Oxidatively modified low-density lipoproteins are potential mediators of proteasome inhibitor resistance in multiple myeloma.

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.

A niche-dependent myeloid transcriptome signature defines dormant myeloma cells.

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.

Inhibiting the osteocyte-specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma.

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.

CCR1 blockade reduces tumor burden and osteolysis in vivo in a mouse model of myeloma bone disease.

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.

Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation.

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 ß (TGF-ß), 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.

Myeloma cells exhibit an increase in proteasome activity and an enhanced response to proteasome inhibition in the bone marrow microenvironment in vivo.

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.

The zinc finger transcription factor Gli2 mediates bone morphogenetic protein 2 expression in osteoblasts in response to hedgehog signaling.

Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation.

Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in mouse models of myeloma.

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-gamma, natural killer cells, and CD8(+) T lymphocytes. Natural killer cells accumulated in tumor draining lymph nodes and showed increased IFN-gamma 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.

The hedgehog signaling molecule Gli2 induces parathyroid hormone-related peptide expression and osteolysis in metastatic human breast cancer cells.

Parathyroid hormone-related peptide (PTHrP) is a major factor involved in tumor-induced osteolysis caused by breast cancers that have metastasized to bone. However, the molecular mechanisms that mediate PTHrP production by breast cancer cells are not entirely clear. We hypothesized that Gli2, a downstream transcriptional effector of the Hedgehog (Hh) signaling pathway, regulates PTHrP expression in metastatic breast cancer because the Hh pathway regulates physiologic PTHrP expression in the developing growth plate. Here, we show that Gli2 is expressed in several human cancer cell lines that cause osteolytic lesions in vivo and produce PTHrP (MDA-MB-231, RWGT2, and PC-3) but is not expressed in nonosteolytic cancer cell lines that do not secrete PTHrP (MCF-7, ZR-75, and T47D). Transient expression of Gli2 in MDA-MB-231 and MCF-7 breast cancer cells increased PTHrP promoter-luciferase activity dose dependently. Stable expression of Gli2 in MDA-MB-231 cells resulted in an increase in PTHrP protein in the conditioned medium. Alternatively, MDA-MB-231 cells stably transfected with Gli2-EnR, a repressor of Gli2 activity, exhibited a 72% to 93% decrease in PTHrP mRNA by quantitative real-time PCR when compared with control cells. To examine the effects of Gli2 on breast cancer-mediated osteolysis in vivo, athymic nude mice were inoculated with MDA-MB-231 cells stably expressing Gli2 or the empty vector. Following tumor cell inoculation via the left cardiac ventricle, Gli2-expressing tumors caused significantly more osteolysis. Together, these data suggest that PTHrP expression and osteolysis in vivo in human breast cancer cells is driven at least in part by Gli2.

Detection of myeloma in skeleton of mice by whole-body optical fluorescence imaging.

Development of new therapies for myeloma has been hindered by the lack of suitable preclinical animal models of the disease in which widespread tumor foci in the skeleton can be detected reliably. Traditional means of detecting skeletal tumor infiltration such as histopathology are cumbersome and labor-intensive and do not allow temporal monitoring of tumor progression or regression in response to therapy. To resolve this problem, we modified the Radl 5TGM1 model of myeloma bone disease such that fluorescent myeloma tumors can be optically imaged in situ. Here, we show that murine myeloma 5TGM1 tumor cells, engineered to express enhanced green fluorescent protein (eGFP; 5TGM1-eGFP cells), can be imaged in a temporal fashion using a fluorescence illuminator and a charge-coupled device camera in skeletons of live C57BL/KaLwRij mice. High-resolution, whole-body images of tumor-bearing mice revealed that myeloma cells homed almost exclusively to the skeleton, with multiple focal tumor foci in the axial skeleton, consistent with myeloma tumor distribution in humans. Finally, the tested antitumor treatment effect of Velcade (bortezomib), a proteasome inhibitor used clinically in myeloma, was readily detected by GFP imaging, suggesting the power of the technique in combination with the Radl 5TGM1-eGFP model for rapid preclinical assessment and sensitive monitoring of novel and potential therapeutics. Whole-body GFP imaging is practical, convenient, inexpensive, and rapid, and these advantages should enable a high throughput when evaluating in vivo efficacy of new potential antimyeloma therapeutics and assessing response to treatment.

Synthesis of a Bone-Targeted Bortezomib with In Vivo Anti-Myeloma Effects in Mice.

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.

Combined treatment with a transforming growth factor beta inhibitor (1D11) and bortezomib improves bone architecture in a mouse model of myeloma-induced bone disease.

Multiple myeloma (MM) patients frequently develop tumor-induced bone destruction, yet no therapy completely eliminates the tumor or fully reverses bone loss. Transforming growth factor-ß (TGF-ß) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-ß inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-ß 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-ß inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4weeks after inoculation of murine 5TGM1 MM cells. TGF-ß 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-ß signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease.

A dominant-negative F-box deleted mutant of E3 ubiquitin ligase, ß-TrCP1/FWD1, markedly reduces myeloma cell growth and survival in mice.

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, SCFß-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-ß-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 ß-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 ß-TrCP inhibitors as an alternative therapy to nonspecific proteasome inhibitors for myeloma patients.

Multiple myeloma induces Mcl-1 expression and survival of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSC) are contributing to an immunosuppressive environment by their ability to inhibit T cell activity and thereby promoting cancer progression. An important feature of the incurable plasma cell malignancy Multiple Myeloma (MM) is immune dysfunction. MDSC were previously identified to be present and active in MM patients, however little is known about the MDSC-inducing and -activating capacity of MM cells. In this study we investigated the effects of the tumor microenvironment on MDSC survival. During MM progression in the 5TMM mouse model, accumulation of MDSC in the bone marrow was observed in early stages of disease development, while circulating myeloid cells were increased at later stages of disease. Interestingly, in vivo MDSC targeting by anti-GR1 antibodies and 5-Fluorouracil resulted in a significant reduced tumor load in 5TMM-diseased mice. In vitro generation of MDSC was demonstrated by increased T cell immunosuppressive capacity and MDSC survival was observed in the presence of MM-conditioned medium. Finally, increased Mcl-1 expression was identified as underlying mechanism for MDSC survival. In conclusion, our data demonstrate that soluble factors from MM cells are able to generate MDSC through Mcl-1 upregulation and this cell population can be considered as a possible target in MM disease.

Wnt/ß-catenin signaling activates bone morphogenetic protein 2 expression in osteoblasts.

The BMP and Wnt/ß-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 ß-catenin/TCF transcription complex in the BMP2 promoter, we hypothesized that the Wnt/ß-catenin pathway stimulates BMP2 expression in osteogenic cells. In this study, we showed that Wnt/ß-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 ß-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 ß-catenin/TCF4 activity with FWD1/ß-TrCP, ICAT, or ΔTCF4. Using a site-directed mutagenesis approach, we confirmed that Wnt/ß-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/ß-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/ß-catenin pathways in osteoblastic differentiation and maintenance of skeletal homeostasis.

Bone marrow-targeted liposomal carriers.

INTRODUCTION: 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. AREA COVERED: 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. EXPERT OPINION: 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.

Microtubule assembly affects bone mass by regulating both osteoblast and osteoclast functions: stathmin deficiency produces an osteopenic phenotype in mice.

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. \

Transcriptional regulation of BMP2 expression by the PTH-CREB signaling pathway in osteoblasts.

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.

TGF-beta promotion of Gli2-induced expression of parathyroid hormone-related protein, an important osteolytic factor in bone metastasis, is independent of canonical Hedgehog signaling.

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-ß, 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-ß-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-ß propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-ß 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-ß to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.

Zerumbone abolishes RANKL-induced NF-kappaB activation, inhibits osteoclastogenesis, and suppresses human breast cancer-induced bone loss in athymic nude mice.

Receptor activator of nuclear factor-kappaB (NF-kappaB) 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-kappaB activation in mouse monocyte, an osteoclast precursor cell, through inhibition of activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, and IkappaBalpha 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-kappaB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss.