RUNX3 Facilitates Growth of Ewing Sarcoma Cells

Authors

Krista L. Bledsoe, Meghan E. McGee-Lawrence, Emily T. Camilleri, Xiaoke Wang, Andre J. van Wijnen, Andre M. Oliveira and Jennifer J. Westendorf

Abstract

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma.

Link To Article

http://dx.doi.org/10.1002/jcp.24663

Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta

Authors

Ingo Grafe, Tao Yang, Stefanie Alexander, Erica P Homan, Caressa Lietman, Ming Ming Jiang, Terry Bertin, Elda Munivez, Yuqing Chen, Brian Dawson, Yoshihiro Ishikawa, Mary Ann Weis, T Kuber Sampath, Catherine Ambrose, David Eyre, Hans Peter Bächinger & Brendan Lee

Abstract

Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations1. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms2. Here, we show that excessive transforming growth factor-β (TGF-β) signaling is a mechanism of OI in both recessive (Crtap−/−) and dominant (Col1a2tm1.1Mcbr) OI mouse models. In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap−/− mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-β activity3, 4. Anti–TGF-β treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap−/− mice. Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

Link To Article

http://dx.doi.org/10.1038/nm.3544

Targeting angiogenesis as a therapeutic means to reinforce osteocyte survival and prevent non-unions in the aftermath of radiotherapy

Authors

Alexis Donneys, Noah S. Nelson, Erin E. Page, Sagar S. Deshpande, Peter A. Felice, Catherine N. Tchanque-Fossuo, Joshua P. Spiegel and Steven R. Buchman

Abstract

Background: Radiotherapy exerts detrimental collateral effects on bone tissue through mechanisms of vascular damage and impediments to osteocytes, ultimately predisposing patients to the debilitating problems of late pathologic fractures and non-unions. We posit that angiogenic therapy will reverse these pathologic effects in a rat model of radiated fracture healing.

Methods: Three groups of rats underwent mandibular osteotomy. Radiated groups received a fractionated 35Gy dose prior to surgery. The deferoxamine group received local injections postoperatively. A 40-day healing period was allowed prior to histology. ANOVA (p<0.05) was used for group comparisons.

Results: Radiated fractures revealed a significantly decreased osteocyte count and corresponding increase in empty lacunae when compared to non-radiated fractures (p=0.001). With the addition of deferoxamine, these differences were not appreciated. Further, a 42% increase in bony unions was observed after deferoxamine therapy.

Conclusions: Targeting angiogenesis is a useful means for promoting osteocyte survival and preventing bone pathology after radiotherapy

Link To Article

http://dx.doi.org/10.1002/hed.23744

Adult Brtl/+ mouse model of osteogenesis imperfecta demonstrates anabolic response to sclerostin antibody treatment with increased bone mass and strength

Authors

B. P. Sinder, L. E. White, J. D. Salemi, M. S. Ominsky, M. S. Caird, J. C. Marini, K. M. Kozloff

Abstract

Summary Treatments to reduce fracture rates in adults with osteogenesis imperfecta are limited. Sclerostin antibody, developed for treating osteoporosis, has not been explored in adults with OI. This study demonstrates that treatment of adult OI mice respond favorably to sclerostin antibody therapy despite retention of the OI-causing defect.

Introduction Osteogenesis imperfecta (OI) is a heritable collagen-related bone dysplasia, characterized by brittle bones with increased fracture risk. Although OI fracture risk is greatest before puberty, adults with OI remain at risk of fracture. Antiresorptive bisphosphonates are commonly used to treat adult OI, but have shown mixed efficacy. New treatments which consistently improve bone mass throughout the skeleton may improve patient outcomes. Neutralizing antibodies to sclerostin (Scl-Ab) are a novel anabolic therapy that have shown efficacy in preclinical studies by stimulating bone formation via the canonical wnt signaling pathway. The purpose of this study was to evaluate Scl-Ab in an adult 6 month old Brtl/+ model of OI that harbors a typical heterozygous OI-causing Gly > Cys substitution on Col1a1.

Methods Six-month-old WT and Brtl/+ mice were treated with Scl-Ab (25 mg/kg, 2×/week) or Veh for 5 weeks. OCN and TRACP5b serum assays, dynamic histomorphometry, microCT and mechanical testing were performed.

Results Adult Brtl/+ mice demonstrated a strong anabolic response to Scl-Ab with increased serum osteocalcin and bone formation rate. This anabolic response led to improved trabecular and cortical bone mass in the femur. Mechanical testing revealed Scl-Ab increased Brtl/+ femoral stiffness and strength.

Conclusion Scl-Ab was successfully anabolic in an adult Brtl/+ model of OI.

Link To Article

http://dx.doi.org/10.1007/s00198-014-2737-y

High fluoride and low calcium levels in drinking water is associated with low bone mass, reduced bone quality and fragility fractures in sheep

Authors

M. J. K. Simon, F. T. Beil, W. Rüther, B. Busse, T. Koehne, M. Steiner, P. Pogoda, A. Ignatius, M. Amling, R. Oheim

Abstract

Summary Chronic environmental fluoride exposure under calcium stress causes fragility fractures due to osteoporosis and bone quality deterioration, at least in sheep. Proof of skeletal fluorosis, presenting without increased bone density, calls for a review of fracture incidence in areas with fluoridated groundwater, including an analysis of patients with low bone mass.

Introduction Understanding the skeletal effects of environmental fluoride exposure especially under calcium stress remains an unmet need of critical importance. Therefore, we studied the skeletal phenotype of sheep chronically exposed to highly fluoridated water in the Kalahari Desert, where livestock is known to present with fragility fractures.

Methods Dorper ewes from two flocks in Namibia were studied. Chemical analyses of water, blood and urine were executed for both cohorts. Skeletal phenotyping comprised micro-computer tomography (μCT), histological, histomorphometric, biomechanical, quantitative backscattered electron imaging (qBEI) and energy-dispersive X-ray (EDX) analysis. Analysis was performed in direct comparison with undecalcified human iliac crest bone biopsies of patients with fluoride-induced osteopathy.

Results The fluoride content of water, blood and urine was significantly elevated in the Kalahari group compared to the control. Surprisingly, a significant decrease in both cortical and trabecular bones was found in sheep chronically exposed to fluoride. Furthermore, osteoid parameters and the degree and heterogeneity of mineralization were increased. The latter findings are reminiscent of those found in osteoporotic patients with treatment-induced fluorosis. Mechanical testing revealed a significant decrease in the bending strength, concurrent with the clinical observation of fragility fractures in sheep within an area of environmental fluoride exposure.

Conclusions Our data suggest that fluoride exposure with concomitant calcium deficit (i) may aggravate bone loss via reductions in mineralized trabecular and cortical bone mass and (ii) can cause fragility fractures and (iii) that the prevalence of skeletal fluorosis especially due to groundwater exposure should be reviewed in many areas of the world as low bone mass alone does not exclude fluorosis.

Link To Article

http://dx.doi.org/10.1007/s00198-014-2707-4

Calcium and vitamin D intake maintained from preovariectomy independently affect calcium metabolism and bone properties in Sprague Dawley rats

Authors

C. Y. Park, W. H. Lee, J. C. Fleet, M. R. Allen, G. P. McCabe, D. M. Walsh, C. M. Weaver

Abstract

Summary The interaction of habitual Ca and vitamin D intake from preovariectomy to 4 months postovariectomy on bone and Ca metabolism was assessed. Higher Ca intake suppressed net bone turnover, and both nutrients independently benefitted trabecular structure. Habitual intake of adequate Ca and ~50 nmol/L vitamin D status is most beneficial.

Introduction Dietary strategies to benefit bone are typically tested prior to or after menopause but not through menopause transition. We investigated the interaction of Ca and vitamin D status on Ca absorption, bone remodeling, Ca kinetics, and bone strength as rats transitioned through estrogen deficiency.

Methods Sprague Dawley rats were randomized at 8 weeks to 0.2 or 1.0 % Ca and 50, 100, or 1,000 IU (1.25, 2.5, or 25 μg) vitamin D/kg diet (2 × 3 factorial design) and ovariectomized at 12 weeks. Urinary 45Ca excretion from deep-labeled bone was used to assess net bone turnover weekly. Ca kinetics was performed between 25 and 28 weeks. Rats were killed at 29 weeks. Femoral and tibiae structure (by μCT), dynamic histomorphometry, and bone Ca content were assessed.

Results Mean 25(OH)D for rats on the 50, 100, 1,000 IU vitamin D/kg diet were 32, 54, and 175 nmol/L, respectively. Higher Ca intake ameliorated net bone turnover, reduced fractional Ca absorption and bone resorption, and increased net Ca absorption. Tibial and femoral trabecular structures were enhanced independently by higher Ca and vitamin D intake. Tibial bone width and fracture resistance were enhanced by higher vitamin D intake. Dynamic histomorphometry in the tibia was not affected by either nutrient. A Ca × vitamin D interaction existed in femur length, tibial Ca content, and mass of the soft tissue/extracellular fluid compartment.

Conclusions Adequate Ca intake and serum 25(OH)D level of 50 nmol/L provided the most benefit for bone health, mostly through independent effects of Ca and vitamin D.

Link To Article

http://dx.doi.org/10.1007/s00198-014-2709-2