Development Of Bone Lesions Research Articles

Trends in mortality from pulmonary tuberculosis before and after antibiotics in the Portuguese sanatorium Carlos Vasconcelos Porto (1918–1991): Archival evidence and its paleopathological relevance

The comparative study of patients' profiles and outcomes from pulmonary tuberculosis (TB), before and after the discovery of antibiotic therapy, using sanatoria archives is an unexplored approach in paleopathology. Although higher mortality rates are assumed before chemotherapy, scarce information exists regarding the disease's duration in institutionalized patients and to what extent tuberculous sufferers lived enough to develop skeletal lesions. To fill this gap, 315 clinical files from the former male Sanatorium Carlos Vasconcelos Porto, located in São Brás de Alportel, Portugal, were studied. Two periods of hospitalization were considered: 1931-1944 (n = 128, Group 1) and 1955-1961 (n = 187, Group 2). The average duration of hospitalization (350.3 days for Group 1 and 371.8 for Group 2) and the crude mortality (18.2% and 11.2%, respectively in Groups 1 and 2) did not differ significantly between groups. However, Cox's regression revealed significant differences between survival curves, after adjusting for age at admission (14-74 years old), with pre-chemotherapy patients presenting a higher risk of dying during hospitalization (p = 0.037, hazard ratio = 1.94, IC95% = 1.03-3.63). This study also confirms poorer prognoses for pulmonary tuberculosis sufferers hospitalized in sanatoria before antibiotics and reveals that a significant number of patients survived enough time to develop bone lesions.

Effects of simvastatin on human breast cancer osteolytic bone metastasis in a nude mice model

Objective To observe the effect of simvastatin on bone metastasis of breast cancer in nude mice model. Methods Sixty mice were divided into three groups randomly with 20 in each group. Mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle. After 7 days, mice were treated with either simvastatin, saline, or nothing twice per week for 19 days. The area of osteolytic metastases was subsequently measured in long bones of all mice using an image analysis system. After sacrifice, parathyroid hormone-related protein (PTHrP) concentrations in bone marrow from all mice were determined using a two-site immunoradiometric assay. Osteoclast number expressed per millimeter of tumor/bone interface was assessed using an OsteoMeasure System. Measured data were compared with analysis of variance, and P<0.05 for the difference was statistically significant. Results The area of osteolytic lesions was significantly lower in mice treated with simvastatin compared with mice receiving saline and no treatment (0.51±0.18 mm2vs 2.13±1.24 mm2vs 2.29±1.22 mm2;F=15.600, P=0.002; F=15.673, P=0.001). In addition, treatment with simvastatin decreased the concentrations of PTHrP in bone marrow plasma (0.98±0.20 pmol/L vs 2.11±0.31 pmol/L vs 1.99±0.29 pmol/L; F=61.469, P<0.001; F=58.274, P<0.001) and the osteoclast numbers per millimeter of tumor/bone interface (4.00±1.73 vs 11.40±4.93 vs 10.91±3.87; F=17.820, P=0.001; F=17.184, P=0.002) compared with controls. Conclusion Simvastatin may reduce the production of PTHrP of breast cancer cells, which suppresses the development of destructive bone lesions as well as the growth of breast cancer cells in bone. Key words: Breast neoplasms; Neoplasm metastasis; Simvastatin; Parathyroid hormone

Abstract 4839: TGF-β signaling in osteoclasts promotes, but in osteoblasts inhibits prostate cancer induced bone lesions

Abstract Bone is the only clinically detected metastatic site in advanced prostate cancer (PCa) patients. Bone metastases were found in 70% of patients who died of PCa. The mechanism of PCa bone metastasis is largely unknown, partially due to different types of bone lesions existing in the same patients. Transforming growth factor beta (TGF-β) is known to be abundant in the bone microenvironment and a key factor driving cancer cell colonization and proliferation in the bone, and inducing the bone lesion development. Previous studies have shown that blocking the pathway systemically or in cancer cells reduces the breast cancer and melanoma bone metastases in animal models, presenting an exciting target for cancer therapy. However, TGF-β is also directly affecting the proliferation and differentiation of all the cells in the bone microenvironment. Blocking TGF-β in bone metastases patients will only be possible until the cell specific contribution of TGF-β signaling in the bone microenvironment to cancer cells were further delineated. We hypothesized that cell specific TGF-β signaling in the bone microenvironment has a distinct role in PCa bone metastasis. Genetically engineered mouse (GEM) models were used to delineate the role of mesenchymal cell or myeloid cell specific TGF-β signaling on PCa-induced osteolytic bone lesion development. Lysozyme M promoter-driven Cre was used to induce the ablation of the TGF-β type II receptor (Tgfbr2) in mature macrophages, granulocytes and osteoclasts, thus to knockout TGF-β signaling in these cells [LysMcre/Tgfbr2floxE2/floxE2/Rosa26/Rag2-/- (Tgfb_off_OC)]. The collagen promoter-driven Cre was used to knockout TGF-β signaling in fibroblasts, chondrocytes and osteoblasts [ColcreERT/Tgfbr2floxE2/floxE2/Rosa26/Rag2-/- (Tgfb_off_OB)]. PC3 PCa cells were injected into the tibiae of the GEMs and their respective Cre- controlled mice. The host mice tibiae were imaged using Faxitron x-ray every week from 2 to 4 weeks post tumor injection; the bone lesion areas were measured and analyzed by Metamorph. We found that the osteolytic bone lesion development was significantly reduced in the Tgfb_off_OC mice compared to the control mice at 4 weeks post tumor inoculation (p&amp;lt;0.05). In contrast, PC3 cells in the Tgfb_off_OB mice compared to the control mice significantly promoted osteolytic bone lesion development started from 2 weeks and up to 4 weeks post tumor injections (p&amp;lt;0.05). All statistics were applied by student's T tests. These results suggest that TGF-β signaling activation is anti-osteolytic in osteoblasts, but pro-osteolytic in osteoclasts in this PC3 induced bone lysis models. Citation Format: Xiangqi Meng, Priscilla Lee, Xiaohong Li. TGF-β signaling in osteoclasts promotes, but in osteoblasts inhibits prostate cancer induced bone lesions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4839. doi:10.1158/1538-7445.AM2014-4839

The role of positron emission tomography-computed tomography and magnetic resonance imaging in diagnosis and follow up of multiple myeloma.

Multiple myeloma is the second most common hematologic malignancy and occurs most commonly in elderly patients. Almost all multiple myeloma patients develop bone lesions in the course of their disease or have evidence of bone loss at initial diagnosis. Whole-body conventional radiography remains the gold standard in the diagnostic evaluation, but computed tomography, magnetic resonance imaging and 18F-fluorodeoxyglucose positron emission tomography are increasingly used as complementary techniques in the detection of bone lesions. Moreover, the number of lesions detected and the presence of extramedullary disease give strong prognostic information. These new techniques may help to assess treatment response in solitary plasmacytoma or in multiple myeloma. In this article, we review recent data on the different imaging techniques used at diagnosis and in the assessment of treatment response, and discuss some current issues.

Molecular Mechanisms of Cadmium-Induced Fibroblast Growth Factor 23 Upregulation in Osteoblast-Like Cells

Itai-itai disease is thought to be the result of chronic cadmium (Cd) intoxication. Renal proximal tubules are a major target of Cd toxicity. The whole mechanism of the adverse effects of Cd remains unresolved, especially how renal damage is related to the development of bone lesions. Fibroblast growth factor 23 (FGF23) is a bone-derived phosphaturic factor that regulates vitamin D and inorganic phosphate metabolism in the kidney. To clarify the role of FGF23 on Cd toxicity, we investigated the mechanisms of Cd-induced FGF23 production in the bone. Cd injection into mice significantly increased plasma FGF23 concentrations, but did not change FGF23 mRNA expression in bone. GalNAc-T3 is involved in secreting intact FGF23. To determine potential roles of GalNAc-T3 in Cd-induced FGF23 production, we examined the effect of Cd on GalNAc-T3 mRNA expression in vivo and in vitro. GalNAc-T3 gene expression was significantly increased in the bones of Cd-injected mice. Cd also enhanced the expression of GalNAc-T3 in cultured osteosarcoma UMR106 cells and primary osteocytes. Cd activated aryl hydrocarbon receptors (AhR) and AhR were required for GalNAc-T3 gene expression induced by Cd. In addition, Cd-dependent FGF23 production was completely inhibited by an AhR antagonist. AhR siRNA markedly suppressed the stimulation of transcriptional activity by Cd. Furthermore, Cd induced AhR activation via phosphorylation of Ser-68 by p38 kinase in the nuclear export signal of AhR. Thus, Cd stimulated GalNAc-T3 gene transcription via enhanced AhR binding to the GalNAc-T3 promoter. These findings suggest that the Cd-induced increase in GalNAc-T3 suppresses proteolytic processing of FGF23 and increases serum FGF23 concentrations.

Biology and Treatment of Skeletal Manifestations in Multiple Myeloma

MM is frequently associated with the development of osteolytic bone lesions, osteoporosis and pathological fractures. Bone destruction in MM is caused by osteoclasts recruited in areas adjacent to myeloma plasma cells; their contact triggers both cell types to secrete soluble factors sustaining one each other’s activation and proliferation. Osteoclasts differentiate and maturate upon binding of the receptor activator of NF-kappaB ligand (RANKL), secreted by bone marrow microenvironmental cells, to its receptor (RANK) on osteoclast progenitors, while osteoprotegerin (OPG), a natural decoy receptor, can block the aforementioned ligation. At the same time osteoblasts are inactivated by the Wnt/β-catenin signaling pathway inhibitor, Dickkopf-1 protein (DKK-1), secreted by malignant plasma cells. Furthermore, DKK-1 deregulates the OPG/RANKL equilibrium, promoting osteoclastogenesis. Myeloma bone disease (MBD) can be treated with myeloma-directed chemotherapy and agents inhibiting bone resorption such as aminobisphosphonates, although new promising biology driven monoclonal antibodies targeting osteoclastogenesis mechanisms are emerging. Palliative MBD treatment includes analgesics, orthotics, radiation therapy, vertebroplasty and kyphoplasty. In case of spinal cord compression, radiation therapy or surgical decompression, should be instantly performed, along with steroid administration. Surgery may also be an option especially in case of weight-bearing bone fractures. MBD is a morbid complication and should be carefully managed because it deteriorates patients’ quality of life and worsens disease outcome.

C950T and C1181G osteoprotegerin gene polymorphisms in myeloma bone disease

ObjectivesBone disease is one of the hallmarks of multiple myeloma (MM). The role of osteoprotegerin (OPG) in the RANK/RANKL/OPG signaling system is well defined in the myeloma bone disease. Polymorphisms of the TNFRSF11B gene encoding OPG have been studied in various bone diseases. However, relationship between the levels of OPG and development of bone lesions regardless of RANKL is yet unknown. In this study, the effects of OPG gene polymorphism on the development of bone lesions in MM were investigated.MethodsC950T and C1181G polymorphisms of the OPG gene were studied in 52 MM patients (36 with bone lesions and 16 without bone lesions) and in another 20 control subjects using DNA sequencing.Results1181 G and 950 T alleles were overrepresented in MM patients having bone lesions. 950 TT/1181 GG haplotype frequency and TT/GG combined haplotype were also higher in MM patients having bone lesions compared to MM patients without bone lesions or to control.DiscussionThis is the first study searching for the relationship between OPG gene variants C950T (promoter), C1181G (exon 1), and myeloma bone disease. It was concluded that the presence of polymorphic 1181 G/950 T alleles and 950 TT/1181 GG genotypes may play a role in the development of bone disease.

C950T and C1181G Osteoprotegerin Gene Polymorphisms In Myeloma Bone Disease

Bone disease is one of the hallmarks of multiple myeloma (MM). The role of osteoprotegerin (OPG) in the RANK/RANKL/OPG signaling system is well defined in the myeloma bone disease. Polymorphisms of the TNFRSF11B gene encoding OPG have been studied in various bone diseases. However, relationship between the levels of OPG and development of bone lesions regardless of RANKL is yet unknown. In this study, the effects of OPG gene polymorphism on the development of bone lesions in MM were investigated.52 MM patients, admitted to Karadeniz Technical University, Medical Faculty, Department of Internal Medicine, Division of Hematology between March 2011 and March 2012, were included into the study. Pathologic fracture and/or destructive bone lesions were confirmed in 36 MM patients using X-ray, computed tomography (CT), or magnetic resonance imaging (MRI). No bone lesions were noted in 16 patients. The control group consisted of 20 age and gender matched volunteers.Exon 1 of OPG gene was sequenced using DNA samples obtained from peripheral blood samples of 52 MM patients and 20 healthy control subjects. OPG gene C950T and C1181G polymorphisms were identified in 45 (33 with bone lesions) MM patients and in 16 control subjects. Polymorphic allele 1181G was higher in MM patients with bone lesions compared to MM patients without bone lesions and control group (χ2=8.629, p=0.013). Other polymorphic allele 950T was also overrepresented (73%; p=0.042) in MM patients with bone lesions compared to MM patients without bone lesions and the control group. The most frequent genotype observed in patients with myeloma bone lesions was 950 TT homozygous variant (56%, p=0.08) followed by 1181 CC homozygous wild type with 50% frequency (p=0.09). The frequency of 950 CT heterozygous genotype was higher in MM patients without bone lesions (50%, p=0.604). 1181 GG genotype showed higher frequency in MM patients with bone lesions compared to patients without bone lesions and control group (χ2=3.853, p=0.145). Of the combined haplotypes, 950 TT/1181 GG haplotype frequency is higher in patients with myeloma bone lesions (36%) compared to MM patients without bone lesions (13%) and controls (15%) (χ2=4.77, p=0.09). The frequency of TT/GG combined haplotype carriers in bone disease patients is higher than of those who do not have either polymorphic homozygous alleles (CT/CC and CC/CC haplotypes) (χ2=6.057, p=0.048). Additionally, 950 CC/1181CC wild type haplotype frequency was reasonably low in patients with myeloma bone lesions (8.3%, p=0.240).This is the first study searching for the relationship between OPG gene variants C950T (promoter), C1181G (exon 1) and myeloma bone disease. It was concluded that the presence of polymorphic 1181 G/950 T alleles and 950 TT/1181 GG genotypes may play a role in the development of bone disease. Disclosures:No relevant conflicts of interest to declare.

Knockdown Of Matrix Metalloproteinase 13 (MMP13) In 5TGM1 Multiple Myeloma Cells Inhibits Development Of Lytic Bone Lesions In Vivo

BackgroundMultiple myeloma (MM) cells secrete osteoclastogenic factors that activate osteoclasts (OCL) and contribute to development of pure lytic bone lesions in MM patients. We have recently shown that i) MMP13 is highly expressed by MM cells and ii) exogenous MMP13 increases OCL fusion and bone resorption (Feng et al, 2009). Further, MMP13 mediates these effects by upregulating dendritic cell-specific transmembrane protein (DC-STAMP), which is critical for OCL fusion and activation (Fu et al, 2012). Here, we investigated the role of MMP13 in MM-related bone disease (MMBD) in vivo and the underlying osteoclastogenic mechanisms. Methods and ResultsThe role of MMP13 in MMBD was examined in vivo by the intratibial 5TGM1-GFP mouse MMBD model. Mouse MM cell line 5TGM1-GFP cells were transduced by pLKO.1-puro empty vector (EV) or sh-MMP13 (MMP13-KD) lentivirus followed by puromycin selection for 2 weeks. MMP13 knockdown in 5TGM1-MMP13-KD cells were confirmed by quantitative RT-PCR. 1×105 5TGM1-GFP-EV and 5TGM1-GFP-MMP13-KD cells were bilaterally intratibially injected into Recombination Activating Gene 2 (Rag2) knockout mice (n=9). After 4 weeks of tumor growth, tibiae were separated for micro quantitative computed tomography (micro-QCT) followed by immunohistochemistry (IHC) analysis.Following 5TGM1-GFP-EV injection, micro-QCT analysis of the tibiae and adjacent femurs indicated severe bone erosions, especially within trabecular bone. By contrast MMP13 KD inhibited the development of MM-induced bone lesions. Bone histomorphologic analysis showed that compared to 5TGM1-GFP-EV, MMP13-KD significantly reduced the MM induced trabecular bone loss with increased relative bone volume (0.069 ± 0.018 vs 0.0499 ± 0.016%; P=0.001), connective density (54.94 ± 33.03 vs 27.33 ± 18.97mm3; P=0.002), trabecular bone numbers (3.26 ± 0.29 vs 3.06 ± 0.33mm-1; P=0.032) and bone mineral density (159.1 ± 20.7 vs 134.2 ± 18.6mg/cm3; P=6E-04); as well as decreased triangulation bone surface to volume ratio (66.12 ± 6.67 vs 73.28 ± 10.07; P=0.017) and triangulation structure model index (3.05 ± 0.36 vs 3.42 ± 0.35 mm-1; P=0.002). In accordance with our finding that MMP13 induced OCL fusion, IHC results confirmed the presence of smaller TRAP+OCLs adjacent to the tumor in mice injected with 5TGM1-GFP-MMP13-KD cells compared with 5TGM1-GFP-EV cells. Although MMP13 knockdown showed no effects on 5TGM1-GFP cell growth in vitro, in vivo tumor progression represented by fluorescence imaging and sera immunoglobin 2G level (0.96 ± 0.12 vs 1.10 ± 0.11 mg/ml) was significantly inhibited (P=0.009 and 0.03 respectively), indicating MMP13 depletion in MM cells impaired OCL activation which, in turn, failed to support MM cell growth in bone marrow microenvironment as effectively in EV control group.In vitro studies demonstrated that MMP13 directly induced ERK1/2 phosphorylation in pre-osteoclasts. Consistent with a critical role for ERK1/2 phosphorylation in regulating OCL formation, U0126 (ERK1/2 inhibitor) blocked MMP13-induced ERK1/2 phosphorylation, ERK1/2-dependent DC-STAMP upregulation and MMP13-induced OCL fusion (P<0.01). ConclusionOur results demonstrate that silencing MMP13 expression in MM cells inhibits MM cell-induced OCL fusion and development of lytic bone lesions in vivo, indicating that MMP13 is essential for MM-induced bone diseases. Further, MMP13 upregulates DC-STAMP expression and OCL fusion via the activation of ERK1/2 signaling. Our data suggest that targeting MMP13 may represent a novel therapeutic approach for the treatment of MMBD. Disclosures:Roodman:Amgen: Membership on an entity's Board of Directors or advisory committees; Lilly: Research Funding. Lentzsch:Celgene: Research Funding.

English

AIM: Hand-Schuller-Christian Disease is a rare proliferative disorder in which pathological Langerhans cells (LCs) accumulate in a variety of organs. It represents triad of diabetes insipidus, bone lesions and exophthalmos. Usually begins at the age of 2 and 6 years. 30%- develop skin lesions and 80%- develop bone lesions (cranium being preferentially involved). MATERIAL & METHOD: A 5-year-old boy was admitted to the Kempegowda Institute of medical sciences hospital, department of orthopaedics with pain in left hip since 2 weeks. The child was small for age, cachexic, mildly lethargic but occasionally irritable. He was referred to skin OPD for seborrheic dermatitis like picture over the scalp and multiple hypopigmented macules over the abdomen. Patient also had multiple boggy defects in the skull and exophthalmos and also history of long term continous ear discharge and on and off fever. Cervical, axillary and inguinal lymphadenopathy were present. RESULT: Skin biopsy findings were consistent with histiocytosis. CD1a and S100 tumor markers were positive. Patient was put on chemotherapy-ETOPOSIDE+CYTOBLASTIN+BETAMETHASONE REGIMEN to which he responded successfully. CONCLUSION: Disorders like HAND-SCHULLER- CHRISTIAN DISEASE detected early can be effectively managed improving the overall survival, quality of life and disability free living.

Cancer pain is not necessarily correlated with spinal overexpression of reactive glia markers

Bone cancer pain is a common and disruptive symptom in cancer patients. In cancer pain animal models, massive reactive astrogliosis in the dorsal horn of the spinal cord has been reported. Because astrocytes may behave as driving partners for pathological pain, we investigated the temporal development of pain behavior and reactive astrogliosis in a rat bone cancer pain model induced by injecting MRMT-1 rat mammary gland carcinoma cells into the tibia. Along with the development of bone lesions, a gradual mechanical and thermal allodynia and hyperalgesia as well as a reduced use of the affected limb developed in bone cancer–bearing animals, but not in sham-treated animals. Dorsal horn Fos expression after nonpainful palpation of the injected limb was also increased in bone cancer–bearing animals. However, at any time during the evolution of tumor, there was no increase in glial fibrillary acidic protein (GFAP) immunoreactivity in the dorsal horn. Further analysis at 21days after injection of the tumor showed no increase in GFAP and interleukin (IL) 1β transcripts, number of superficial dorsal horn S100β protein immunoreactive astrocytes, or immunoreactivity for microglial markers (OX-42 and Iba-1). In contrast, all these parameters were increased in the dorsal horn of rats 2weeks after sciatic nerve ligation. This suggests that in some cases, bone cancer pain may not be correlated with spinal overexpression of reactive glia markers, whereas neuropathic pain is. Glia may thus play different roles in the development and maintenance of chronic pain in these 2 situations.

Breast Cancer Cells Induce Osteolytic Bone Lesions In vivo through a Reduction in Osteoblast Activity in Mice

Bone metastases are severely debilitating and have a significant impact on the quality of life of women with metastatic breast cancer. Treatment options are limited and in order to develop more targeted therapies, improved understanding of the complex mechanisms that lead to bone lesion development are warranted. Interestingly, whilst prostate-derived bone metastases are characterised by mixed or osteoblastic lesions, breast-derived bone metastases are characterised by osteolytic lesions, suggesting unique regulatory patterns. This study aimed to measure the changes in bone formation and bone resorption activity at two time-points (18 and 36 days) during development of the bone lesion following intratibial injection of MDA-MB-231 human breast cancer cells into the left tibiae of Severely Combined Immuno-Deficient (SCID) mice. The contralateral tibia was used as a control. Tibiae were extracted and processed for undecalcified histomorphometric analysis. We provide evidence that the early bone loss observed following exposure to MDA-MB-231 cells was due to a significant reduction in mineral apposition rate, rather than increased levels of bone resorption. This suggests that osteoblast activity was impaired in the presence of breast cancer cells, contrary to previous reports of osteoclast-dependent bone loss. Furthermore mRNA expression of Dickkopf Homolog 1 (DKK-1) and Noggin were confirmed in the MDA-MB-231 cell line, both of which antagonise osteoblast regulatory pathways. The observed bone loss following injection of cancer cells was due to an overall thinning of the trabecular bone struts rather than perforation of the bone tissue matrix (as measured by trabecular width and trabecular separation, respectively), suggesting an opportunity to reverse the cancer-induced bone changes. These novel insights into the mechanisms through which osteolytic bone lesions develop may be important in the development of new treatment strategies for metastatic breast cancer patients.

Oleocanthal Inhibits Proliferation and MIP-1&amp;#945; Expression in Human Multiple Myeloma Cells

Multiple myeloma (MM) is a plasma cell malignancy that causes devastating bone destruction by activating osteoclasts in the bone marrow milieu. MM is the second of all hematological malignancies. Thus, the search for new pharmacological weapons is under intensive investigation being MM a critically important public health goal. Recently, it has been demonstrated that macrophage inflammatory protein 1- alpha (MIP-1 α) is crucially involved in the development of osteolytic bone lesions in MM. Phenolic components of extra virgin olive oil are reported to have anti tumor activity. However, the underlying mechanisms and specific targets of extra virgin olive oil remain to be elucidated. In the present study, we investigated the effects of a recently isolated novel extra virgin olive oil polyphenol, oleocanthal, on the human multiple myeloma cell line ARH-77. Here we report that this natural compound has a remarkable in vitro activity by inhibiting MIP-1 α expression and secretion in MM cells. In addition, we also demonstrated that oleocanthal inhibits MM cells proliferation by inducing the activation of apoptosis mechanisms and by down-regulating ERK1/2 and AKT signal transduction pathways. This in vitro study suggests a therapeutic potential of oleocanthal in treating multiple myeloma.

Measles virus nucleocapsid protein, a key contributor to Paget's disease, increases IL-6 expression via down-regulation of FoxO3/Sirt1 signaling

Measles virus plays an important role as an environmental factor in the pathogenesis of Paget's disease (PD). Previous studies have shown that IL-6 is increased in the bone marrow of Paget's patients and that measles virus nucleocapsid protein (MVNP) induces IL-6 secretion by pagetic osteoclasts. Further, IL-6 plays a critical role in the development of pagetic osteoclasts and bone lesions induced by PD, but the mechanisms regulating IL-6 production by MVNP remain unclear. Our current studies revealed that MVNP expression in osteoclast precursors down-regulated Sirt1 mRNA and protein, a negative regulator of NF-κB activity, which is a key factor for IL-6 expression. MVNP expression in NIH3T3 cells also elevated Il-6 transcription and impaired the expression of Sirt1 mRNA both under basal conditions and upon activation of the Sirt1 upstream regulator FoxO3 by LY294002 (a PI3K/AKT inhibitor). Luciferase activity assays showed that constitutively active FoxO3 abolished the repressive effect of MVNP on reporters driven by either FoxO3 response elements or the Sirt1 promoter. Further, protein stability assays revealed that FoxO3 was degraded more rapidly in MVNP-expressing cells than in control cells following the addition of cycloheximide. Similarly, co-transfection of MVNP and FoxO3 into HEK293 cells demonstrated that MVNP decreased the protein levels of over-expressed FoxO3 in a dose-dependent manner. Treatment with the proteasome inhibitor, MG132, blocked the MVNP-triggered decrease of FoxO3, and the treatment with the serine/threonine phosphatase inhibitor, calyculin A, revealed that MVNP increased phosphorylation of FoxO3. Further, over-expression of Sirt1 or treatment with the Sirt1 activator resveratrol blocked the increase in Il-6 transcription by MVNP. Finally, resveratrol reduced the numbers of TRAP positive multi-nuclear cells in bone marrow cultures from TRAP-MVNP transgenic mice to wild type levels. These results indicate that MVNP decreases FoxO3/Sirt1 signaling to enhance the levels of IL-6, which in part mediate MVNP's contribution to the development of Paget's disease.

Matrix Metalloproteinase 13 (MMP13) Upregulation Is Essential for Multiple Myeloma Related Bone Lytic Lesion

AbstractAbstract 4025 Background:MM cells produce several osteoclast-activating factors, which result in highly activated osteoclasts and cause dysregulated bone remodeling with excessive bone resorption. Our previous data showed that matrix metalloproteinase 13 (MMP13) is highly expressed in MM cells, and co-culture with stromal cells further increased MMP13 levels. Most importantly, exogenous MMP13 increased OCL fusion and bone resorption activity induced by nuclear factor kappa-B ligand (RANKL). Here, we further addressed the mechanism of MMP13 upregulation in MM and its role in MM-related bone disease in vivo. Methods and Results:Based upon the previous results that IL-6 neutralizing antibody blocks MMP13 upregulation in MM cells co-cultured with stromal cells, we further investigated the role of IL-6 on MMP13 induction in MM cells. Human RPMI8266 MM cells were serum-starved for 24 hours, then treated with IL-6 for up to 96 hours. RT-PCR and western blotting showed that MMP13 expression and secretion by MM cells were upregulated after 24h and prolonged through 96h. AP-1 binding sites were identified in the MMP13 promoter, and we found that IL-6 induced upregulation of the AP-1 members c-Jun and c-fos in MM cells by RT-PCR and western blotting, which correlated with MMP13 induction. To address the role of heightened MMP13 expression/secretion of MM on OCL formation and development of lytic lesions, we silenced MMP13 expression in MM cells by lentiviral mediated shRNA transduction. 5TGM1 mouse MM cells were infected with the pKLO.1-puro-vector (EV) or pKLO.1-puro-sh-MMP13 (MMP13 Knock down [KD]) lentivirus particles and cells were selected by puromycin. MMP13 KD was confirmed by RT-PCR and western blotting. To investigate the effects of MMP13 KD on OCL formation, we co-cultured mouse bone marrow cells (BMC) with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells using a transwell system to permit only soluble molecule exchange. Co-culture with 5TGM1-EV MM cells significantly (p<0.01) induced OCL fusion, while this effect was largely impaired in the co-culture group with 5TGM1-MMP13 KD MM cells. Treatment with IL-6 further enhanced the OCL fusion activity in both groups. However, in 5TGM1-MMP13 KD group, IL-6 did not compensate for impaired OCL formation due to MMP13 silencing. This indicates that MMP13 secreted from MM cells is one of the key factors facilitating OCL formation/activity in MM. We further investigated the effects of MMP13 silencing on MM tumor progression and bone disease in vivo using the 5TGM1 intratibial tumor model. Recombination activating genes 2 (RAG2) knockout mice were intratibially injected with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells, and 3 weeks later, mice were sacrificed and tumor growth and bone lytic lesion were monitored by serum IgG2b level (by Elisa) and micro-QCT respectively. Preliminary results show that MMP13 KD in MM cells inhibited tumor growth and lytic lesions in trabecular bone. Conclusion:Our results demonstrate that IL-6-induced high level of MMP13 in MM cells is essential for multiple myeloma tumor growth, OCL induction and development of lytic bone lesions. Disclosures:Lentzsch:Celgene: Consultancy, Research Funding.

P38 MAPK Activity in Myeloma Cells Regulates Osteoclast and Osteoblast Activity and Induces Bone Destruction in Vivo

Abstract 566Bone destruction is a hallmark of multiple myeloma (MM). More than 80% of MM patients have osteolysis, which is characterized by pathological fractures, severe bone pain, spinal cord compression, and hypercalcemia. These symptoms can severely compromise a patient's quality of life and performance status. It has been proposed that MM cells activate osteoclast (OC)-mediated bone resorption and inhibit osteoblast (OB)-mediated bone formation. However, the mechanism underlying the association of MM cells with development of bone lesions remains poorly elucidated. Our previous studies showed that p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in MM cells, is a master regulator of MM-mediated bone destruction. Knocking down or inhibiting p38 MAPK activity in MM cells prevented MM-induced bone destruction in vivo. In the present study, we further investigated the mechanism of MM cell p38 MAPK-induced bone destruction. We hypothesized that p38 MAPK activity in MM cells can regulate OB and OC differentiation and activity by upregulating cytokine production by MM cells. In a cytokine array analysis, we examined the expression and secretion of MM-derived cytokines that regulate OB and OC differentiation. Our results showed for the first time that either knockdown or inhibition of p38 MAPK activity by p38 MAPK short hairpin RNAs or inhibitors significantly downregulated the production of dickkopf-1 (DKK-1) and monocyte chemotactic protein-1 (MCP-1) by MM cells. Real-time PCR and ELISA quantified and confirmed the array analysis results. To determine the role of p38 MAPK-upregulated DKK-1 and MCP-1 production in bone destruction, we administered treatment with neutralizing antibodies to SCID mice injected intravenously with ARP-1 or MM.1S cells. Our results showed that neutralization of DKK-1 and MCP-1 led to fewer bone lesions in these mice. Furthermore, we examined the impact of MM cell p38 MAPK activity on OB and/or OC differentiation. Our results showed that knockdown or inhibition of MM cell p38 MAPK significantly downregulated osteoclastogenesis but upregulated osteoblastogenesis in vitro and in vivo. Although DKK-1 is well known to inhibit OB differentiation, we found that DKK-1, together with MCP-1, promoted OC differentiation and bone resorption. Mechanistic studies further showed that MCP-1 upregulated RANK expression in OC precursors and that DKK-1 increased RANKL secretion from stromal cells and mature OBs, all of which led to activation of the NF-kB and MAPK signaling pathways in OCs. Thus, our study uncovered a novel mechanism by which p38 MAPK signaling in MM cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction in patients with this disease. These findings strongly suggest that disrupting and targeting MM cell p38 signaling are effective approaches to treating osteolytic bone lesions in MM patients. Disclosures:No relevant conflicts of interest to declare.

Extensive bone marrow infiltration and abnormal free light chain ratio identifies patients with asymptomatic myeloma at high risk for progression to symptomatic disease

Asymptomatic multiple myeloma (AMM) is characterized by a constant risk of progression to symptomatic myeloma. To evaluate previously recognized risk factors and to identify high-risk features we analyzed 96 patients with AMM and at least 18 months of follow-up. The progression rate at 1,2, and 3 years was 8%, 15% and 26%, respectively, and the projected 5-year progression rate was 38%. Extensive bone marrow (BM) infiltration, abnormal free light chain (FLC) ratio and serum monoclonal (M)-protein ≥ 3 gr/dl were the most significant factors for progression, whereas the type of heavy (IgG vs IgA) or light chain or immunoparesis of the uninvolved immunoglobulins were not. Abnormal marrow signal of magnetic resonance imaging of the spine was associated with a significant risk of progression (median 15 months, P=0.001). Extensive BM infiltration ≥ 60% (hazard ratio, HR: 13.7, P<0.001) and FLC ratio ≥ 100 (HR: 9, P=0.003) independently identified a 'very high-risk' group, which included 12.5% of patients with AMM and who progressed ≤ 18 months from initial diagnosis. Development of anemia and/or lytic bone lesions were the most common features of symptomatic progression. In conclusion, there is a subgroup of patients who have a substantial risk of progression to symptomatic disease that can be detected at diagnosis (either by extensive BM infiltration ≥ 60% or FLC ratio ≥ 100) and may be considered for immediate treatment.

Effect of matrix metallopeptidase 13 (MMP13) on multiple myeloma (MM) cells, osteoclast (OCL) activity, and bone resorption.

8099 Background: MM cells produce OCL-activating factors that induce excessive bone resorption resulting in lytic lesions. The role of MMPs in invasion/progression of solid tumors is well-known, but its function in MM has not been well elucidated. Our group has shown that MMP13 is highly expressed in primary MM cells and in sera of MM patients. Levels of MMP13 significantly correlate with the extent of bone disease. MMP13 is induced by IL-6 via AP-1 activation in MM cells and enhances fusion of OCL precursors resulting in excessive bone resorption. OCL formation using MNCs of mmp-13-/- mice resulted in a fusion defect, significantly decreased OCL size and activity, which could be reversed by exogenous MMP13 (ASH 2009, IMW 2011). Methods: Methods will be presented in the Results section. Results: RT-PCR and western blotting revealed that IL-6 treatment of MM cells induced MMP13 transcription (30-fold) and secretion (&gt;1000-fold). Protein expression of the AP-1 members c-Jun and c-Fos was induced by IL-6, which correlated with MMP13 upregulation. Our data further indicate that the catalytic activity of MMP13 is not required to enhance OCL formation and bone resorption. To prove this, we generated the MMP13 activity-dead mutation MMP13-E223A construct by site-directed mutagenesis PCR-based cloning. The mutated protein was overexpressed in HEK293 cells and purified from the supernatant to confirm whether loss of catalytic activity blocks MMP13 function. To further investigate the in vivo role of MMP13 in MM bone disease, MMP13 expression was knocked down (KD) in murine 5TGM1-MM cells by pKLO. 1 puro lentiviral infection containing sh-RNA targeting mouse MMP13 sequence. MMP13-KD 5TGM1-MM cells or WT-5TGM1-MM cells were intratibially injected into RAG2-/- mice. Development of lytic bone lesions are monitored by micro-QCT and data will be available at the time of presentation. Conclusions: Our data suggest that MMP13, secreted by MM cells, plays a critical role in the development of lytic lesions. Targeting MMP13 represents a promising approach to treat or to prevent bone disease in MM.

Predictive value of baseline serum MIP-1α and CRP on symptom burden and tumor response to induction therapy in patients with multiple myeloma.

8091 Background: Macrophage inflammatory protein-1α(MIP-1α) is a growth factor for human multiple myeloma (MM) cells. As an osteoclastic factor, its presence provides pathophysiological evidence of the development of lytic bone lesions in MM. C-reactive protein (CRP) indicates systemic inflammation. The relationship between disease-driven inflammatory markers and both patient-experienced symptoms and tumor response to induction therapy is unknown. Methods: MM patients (N=39) who were either newly diagnosed or had received fewer than 2 cycles of chemotherapy, and who also were to receive induction therapy, were enrolled. To test concentrations of MIP-1α and CRP, serum samples were collected before and after induction and assayed by Luminex. Multiple symptoms were measured twice a week via the M. D. Anderson Symptom Inventory MM module (MDASI-MM) from -8 to +112 days of induction. The MM-specific items of the MDASI-MM are bone aches, constipation, muscle weakness, diarrhea, sore mouth or throat, rash, and difficulty paying attention. Correlation between symptom severity and inflammatory markers at baseline was examined by linear regression modeling. Kruskal-Wallis significance test and Wilcoxon test were used to examine association between MIP-1α and tumor response. Results: Patients received either bortezomib-based (89%) or lenalidomide-based induction therapy. Baseline MIP-1α and CRP were significantly inversely related to the mean severity component score of the 5 most-severe symptoms (fatigue, pain, bone aches, poor sleep, drowsiness) (p=.03; p=.02), and significantly inversely related to the severity of a component score of the module-specific symptoms (p=0.04; p=.002). Change over time in MIP-1α differed significantly by tumor response category (p=.04), with the partial response group having a higher median score than the complete response group (1.483 vs. 0.016, p=.01). Conclusions: Our data suggest that higher baseline levels of serum MIP-1α and CRP predict effective chemotherapy-induced reduction of disease-related symptoms in MM patients. Higher serum MIP-1α expression after induction therapy was related to less-ideal tumor response.

Prophylaxe und Therapie beim multiplen Myelom und anderen tumorbedingten Knochenveränderungen

Multiple myeloma (MM) ranges second of all hematological malignancies and occurs most commonly in elderly patients. Almost all MM patients develop bone lesions in the course of their disease or have evidence of bone loss at initial diagnosis. Whole-body conventional radiography remains the gold standard in the diagnostic evaluation, albeit computed tomography (CT) and magnetic resonance imaging (MRI) are increasingly used as complementary techniques in the more sensitive detection of osteolytic processes. Bisphosphonates like zoledronate or pamidronate represent the cornerstone therapeutics in osteolytic disease, and are effective supportives to potent anti-myeloma therapies, including novel agents such as the proteasome inhibitor bortezomib or immunomodulatory drugs (IMIDs, e. g. thalidomide or lenalidomide). Several studies are ongoing to investigate the effects of alternative bone-seeking agents and their therapeutic potential for the management of myeloma bone disease, such as denosumab (RANKL-neutralizing antibody), anti-sclerostin (monoclonal antibody, generated against sclerostin) or sotatercept (potent activin-A inhibitor). This review summarizes the most prominent data on myeloma bone disease pathogenesis, the role of imaging techniques as well as therapy and prevention of lytic complications in myeloma which may similarly or equally be true for other bone metastases-inducing solid tumors.