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Supplementary Figure Legend from Zerumbone Abolishes RANKL-Induced NF-κB Activation, Inhibits Osteoclastogenesis, and Suppresses Human Breast Cancer–Induced Bone Loss in Athymic Nude Mice

Supplementary Figure 1 from TGF-β Promotion of Gli2-Induced Expression of Parathyroid Hormone-Related Protein, an Important Osteolytic Factor in Bone Metastasis, Is Independent of Canonical Hedgehog Signaling

Supplementary Figure Legends 1-2 from TGF-β Promotion of Gli2-Induced Expression of Parathyroid Hormone-Related Protein, an Important Osteolytic Factor in Bone Metastasis, Is Independent of Canonical Hedgehog Signaling

Supplementary Figure 1 from Zerumbone Abolishes RANKL-Induced NF-κB Activation, Inhibits Osteoclastogenesis, and Suppresses Human Breast Cancer–Induced Bone Loss in Athymic Nude Mice

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone–related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β–stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep–expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction. Cancer Res; 71(3); 822–31. ©2010 AACR.

Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) has emerged as a major mediator of bone resorption, commonly associated with cancer and other chronic inflammatory diseases. Inhibitors of RANKL signaling thus have potential in preventing bone loss. In the present report, the potential of zerumbone, a sesquiterpene derived from subtropical ginger, to modulate osteoclastogenesis induced by RANKL and breast cancer was examined. We found that zerumbone inhibited RANKL-induced NF-κB activation in mouse monocyte, an osteoclast precursor cell, through inhibition of activation of IκBα kinase, IκBα phosphorylation, and IκBα degradation. Zerumbone also suppressed RANKL-induced differentiation of these cells to osteoclasts. This sesquiterpene also inhibited the osteoclast formation induced by human breast tumor cells and by multiple myeloma cells. Finally, we examined whether zerumbone could prevent human breast cancer–induced bone loss in animals. We found that zerumbone decreased osteolysis in a dose-dependent manner in MDA-MB-231 breast cancer tumor-bearing athymic nude mice. These results indicate that zerumbone is an effective blocker of RANKL-induced NF-κB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss. [Cancer Res 2009;69(4):1477–84]

Proteasome inhibitor (PI) therapy has improved the survival of multiple myeloma (MM) patients. However, inevitably, primary or acquired resistance to PIs leads to disease progression; resistance mechanisms are unclear. Obesity is a risk factor for MM mortality. Oxidized LDL (OxLDL), a central mediator of atherosclerosis that is elevated in metabolic syndrome (co-occurrence of obesity, insulin resistance, dyslipidemia and hypertension), has been linked to an increased risk of solid cancers and shown to stimulate pro-oncogenic/survival signaling. We hypothesized that OxLDL is a mediator of chemoresistance, and evaluated its effects on MM cell killing by PIs. OxLDL potently suppressed the ability of the boronic-acid based PIs bortezomib (BTZ) and ixazomib, but not the epoxyketone-based PI carfilzomib, to kill human MM cell lines and primary cells. OxLDL suppressed BTZ-induced inhibition of proteasome activity and induction of pro-apoptotic signaling. These cytoprotective effects were abrogated when lipid hydroperoxides (LOOHs) associated with OxLDL were enzymatically reduced. We also demonstrated the presence of OxLDL in the MM bone marrow microenvironment as well as numerous granulocytes and monocytes capable of cell-mediated LDL oxidation through myeloperoxidase. Our findings suggest that OxLDL may be a potent mediator of boronic acid-based PI resistance, particularly for MM patients with metabolic syndrome, given their elevated systemic levels of OxLDL. LDL cholesterol-lowering therapy to reduce circulating OxLDL, and pharmacologic targeting of LOOH levels or resistance pathways induced by the modified lipoprotein, could deepen the response to these important agents and offer clinical benefit to MM patients with metabolic syndrome. This article is protected by copyright. All rights reserved.

BackgroundMultiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported.MethodsThis study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment.ResultsUsing the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8+ T cell numbers; (ii) activated NK cells and CD8+ T cells and (iii) upregulated effector-memory CD8+ T cells. Moreover, increased effector-memory CD8+ T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes.ConclusionThese data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority.

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.

Multiple myeloma (MM) is the most common cancer affecting the bone and bone marrow and remains incurable for most patients, novel therapies are therefore needed. Bortezomib (Btz) is an FDA-approved drug for the treatment of patients with MM. However, its severe side effects require a dose reduction or the potential discontinuation of treatment. To overcome this limitation, we conjugated Btz to a bisphosphonate (BP) residue lacking anti-osteoclastic activity using a novel chemical linker and generated a new bone-targeted Btz-based (BP-Btz) proteasome inhibitor. We demonstrated that BP-Btz, but not Btz, bound to bone slices and inhibited the growth of MM cells in vitro. In a mouse model of MM, BP-Btz more effectively reduced tumor burden and bone loss with less systemic side effects than Btz. Thus, BP-Btz may represent a novel therapeutic approach to treat patients with MM.

// Ramaswamy Sharma 1,* , Paul J. Williams 1,* , Anjana Gupta 1 , Brandon McCluskey 1 , Shylesh Bhaskaran 1,2 , Steve Munoz 1,3 and Babatunde O. Oyajobi 1,4 1 Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 2 Current address: Oklahoma Medical Research Foundation, Oklahoma City, OK, USA 3 Current address: Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, Nashville, TN, USA 4 Cancer Therapy and Research Center, San Antonio, TX, USA * These authors contributed equally to this work Correspondence to: Babatunde O. Oyajobi, email: // Keywords : FWD1/β-TrCP1, 5TGM1, myeloma, plasmacytoma, proteasome Received : December 23, 2014 Accepted : April 30, 2015 Published : May 12, 2015 Abstract Treatment of multiple myeloma with bortezomib can result in severe adverse effects, necessitating the development of targeted inhibitors of the proteasome. We show that stable expression of a dominant-negative F-box deleted (∆F) mutant of the E3 ubiquitin ligase, SCF β-TrCP/FWD1 , in murine 5TGM1 myeloma cells dramatically attenuated their skeletal engraftment and survival when inoculated into immunocompetent C57BL/KaLwRij mice. Similar results were obtained in immunodeficient bg-nu-xid mice, suggesting that the observed effects were independent of host recipient immune status. Bone marrow stroma offered no protection for 5TGM1-∆F cells in cocultures treated with tumor necrosis factor (TNF), indicating a cell-autonomous anti-myeloma effect. Levels of p100, IκBα, Mcl-1, ATF4, total and cleaved caspase-3, and phospho-β-catenin were elevated in 5TGM1-∆F cells whereas cIAP was down-regulated. TNF also activated caspase-3 and downregulated Bcl-2, correlating with the enhanced susceptibility of 5TGM1-∆F cells to apoptosis. Treatment of 5TGM1 tumor-bearing mice with a β-TrCP1/FWD1 inhibitor, pyrrolidine dithiocarbamate (PDTC), significantly reduced tumor burden in bone. PDTC also increased levels of cleaved Mcl-1 and caspase-3 in U266 human myeloma cells, correlating with our murine data and validating the development of specific β-TrCP inhibitors as an alternative therapy to nonspecific proteasome inhibitors for myeloma patients.

Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.

The BMP and Wnt/β-catenin signaling pathways cooperatively regulate osteoblast differentiation and bone formation. Although BMP signaling regulates gene expression of the Wnt pathway, much less is known about whether Wnt signaling modulates BMP expression in osteoblasts. Given the presence of putative Tcf/Lef response elements that bind β-catenin/TCF transcription complex in the BMP2 promoter, we hypothesized that the Wnt/β-catenin pathway stimulates BMP2 expression in osteogenic cells. In this study, we showed that Wnt/β-catenin signaling is active in various osteoblast or osteoblast precursor cell lines, including MC3T3-E1, 2T3, C2C12, and C3H10T1/2 cells. Furthermore, crosstalk between the BMP and Wnt pathways affected BMP signaling activity, osteoblast differentiation, and bone formation, suggesting Wnt signaling is an upstream regulator of BMP signaling. Activation of Wnt signaling by Wnt3a or overexpression of β-catenin/TCF4 both stimulated BMP2 transcription at promoter and mRNA levels. In contrast, transcription of BMP2 in osteogenic cells was decreased by either blocking the Wnt pathway with DKK1 and sFRP4, or inhibiting β-catenin/TCF4 activity with FWD1/β-TrCP, ICAT, or ΔTCF4. Using a site-directed mutagenesis approach, we confirmed that Wnt/β-catenin transactivation of BMP2 transcription is directly mediated through the Tcf/Lef response elements in the BMP2 promoter. These results, which demonstrate that the Wnt/β-catenin signaling pathway is an upstream activator of BMP2 expression in osteoblasts, provide novel insights into the nature of functional cross talk integrating the BMP and Wnt/β-catenin pathways in osteoblastic differentiation and maintenance of skeletal homeostasis.

Deer antler growth provides a unique natural model of rapid and complete bone regeneration. In this study, the distal antler tips of male red deer (Cervus elaphus) were collected post-mortem during the annual growth period (April-August), and an in vitro system established for the culture of cells from three regions; the inner layer of the perichondrium, the reserve mesenchyme and the cartilage zone. Alkaline phosphatase (ALP) expression by cultured cells, as demonstrated by enzyme histochemistry and biochemical assay, reflected the stage of cellular differentiation. ALP activity was highest in cells cultured from the hypertrophic cartilage region (3.6 ± 0.2 μmol/μg cell protein/minute), and lowest in undifferentiated mesenchymal cells (0.3 ± 0.01 μmol/μg cell protein/minute). ALP expression was lost with passage in culture. Levels of ALP activity in cultured cells correlated with the pattern and extent of enzyme expression in tissue sections as demonstrated by histochemical staining. Insulin-like growth factor (IGF)-I (10−9M-10−7M) was found to be mitogenic for cultured cells from all three zones as shown by increased incorporation of [3H]thymidine into DNA. These results demonstrate that cells from three different regions of the antler tip can be maintained in culture, and that antler cells share certain phenotypic characteristics of growth plate chondrocytes. These data provide further evidence of a role for IGF-1 in the regulation of antler growth. Antler regrowth is a potentially useful model for the study of the factors that regulate bone formation.

Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.

Multiplemyeloma (MM) patients frequently develop tumor-induced bone destruction, yet no therapy completely eliminates the tumor or fully reverses bone loss. Transforming growth factor-β (TGF-β) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-β inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-β signaling also reduces tumor growth, increases bone volume, and improves vertebral body strength in MM-bearing mice. We treated myeloma tumor-bearing (immunocompetent KaLwRij and immunocompromised Rag2 −/−) mice with a TGF-β inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4 weeks after inoculation of murine 5TGM1 MM cells. TGF-β inhibition increased trabecular bone volume, improved trabecular architecture, increased tissue mineral density of the trabeculae as assessed by ex vivo micro-computed tomography, and was associated with significantly greater vertebral body strength in biomechanical compression tests. Serum monoclonal paraprotein titers and spleen weights showed that 1D11 monotherapy did not reduce overall MM tumor burden. Combination therapy with 1D11 and bortezomib increased vertebral body strength, reduced tumor burden, and reduced cortical lesions in the femoral metaphysis, although it did not significantly improve cortical bone strength in three-point bending tests of the mid-shaft femur. Overall, our data provides rationale for evaluating inhibition of TGF-β signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease.

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor b (TGF-β), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.

Oncolytic virotherapy with reovirus has demonstrated anti-cancer activity and minimal toxicity in clinical trials, but the mechanisms underlying these effects have not been fully elucidated. Reolysin, a proprietary formulation of reovirus for cancer therapy, stimulated selective viral replication and apoptosis in multiple myeloma (MM) cells. Reolysin-mediated apoptosis was associated with an induction of endoplasmic reticular (ER) stress-related gene expression, swelling of the endoplasmic reticulum, increases in intracellular calcium levels and a strong induction of the Bcl-2 homology 3 (BH3)-only pro-apoptotic protein NOXA. Knockdown of NOXA expression by short hairpin RNA significantly reduced the pro-apoptotic effects of Reolysin. We next showed that co-administration of Reolysin and bortezomib resulted in the dual accumulation of viral and ubiquitinated proteins, which led to enhanced ER stress, NOXA induction and apoptosis. Importantly, the combination of reovirus infection and proteasomal inhibition significantly decreased tumor burden in a xenograft and syngeneic bone disease model of MM without exhibiting adverse side effects. Our study establishes ER stress stimulation and NOXA induction as novel mediators of reovirus-induced apoptosis. Furthermore, reovirus infection can be used as a promising approach to augment the anti-myeloma activity of bortezomib by promoting additional stress to the endoplasmic reticulum of MM cells.

Bone marrow-targeted drug delivery systems appear to offer a promising strategy for advancing diagnostic, protective and/or therapeutic medicine for the hematopoietic system. Liposome technology can provide a drug delivery system with high bone marrow targeting that is mediated by specific phagocytosis in bone marrow.This review focuses on a bone marrow-specific liposome formulation labeled with technetium-99 m. Interspecies differences in bone marrow distribution of the bone marrow-targeted formulation are emphasized. This review provides a liposome technology to target bone marrow. In addition, the selection of proper species for the investigation of bone marrow targeting is suggested.It can be speculated that the bone marrow macrophages have a role in the delivery of lipids to the bone marrow as a source of energy and for membrane biosynthesis or in the delivery of fat-soluble vitamins for hematopoiesis. This homeostatic system offers a potent pathway to deliver drugs selectively into bone marrow tissues from blood. High selectivity of the present bone marrow-targeted liposome formulation for bone marrow suggests the presence of an active and specific mechanism, but specific factors affecting the uptake of the bone marrow mononuclear phagocyte system are still unknown. Further investigation of this mechanism will increase our understanding of factors required for effective transport of agents to the bone marrow, and may provide an efficient system for bone marrow delivery for therapeutic purposes.

Transforming growth β (TGF-β) has been proposed to have a role in bone remodeling by affecting the differentiation and activity of osteoblasts and osteoclasts and by inhibiting the production of proteinases, such as plasminogen activators (PAs). Studies on PAs have largely been based on data from nonhuman and fetal cell lines, however. The purpose of this study was to investigate the effect of TGF-β on the PA activity of normal human osteoblast-like cells and to compare this with its action on the human osteosarcoma cell line MG-63. The action of interleukin-1β (IL-1β) was also assessed because it has been shown to increase PA activity in other connective tissue cell types. Normal osteoblast-like cells had low to undetectable basal urokinase (uPA) and tissue plasminogen activator (tPA) activity, which was significantly stimulated by TGF-β1. This action was shown to be dependent on transcription and new protein synthesis. TGY-β2 had a similar action. IL-1β did not stimulate PA activity. In contrast, the MG-63 cell line had high basal tPA and uPA activities. TGF-β1 decreased basal PA activity, the effect being most marked for uPA activity. IL-1β stimulated uPA and tPA activity. TGF-β1 inhibited IL-1β-stimulated uPA activity, but the effect on tPA was more variable. This study has shown that TGF-β has opposite effects on the PA activity of the two osteoblast-like cell types studied. Care must therefore be used before extrapolating data from one cell type to another. It is suggested that under certain circumstances TGF-β may be involved in bone resorption by modulating PA activity.

Polymer-conjugated nanoparticles are an important technology to control the stability, safety, and efficacy in drug delivery systems. Herein, we investigate self-organized mixed assemblies of a lipophilic drug candidate, curcumin (Cm), and a poly(oxyethylene) cholesteryl ether (PEG-Chol). Cm was assembled together with PEG-Chol to form nano-sized assemblies (around 10 nm) of assumed micelles. In contrast with the rapid decomposition of free Cm due to the hydrolysis, the Cm was highly stabilized in the nanoparticles, especially at below 40 mol% Cm. Cell viability assay revealed that the cytotoxic activity of the Cm/PEG-Chol nanoparticles against myeloma cells is higher than those of free Cm in a comparison at 1 microM. On the other hand, both the Cm/PEG-Chol nanoparticles and PEG-Chol micelles had significant cytotoxicity to the myeloma cells at 5 microM. Taken together, the present Cm/PEG-Chol system offers a stable nanoparticle encapsulating Cm which can be injected as a liquid. Cm and vehicle micelles will damage the cancer cells cooperatively.

There is increasing evidence to suggest that the Wnt signaling pathway plays a critical role in the pathogenesis of myeloma bone disease. In the present study, we determined whether increasing Wnt signaling within the bone marrow microenvironment in myeloma counteracts development of osteolytic bone disease. C57BL/KaLwRij mice were inoculated intravenously with murine 5TGM1 myeloma cells, resulting in tumor growth in bone and development of myeloma bone disease. Lithium chloride (LiCl) treatment activated Wnt signaling in osteoblasts, inhibited myeloma bone disease, and decreased tumor burden in bone, but increased tumor growth when 5TGM1 cells were inoculated subcutaneously. Abrogation of β-catenin activity and disruption of Wnt signaling in 5TGM1 cells by stable overexpression of a dominant-negative TCF4 prevented the LiCl-induced increase in subcutaneous growth but had no effect on LiCl-induced reduction in tumor burden within bone or on osteolysis in myeloma-bearing mice. Together, these data highlight the importance of the local microenvironment in the effect of Wnt signaling on the development of myeloma bone disease and demonstrate that, despite a direct effect to increase tumor growth at extraosseous sites, increasing Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease and inhibit myeloma growth within bone in vivo.

Impaired bone formation contributes to the lack of bone healing in multiple myeloma and there is a need for agents with bone anabolic properties to reverse the bone deficit in patients. Bortezomib, a proteasome inhibitor with antitumour efficacy in myeloma patients, enhanced new bone formation in mouse calvarial cultures; this effect was blocked by dickkopf 1(Dkk1), an antagonist of Wnt signalling implicated in myeloma bone disease. Bortezomib inhibited Dkk1 expression in calvariae and bone marrow-derived stromal cells, suggesting a novel mechanism by which bortezomib exerts its effects in bone. Clinical trials in patients with myeloma bone disease are needed to validate these results.

Despite remarkable therapeutic improvement in multiple myeloma (MM), prognosis is very poor once patients become refractory to or ineligible for proteasome inhibitors and immunomodulatory drugs. Accordingly, new drugs are clearly needed for this patient population, and extensive efforts are leading to a new breed of antimyeloma drugs.

Bone morphogenetic proteins (BMPs) are required for normal postnatal bone formation and osteoblast differentiation. There is evidence from recent studies that BMP signaling in osteoblasts is controlled by an ubiquitin-proteasome regulatory mechanism involving a cascade of enzymatic reactions. The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes. We have examined the role of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1), a member of the Hect domain family of E3 ubiquitin ligases in osteoblast function. Smurf1 has been found to interact with BMP-activated Smad1 and -5 and to mediate degradation of these Smad proteins. Recently we have found that Smurf1 mediates the protein degradation of the osteoblast-specific transcription factor Runx2/Cbfa1. To determine the role of Smurf1 in osteoblast differentiation, in the present studies we transfected a Smurf1 expression plasmid into 2T3 osteoblast precursor cells and found that Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation. To further investigate the role of Smurf1 in bone formation in vivo, we generated transgenic mice in which expression of the epitope-tagged Smurf1 transgene was targeted to osteoblasts using the murine 2.3-kb osteoblast-specific type I collagen promoter. In these transgenic mice, bone formation was significantly reduced during postnatal life. Our results demonstrate for the first time that Smurf1 plays a specific role in osteoblast differentiation and bone formation in vivo.

Osteoblast differentiation and bone formation is stimulated by bone morphogenetic protein (BMP)-2 and its downstream signaling molecules Smad1 and -5 and the osteoblast-specific transcription factor core-binding factor alpha1 (Cbfa1). Proteolytic degradation of Smad1 and Cbfa1 is proteasome-dependent, and intracellular concentrations of Smad1 and Cbfa1 are enhanced by inhibition of the 26 S proteasome. Smad1 degradation is mediated by the E3 ubiquitin ligase Smurf1 (Smad ubiquitin regulatory factor 1), but the specific E3 ligase responsible for Cbfa1 degradation has not been identified. Because Cbfa1 interacts with Smad1, whose degradation is mediated by Smurf1, we examined the effect of Smurf1 on Cbfa1 degradation in osteoblast precursor cells. Smurf1 interacts directly with Cbfa1 and mediates Cbfa1 degradation in a ubiquitin- and proteasome-dependent manner. Because Smurf1 controls the intracellular concentrations of several key molecules in the bone formation cascade, we examined the effect of a mutant form of Smurf1 in osteoblasts and found that expression of mutant Smurf1 markedly enhanced osteoblast differentiation. Smurf1 therefore appears to be an important regulatory factor in osteoblast differentiation and a potential molecular target for identification of bone anabolic agents.

Recent data have implicated macrophage inflammatory protein-1α (MIP-1α) in multiple myeloma (MM)-associated osteolysis. However, it is unclear whether the chemokine's effects are direct, to enhance osteolysis, or indirect and mediated through a reduction in tumor burden, or both. It is also unclear whether MIP-1α requires other factors such as receptor activator of nuclear factor-κB ligand (RANKL) for its effects on bone. In murine 5TGM1 (Radl) myeloma-bearing mice, administration of neutralizing anti-MIP-1α antibodies reduced tumor load assessed by monoclonal paraprotein titers, prevented splenomegaly, limited development of osteolytic lesions, and concomitantly reduced tumor growth in bone. To determine the effects of MIP-1α on bone in vivo, Chinese hamster ovary (CHO) cells secreting human MIP-1α (CHO/MIP-1α) were inoculated into athymic mice. Mice bearing intramuscular CHO/MIP-1α tumors developed lytic lesions at distant skeletal sites, which occurred earlier and were larger than those in mice with CHO/empty vector (EV) tumors. When experimental metastases were induced via intracardiac inoculation, mice bearing CHO/MIP-1α tumors developed hypercalcemia and significantly more osteolytic lesions than mice bearing CHO/EV tumors, with intramedullary CHO/MIP-1α tumors associated with significantly more tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts. Injection of recombinant MIP-1α over calvariae of normal mice evoked a striking increase in osteoclast formation, an effect dependent on RANK/RANKL signaling because MIP-1α had no effect in RANK-/- mice. Together, these results establish that MIP-1α is sufficient to induce MM-like destructive lesions in bone in vivo. Because, in the 5TGM1 model, blockade of osteoclastic resorption in other situations does not decrease tumor burden, we conclude that MIP-1α exerts a dual effect in myeloma, on osteoclasts, and tumor cells. (Blood. 2003;102:311-319)

The current introduction summarizes the five scientific sessions (bone marrow microenvironment and animal models of bone metastasis; prostate carcinoma; multiple myeloma; breast carcinoma; and preclinical and clinical studies on metastasis: future directions) and provides an overview of the proceedings of the Third North American Symposium on Skeletal Complications of Malignancy, Bethesda, Maryland, April 25-27, 2002.

The regulation of human bone marrow stromal precursor cell differentiation toward the chondrocyte, osteoblast or adipocyte lineages is not known. In this study, we assessed the lineage-specific differentiation and conversion of immortalized clonal F/STRO-1(+) A human fetal bone marrow stromal cells under the control of dexamethasone (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F/STRO-1(+) A cells expressed markers mRNAs or proteins of the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline phosphatase (ALP), type 1 collagen], of the chondrocyte lineage (aggrecan, types 2, 9 and 10 collagen), and of the adipocyte lineage (PPARgamma2, C/EBPalpha, aP2, G3PDH, lipoprotein lipase, leptin). Treatment with Dex increased CBFA1, OC and ALP mRNA and protein levels. Exposure to LA enhanced expression of adipocytic genes and cytoplasmic triglycerides accumulation, and suppressed the Dex-induced stimulation of osteoblast marker genes. Indo/Ins stimulated the synthesis of aggrecan and type 2 collagen and increased types 9 and 10 collagen mRNA levels, and suppressed both basal and Dex-promoted expression of osteoblast markers. Conversely, stimulation of osteoblastogenesis by Dex suppressed both basal and Indo/Ins-stimulated chondrocyte genes. Thus, the clonal human fetal bone marrow stromal F/STRO-1(+) A cell line is a lineage-unrestricted common progenitor that expresses tripotential adipocyte, osteoblast or chondrocyte characteristics. Our data also show that differentiation towards one pathway in response to Dex, Indo/Ins and LA restricts expression of other lineage-specific genes, and provide evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of clonal F/STRO-1(+) human bone marrow stromal cells.

Obesity increases the risk of developing multiple myeloma (MM). Adiponectin is a cytokine produced by adipocytes, but paradoxically decreased in obesity, that has been implicated in MM progression. Herein, we evaluated how prolonged exposure to adiponectin affected the survival of MM cells as well as putative signaling mechanisms. Adiponectin activates protein kinase A (PKA), which leads to decreased AKT activity and increased AMP-activated protein kinase (AMPK) activation. AMPK, in turn, induces cell cycle arrest and apoptosis. Adiponectin-induced apoptosis may be mediated, at least in part, by the PKA/AMPK-dependent decline in the expression of the enzyme acetyl-CoA-carboxylase (ACC), which is essential to lipogenesis. Supplementation with palmitic acid, the preliminary end product of fatty acid synthesis, rescues MM cells from adiponectin-induced apoptosis. Furthermore, 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), an ACC inhibitor, exhibited potent antiproliferative effects on MM cells that could also be inhibited by fatty acid supplementation. Thus, adiponectin's ability to reduce survival of MM cells appears to be mediated through its ability to suppress lipogenesis. Our findings suggest that PKA/AMPK pathway activators, or inhibitors of ACC, may be useful adjuvants to treat MM. Moreover, the antimyeloma effect of adiponectin supports the concept that hypoadiponectinemia, as occurs in obesity, promotes MM tumor progression.

Kaposi's sarcoma-associated herpesvirus (KSHV, also known as HHV-8) has been found in patients with multiple myeloma (MM) and postulated to be aetiologically associated with the development of this common plasma cell malignancy. A murine model of MM was previously established in which intravenous transfer of 5T myeloma cells into C57BL/KaLwRij mice resulted in characteristic features of human MM. In the present study, we sought to identify herpesvirus DNA sequences in this murine model of MM through polymerase chain reaction (PCR) analysis using primers specific for KSHV, murine herpesvirus 68 (MHV68) and murine cytomegalovirus (MCMV) as well as consensus primers designed from the highly conserved DNA polymerase genes of the Herpesviridae family. None of the DNA samples from whole bone marrow (n = 6) or dendritic cells enriched by long-term culture (n = 8) of 5T myeloma-bearing mice as well as the 5T myeloma cell lines (n = 3) maintained in long-term culture yielded specific amplification products in any of the PCR assays. Two KSHV-specific serological assays measuring antibodies to KSHV latent and lytic antigens also failed to detect the presence of anti-KSHV antibodies in mice that developed MM. These results suggest that the development of 5T murine MM is unlikely to be involved with KSHV or a KSHV-like murine herpesvirus.

Interleukin-6 (IL-6) is an important growth and survival factor for myeloma cells. However, the identity of the cells producing IL-6 in vivo remains unclear. Myeloma cells are found closely associated with sites of active bone turnover, and cells of the osteogenic lineage, including bone marrow osteoprogenitors, osteoblasts and bone lining cells, may therefore be ideally placed to synthesize IL-6. We have examined the possibility that human osteogenic cells may produce IL-6 in response to stimulation by myeloma cells. Primary human osteoblasts (hOBs) were isolated from normal donors, co-cultured with the human myeloma cell lines, JJN-3, RPMI-8226 and NCI-H929, and the amount of IL-6 released was determined by enzyme-linked immunosorbent assay (ELISA). All myeloma cells stimulated a significant increase in the production of IL-6 when cultured with hOBs (P < 0.05). Prior fixation of hOBs completely abrogated release of IL-6 in the co-cultures. In contrast, fixed myeloma cells retained the ability to induce IL-6 production, suggesting that hOBs were the principal source of IL-6. Physical separation of myeloma cells from hOBs using transwell inserts caused a partial inhibition of IL-6 release (P < 0.05), whereas the addition of media conditioned by myeloma cells to cultures of hOBs stimulated a significant increase in IL-6 production (P < 0.05). hOBs secreted greater amounts of IL-6 than human bone marrow stromal cells (hBMSCs) (2.2- to 3.5-fold, P < 0.05), but incubating hBMSCs with dexamethasone to stimulate osteoblastic differentiation resulted in an increase in their ability to produce IL-6 (1.7- to 4. 8-fold, P < 0.05) and to respond to myeloma cells (P < 0.05). These data clearly indicate that cells of the osteoblast lineage release significant amounts of IL-6 in response to stimulation by myeloma cells and may contribute to the IL-6 that promotes the proliferation and survival of myeloma cells in vivo.

We determined the effects of the potent bisphosphonate ibandronate in a murine model of human myeloma bone disease. In this model, bone lesions typical of the human disease develop in mice following inoculation of myeloma cells via the tail vein. Treatment with ibandronate (4 μg per mouse per day) significantly reduced the occurrence of osteolytic bone lesions in myeloma-bearing mice. However, ibandronate did not prevent the mice from developing hindlimb paralysis and did not produce a detectable effect on survival. There was no significant effect of ibandronate on total myeloma cell burden, as assessed by morphometric measurements of myeloma cells in the bone marrow, liver, and spleen, or by measurement of serum IgG2b levels. These results support clinical findings that bisphosphonates may be useful for the treatment of myeloma-associated bone destruction, but suggest that other therapies are also required to reduce tumor growth.

Osteoprogenitor cells present in human fetal bone marrow (BM) stroma have not been characterized. We used density gradient centrifugation, aggregation on binding lectin, and enrichment by magnetic activated cell sorting with STRO-1 antibody to isolate STRO-1+ cells from nonadherent human fetal BM stromal cells. Immunoselected STRO-1+ cells were immortalized using SV-40 large T antigen and a clone, F/STRO-1+ A, with weak alkaline phosphatase (ALP) activity was selected. The cloned cells proliferated rapidly but were not tumorigenic. Preconfluent F/STRO-1+ A cells showed immunoreactivity for osteopontin, alpha1(I) procollagen, and parathyroid hormone-related peptide, but not for the late osteoblast differentiation markers, osteocalcin (OC), or bone sialoprotein. However, differentiation of F/STRO-1+ A cells was induced by dexamethasone and 1,25-dihydroxyvitamin D3, as shown by increased ALP activity. In addition, osteogenesis occurred in F/STRO-1+ A cells cultured in three-dimentional aggregates, as assessed morphologically, histologically, and biochemically. Moreover, reverse transcription-polymerase chain reaction analysis showed that OC expression was silent in exponentially growing cells and occurred when cell-cell contacts were established in monolayer and in aggregates, showing induction of mature osteoblast phenotype by cell-cell contacts. Thus, clonal F/STRO-1+ A cells immunoselected from human fetal BM stroma display features of immature osteoprogenitor cells which can differentiate into mature osteogenic cells by cell-cell interactions or inducing agents. The generation by immunoselection of an immortalized clonogenic human fetal BM stroma-derived cell line which behaves like an osteoprogenitor cell provides a novel model system for identifying the signals required for the commitment of osteoprogenitors in the human fetal BM stroma.

We determined the effects of the potent bisphosphonate ibandronate in a murine model of human myeloma bone disease. In this model, bone lesions typical of the human disease develop in mice following inoculation of myeloma cells via the tail vein. Treatment with ibandronate (4 μg per mouse per day) significantly reduced the occurrence of osteolytic bone lesions in myeloma-bearing mice. However, ibandronate did not prevent the mice from developing hindlimb paralysis and did not produce a detectable effect on survival. There was no significant effect of ibandronate on total myeloma cell burden, as assessed by morphometric measurements of myeloma cells in the bone marrow, liver, and spleen, or by measurement of serum IgG2b levels. These results support clinical findings that bisphosphonates may be useful for the treatment of myeloma-associated bone destruction, but suggest that other therapies are also required to reduce tumor growth.

The etiology of osteoporosis is multifactorial, but there is evidence from both animal and human studies that the volume of marrow adipose tissue increases when bone volume is reduced in osteoporosis. The cell-related mechanism that may account for this inverse relationship between the volume of marrow adipose tissue and bone remains to be clarified, although it is known that both adipocytes and osteoblasts are derived from stromal cell precursors in bone marrow. We report that retroviral transduction with a temperature-sensitive oncogene (SV40 large T antigen) can generate bipotential cell lines from human marrow stroma that are capable of directed differentiation, in vitro, down either an osteogenic or adipocytic lineage pathway. One such clone, designated hOP 7, expresses type α1(I) procollagen and has low alkaline phosphatase (AP) activity under basal culture conditions that is reminiscent of an osteoprogenitor cell. Exposure of hOP 7 cells to dexamethasone upregulates AP activity and enables the cells to mineralize their extracellular matrix. Also, treatment with calcitriol induces osteocalcin expression and both PTH and PGE 2 induce/augment cAMP formation. Incubation with normal rabbit serum, however, causes the cells to become adipogenic as demonstrated by histological staining with Oil-red-O, expression of mRNA for the early and late adipocyte markers lipoprotein lipase and glycerol 3-phosphate dehydrogenase, respectively, and loss of type α1(I) procollagen mRNA. The generation of homogeneous populations of these cells, as confirmed by Southern blot analysis, demonstrates the capacity of a human clonal cell line to differentiate in either an osteogenic or adipogenic direction.

The aim of this study was to investigate the effects of insulin-like growth factor-I, transforming growth factor-beta (TGF-beta), and interluekin-1 alpha (IL-1 alpha) on the deposition and degradation of a cartilage-like matrix in high-density pellet cultures of adult bovine chondrocytes. Proteoglycan was determined by toluidine blue staining and colorimetric assay. Type II collagen was determined by immunohistochemical staining and its unwinding in situ by a recently developed immunoassay. Bovine nasal chondrocytes cultured as pellets deposited a well-organized extracellular matrix of proteoglycan and type II collagen. Insulin-like growth factor-I (2-10 ng/ml) increased the synthesis and incorporation into the matrix of both these proteins. TGF-beta (2-10 ng/ml) also increased proteoglycan synthesis. However it inhibited proteoglycan deposition, presumably through increased degradation of the molecule, as shown by increased release of aggrecan fragments into the tissue culture medium. TGF-beta had no effect on type II collagen deposition. In pellet cultures of bovine nasal or articular chondrocytes, 20 ng/ml IL-1 alpha induced a significant degradation of both proteoglycan and type II collagen. The effect on collagen clearly involved proteolytic cleavage of its triple helix because there was an increase in the proportion of unwound type II collagen in the matrix, as well as a loss of total type II collagen. In explant cultures of intact bovine articular cartilage, incubation with 50 ng/ml IL-1 alpha stimulated significant degradation of the proteoglycan but no degradation of the type II collagen. These results demonstrate that although the articular chondrocytes are capable of degrading type II collagen when isolated, they do not do so in situ, presumably because of some inherent property of the mature extracellular matrix. This study demonstrates the utility of pellet cultures when investigating chondrocyte-mediated turnover of cartilage matrix and its modulation by cytokines and growth factors.

The annual regrowth of antlers in male deer is a unique example of complete bone regeneration occurring in an adult animal. Growth is initiated at the distal antler tip, which is similar to the epiphyseal growth plate in some respects. However, there is some debate as to whether this process represents “true” endochondral ossification. As part of the characterization of the developmental process in pre-osseus antler tissue, we have studied, by in situ hybridization, the spatial expression of mRNAs for types I, II, and X collagen. Viewed in a coronal plane, type I procollagen mRNA was observed in skin, the fibrous perichondrium, and the densely cellular area immediately adjacent to the perichondrium. Below this area, as cells began to assume a columnar arrangement and coincident with the appearance of a vasculature and synthesis of a cartilaginous matrix, transcripts for types I, IIA, IIB procollagen and X collagen were detected. Further down in the cartilage zone, the pattern of type I procollagen mRNA expression was altered. Here, the signal was detected only in a morphologically distinct subpopulation of small, flattened cells within the intercellular matrix at the periphery of the columns of chondrocytes. The alternative splice form of type II procollagen mRNA (IIA), characteristic of chondroprogenitor cells (Sandell et al. [1991] J. Cell Biol. 114:1307-1319), was expressed by a subset of cells in the upper region of the columns, indicating that this zone contains a population of prechondrocytic cells. Positive hybridization to type IIA was most abundant in these cells. In contrast, transcripts for the other procollagen splice form (IIB) and type X collagen were expressed by chondrocytes throughout the whole of the cartilage region studied. The translation and export of type II collagen and type X collagen were confirmed by detecting specific immunoreactivity for each. The spatial distribution of immunoreactivity for collagen types II and X was consistent with that of corresponding mRNAs. These data demonstrate for the first time the distinct pattern of expression of genes for major cartilage matrix macromolecules, the expression of the differentially spliced form of type II procollagen mRNA (IIA), and specifically the colocalization of types II and X collagen in the developing antler tip. Taken together, they strongly indicate that antler growth involves an endochondral process. © 1996 Wiley-Liss, Inc.

The chemokine CCL3/MIP-1α is a risk factor in the outcome of multiple myeloma (MM), particularly in the development of osteolytic bone disease. This chemokine, highly overexpressed by MM cells, can signal mainly through 2 receptors, CCR1 and CCR5, only 1 of which (CCR1) is responsive to CCL3 in human and mouse osteoclast precursors. CCR1 activation leads to the formation of osteolytic lesions and facilitates tumor growth. Here we show that formation of mature osteoclasts is blocked by the highly potent and selective CCR1 antagonist CCX721, an analog of the clinical compound CCX354. We also show that doses of CCX721 selected to completely inhibit CCR1 produce a profound decrease in tumor burden and osteolytic damage in the murine 5TGM1 model of MM bone disease. Similar effects were observed when the antagonist was used prophylactically or therapeutically, with comparable efficacy to that of zoledronic acid. 5TGM1 cells were shown to express minimal levels of CCR1 while secreting high levels of CCL3, suggesting that the therapeutic effects of CCX721 result from CCR1 inhibition on non-MM cells, most likely osteoclasts and osteoclast precursors. These results provide a strong rationale for further development of CCR1 antagonists for the treatment of MM and associated osteolytic bone disease.

Bone loss is one of the major complications of advanced cancers such as breast cancer, prostate cancer, and multiple myeloma; agents that can suppress this bone loss have therapeutic potential. Extensive research within the last decade has revealed that RANKL, a member of the tumor necrosis factor superfamily, plays a major role in cancer-associated bone resorption and thus is a therapeutic target. We investigated the potential of vitamin K3 analogue plumbagin (derived from Chitrak, an Ayurvedic medicinal plant) to modulate RANKL signaling, osteoclastogenesis, and breast cancer-induced osteolysis. Plumbagin suppressed RANKL-induced NF-κB activation in mouse monocytes, an osteoclast precursor cell, through sequential inhibition of activation of IκBα kinase, IκBα phosphorylation, and IκBα degradation. Plumbagin also suppressed differentiation of these cells into osteoclasts induced either by RANKL or by human breast cancer or human multiple myeloma cells. When examined for its ability to prevent human breast cancer-induced bone loss in animals, plumbagin (2 mg/kg body weight) administered via the intraperitoneal route significantly decreased osteolytic lesions, resulting in preservation of bone volume in nude mice bearing human breast tumors. Overall, our results indicate that plumbagin, a vitamin K analogue, is a potent inhibitor of osteoclastogenesis induced by tumor cells and of breast cancer-induced osteolytic metastasis through suppression of RANKL signaling. Mol Cancer Ther; 11(2); 350–9. ©2011 AACR.

Cytoskeleton microtubules regulate various cell signaling pathways that are involved in bone cell function. We recently reported that inhibition of microtubule assembly by microtubule-targeting drugs stimulates osteoblast differentiation and bone formation. To further elucidate the role of microtubules in bone homeostasis, we characterized the skeletal phenotype of mice null for stathmin, an endogenous protein that inhibits microtubule assembly. In vivo micro–computed tomography (µCT) and histology revealed that stathmin deficiency results in a significant reduction of bone mass in adult mice concurrent with decreased osteoblast and increased osteoclast numbers in bone tissues. Phenotypic analyses of primary calvarial cells and bone marrow cells showed that stathmin deficiency inhibited osteoblast differentiation and induced osteoclast formation. In vitro overexpression studies showed that increased stathmin levels enhanced osteogenic differentiation of preosteoblast MC3T3-E1 cells and mouse bone marrow–derived cells and attenuated osteoclast formation from osteoclast precursor Raw264.7 cells and bone marrow cells. Results of immunofluorescent studies indicated that overexpression of stathmin disrupted radial microtubule filaments, whereas deficiency of stathmin stabilized the microtubule network structure in these bone cells. In addition, microtubule-targeting drugs that inhibit microtubule assembly and induce osteoblast differentiation lost these effects in the absence of stathmin. Collectively, these results suggest that stathmin, which alters microtubule dynamics, plays an essential role in maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone. © 2011 American Society for Bone and Mineral Research

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone–related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β–stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep–expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction. Cancer Res; 71(3); 822–31. ©2010 AACR.

Publisher Summary Bone is continuously remodeled in normal individuals and this is achieved via a finely regulated balance between the processes of bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. This bone remodeling is regulated, in part, by local factors including cytokines generated in the bone microenvironment. The purpose of this chapter is to summarize what is currently known about the role of cytokines and their receptors in bone remodeling. Recent advances in molecular biological techniques have meant that most of the biological activities ascribed to cytokines have now been associated with specific molecules, and their receptors identified and molecularly cloned. Several cytokines and their cognate receptors have been shown to be expressed by bone cells, marrow cells, or accessory cells in the bone microenvironment. Moreover, studies using knockout and transgenic mice have increased the understanding of the complex signal transduction mechanisms utilized by cytokines and are opening up new and exciting areas of study. Cytokines tend to be pleiotropic and multifactorial, and may have overlapping and seemingly redundant biological effects. Some of this redundancy is apparent in the receptor mechanisms and signal transduction pathways used by groups of cytokines. Classic examples that illustrate this vividly are the various cytokines belonging to the interleukin (IL)-6 family, such as IL-6, leukemia inhibitory factor, oncostatin-M, and IL-11, which utilize a common signal transduction protein known as gp130. These cytokines bind to distinct membrane-associated receptors, which form hetero- or homo-dimers upon binding to the ligand. Further, there is now a body of data derived from in vivo studies in animals which show that over- or under-production of certain cytokines cause profound effects on bone. These fundamental observations have the potential of not only increasing the understanding of the pathophysiology of osteoporosis, but also leading to new and better forms of therapy using these molecules as targets for drug discovery programs.

The proteasome inhibitor bortezomib has a striking clinical benefit in patients with multiple myeloma. It is unknown whether the bone marrow microenvironment directly contributes to the dramatic response of myeloma cells to proteasome inhibition in vivo. We have used the well-characterized 5TGM1 murine model of myeloma to investigate myeloma growth within bone and response to the proteasome inhibitor bortezomib in vivo. Myeloma cells freshly isolated from the bone marrow of myeloma-bearing mice were found to have an increase in proteasome activity and an enhanced response to in vitro proteasome inhibition, as compared with pre-inoculation myeloma cells. Treatment of myeloma-bearing mice with bortezomib resulted in a greater reduction in tumor burden when the myeloma cells were located within the bone marrow when compared with extra-osseous sites. Our results demonstrate that myeloma cells exhibit an increase in proteasome activity and an enhanced response to bortezomib treatment when located within the bone marrow microenvironment in vivo.

Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) has emerged as a major mediator of bone resorption, commonly associated with cancer and other chronic inflammatory diseases. Inhibitors of RANKL signaling thus have potential in preventing bone loss. In the present report, the potential of zerumbone, a sesquiterpene derived from subtropical ginger, to modulate osteoclastogenesis induced by RANKL and breast cancer was examined. We found that zerumbone inhibited RANKL-induced NF-κB activation in mouse monocyte, an osteoclast precursor cell, through inhibition of activation of IκBα kinase, IκBα phosphorylation, and IκBα degradation. Zerumbone also suppressed RANKL-induced differentiation of these cells to osteoclasts. This sesquiterpene also inhibited the osteoclast formation induced by human breast tumor cells and by multiple myeloma cells. Finally, we examined whether zerumbone could prevent human breast cancer–induced bone loss in animals. We found that zerumbone decreased osteolysis in a dose-dependent manner in MDA-MB-231 breast cancer tumor-bearing athymic nude mice. These results indicate that zerumbone is an effective blocker of RANKL-induced NF-κB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss. [Cancer Res 2009;69(4):1477–84]

Purpose: Eradication of post-treatment residual myeloma cells is needed to prevent relapses, and immunostimulatory monoclonal antibodies (mAb) such as anti-CD137, CTLA-4, CD40, etc., which enhance the immune response against malignancies, represent a means of achieving this purpose. This study explores anti-CD137 mAbs for multiple myeloma treatment in preclinical models of the disease because they safely augment tumor immunity and are in clinical trials for other cancers.Experimental Design: The antitumor effect of anti-CD137 mAb on mouse plasmacytomas derived from HOPC and NS0 cell lines was studied and compared with that of anti-CTLA-4, anti-CD40, and anti-ICAM-2 mAbs. The antitumor effect of anti-CD137 mAb was also examined in a mouse syngeneic disseminated myeloma (5TGM1) model, which more closely resembles human multiple myeloma. Depletions of specific cell populations and gene-targeted mice were used to unravel the requirements for tumor rejection.Results: Agonistic mAb against CD137 and blocking anti-CTLA-4 mAb showed activity against i.p. HOPC tumors, resulting in extended survival of mice that also became immune to rechallenge. Anti-CD137 mAbs induced complete eradications of established s.c. NS0-derived tumors that were dependent on IFN-γ, natural killer cells, and CD8+ T lymphocytes. Natural killer cells accumulated in tumor draining lymph nodes and showed increased IFN-γ production. Antitumor efficacy of anti-CD137 mAb was preserved in CD28-deficient mice despite the fact that CD28 signaling increases the expression of CD137 on CD8+ T cells. Importantly, anti-CD137 mAb treatment significantly decreased systemic tumor burden in the disseminated 5TGM1 model.Conclusions: The immune-mediated antitumor activity of anti-CD137 mAb in mouse models holds promise for myeloma treatment in humans.

Impaired bone formation contributes to the lack of bone healing in multiple myeloma and there is a need for agents with bone anabolic properties to reverse the bone deficit in patients. Bortezomib, a proteasome inhibitor with antitumour efficacy in myeloma patients, enhanced new bone formation in mouse calvarial cultures; this effect was blocked by dickkopf 1(Dkk1), an antagonist of Wnt signalling implicated in myeloma bone disease. Bortezomib inhibited Dkk1 expression in calvariae and bone marrow-derived stromal cells, suggesting a novel mechanism by which bortezomib exerts its effects in bone. Clinical trials in patients with myeloma bone disease are needed to validate these results.