. Addition of the sPLA2 substrate, palmitoyl-arachidonoyl-phosphatidic acid (PAPA), to AJ02nm0 cells resulted in a modest (12%-24%), but significant (P < 0.01), inhibition of growth that was dependent on sPLA2-IIA expression. However, when AJ02nm0 and HCT-116 cells were injected subcutaneously (sc) into nude mice, Pla2g2a expression resulted in a 2.5-fold increase in tumor size. In addition, sPLA2-IIA expressing HCT-116 tumors were found to be more infiltrative than controls. We conclude that the ability of sPLA2-IIA to slow tumor cell growth is dependent upon the availability of substrate, and that in some instances sPLA2-IIA may actually enhance tumor growth. Mechanisms that may account for differences between the tumor explant model versus the Apc(Min) model of intestinal cancer are discussed.