Osteolytic Bone Destruction Research Articles

Potency of bortezomib in combination with MP0250, a bispecific VEGF- and HGF-targeting darpin, in a preclinical multiple myeloma model.

e19574 Background: In multiple myeloma, tumor cells and stroma exert a reciprocal effect on each other that leads to tumor expansion in the bone marrow and bone destruction. Hepatocyte growth factor (HGF) is a main player in osteolytic bone destruction and also stimulates myeloma cell proliferation, migration and adhesion in the bone marrow. Additionally, VEGF plays a key neovascularization role in multiple myeloma. Therefore, we investigated whether MP0250, a bispecific DARPin simultaneously inhibiting tumor stroma HGF and VEGF, could potentiate the anti-tumor effect of the standard of care proteasome inhibitor bortezomib. Methods: MP0250 was tested as monotherapy and in combination with 0.6 mg/kg of bortezomib in an orthotopic murine model, where human multiple myeloma cells are implanted in the murine bone marrow. Tumor growth and invasion into the muscle were monitored by X-ray, whereas bone destruction was analyzed by microCT. Results: MP0250 significantly inhibited bone lysis and tumor invasion both...

CD14+CD16+ monocytes rather than CD14+CD51/61+ monocytes are a potential cytological marker of circulating osteoclast precursors in multiple myeloma. A preliminary study

Osteolytic bone destruction is a major clinical problem in multiple myeloma patients. Osteoclasts can differentiate in vitro from bone marrow-resident monocyte progenitors, such as common monocyte progenitors, as well as circulating monocytes. Various types of monocytes, including osteoclast precursors, appear to circulate systemically. We investigated the possibility of demonstrating, by in vitro differentiation and flow cytometry, a circulating osteoclast precursor population in multiple myeloma (MM) patients by studying the distribution of CD14(+/++) CD11b(+) CD51/61(+) and CD14(+/++) CD16(+/-) populations. Under short-term in vitro osteoclastic differentiation conditions, almost all CD14 monocytes acquired CD51/61 and CD16 expression. Flow cytometry studies failed to demonstrate a statistically significant increase in circulating CD14(+/++) CD11b(+) CD51/61(+) populations in 20 MM patients with osteolytic lesions. However, the minor circulating CD14(+/++) CD16(+) fraction was significantly increased in MM patients compared with healthy volunteers (109.3±63.1/mm(3) vs. 65.3±34.9/mm(3) ; P=0.005), but with no correlation with markers of tumour burden. The CD14(+/++) CD16(+) to CD14(+/++) CD16(-) ratio was higher in MM patients. The circulating CD14(+/++) CD11b(+) CD51/61(+) fraction was not correlated with bone lesions in MM patients. However, CD14(+/++) CD16(+) monocytes may be a candidate marker. A larger study must be conducted to confirm these promising results for the diagnosis and follow-up of MM patients.

RANKL Inhibition Blocks Osteolytic Lesions and Reduces Skeletal Tumor Burden in Models of Non–Small-Cell Lung Cancer Bone Metastases

Bone metastasis is a serious complication in patients with lung cancer, occurring in up to 40% of patients. Tumor cell-mediated osteolysis occurs ultimately through induction of RANK ligand (RANKL) within the bone stroma although this hypothesis has not been tested extensively in the setting of non-small-cell lung cancer (NSCLC). By using two novel NSCLC bone metastasis mouse models, we examined the effects of RANKL inhibition on osteolysis and tumor progression. We treated mice bearing skeletal NSCLC tumors with osteoprotegerin-Fc (OPG-Fc) to assess whether osteoclast inhibition through RANKL inhibition would affect bone metastases at early or late stages of bone colonization. Progression of skeletal tumor was determined by radiography, longitudinal bioluminescent imaging, and histological analyses. OPG-Fc reduced development and progression of radiographically evident osteolytic lesions and also significantly reduced skeletal tumor progression in both NSCLC bone metastasis models. In the H1299 human NSCLC bone metastasis model, OPG-Fc plus docetaxel in combination resulted in significantly greater inhibition of skeletal tumor growth compared with either single agent alone. The observed ability of RANKL inhibition to reduce NSCLC osteolytic bone destruction or skeletal tumor burden was associated with decreases in tumor-associated osteoclasts. These results demonstrate that RANKL is required for the development of tumor-induced osteolytic bone destruction caused by NSCLC cells in vivo. RANKL inhibition also reduced skeletal tumor burden, presumably through the indirect mechanism of blocking tumor-induced osteoclastogenesis and resultant production of growth factors and calcium from the bone microenvironment. RANKL inhibition also provided an additive benefit to docetaxel treatment by augmenting the reduction of tumor burden.

Cancer-Associated Osteoclast Differentiation Takes a Good Look in the miR(NA)ror

Tumor-bone cell interactions are critical for the development of metastasis-related osteolytic bone destruction. In this issue of Cancer Cell, Ell and colleagues show how a discrete miRNA network regulates osteoclastogenesis during breast cancer bone metastasis. A signature of upregulated miRNAs may have diagnostic and therapeutic implications for bone metastases.

Abstract 2161: Targeting Bruton's tyrosine kinase (BTK) in multiple myeloma with novel BTK inhibitors.

Abstract Multiple myeloma (MM) is a malignant disease that is characterized by an excess of monotypic plasma cells in the bone marrow (BM). A key clinical characteristic of MM is the localization of the MM cells to the bone marrow where they promote osteolytic bone destruction and impaired hematopoietic function. As a consequence MM patients experience bone pain, hypocalcaemia, anemia. Although there has been some progress in recent years in the development of novel drugs, such as proteasome inhibitors and derivatives of thalidomide, MM remains incurable and the majority of patients eventually succumb to their cancer. We have recently identified BTK over-expression in cancer cells taken from patients with multiple myeloma. In addition we have observed that BTK is also expressed in the activated osteoclasts of MM patients indicating that targeted inhibition of BTK might not only effect the MM cancer cells but may also influence the activity of osteoclasts and the associated osteolytic lesions. Bruton's tyrosine kinase (BTK) is a cytoplasmic nonreceptor tyrosine kinase belonging to the Tec family of kinases. BTK has been extensively studied for its role in B-cell maturation and activation of B-cells by various ligands is accompanied by the translocation of BTK to the cell membrane where it binds phosphatidylinositol-3,4,5-trisphosphate through its PH domain. Activation of BTK results in downstream signaling through the PI3K/AKT, PLCγ1/2, NFκB, and other signaling pathways important for B-cell development and function. We propose that BTK plays an important role in multiple myeloma pathophysiology and that therapeutically targeting BTK will inhibit the growth of cancer cells and alter the tumor microenvironment in the bone marrow of multiple myeloma cancer patients. Using a structure-based approach we have developed a series of irreversible BTK inhibitors with selective and potent low nanomolar activity. In preclinical studies to date, our compounds have demonstrated promising activity in biochemical and cell-based experiments. Our BTK-targeted agents have shown activity in MM cells and have good pharmacokinetics when delivered IV and oral. MM cells create an adverse microenvironment from a pathophysiologic and clinical perspective because of the disruption of bone remodeling and this disruption of normal bone function has been shown to inhibit the response of MM cell to drug treatment. Therefore, a strategy to develop new drugs for MM must take into account the ability of the new agents to partition and distribute to the bone and function in the bone microenviroment of MM. Our compounds are being optimized for their ability to partition to bone and remain active in the BM microenvironment. By inhibiting BTK we seek to block BTK-dependent growth and migration of multiple myeloma cells and inhibit of the production of differentiated activated osteoclasts thereby disrupting the bone marrow microenvironment in MM. Citation Format: Destinee Bushman, Jared J. Bearss, Venkataswamy Sorna, Hariprasad Vankayalapati, Sunil Sharma, Fenghuang Zhan, David Bearss. Targeting Bruton's tyrosine kinase (BTK) in multiple myeloma with novel BTK inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2161. doi:10.1158/1538-7445.AM2013-2161

Gene silencing of the BDNF/TrkB axis in multiple myeloma blocks bone destruction and tumor burden in vitro and in vivo

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF-κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as a MM-derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma-osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM-derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time- and dose-dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID-RPMI8226 mice model was constructed using lentiviral short hairpin RNA-transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma-myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.

Notch-directed microenvironment reprogramming in myeloma: a single path to multiple outcomes

Multiple myeloma is a deadly hematopoietic malignancy. Despite therapeutic advances such as autologous stem cell transplantation and novel chemotherapeutics, multiple myeloma remains incurable. Multiple myeloma cell localization in the bone marrow and the cross-talk with the bone niche trigger dramatic alterations in the bone marrow microenvironment critical for tumor progression, resistance to therapies and osteolytic bone destruction. It does not surprise that the molecular bases of such fatal interaction are under examination as source of novel potential pharmacological targets. Among these, the Notch family of receptors and ligands has gained growing interest in the recent years because of their early deregulation in multiple myeloma and their ability to affect multiple features of the disease, including tumor cell growth, drug resistance, angiogenesis and bone lesions. This review will explore the evidences of Notch deregulation in multiple myeloma, the state of the art of the currently known roles of its signaling in the fatal interaction between multiple myeloma cells, extracellular matrix and cells in the bone marrow stroma. Finally, we will present recent findings concerning the arguments for or against a therapy addressed to Notch signaling inhibition in the cure of multiple myeloma.

Role of TGF-<i>β</i> in breast cancer bone metastases

Breast cancer is the most prevalent cancer among females worldwide leading to approximately 350,000 deaths each year. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ~70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by increased fracture risk, pain, nerve compression and hypercalcemia, causing severe morbidity. In the bone matrix, transforming growth factor-β (TGF-β) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-β, in turn, stimulates bone metastatic tumor cells to secrete factors that further drive osteolytic bone destruction adjacent to the tumor. Thus, TGF-β is a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-β activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-β signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-β in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-β inhibitors in clinical practice to treat breast cancer bone metastases.

ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone

IntroductionBone is the most common site of breast cancer metastasis, and complications associated with bone metastases can lead to a significantly decreased patient quality of life. Thus, it is essential to gain a better understanding of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases.MethodsTo search for novel molecular mediators that influence breast cancer bone metastasis, we generated gene-expression profiles from laser-capture microdissected trephine biopsies of both breast cancer bone metastases and independent primary breast tumors that metastasized to bone. Bioinformatics analysis identified genes that are differentially expressed in breast cancer bone metastases compared with primary, bone-metastatic breast tumors.ResultsABCC5, an ATP-dependent transporter, was found to be overexpressed in breast cancer osseous metastases relative to primary breast tumors. In addition, ABCC5 was significantly upregulated in human and mouse breast cancer cell lines with high bone-metastatic potential. Stable knockdown of ABCC5 substantially reduced bone metastatic burden and osteolytic bone destruction in mice. The decrease in osteolysis was further associated with diminished osteoclast numbers in vivo. Finally, conditioned media from breast cancer cells with reduced ABCC5 expression failed to induce in vitro osteoclastogenesis to the same extent as conditioned media from breast cancer cells expressing ABCC5.ConclusionsOur data suggest that ABCC5 functions as a mediator of breast cancer skeletal metastasis. ABCC5 expression in breast cancer cells is important for efficient osteoclast-mediated bone resorption. Hence, ABCC5 may be a potential therapeutic target for breast cancer bone metastasis.

Study of treatment using percutaneous acetabuloplasty and interstitial implantation of 125I seeds for patients with metastatic periacetabular tumors

BackgroundThe periacetabular area is one of the primary sites of metastatic tumors, which often present as osteolytic bone destruction. Bone destruction in the acetabulum caused by metastatic tumors will cause hip pain and joint dysfunction. It results in decreased quality of life for patients. The aim of our study was to explore the clinical effect of metastatic periacetabular tumors treated with percutaneous cementoplasty and interstitial implantation of 125I seeds.MethodsA retrospective analysis was performed on 24 patients with metastatic periacetabular tumors who underwent combined therapy of percutaneous acetabuloplasty and interstitial implantation of 125I seeds between February 2003 and June 2011. There were 13 males and 11 females aged 19–80 years with a mean age of 57.3. The primary tumor site was the lung in eight cases, the breast in six, the prostate cancer in eight, and the liver in two. The amount of implanted 125I seeds was 12–20 seeds/person, with a mean of 16.5 seeds/person, and the matching peripheral dosage (MPD) was 80~100Gy. Routine postoperative chemotherapy and other combined treatments were applied to patients after the surgical operation. Changes in the Karnofsky Score(KPS), Harris Hip Score(Harris), and Visual Analog Scale(VAS) were observed during the follow-up period.ResultsThe 24 patients’ operations were all successful. No major complications occurred. Complete pain relief was achieved in 58% (14 of 24) of patients, and pain reduction was achieved in the 42% remaining (10) patients. The mean duration of pain relief was 8.3 months. Pain recurred in one patient 3 months after surgery. Six patients had died and 18 patients were alive at the time of the 1-year follow-up. Comparing the KPS, Harris and VAS scores pre- and postoperativelyat 1, 6, and 12 months, the combined therapy method was significantly effective in metastatic periacetabular tumor patients (P<0.05).ConclusionsPercutaneous cementoplasty with interstitial implantation of 125I seeds is an effective treatment method for metastatic periacetabular tumor patients, providing tumor resistance, pain relief, increased bone stability, and improved quality of life for patients.

Brain-Derived Neurotrophic Factor Enhances Osteoclastogenesis in Multiple Myeloma via Upregulation of RANKL/OPG Ratio Both in Vitro and in a Murine Model of Myeloma Bone Disease

Abstract 568Osteolytic bone disease is a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption uncoupled with osteoblastic bone formation. Myeloma-induced osteoclastogenesis is largely depending on the increase of receptor activator of NF-κB ligand (RANKL) and decrease of osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as an MM-derived factor correlated with increased RANKL level and contributed to myeloma bone destruction. On the other hand, tyrosine receptor kinase B (TrkB), the receptor of BDNF, was found to be abundantly expressed by osteoblasts (OBs). Since OBs are the main source of RANKL and OPG in bone, here we sought to evaluate the involvement of BDNF/TrkB in the crosstalk between myeloma cells and OBs, as well as the effects of BDNF on RANKL/OPG ratio and myeloma bone disease.Co-cultures of OBs with pre-osteoclasts were performed in a non-contacted transwell system and treated with various concentration of BDNF. Osteoclast formation was detected with a tartrate-resistant acid phosphatase (TRAP) staining kit. Then, RANKL and OPG levels were measured when OBs cultures were exposed to BDNF or co-cultured with three human myeloma cell lines (RPMI8226, ARH-77 and U266). K252a (an inhibitor of TrkB) was present or absent in these systems to assess the effects of BDNF on RANKL/OPG expression in OBs. The involvement of downstream signaling molecules activated by BDNF in OBs was also investigated in this study, with the use of U0126 and a specific small interfering RNA (siRNA) for TrkB. For in vivo study, ARH-77 cells were stably transfected with an antisense short-hairpin RNA construct to BDNF (AS-ARH) or empty vector (EV-ARH). These cells were then intravenously injected to severe combined immunodeficiency (SCID) mice, to test their capacity to induce MM bone disease. Radiographs of mice tibiae and vertebrae were taken weekly by X ray. Changes in total body bone mineral density (BMD) of mice skeleton were recorded. At the end of the experiment, bone sections were stained with hematoxylin and eosin staining or TRAP staining. Secretion levels of RANKL and OPG in mice bone marrow were measured by ELISA.We showed that BDNF increased RANKL and decreased OPG production in OBs in a time- and dose-dependent manner, thus contributing to osteoclast formation in vitro. In addition, these effects were completely abolished by K252a and TrkB-siRNA (P < 0.05). BDNF regulates RANKL/OPG expression in OBs through the TrkB/ERK signaling pathway. Our in vivo results indicated that mice injected with AS-ARH cells, which expressed low levels of endogenous BDNF, were preserved and exhibited no radiologically identifiable osteolytic lesions. In addition, mice in AS-ARH group also had a lower incidence of vertebral compression deformities and paralysis in comparison with mice in EV-ARH group (P < 0.05). Further more, bones harboring AS-ARH cells showed marked reduction of RANKL/OPG ratio and osteoclast density when compared to the controls harboring EV-ARH cells (P < 0.05).Our results demonstrate that BDNF is an important contributor to osteoclastogenesis in MM. Antisense inhibition of BDNF in MM cells remarkably inhibited osteolytic bone destruction in SCID-ARH mice model. BDNF-induced bone destruction is partially mediated by MM-OB interactions via upregulation of RANKL/OPG ratio in the bone marrow milieu. These findings suggest targeting BDNF may become a new therapeutic strategy to improve patient outcome in MM. Disclosures:No relevant conflicts of interest to declare.

Curcuminoids Block TGF-β Signaling in Human Breast Cancer Cells and Limit Osteolysis in a Murine Model of Breast Cancer Bone Metastasis

Effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide parathyroid hormone-related protein (PTHrP) and on lytic breast cancer bone metastasis were evaluated. In vitro, transforming growth factor (TGF)-β-stimulated PTHrP secretion was inhibited by curcuminoids (IC50 = 24 μM) in MDA-MB-231 human breast cancer cells independent of effects on cell growth inhibition. Effects on TGF-β signaling revealed decreases in phospho-Smad2/3 and Ets-1 protein levels with no effect on p-38 MAPK-mediated TGF-β signaling. In vivo, mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle and treated ip every other day with curcuminoids (25 or 50 mg/kg) for 21 days. Osteolytic bone lesion area was reduced up to 51% (p < 0.01). Consistent with specific effects on bone osteolysis, osteoclast number at the bone-tumor interface was reduced up to 53% (p < 0.05), while tumor area within bone was unaltered. In a separate study, tumor mass in orthotopic mammary xenografts was also unaltered by treatment. These data suggest that curcuminoids prevent TGF-β induction of PTHrP and reduce osteolytic bone destruction by blockade of Smad signaling in breast cancer cells.

1108TiP - Denosumab Compared with Zoledronic Acid for Preventing Skeletal Complications in Patients with Multiple Myeloma: A Randomized, Phase 3, Double-Blind, Double-Dummy Trial

ABSTRACT Patients with multiple myeloma (MM) experience osteolytic bone destruction resulting in skeletal-related events (SREs: pathologic fracture, spinal cord compression, surgery or radiation to bone). Severity of bone destruction correlates with significant morbidity and is associated with decreased survival. Osteoclast activity is enhanced in MM and is primarily regulated by RANK Ligand (RANKL) and osteoprotegerin. Denosumab is a fully human monoclonal antibody which binds RANKL, preventing activation of RANK and inhibiting osteoclast differentiation, activation and survival. Bone resorption and cancer-induced bone destruction are thereby reduced. Denosumab was superior to zoledronic acid (ZA) in 3 large randomized trials in patients with breast, prostate, or solid tumors-only analysis. Denosumab is approved for the prevention of SREs in patients with bone metastases from solid tumors in many regions worldwide. The current trial will evaluate the efficacy and safety of denosumab compared with ZA in preventing skeletal complications in patients with MM. The primary endpoint will determine if denosumab is non-inferior to ZA in prevention of the first on-study SRE. If denosumab is found to be non-inferior to ZA, superiority in time to first on-study SRE and time to first-and-subsequent SRE will be assessed as secondary endpoints. Patients will be randomly assigned 1:1 to receive subcutaneous (SC) denosumab 120 mg and intravenous (IV) placebo or IV ZA 4 mg and SC placebo every 4 weeks (Q4W). Randomization will be stratified by planned autologous transplantation (yes/no), anti-myeloma therapy (novel therapy-based vs non-novel therapy-based), disease stage (International Staging System 1, 2, or 3), previous SRE (yes/no), and region (Japan vs other). Daily supplements of calcium ≥500 mg and vitamin D ≥400 IU will be strongly recommended. Patient screening and enrollment is underway. In this event-driven study, data cutoff for the primary efficacy analysis is planned for when approximately 800 patients are anticipated to experience an on-study SRE. The trial is sponsored by Amgen Inc. and registered with ClinicalTrials.gov (NCT01345019). Disclosure A. Palumbo: Advisory Board Member: Celgene and Janssen-Cilag Honoraria: Celgene, Janssen-Cilag, Bristol-Myers Squibb, Millenium, Merck, Onyx and Amgen Consultancy: Celgene and Janssen-Cilag B.G. Durie: Advisory Board Member: Celgene Corporation, Millenium Pharmaceuticals, and Onyx Pharmaceuticals N. Raje: Advisory Board Member: Celgene, Millenium, Amgen Corporate-sponsored Research: Eli-Lilly, Acetylon R. Garcia Sanz: Honoraria: Amgen, Jansen Cilag, Celgene, Novartis O. Sezer: Advisory Board Member: Amgen, Janssen E. Terpos: Advisory Board Member: Amgen, Novartis W. Willenbacher: Advisory Board Member: Amgen Y. Qian: Employee and Stock Ownership: Amgen A. Balakumaran: Employee and Stock Ownership: Amgen All other authors have declared no conflicts of interest.

Recurrence in giant cell tumour of bone: imaging features and risk factors

This study was done to investigate X-ray, computed tomography (CT) and magnetic resonance (MR) imaging features of recurrence in giant cell tumour of bone (GCTB) and to evaluate risk factors. Medical records and imaging data were reviewed for 55 cases of recurrent GCTB. All images were reviewed retrospectively and independently by two radiologists experienced in skeletal musculature. The common radiological findings; factors related to tumour recurrence such as gender, age, location; pathological fracture, Campanacci grading and surgical procedure were analysed by nonparametric test (Mann-Whitney U test for two independent samples test and Kruskal-Wallis H test for multiple independent samples test). p values <0.05 were considered to indicate a statistically significant difference. The imaging features of recurrent GCTB were as follows: osteolytic destruction or bone resorption of graft bone or around the polymethylmethacrylate (PMMA), soft tissue mass formation and expansile change. Tumour parenchyma showed markedly heterogeneous enhancement, except for necrotic cystic cavities, on contrast-enhanced MR images. Wide resection had a smaller (p=0.031) risk of local recurrence than did intralesional curettage. There was no statistical significance in gender, age, location, pathological fracture and Campanacci staging (p>0.05). The risk of recurrence in GCTB was influenced by the type of surgery and adjuvants. Bone transformaresorption, soft tissue mass formation and aggravated expansile change are reliable signs of recurrence on imaging.

Heparin-like Polysaccharides Reduce Osteolytic Bone Destruction and Tumor Growth in a Mouse Model of Breast Cancer Bone Metastasis

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.

Effect of Sangu Decoction (三骨汤) on metastatic bone destruction in rats with mammary cancer

To study and evaluate the effect of Sangu Decoction (SGD, ) on the bone destruction due to mammary cancer metastasis. Metastasis rat mammary tumor-1 cells were transplanted into the left hind limb tibia of SD rats to establish the bone metastasis of the mammary cancer model. The modeled rats were treated with SGD for observing its effect on rats' pain behavior, including 50% paw withdrawal threshold (50% PWT) after von Frey fiber stimulation, burden difference of bilateral feet, and thermal withdrawal latency (TWL), with zoledronic acid as the positive control. Moreover, the damage in the tibia sample of rats was scored by an iconographic method, and the bone mineral density (BMD) as well as the bone mineral content (BMC) were estimated. The model established showed characteristics of mixed metastasis, revealing the manifestations of tumor development, bone destruction, cancerous pain, etc. In the SGD-treated group, 50% PWT was prolonged (8.13 ± 4.76 vs. 2.30 ± 2.19), and TWL was longer (3.48 ± 0.62 s vs. 2.89 ± 0.26 s) than those in the control group, respectively (P<0.05 or P<0.01). Iconographic scoring also showed improvement of BMD (0.134 ± 0.009 vs. 0.120 ± 0.007, P<0.01) and an elevating trend of BMC in the SGD-treated group. SGD could effectively alleviate the cancerous pain of bone metastasis and mitigate the metastasis that cause osteolytic destruction of bone.

Regulation of Breast Cancer-induced Bone Lesions by β-Catenin Protein Signaling

Breast cancer patients have an extremely high rate of bone metastases. Morphological analyses of the bones in most of the patients have revealed the mixed bone lesions, comprising both osteolytic and osteoblastic elements. β-Catenin plays a key role in both embryonic skeletogenesis and postnatal bone regeneration. Although this pathway is also involved in many bone malignancy, such as osteosarcoma and prostate cancer-induced bone metastases, its regulation of breast cancer bone metastases remains unknown. Here, we provide evidence that the β-catenin signaling pathway has a significant impact on the bone lesion phenotype. In this study, we established a novel mouse model of mixed bone lesions using intratibial injection of TM40D-MB cells, a breast cancer cell line that is highly metastatic to bone. We found that both upstream and downstream molecules of the β-catenin pathway are up-regulated in TM40D-MB cells compared with non-bone metastatic TM40D cells. TM40D-MB cells also have a higher T cell factor (TCF) reporter activity than TM40D cells. Inactivation of β-catenin in TM40D-MB cells through expression of a dominant negative TCF4 not only increases osteoclast differentiation in a tumor-bone co-culture system and enhances osteolytic bone destruction in mice, but also inhibits osteoblast differentiation. Surprisingly, although tumor cells overexpressing β-catenin did induce a slight increase of osteoblast differentiation in vitro, these cells display a minimal effect on osteoblastic bone formation in mice. These data collectively demonstrate that β-catenin acts as an important determinant in mixed bone lesions, especially in controlling osteoblastic effect within tumor-harboring bone environment.

Dual Inhibition of the STAT3 and Src Pathways by NS-018, a JAK2 and Src-Family Kinase Inhibitor, Reduces Myeloma Cell Proliferation and Adhesion and Suppresses Osteoclast Formation

Abstract 2900Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of malignant plasma cells in the bone marrow, and it finally leads to osteolytic bone destruction and impaired hematopoiesis. The pathophysiology of MM is closely linked with the bone marrow microenvironment, which consists of various cell components including bone marrow stromal cells (BMSCs) and bone marrow endothelial cells as well as osteoclasts and osteoblasts. Interaction between MM cells and BMSCs through cell-adhesion molecules confers drug resistance to myeloma cells and stimulates the release of cytokines such as interleukin-6 (IL-6) and receptor activator of NF-kappaB ligand (RANKL) from BMSCs. IL-6 is a major cytokine which enhances cell proliferation and promotes the survival of MM cells by downstream signaling through Janus kinase (JAK) and signal transducer and activator of transcription (STAT). RANKL triggers osteoclast differentiation and activation leading to bone resorption, lytic bone lesion, and osteopenia. The Src family kinases (SFKs), c-Src and Fyn, mediate signaling by cell-adhesion molecules and the RANKL receptor, and they play important roles in cell adhesion and osteoclast differentiation.NS-018 is a potent and selective dual JAK2/SFKs inhibitor which is under phase1/2 clinical development for the treatment of myelofibrosis. A previous study has shown that NS-018 inhibits JAK2, c-Src and Fyn kinases with IC50 values of 0.72, 6.0 and 7.3 nmol/L, respectively. NS-018 also inhibits JAK2 and SFKs in cells as evidenced by its antiproliferative effect against Ba/F3 cells expressing constitutively activated JAK2 and NIH3T3 cells transformed by v-Src.In the present study, the ability of NS-018 to inhibit JAK2/STAT3 signaling was examined in IL-6-responsive human MM cell lines such as U266, RPMI 8226, and PCM6. NS-018 suppressed IL-6-induced phosphorylation of STAT3 in a dose-dependent manner at concentrations greater than 100 nmol/L. In addition, NS-018 inhibited the IL-6-enhanced the proliferation of PCM6 cells at concentrations similar to those required to inhibit STAT3 phosphorylation.To assess whether SFKs inhibition by NS-018 could contribute to an improvement in MM pathology, we next investigated the effect of NS-018 on the adhesion of myeloma cells to cell-adhesion molecules and on osteoclast formation. NS-018 (100 nmol/L) inhibited the adhesion of RPMI 8226 cells to collagen type 1 and VCAM-1 by about 40%. NS-018 also suppressed RANKL-induced differentiation of human osteoclast precursor cells to mature osteoclasts. The numbers of TRAP-positive multinucleated osteoclasts were reduced to about one-half at 100 nmol/L NS-018 and none were observed at 1000 nmol/L. NS-018 similarly suppressed the differentiation of murine RAW264.7 cells to mature osteoclasts. The suppression of cell adhesion and osteoclast formation by NS-018 could both be mediated by c-Src and/or Fyn inhibition, because both inhibitory effects were observed with a typical SFKs inhibitor but not with a typical JAK2 inhibitor.In conclusion, NS-018 reduced IL-6-enhanced myeloma cell proliferation through inhibition of the JAK2/STAT3 signaling pathway and suppressed cell adhesion and osteoclast formation through inhibition of c-Src and/or Fyn. These results suggest that NS-018 has a dual mechanism of action in MM by simultaneously blocking the JAK2/STAT3 and SFKs pathways. Treatment with NS-018 is a potential new therapeutic option to improve the complex pathological condition of patients with MM. Disclosures:No relevant conflicts of interest to declare.

Targeting HSP 90 Induces Apoptosis and Inhibits Critical Survival and Proliferation Pathways in Multiple Myeloma

The second most commonly diagnosed hematologic malignancy, multiple myeloma, affects predominantly older patients (>60s) and is characterized by paraprotein in the serum or urine. Clinical manifestations include anemia, hypercalcaemia, progressive renal impairment, and osteolytic bone destruction. Despite promising new therapies, multiple myeloma eventually relapses in almost all patients. HSP are ubiquitous and highly conserved in prokaryotes and eukaryote organisms. Exposure to a broad range of stimuli results in increased HSP protein expression. These chaperone proteins are involved in protein transportation, prevent protein aggregation, and ensure correct folding of nascent and stress-accumulated misfolded proteins. In cancer, HSP expression is dysregulated, resulting in elevated expression, which promotes cancer by preventing programmed cell death and supporting autonomous cells growth, ultimately leading to resistance to heat, chemotherapy, and other stresses. Client proteins of HSP90 such as AKT, p53, MEK, STAT3, and Bcr-Abl are vital in tumor progression, including multiple myeloma, and their maturation and stability is dependent on HSP90. Therefore, inhibition of HSP90 via a HSP90 inhibitor (such as NVP-HSP990) should interrupt multiple signaling pathways essential for oncogenesis and growth in multiple myeloma. Our study showed that NVP-HSP990 triggered apoptosis in a panel of human multiple myeloma cells, induced cell-cycle arrest, PARP cleavage, downregulation of client proteins, the inability to reactivate phospho-STAT3 following exogenous IL-6 stimulation, and it synergized with azacytidine and bortezomib in cell lines and primary multiple myeloma samples. The mechanism of HSP90 inhibition in multiple myeloma warrants further evaluation.

Metastatic breast cancer cells inhibit osteoblast differentiation through the Runx2/CBFβ-dependent expression of the Wnt antagonist, sclerostin

IntroductionBreast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction by stimulating osteoclasts to resorb bone and by preventing osteoblasts from producing new bone. The Runt-related transcription factor 2, Runx2, is an important determinant of bone metastasis in breast cancer. Runx2 is known to mediate activation of osteoclast activity and inhibition of osteoblast differentiation by metastatic breast cancer cells. However, while Runx2-regulated genes that mediate osteoclast activation have been identified, how Runx2 determines inhibition of osteoblasts is unknown.MethodsThe aim of this study was to determine how Runx2 mediates the ability of metastatic breast cancer cells to modulate the activity of bone cells. We have previously demonstrated that Runx2 requires the co-activator core binding factor beta (CBFβ) to regulate gene expression in breast cancer cells. We, therefore, performed independent microarray analyses to identify target genes whose expression is dependent upon both Runx2 and CBFβ. Common target genes, with a role in modulating bone-cell function, were confirmed using a combination of siRNA, quantitative reverse transcriptase PCR (qRT-PCR), ELISA, promoter reporter analysis, Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The function of Runx2/CBFβ-regulated genes in mediating the ability of MDA-MB-231 to inhibit osteoblast differentiation was subsequently established in primary bone marrow stromal cell cultures and MC-3T3 osteoblast cells.ResultsWe show that Runx2/CBFβ mediates inhibition of osteoblast differentiation by MDA-MB-231 cells through induction of the Wnt signaling antagonist, sclerostin. We demonstrate that MDA-MB-231 cells secrete sclerostin and that sclerostin-expression is critically dependent on both Runx2 and CBFβ. We also identified the osteoclast activators IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as new target genes of Runx2/CBFβ in metastatic breast cancer cells.ConclusionsThis study demonstrates that Runx2 and CBFβ are required for the expression of genes that mediate the ability of metastatic breast cancer cells to directly modulate both osteoclast and osteoblast function. We also show that Runx2-dependent inhibition of osteoblast differentiation by breast cancer cells is mediated through the Wnt antagonist, sclerostin.