The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1-bone morphogenic protein 2 longevity axis.

BACKGROUND AND PURPOSE: The polyphenol resveratrol (RSV) exists in high quantities in certain foods (e.g. grapes and nuts). However, the capacity of RSV to confer physiological health benefits and a biological mechanism through which this might occur remains unclear. EXPERIMENTAL APPROACH: Aged, RSV-treated (300 mg·kg-1 ·day-1 ) and genetically modified [endothelial NOS (eNOS-/- )] female mice were assessed using histomorphometric and µCT analysis. Alongside in vivo analysis, molecular siRNA knockdown and pharmacological manipulation of eNOS, BMP2 and sirtuin 1 (SIRT1) and functional cellular assays in an osteoblast cell line panel, explored the mechanism through which RSV might impact overall bone volume. KEY RESULTS: RSV promoted osteoblast activity and bone growth in vivo. RSV dose-dependently and simultaneously increased alkaline phosphatase (ALP) and eNOS levels. Similarly, NO-donor treatment increased ALP, runt homology transcription factor 2, BMP2 and stimulated bone formation, whilst eNOS-deficient mice displayed a bone loss phenotype. Moreover, RSV-induced increase in ALP and BMP2 expression was blocked in eNOS-/- osteoblasts and by BMP-inhibitor noggin. The longevity-linked SIRT1 enzyme was positively regulated by RSV and SIRT1 deletion reduced eNOS, BMP2 and ALP. Like eNOS deletion, loss of SIRT1 blocked RSV-induced osteoblast activity; however, SIRT1 levels remained unchanged in eNOS-/- mice, indicating RSV activation of SIRT1 stimulates BMP2 release via eNOS. This signalling axis is supported by decreased SIRT1, eNOS and BMP2 confirmed in old versus young bone. CONCLUSIONS AND IMPLICATIONS: These findings suggest a new mechanism of action in bone remodelling and the ageing skeleton, where RSV positively impacts bone homeostasis via SIRT1 activation of BMP2.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells.

We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

Silent information regulator (Sir)T1 inhibits NF-κB signaling to maintain normal skeletal remodeling.

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF-κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF-κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast- or osteoblast-specific SirT1 deletion using the Sirt(flox/flox) mice crossed to lysozyme M-cre and the 2.3 kb col1a1-cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF-κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF-κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF-κB. In further support of the critical role of NF-κB signaling in bone remodeling, elevated NF-κB activity in IκBα(+/-) mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell-autonomous manner in both osteoblasts and osteoclasts, through control of NF-κB and bone cell differentiation.

Osteoclasts in multiple myeloma are derived from Gr-1+CD11b+myeloid-derived suppressor cells.

Osteoclasts play a key role in the development of cancer-associated osteolytic lesions. The number and activity of osteoclasts are often enhanced by tumors. However, the origin of osteoclasts is unknown. Myeloid-derived suppressor cells (MDSCs) are one of the pre-metastatic niche components that are induced to expand by tumor cells. Here we show that the MDSCs can differentiate into mature and functional osteoclasts in vitro and in vivo. Inoculation of 5TGM1-GFP myeloma cells into C57BL6/KaLwRij mice led to a significant expansion of MDSCs in blood, spleen, and bone marrow over time. When grown in osteoclastogenic media in vitro, MDSCs from tumor-challenged mice displayed 14 times greater potential to differentiate into mature and functional osteoclasts than those from non-tumor controls. Importantly, MDSCs from tumor-challenged LacZ transgenic mice differentiated into LacZ+osteoclasts in vivo. Furthermore, a significant increase in tumor burden and bone loss accompanied by increased number of osteoclasts was observed in mice co-inoculated with tumor-challenged MDSCs and 5TGM1 cells compared to the control animals received 5TGM1 cells alone. Finally, treatment of MDSCs from myeloma-challenged mice with Zoledronic acid (ZA), a potent inhibitor of bone resorption, inhibited the number of osteoclasts formed in MDSC cultures and the expansion of MDSCs and bone lesions in mice. Collectively, these data provide in vitro and in vivo evidence that tumor-induced MDSCs exacerbate cancer-associated bone destruction by directly serving as osteoclast precursors.

Bone-derived IGF mediates crosstalk between bone and breast cancer cells in bony metastases.

The continuous release of bone-stored growth factors after bone resorption promotes the colonization of circulating cancer cells. However, the precise role of each of the various growth factors remains unclear. In this study, we investigated the role of bone-derived insulin-like growth factor (IGF) in the development of bone metastases in an animal model of breast cancer. We found that local stimulation of calvarial bone resorption before cell inoculation stimulated subsequent bone metastases to that site in vivo, although inhibition of bone resorption inhibited bone metastases. Anchorage-independent growth of cancer cells was stimulated by the culture supernatants from resorbed bones, which contained elevated levels of IGF-I. This stimulation was blocked by IGF type I receptor (IGF-IR) neutralizing antibody, but not antibody targeting other bone-stored growth factors including TGF-ß, fibroblast growth factors, and platelet-derived growth factors. Although recombinant human IGF-I caused IGF-IR tyrosine autophosphorylation, followed by activation of Akt and NF-κB in cancer cells, dominant-negative inhibition of IGF-IR, Akt, or NF-κB significantly reduced bone metastases with increased apoptosis and decreased mitosis in metastatic cells. Together, our findings suggest that bone-derived IGF-I bridges the crosstalk between bone and metastasized cancer cells via activation of the IGF-IR/Akt/NF-κB pathway. Disruption of this pathway therefore may represent a promising therapeutic intervention for bone metastasis.

Bone marrow stromal cells create a permissive microenvironment for myeloma development: a new stromal role for Wnt inhibitor Dkk1.

The rapid progression of multiple myeloma is dependent upon cellular interactions within the bone marrow microenvironment. In vitro studies suggest that bone marrow stromal cells (BMSC) can promote myeloma growth and survival and osteolytic bone disease. However, it is not possible to recreate all cellular aspects of the bone marrow microenvironment in an in vitro system, and the contributions of BMSCs to myeloma pathogenesis in an intact, immune competent, in vivo system are unknown. To investigate this, we used a murine myeloma model that replicates many features of the human disease. Coinoculation of myeloma cells and a BMSC line, isolated from myeloma-permissive mice, into otherwise nonpermissive mice resulted in myeloma development, associated with tumor growth within bone marrow and osteolytic bone disease. In contrast, inoculation of myeloma cells alone did not result in myeloma. BMSCs inoculated alone induced osteoblast suppression, associated with an increase in serum concentrations of the Wnt signaling inhibitor, Dkk1. Dkk1 was highly expressed in BMSCs and in myeloma-permissive bone marrow. Knockdown of Dkk1 expression in BMSCs decreased their ability to promote myeloma and the associated bone disease in mice. Collectively, our results show novel roles of BMSCs and BMSC-derived Dkk1 in the pathogenesis of multiple myeloma in vivo.

Anti-transforming growth factor ß antibody treatment rescues bone loss and prevents breast cancer metastasis to bone.

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFß. Because TGFß favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFß antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFß antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFß antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFß treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFß antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFß antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFß antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFß treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.

Host-derived adiponectin is tumor-suppressive and a novel therapeutic target for multiple myeloma and the associated bone disease.

The contributions of the host microenvironment to the pathogenesis of multiple myeloma, including progression from the non-malignant disorder monoclonal gammopathy of undetermined significance, are poorly understood. In the present study, microarray analysis of a murine model requiring a unique host microenvironment for myeloma development identified decreased host-derived adiponectin compared with normal mice. In support, clinical analysis revealed decreased serum adiponectin concentrations in monoclonal gammopathy of undetermined significance patients who subsequently progressed to myeloma. We investigated the role of adiponectin in myeloma pathogenesis and as a treatment approach, using both mice deficient in adiponectin and pharmacologic enhancement of circulating adiponectin. Increased tumor burden and bone disease were observed in myeloma-bearing adiponectin-deficient mice, and adiponectin was found to induce myeloma cell apoptosis. The apolipoprotein peptide mimetic L-4F was used for pharmacologic enhancement of adiponectin. L-4F reduced tumor burden, increased survival of myeloma-bearing mice, and prevented myeloma bone disease. Collectively, our studies have identified a novel mechanism whereby decreased host-derived adiponectin promotes myeloma tumor growth and osteolysis. Furthermore, we have established the potential therapeutic benefit of increasing adiponectin for the treatment of myeloma and the associated bone disease.

6-Thioguanine inhibition of parathyroid hormone-related protein expression is mediated by GLI2.

BACKGROUND: Breast cancer cells frequently metastasize to bone, where they up-regulate their expression of the transcription factor GLI2 and the downstream osteolytic factor parathyroid hormone-related protein (PTHrP). The guanosine nucleotide 6-thioguanine (6-TG) inhibits PTHrP expression and blocks osteolytic bone destruction in mice inoculated with bone metastatic cells; however, the mechanism by which 6-TG inhibits PTHrP remains unclear. We hypothesized that 6-TG inhibition of PTHrP is mediated through GLI2 signaling. MATERIALS AND METHODS: Human MDA-MB-231 breast cancer cells and RWGT2 squamous-cell lung carcinoma cells were treated with 100 µM 6-TG and examined for GLI2 mRNA expression and stability by Q-PCR, promoter activity by luciferase assay, and protein expression by Western blot. RESULTS: 6-TG significantly blocked GLI2 mRNA and protein expression, but did not affect stability. Additionally, 6-TG directly inhibited GLI2 promoter activity, and when cells were transfected with constitutively expressed GLI2, the inhibitory effect of 6-TG on PTHrP expression was abolished. CONCLUSION: Taken together, these data indicate that 6-TG regulates PTHrP in part through GLI2 transcription, and therefore the clinical use of 6-TG or other guanosine nucleotides may be a viable therapeutic option in tumor types expressing elevated levels of GLI proteins.

Differential effects between the loss of MMP-2 and MMP-9 on structural and tissue-level properties of bone.

Matrix metalloproteinases (MMPs) are capable of processing certain components of bone tissue, including type 1 collagen, a determinant of the biomechanical properties of bone tissue, and they are expressed by osteoclasts and osteoblasts. Therefore, we posit that MMP activity can affect the ability of bone to resist fracture. To explore this possibility, we determined the architectural, compositional, and biomechanical properties of bones from wild-type (WT), Mmp2(-/-) , and Mmp9(-/-) female mice at 16 weeks of age. MMP-2 and MMP-9 have similar substrates but are expressed primarily by osteoblasts and osteoclasts, respectively. Analysis of the trabecular compartment of the tibia metaphysis by micro-computed tomography (µCT) revealed that these MMPs influence trabecular architecture, not volume. Interestingly, the loss of MMP-9 improved the connectivity density of the trabeculae, whereas the loss of MMP-2 reduced this parameter. Similar differential effects in architecture were observed in the L(5) vertebra, but bone volume fraction was lower for both Mmp2(-/-) and Mmp9(-/-) mice than for WT mice. The mineralization density and mineral-to-collagen ratio, as determined by µCT and Raman microspectroscopy, were lower in the Mmp2(-/-) bones than in WT control bones. Whole-bone strength, as determined by three-point bending or compression testing, and tissue-level modulus and hardness, as determined by nanoindentation, were less for Mmp2(-/-) than for WT bones. In contrast, the Mmp9(-/-) femurs were less tough with lower postyield deflection (more brittle) than the WT femurs. Taken together, this information reveals that MMPs play a complex role in maintaining bone integrity, with the cell type that expresses the MMP likely being a contributing factor to how the enzyme affects bone quality.

Advances in osteoclast biology: old findings and new insights from mouse models.

The maintenance of adequate bone mass is dependent upon the controlled and timely removal of old, damaged bone. This complex process is performed by the highly specialized, multinucleated osteoclast. Over the past 15 years, a detailed picture has emerged describing the origins, differentiation pathways and activation stages that contribute to normal osteoclast function. This information has primarily been obtained by the development and skeletal analysis of genetically modified mouse models. Mice harboring mutations in specific genetic loci exhibit bone defects as a direct result of aberrations in normal osteoclast recruitment, formation or function. These findings include the identification of the RANK-RANKL-OPG system as a primary mediator of osteoclastogenesis, the characterization of ion transport and cellular attachment mechanisms and the recognition that matrix-degrading enzymes are essential components of resorptive activity. This Review focuses on the principal observations in osteoclast biology derived from genetic mouse models, and highlights emerging concepts that describe how the osteoclast is thought to contribute to the maintenance of adequate bone mass and integrity throughout life.

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.

Advances in the biology of bone metastasis: how the skeleton affects tumor behavior.

It is increasingly evident that the microenvironment of bone can influence the cancer phenotype in many ways that favor growth in bone. The ability of cancer cells to adhere to bone matrix and to promote osteoclast formation are key requirements for the establishment and growth of bone metastases. Several cytokine products of breast cancers (e.g. PTHrP, IL-11, IL-8) have been shown to act upon host cells of the bone microenvironment to promote osteoclast formation, allowing for excessive bone resorption. The increased release of matrix-derived growth factors, especially TGF-ß, acts back upon the tumor to facilitate further tumor expansion and enhance cytokine production, and also upon osteoblasts to suppress bone formation. This provides a self-perpetuating cycle of bone loss and tumor growth within the skeleton. Other contributing factors favoring tumor metastasis and colonization in bone include the unique structure and stiffness of skeletal tissue, along with the diverse cellular composition of the marrow environment (e.g. bone cells, stromal fibroblasts, immune cells), any of which can contribute to the phenotypic changes that can take place in metastatic deposits that favor their survival. Additionally, it is also apparent that breast cancer cells begin to express different bone specific proteins as well as proteins important for normal breast development and lactation that allow them to grow in bone and stimulate bone destruction. Taken together, these continually emerging areas of study suggest new potential pathways important in the pathogenesis of bone metastasis and potential areas for targeting therapeutics.

Longitudinal live animal micro-CT allows for quantitative analysis of tumor-induced bone destruction.

The majority of breast cancer and prostate cancer patients with metastatic disease will go on to develop bone metastases, which contribute largely to the patient's morbidity and mortality. Numerous small animal models of cancer metastasis to bone have been developed to study tumor-induced bone destruction, but the advancement of imaging modalities utilized for these models has lagged significantly behind clinical imaging. Therefore, there is a significant need for improvements to live small animal imaging, particularly when obtaining high-resolution images for longitudinal quantitative analyses. Recently, live animal micro-computed tomography (µCT) has gained popularity due to its ability to obtain high-resolution 3-dimensional images. However, the utility of µCT in bone metastasis models has been limited to end-point analyses due to off-target radiation effects on tumor cells. We hypothesized that live animal in vivo µCT can be utilized to perform reproducible and quantitative longitudinal analyses of bone volume in tumor-bearing mice, particularly in a drug treatment model of breast cancer metastasis to bone. To test this hypothesis, we utilized the MDA-MB-231 osteolytic breast cancer model in which the tumor cells are inoculated directly into the tibia of athymic nude mice and imaged mice weekly by Faxitron (radiography), Imtek µCT (in vivo), and Maestro (GFP-imaging). Exvivo µCT and histology were performed at end point for validation. After establishing a high-resolution scanning protocol for the Imtek CT, we determined whether clear, measurable differences in bone volume were detectable in mice undergoing bisphosphonate drug treatments. We found that in vivo µCT could be used to obtain quantifiable and longitudinal images of the progression of bone destruction over time without altering tumor cell growth. In addition, we found that we could detect lesions as early as week 1 and that this approach could be used to monitor the effect of drug treatment on bone. Taken together, these data indicate that in vivo µCT is an effective and reproducible method for longitudinal monitoring of tumor-associated bone destruction in mouse models of tumor-induced bone disease.

Matrix rigidity induces osteolytic gene expression of metastatic breast cancer cells.

Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP). While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK) using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung) preferentially metastasize to bone.

Gr-1+CD11b+ myeloid-derived suppressor cells: formidable partners in tumor metastasis.

The growth and metastasis of solid tumors not only depends on their ability to escape from immune surveillance but also hinges on their ability to invade the vasculature system as well as to induce the formation of new blood vessels. Gr-1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs), overproduced in tumor-bearing hosts, contribute significantly to all these aspects. They also have a potential role in the osteolysis associated with bone metastases. They are formidable partners in tumor metastasis.

Targeting the Transforming Growth Factor-beta pathway inhibits human basal-like breast cancer metastasis.

BACKGROUND: Transforming Growth Factor beta (TGF-beta) plays an important role in tumor invasion and metastasis. We set out to investigate the possible clinical utility of TGF-beta antagonists in a human metastatic basal-like breast cancer model. We examined the effects of two types of the TGF-beta pathway antagonists (1D11, a mouse monoclonal pan-TGF-beta neutralizing antibody and LY2109761, a chemical inhibitor of TGF-beta type I and II receptor kinases) on sublines of basal cell-like MDA-MB-231 human breast carcinoma cells that preferentially metastasize to lungs (4175TR, 4173) or bones (SCP2TR, SCP25TR, 2860TR, 3847TR). RESULTS: Both 1D11 and LY2109761 effectively blocked TGF-beta-induced phosphorylation of receptor-associated Smads in all MDA-MB-231 subclones in vitro. Moreover, both antagonists inhibited TGF-beta stimulated in vitro migration and invasiveness of MDA-MB-231 subclones, indicating that these processes are partly driven by TGF-beta. In addition, both antagonists significantly reduced the metastatic burden to either lungs or bones in vivo, seemingly independently of intrinsic differences between the individual tumor cell clones. Besides inhibiting metastasis in a tumor cell autonomous manner, the TGF-beta antagonists inhibited angiogenesis associated with lung metastases and osteoclast number and activity associated with lytic bone metastases. In aggregate, these studies support the notion that TGF-beta plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-beta signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-beta pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer. CONCLUSIONS: In aggregate, these studies support the notion that TGF-beta plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-beta signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-beta pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.

Inhibition of TGF-ß signaling by 1D11 antibody treatment increases bone mass and quality in vivo.

Transforming growth factor ß (TGF-ß) is an abundant bone matrix protein that influences osteoblast and osteoclast interactions to control bone remodeling. As such, TGF-ß represents an obvious pharmacologic target with the potential to regulate both bone formation and resorption to improve bone volume and strength. To investigate the skeletal effect of TGF-ß inhibition in vivo, we used an antibody (1D11) specifically directed at all three isoforms of TGF-ß. Normal mice were treated with 1D11 or control antibody (4 weeks), and cortical and trabecular bone was assessed by micro-computed tomographic (µCT) scanning. Bone volume and cellular distribution were determined by histomorphometric analysis of vertebrae and long bones. Also, whole-bone strength was assessed biomechanically by three-point bend testing, and tissue-level modulus and composition were analyzed by nanoindentation and Raman microspectroscopy, respectively. TGF-ß blockade by 1D11 increased bone mineral density (BMD), trabecular thickness, and bone volume by up to 54%, accompanied by elevated osteoblast numbers and decreased osteoclasts. Biomechanical properties of bone also were enhanced significantly by 1D11 treatment, with increased bending strength and tissue-level modulus. In addition, Raman microspectroscopy demonstrated that 1D11-mediated TGF-ß inhibition in the bone environment led to an 11% increase in the mineral-to-collagen ratio of trabecular bone. Together these studies demonstrate that neutralizing TGF-ß with 1D11 increases osteoblast numbers while simultaneously decreasing active osteoclasts in the marrow, resulting in a profound increase in bone volume and quality, similar to that seen in parathyroid hormone (PTH)-treated rodent studies.

Metastasis and bone loss: advancing treatment and prevention.

Tumor metastasis to the skeleton affects over 400,000 individuals in the United States annually, more than any other site of metastasis, including significant proportions of patients with breast, prostate, lung and other solid tumors. Research on the bone microenvironment and its role in metastasis suggests a complex role in tumor growth. Parallel preclinical and clinical investigations into the role of adjuvant bone-targeted agents in preventing metastasis and avoiding cancer therapy-induced bone loss have recently reported exciting and intriguing results. A multidisciplinary consensus conference convened to review recent progress in basic and clinical research, assess gaps in current knowledge and prioritize recommendations to advance research over the next 5 years. The program addressed three topics: advancing understanding of metastasis prevention in the context of bone pathophysiology; developing therapeutic approaches to prevent metastasis and defining strategies to prevent cancer therapy-induced bone loss. Several priorities were identified: (1) further investigate the effects of bone-targeted therapies on tumor and immune cell interactions within the bone microenvironment; (2) utilize and further develop preclinical models to study combination therapies; (3) conduct clinical studies of bone-targeted therapies with radiation and chemotherapy across a range of solid tumors; (4) develop biomarkers to identify patients most likely to benefit from bone-targeted therapies; (5) educate physicians on bone loss and fracture risk; (6) define optimal endpoints and new measures of efficacy for future clinical trials; and (7) define the optimum type, dose and schedule of adjuvant bone-targeted therapy.

Local low-dose lovastatin delivery improves the bone-healing defect caused by Nf1 loss of function in osteoblasts.

Postfracture tibial nonunion (pseudoarthrosis) leads to lifelong disability in patients with neurofibromatosis type I (NF1), a disorder caused by mutations in the NF1 gene. To determine the contribution of NF1 in bone healing, we assessed bone healing in the Nf1(ob) (-/-) conditional mouse model lacking Nf1 specifically in osteoblasts. A closed distal tibia fracture protocol and a longitudinal study design were used. During the 21- to 28-day postfracture period, callus volume, as expected, decreased in wild-type but not in Nf1(ob) (-/-) mice, suggesting delayed healing. At these two time points, bone volume (BV/TV) and volumetric bone mineral density (vBMD) measured by 3D micro-computed tomography were decreased in Nf1(ob) (-/-) callus-bridging cortices and trabecular compartments compared with wild-type controls. Histomorphometric analyses revealed the presence of cartilaginous remnants, a high amount of osteoid, and increased osteoclast surfaces in Nf1(ob) (-/-) calluses 21 days after fracture, which was accompanied by increased expression of osteopontin, Rankl, and Tgfbeta. Callus strength measured by three-point bending 28 days after fracture was reduced in Nf1(ob) (-/-) versus wild-type calluses. Importantly, from a clinical point of view, this defect of callus maturation and strength could be ameliorated by local delivery of low-dose lovastatin microparticles, which successfully decreased osteoid volume and cartilaginous remnant number and increased callus BV/TV and strength in mutant mice. These results thus indicate that the dysfunctions caused by loss of Nf1 in osteoblasts impair callus maturation and weaken callus mechanical properties and suggest that local delivery of low-dose lovastatin may improve bone healing in NF1 patients.