An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment

Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment.To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays).Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases

Multiple mechanisms mediating carbon monoxide inhibition of the voltage-gated K+ channel Kv1.5

The voltage-gated K+ channel plays key roles in the vasculature and in atrial excitability, and contributes to apoptosis in various tissues. In this study, we have explored its regulation by carbon monoxide (CO), a product of the cytoprotective heme oxygenase enzymes, and a recognized toxin. CO inhibited recombinant Kv1.5 expressed in HEK293 cells in a concentration-dependent manner which involved multiple signalling pathways. CO inhibition was partially reversed by superoxide dismutase mimetics, and by suppression of mitochondrial reactive oxygen species. CO also elevated intracellular nitric oxide (NO) levels. Prevention of NO formation also partially reversed CO inhibition of Kv1.5, as did inhibition of soluble guanylyl cyclase. CO also elevated intracellular peroxynitrite levels, and a peroxynitrite scavenger markedly attenuated the ability of CO to inhibit Kv1.5. CO caused nitrosylation of Kv1.5, an effect which was also observed in C331A and C346A mutant forms of the channel, which had previously been suggested as nitrosylation sites within Kv1.5. Augmentation of Kv1.5 via exposure to hydrogen peroxide was fully reversed by CO. Native Kv1.5 recorded in HL-1 murine atrial cells was also inhibited by CO. Action potentials recorded in HL-1 cells were increased in amplitude and duration by CO, an effect mimicked and occluded by pharmacological inhibition of Kv1.5. Our data indicate that Kv1.5 is a target for modulation by CO via multiple mechanisms. This regulation has important implications for diverse cellular functions, including excitability, contractility and apoptosis

Exposure to general anesthesia and risk of alzheimer's disease: a systematic review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) is common among older adults and leads to significant disability. Volatile anesthetic gases administered during general anesthesia (GA) have been hypothesized to be a risk factor for the development of AD. The objective of this study is to systematically review the association between exposure to GA and risk of AD.</p> <p>Methods</p> <p>We searched electronic databases including MEDLINE, Embase, and Google scholar for observational studies examining the association between exposure to GA and risk of AD. We examined study quality using a modified version of the Newcastle-Ottawa risk of bias assessment for observational studies. We used standard meta-analytic techniques to estimate pooled odds ratios (OR) and 95% confidence intervals (CI). Subgroup and sensitivity analyses were undertaken to evaluate the robustness of the findings.</p> <p>Results</p> <p>A total of 15 case-control studies were included in the review. No cohort studies were identified that met inclusion criteria. There was variation in the methodological quality of included studies. There was no significant association between any exposure to GA and risk of AD (pooled OR: 1.05; 95% CI: 0.93 - 1.19, Z = 0.80, <it>p </it>= 0.43). There was also no significant association between GA and risk of AD in several subgroup and sensitivity analyses.</p> <p>Conclusions</p> <p>A history of exposure to GA is not associated with an increased risk of AD although there are few high-quality studies in this area. Prospective cohort studies with long-term follow-up or randomized controlled trials are required to further understand the association between GA and AD.</p

Effects of G/A polymorphism, rs266882, in the androgen response element 1 of the PSA gene on prostate cancer risk, survival and circulating PSA levels

Prostate-specific antigen (PSA) is a protease produced in the prostate that cleaves insulin-like growth factor binding protein-3 and other proteins. Production is mediated by the androgen receptor (AR) binding to the androgen response elements (ARE) in the promoter region of the PSA gene. Studies of a single nucleotide polymorphism (PSA −158 G/A, rs266882) in ARE1 of the PSA gene have been conflicting for risk of prostate cancer and effect on plasma PSA levels. In this nested case–control analysis of 500 white cases and 676 age- and smoking-matched white controls in the Physicians' Health Study we evaluated the association of rs266882 with risk and survival of prostate cancer and prediagnostic total and free PSA plasma levels, alone or in combination with AR CAG repeats. We used conditional logistic regression, linear regression and Cox regression, and found no significant associations between rs266882 (GG allele vs AA allele) and overall prostate cancer risk (RR=1.21, 95% confidence intervals (CI): 0.88–1.67) or prostate cancer-specific survival (RR=0.94, 95%CI: 0.56–1.58). Similarly, no associations were found among high grade or advanced stage tumours, or by calendar year of diagnosis. There was no significant association between rs266882 and baseline total or free PSA levels or the AR CAG repeats, nor any interaction associated with prostate cancer risk. Meta-analysis of 12 studies of rs266882 and overall prostate cancer risk was null

Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line

<p>Abstract</p> <p>Background</p> <p>The mammalian FOXO (forkhead box, O subclass) proteins are a family of pleiotropic transcription factors involved in the regulation of a broad range of cellular processes critical for survival. Despite the essential and diverse roles of the FOXO family members in human cells and their involvement in tumor pathogenesis, the regulation of <it>FOXO </it>expression remains poorly understood. We have addressed the mechanisms underlying the high level of expression of the <it>FOXO1A </it>gene in a cell line, PER-453, derived from a primitive neuroectodermal tumor of the central nervous system (CNS-PNET).</p> <p>Methods</p> <p>The status of the <it>FOXO1A </it>locus in the PER-453 CNS-PNET cell line was investigated by Southern blotting and DNA sequence analysis of the proximal promoter, 5'-UTR, open reading frame and 3'-UTR. FOXO1A expression was assessed by conventional and quantitative RT-PCR, Northern and Western blotting.</p> <p>Results</p> <p>Quantitative real-time RT-PCR (qRT-PCR) data indicated that after normalization to <it>ACTB </it>mRNA levels, canonical <it>FOXO1A </it>mRNA expression in the PER-453 cell line was 124-fold higher than the average level of five other CNS-PNET cell lines tested, 24-fold higher than the level in whole fetal brain, and 3.5-fold higher than the level in fetal brain germinal matrix cells. No mutations within the <it>FOXO1A </it>open reading frame or gross rearrangements of the <it>FOXO1A </it>locus were detected. However, a single nucleotide change within the proximal promoter and several nucleotide changes within the 3'-UTR were identified. In addition, two novel <it>FOXO1A </it>transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR.</p> <p>Conclusion</p> <p>The CNS-PNET cell line, PER-453, expresses <it>FOXO1A </it>at very high levels relative to most normal and cancer cells from a broad range of tissues. The <it>FOXO1A </it>open reading frame is wild type in the PER-453 cell line and the abnormally high <it>FOXO1A </it>mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter. Over expression of <it>FOXO1A </it>may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.</p

Very Late Antigen-4 (α<inf>4</inf>β<inf>1</inf> Integrin) Targeted PET Imaging of Multiple Myeloma

Biomedical imaging techniques such as skeletal survey and 18F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30-50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α4β1 integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, 64Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of 64Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with 64Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, 64Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using 64Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors. © 2013 Soodgupta et al

Osteocyte deficiency in hip fractures

Osteocytes play a central role in the regulation of bone remodeling. The aim of this study was to explore osteocyte function, and particularly the expression of SOST, a Wnt inhibitor, in patients with hip fractures. Serum sclerostin levels were measured by ELISA. The expression of several osteocytic genes was studied by quantitative PCR in trabecular samples of the femoral head of patients with hip fractures, hip osteoarthritis and control subjects. The presence of sclerostin protein and activated caspase 3 was revealed by immunostaining. There were no significant differences in serum sclerostin between the three groups. Patients with fractures have fewer lacunae occupied by osteocytes (60 ± 5% vs. 64 ± 6% in control subjects, P = 0.014) and higher numbers of osteocytes expressing activated caspase 3, a marker of apoptosis. The proportion of sclerostin-positive lacunae was lower in patients with fractures than in control subjects (34 ± 11% vs. 69 ± 10%, P = 2 × 10(-8)). The proportion of sclerostin-positive osteocytes was also lower in patients. RNA transcripts of SOST, FGF23 and PHEX were also less abundant in fractures than in control bones (P = 0.002, 5 × 10(-6), and 0.04, respectively). On the contrary, in patients with osteoarthritis, there was a decreased expression of SOST and FGF23, without differences in PHEX transcripts or osteocyte numbers. Osteocyte activity is altered in patients with hip fractures, with increased osteocyte apoptosis and reduced osteocyte numbers, as well as decreased transcription of osteocytic genes. Therefore, these results suggest that an osteocyte deficiency may play a role in the propensity to hip fractures

Medulloblastoma outcome is adversely associated with overexpression of EEF1D, RPL30, and RPS20 on the long arm of chromosome 8

BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood. Improvements in clinical outcome require a better understanding of the genetic alterations to identify clinically significant biological factors and to stratify patients accordingly. In the present study, we applied cytogenetic characterization to guide the identification of biologically significant genes from gene expression microarray profiles of medulloblastoma. METHODS: We analyzed 71 primary medulloblastomas for chromosomal copy number aberrations (CNAs) using comparative genomic hybridization (CGH). Among 64 tumors that we previously analyzed by gene expression microarrays, 27 were included in our CGH series. We analyzed clinical outcome with respect to CNAs and microarray results. We filtered microarray data using specific CNAs to detect differentially expressed candidate genes associated with survival. RESULTS: The most frequent lesions detected in our series involved chromosome 17; loss of 16q, 10q, or 8p; and gain of 7q or 2p. Recurrent amplifications at 2p23-p24, 2q14, 7q34, and 12p13 were also observed. Gain of 8q is associated with worse overall survival (p = 0.0141), which is not entirely attributable to MYC amplification or overexpression. By applying CGH results to gene expression analysis of medulloblastoma, we identified three 8q-mapped genes that are associated with overall survival in the larger group of 64 patients (p < 0.05): eukaryotic translation elongation factor 1D (EEF1D), ribosomal protein L30 (RPL30), and ribosomal protein S20 (RPS20). CONCLUSION: The complementary use of CGH and expression profiles can facilitate the identification of clinically significant candidate genes involved in medulloblastoma growth. We demonstrate that gain of 8q and expression levels of three 8q-mapped candidate genes (EEF1D, RPL30, RPS20) are associated with adverse outcome in medulloblastoma

The Appearance and Modulation of Osteocyte Marker Expression during Calcification of Vascular Smooth Muscle Cells

Vascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment.In the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1(-/-) mouse aortic tissue.This study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention