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Paget's disease (PD) is characterized by increased numbers of abnormal osteoclasts (OCLs) that drive exuberant bone formation, but the mechanisms responsible for the increased bone formation remain unclear. We previously reported that OCLs from 70% of PD patients express measles virus nucleocapsid protein (MVNP), and that transgenic mice with targeted expression of MVNP in OCLs (MVNP mice) develop bone lesions and abnormal OCLs characteristic of PD. In this report, we examined if OCL‐derived sphingosine‐1‐phosphate (S1P) contributed to the abnormal bone formation in PD, since OCL‐derived S1P can act as a coupling factor to increase normal bone formation via binding S1P‐receptor‐3 (S1PR3) on osteoblasts (OBs). We report that OCLs from MVNP mice and PD patients expressed high levels of sphingosine kinase‐1 (SphK‐1) compared with wild‐type (WT) mouse and normal donor OCLs. SphK‐1 production by MVNP‐OCLs was interleukin‐6 (IL‐6)‐dependent since OCLs from MVNP/IL‐6 −/− mice expressed lower levels of SphK‐1. Immunohistochemistry of bone biopsies from a normal donor, a PD patient, WT and MVNP mice confirmed increased expression levels of SphK‐1 in OCLs and S1PR3 in OBs of the PD patient and MVNP mice compared with normal donor and WT mice. Further, MVNP‐OCLs cocultured with OBs from MVNP or WT mice increased OB‐S1PR3 expression and enhanced expression of OB differentiation markers in MVNP‐OBs precursors compared with WT‐OBs, which was mediated by IL‐6 and insulin‐like growth factor 1 secreted by MVNP‐OCLs. Finally, the addition of an S1PR3 antagonist (VPC23019) to WT or MVNP‐OBs treated with WT and MVNP‐OCL‐conditioned media (CM) blocked enhanced OB differentiation of MVNP‐OBs treated with MVNP‐OCL‐CM. In contrast, the addition of the SIPR3 agonist, VPC24191, to the cultures enhanced osterix and Col‐1A expression in MVNP‐OBs treated with MVNP‐OCL‐CM compared with WT‐OBs treated with WT‐OCL‐CM. These results suggest that IL‐6 produced by PD‐OCLs increases S1P in OCLs and S1PR3 on OBs, to increase bone formation in PD., Model for the effects of sphingosine kinase‐1/sphingosine‐1‐phosphate/S1P‐receptor‐3 (SphK‐1/S1P/S1PR3) in abnormal bone remodeling in Paget's disease (PD). Measles virus nucleocapsid protein (MVNP) in PD‐osteoclasts (OCLs) induces interleukin‐6 (IL‐6), which upregulates insulin‐like growth factor 1 (IGF‐1) and Sphk1. SphK‐1 enhances S1P levels in OCLs, and IL‐6 and IGF‐1 increase S1PR3 on osteoblast (OBs). S1P then increases OB differentiation and bone formation via enhanced S1PR3 expression on OBs.

The bone marrow (BM) microenvironment/niche plays a key role in regulating hematopoietic stem and progenitor cell (HSPC) activities; however, mechanisms regulating niche cell function are not well understood. In this study, we show that niche intrinsic expression of the CXCR4 chemokine receptor critically regulates HSPC maintenance during steady state, and promotes early hematopoietic regeneration after myeloablative irradiation. At steady state, chimeric mice with wild-type (WT) HSPC and marrow stroma that lack CXCR4 show decreased HSPC quiescence, and their repopulation capacity was markedly reduced. Mesenchymal stromal cells (MSC) were significantly reduced in the BM of CXCR4 deficient mice, which was accompanied by decreased levels of the HSPC supporting factors stromal cell-derived factor-1 (SDF-1) and stem cell factor (SCF). CXCR4 also plays a crucial role in survival and restoration of BM stromal cells after myeloablative irradiation, where the loss of BM stromal cells was more severe in CXCR4-deficient mice compared to WT mice. In addition, transplantation of WT donor HSPC into CXCR4-deficient recipient mice demonstrated reduced HSPC homing and early hematopoietic reconstitution. We found that CXCR4 signaling attenuates irradiation-induced BM stromal cell loss by upregulating the expression of the antiapoptotic protein Survivin via the PI3K pathway. Our study suggests that SDF-1-CXCR4 signaling in the stromal microenvironment cells plays a crucial role in maintenance of HSPCs during homeostasis, and promotes niche regeneration and early hematopoietic reconstitution after transplantation. Modulation of CXCR4 signaling in the HSPC microenvironment could be a means to enhance hematopoietic recovery after clinical hematopoietic cell transplantation.

Exposure to potentially lethal high-dose ionizing radiation results in bone marrow suppression, known as the hematopoietic acute radiation syndrome (H-ARS), which can lead to pancytopenia and possible death from hemorrhage or infection. Medical countermeasures to protect from or mitigate the effects of radiation exposure are an ongoing medical need. We recently reported that 16,16 dimethyl prostaglandin E

Metabolic Syndrome (MetS) has detrimental effects on the bladder, including detrusor underactivity. The progression and mechanism of disease are poorly understood. A swine model for diabetic bladder dysfunction (DBD) was established because of the pig's human-sized bladder and its ability to develop MetS by dietary modification alone. The hypothesis of this study is that this swine model will demonstrate oxidative stress associated with MetS, which contributes to both bladder fibrosis and detrusor underactivity (DU). Ossabaw pigs underwent dietary modification consisting of a hypercaloric, atherogenic diet for 10 mo to induce MetS, and were compared with a group of control (lean) pigs. Urodynamic studies were performed in both groups to confirm DU. Thiobarbituric acid reactive substances (TBARS) detected in the urine were used to measure oxidative stress activity in the urinary tract, and urinary IL17a was used to detect profibrotic activity. MetS was confirmed by assessing body weight, blood pressure, glucose tolerance, total cholesterol, and triglycerides. The MetS group exhibited an increase in the relative levels of urinary TBARS and IL17a. Bladder pressures at capacity were lower in the MetS group, suggesting DU. Histologic analysis of a cohort of control (lean) and MetS pigs revealed that as compared with the control pigs, the MetS pigs had significantly more collagen in the muscularis layer, but not in the submucosa or mucosa layer. In conclusion, the Ossabaw pig model for diet-induced MetS is associated with oxidative stress and profibrotic activity in the bladder, which results in DU. This has previously been shown in mice and rats, but never in pigs. This novel model will better represent human MetS and DBD because the mechanism and size of the pig bladder more closely resemble that of a human, resulting in a more valid model and facilitating further study into the signaling mechanisms responsible for this impairment.

Endothelial cells (ECs) are components of the hematopoietic microenvironment and regulate hematopoietic stem and progenitor cell (HSPC) homeostasis. Cytokine treatments that cause HSPC trafficking to peripheral blood are associated with an increase in dipeptidylpeptidase 4/CD26 (DPP4/CD26), an enzyme that truncates the neurotransmitter neuropeptide Y (NPY). Here, we show that enzymatically altered NPY signaling in ECs caused reduced VE-cadherin and CD31 expression along EC junctions, resulting in increased vascular permeability and HSPC egress. Moreover, selective NPY2 and NPY5 receptor antagonists restored vascular integrity and limited HSPC mobilization, demonstrating that the enzymatically controlled vascular gateway specifically opens by cleavage of NPY by CD26 signaling via NPY2 and NPY5 receptors. Mice lacking CD26 or NPY exhibited impaired HSPC trafficking that was restored by treatment with truncated NPY. Thus, our results point to ECs as gatekeepers of HSPC trafficking and identify a CD26-mediated NPY axis that has potential as a pharmacologic target to regulate hematopoietic trafficking in homeostatic and stress conditions.

// Trupti Trivedi 1, 2 , Yu Zheng 1 , Pierrick G.J. Fournier 2, 5 , Sreemala Murthy 2 , Sutha John 2 , Suzanne Schillo 1 , Colin R. Dunstan 3 , Khalid S. Mohammad 2 , Hong Zhou 1 , Markus J. Seibel 1, 4 , Theresa A. Guise 2 1 Bone Research Program, ANZAC Research Institute, University of Sydney, Sydney, Australia 2 Division of Endocrinology, Department of Medicine, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana, USA 3 Department of Biomedical Engineering, University of Sydney, Sydney, Australia 4 Department of Endocrinology and Metabolism, Concord Hospital, Concord, Sydney, Australia 5 Biomedical Innovation Department, Scientific Research and High Education Center from Ensenada (CICESE), Ensenada, Baja California, Mexico Correspondence to: Theresa A. Guise, email: tguise@iu.edu Markus J. Seibel, email: markus.seibel@sydney.edu.au Keywords: breast cancer, vitamin D, vitamin D receptor, ligand independent, bone metastasis Received: November 04, 2016 Accepted: February 15, 2017 Published: March 01, 2017 ABSTRACT Vitamin D has pleiotropic effects on multiple tissues, including malignant tumors. Vitamin D inhibits breast cancer growth through activation of the vitamin D receptor (VDR) and via classical nuclear signaling pathways. Here, we demonstrate that the VDR can also function in the absence of its ligand to control behaviour of human breast cancer cells both outside and within the bone microenvironment. Stable shRNA expression was used to knock down VDR expression in MCF-7 cells, generating two VDR knockdown clonal lines. In ligand-free culture, knockdown of VDR in MCF-7 cells significantly reduced proliferation and increased apoptosis, suggesting that the VDR plays a ligand-independent role in cancer cell growth. Implantation of these VDR knockdown cells into the mammary fat pad of nude mice resulted in reduced tumor growth in vivo compared with controls. In the intra-tibial xenograft model, VDR knockdown greatly reduced the ability of the cells to form tumors in the bone microenvironment. The in vitro growth of VDR knockdown cells was rescued by the expression of a mutant form of VDR which is unable to translocate to the nucleus and hence accumulates in the cytoplasm. Thus, our data indicate that in the absence of ligand, the VDR promotes breast cancer growth both in vitro and in vivo and that cytoplasmic accumulation of VDR is sufficient to produce this effect in vitro . This new mechanism of VDR action in breast cancer cells contrasts the known anti-proliferative nuclear actions of the VDR-vitamin D ligand complex.

Summary De novo ASXL1 mutations are found in patients with Bohring-Opitz syndrome, a disease with severe developmental defects and early childhood mortality. The underlying pathologic mechanisms remain largely unknown. Using Asxl1-targeted murine models, we found that Asxl1 global loss as well as conditional deletion in osteoblasts and their progenitors led to significant bone loss and a markedly decreased number of bone marrow stromal cells (BMSCs) compared with wild-type littermates. Asxl1−/− BMSCs displayed impaired self-renewal and skewed differentiation, away from osteoblasts and favoring adipocytes. RNA-sequencing analysis revealed altered expression of genes involved in cell proliferation, skeletal development, and morphogenesis. Furthermore, gene set enrichment analysis showed decreased expression of stem cell self-renewal gene signature, suggesting a role of Asxl1 in regulating the stemness of BMSCs. Importantly, re-introduction of Asxl1 normalized NANOG and OCT4 expression and restored the self-renewal capacity of Asxl1−/− BMSCs. Our study unveils a pivotal role of ASXL1 in the maintenance of BMSC functions and skeletal development., Graphical Abstract, Highlights • Asxl1 loss impairs BMSC self-renewal and cell fate • Asxl1 loss leads to dramatic bone loss • Asxl1 loss alters the expression of genes critical for cell fates of BMSCs • Re-introducing Asxl1 restores self-renewal and lineage commitment in Asxl1−/− BMSCs, In this article, Yang, Wang, and colleagues show that loss of Asxl1 led to multiple skeletal developmental defects, closely reminiscent of Bohring-Opitz syndrome. They identified that the skeletal defects were associated with an impaired self-renewal and skewed lineage commitment of bone marrow stromal cells (BMSCs). These findings indicate a pivotal role of ASXL1 in the maintenance of BMSC functions and skeletal development.

Background In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown. Methods Human primary CD138+ and BMSC were purified from normal donors and MM patients’ bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo. Results We found that Gfi1 expression is increased in patient’s MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth. Conclusions These results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM. Electronic supplementary material The online version of this article (10.1186/s13045-018-0666-5) contains supplementary material, which is available to authorized users.

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

© 2017 Poor bone quality contributes to bone fragility in diabetes, aging, and osteogenesis imperfecta. However, the mechanisms controlling bone quality are not well understood, contributing to the current lack of strategies to diagnose or treat bone quality deficits. Transforming growth factor beta (TGF-β) signaling is a crucial mechanism known to regulate the material quality of bone, but its cellular target in this regulation is unknown. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix led us to hypothesize that TGF-β controls bone quality through perilacunar/canalicular remodeling (PLR). Using inhibitors and mice with an osteocyte-intrinsic defect in TGF-β signaling (TβRIIocy−/−), we show that TGF-β regulates PLR in a cell-intrinsic manner to control bone quality. Altogether, this study emphasizes that osteocytes are key in executing the biological control of bone quality through PLR, thereby highlighting the fundamental role of osteocyte-mediated PLR in bone homeostasis and fragility. Resistance to fracture requires healthy bone mass and quality. However, the cellular mechanisms regulating bone quality are unclear. Dole et al. show that osteocyte-intrinsic TGF-β signaling maintains bone quality through perilacunar/canalicular remodeling. Thus, osteocytes mediate perilacunar/canalicular remodeling and osteoclast-directed remodeling to cooperatively maintain bone quality and mass and prevent fragility.

Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for malignant and nonmalignant hematologic diseases and metabolic disorders. Although successful, hematopoietic transplantation can be hindered by inadequate stem cell number or poor engrafting efficiency. To overcome these deficits, we and others have previously reported the HSC-enhancing ability of a short-term exposure of prostaglandin E2 (PGE2); this strategy has now progressed to phase 1 clinical trials in double cord blood transplantation. To further analyze the short- and long-term effects of HSC exposure to PGE2, we followed the repopulation kinetics of PGE2-treated hematopoietic grafts through 5 serial transplantations and compared inherent long-term competitiveness in a HSC head-to-head secondary transplantation model. Treatment with PGE2 did not result in a long-term increase in HSC competitiveness, lineage bias, or enhanced proliferative potential, demonstrating that pulse exposure to PGE2 results in transient increases in HSC homing and engraftment potential.

Multiple myeloma is incurable once osteolytic lesions have seeded at skeletal sites, but factors mediating this deadly pathogenic advance remain poorly understood. Here, we report evidence of a major role for the cell adhesion molecule CD166, which we discovered to be highly expressed in multiple myeloma cell lines and primary bone marrow cells from patients. CD166+ multiple myeloma cells homed more efficiently than CD166− cells to the bone marrow of engrafted immunodeficient NSG mice. CD166 silencing in multiple myeloma cells enabled longer survival, a smaller tumor burden, and less osteolytic lesions, as compared with mice bearing control cells. CD166 deficiency in multiple myeloma cell lines or CD138+ bone marrow cells from multiple myeloma patients compromised their ability to induce bone resorption in an ex vivo organ culture system. Furthermore, CD166 deficiency in multiple myeloma cells also reduced the formation of osteolytic disease in vivo after intratibial engraftment. Mechanistic investigation revealed that CD166 expression in multiple myeloma cells inhibited osteoblastogenesis of bone marrow–derived osteoblast progenitors by suppressing Runx2 gene expression. Conversely, CD166 expression in multiple myeloma cells promoted osteoclastogenesis by activating TRAF6-dependent signaling pathways in osteoclast progenitors. Overall, our results define CD166 as a pivotal director in multiple myeloma cell homing to the bone marrow and multiple myeloma progression, rationalizing its further study as a candidate therapeutic target for multiple myeloma treatment. Cancer Res; 76(23); 6901–10. ©2016 AACR.

TGF-β derived from bone fuels melanoma bone metastases by inducing tumor secretion of prometastatic factors that act on bone cells to change the skeletal microenvironment. Halofuginone is a plant alkaloid derivative that blocks TGF-β signaling with antiangiogenic and antiproliferative properties. Here, we show for the first time that halofuginone therapy decreases development and progression of bone metastasis caused by melanoma cells through the inhibition of TGF-β signaling. Halofuginone treatment of human melanoma cells inhibited cell proliferation, phosphorylation of SMAD proteins in response to TGF-β, and TGF-β–induced SMAD-driven transcription. In addition, halofuginone reduced expression of TGF-β target genes that enhance bone metastases, including PTHrP, CTGF, CXCR4, and IL11. Also, cell apoptosis was increased in response to halofuginone. In nude mice inoculated with 1205Lu melanoma cells, a preventive protocol with halofuginone inhibited bone metastasis. The beneficial effects of halofuginone treatment were comparable with those observed with other anti–TGF-β strategies, including systemic administration of SD208, a small-molecule inhibitor of TGF-β receptor I kinase, or forced overexpression of Smad7, a negative regulator of TGF-β signaling. Furthermore, mice with established bone metastases treated with halofuginone had significantly less osteolysis than mice receiving placebo assessed by radiography. Thus, halofuginone is also effective in reducing the progression of melanoma bone metastases. Moreover, halofuginone treatment reduced melanoma metastasis to the brain, showing the potential of this novel treatment against cancer metastasis. Cancer Res; 72(23); 6247–56. ©2012 AACR.

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

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β–induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β–induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts. Mol Cancer Res; 10(5); 597–604. ©2012 AACR.

Endothelin-1 (ET-1) is a potent vasoconstrictor that also stimulates cells in the osteoblast lineage by binding to the endothelin A receptor (ETAR). ET-1 ligand is widely secreted, particularly by the vasculature. However, the contributions of ETAR signaling to adult bone homeostasis have not been defined. ETAR was inactivated in osteoblasts by crossing ETAR-floxed and osteocalcin-Cre mice. Histomorphometric analyses were performed on 4-, 8-, and 12-week-old osteoblast-targeted ETAR knockout (KO) and wild-type (WT) male and female mice. Tibial trabecular bone volume was significantly lower from 12 weeks in KO versus WT mice in both males and females. Bone-formation rate, osteoblast density, and in vitro osteoblast differentiation were reduced by targeted inactivation of ETAR. A separate longitudinal analysis was performed between 8 and 64 weeks to examine the effect of aging and castration on bone metabolism in ETAR KO mice. Hypogonadism did not change the rate of bone accrual in WT or KO females. However, eugonadal KO males had a significantly larger increase in tibial and femoral bone acquisition than WT mice. Male mice castrated at 8 weeks of age showed the reverse: KO mice had reduced rates of tibial and femoral BMD acquisition compared with WT mice. In vitro, ET-1 increased osteoblast proliferation, survival, and differentiation. Dihydrotestosterone also increased osteoblast differentiation using a mechanism distinct from the actions of ET-1. These results demonstrate that endothelin signaling in osteoblasts is an important regulator of postnatal trabecular bone remodeling and a modulator of androgen effects on bone. © 2011 American Society for Bone and Mineral Research

Bone development and homeostasis are achieved through an intricate balance between osteoblastic bone formation and osteoclastic bone resorption (Zaidi 2007; Khosla et al. 2008). Mammalian bone develops through endochondral ossification, a process that requires the coordination of osteoblasts and blood vessel invasion into the cartilaginous template to trigger the formation of the primary ossification center, highlighting the importance of angiogenesis during bone formation (Kronenberg 2003; Provot and Schipani 2005; Maes 2013). Maintenance of bone homeostasis is achieved by coupling bone formation with osteoblastic bone resorption. In particular, the RANK/RANKL/osteoprotegerin (OPG) axis contributes to this process by facilitating osteoblast-mediated osteoclastogenesis (Boyle et al. 2003). Deregulation of bone homeostasis is a hallmark of pathophysiological bone disease and occurs due to imbalances in osteogenic–angiogenic coupling or between bone-forming osteoblasts and bone resorption by osteoclasts. Increased bone results in osteopetrosis, whereas osteoporosis is associated with bone loss (Teitelbaum and Ross 2003; Seeman and Delmas 2006). Elucidating the molecular mechanisms that control bone homeostasis within the bone microenvironment is needed in order to identify novel therapeutic strategies to generate functional bone to treat chronic metabolic bone disorders where the options for treatment are still limited. The bone microenvironment is a particularly hypoxic microenvironment. Most adult tissues are well vascularized, allowing for the promotion of oxygen transport with normal median interstitial oxygen tension (pO2) values ranging from 3% to 9% (Vaupel et al. 1989). In contrast, pO2 levels in the bone range from 5%, the PHD enzymes regulate HIF protein stability by catalyzing the hydroxylation of HIF transcription factors on conserved proline residues, targeting them for proteosomal degradation by the von Hippel-Lindau (pVHL) ubiquitin ligase complex. When oxygen tensions fall below 5%, PHD enzyme activity is diminished, and HIF-1α and HIF-2α are stabilized and activate the expression of genes involved in diverse physiological conditions ranging from erythropoiesis to metabolism to angiogenesis (Semenza 2012). Thus, PHD enzymes provide a direct link between oxygen availability and the regulation of HIF transcriptional activity. While the PHD isoforms have nearly ubiquitous tissue expression, the tissue-specific role of each isoform in regulating HIF protein levels remains poorly defined. Here, we sought to dissect the physiologic role of oxygen-sensing PHD1, PHD2, and PHD3 enzymes in the regulation of bone homeostasis. For this purpose, we used a genetic approach to specifically inactivate Phd1, Phd2, and Phd3 in cells of the osteoblastic lineage using Cre-loxP-mediated recombination. In addition to coordinating osteoblastic–angiogenic coupling, we identified a previously unknown role for the PHD/HIF signaling pathway in the osteoblastic regulation of osteoclastogenesis through the direct regulation of OPG. Importantly, inactivation of selective PHD isoforms within the cells of the osteoblastic lineage inhibited bone loss in ovariectomized mice. Thus, molecular oxygen, through the PHD/HIF signaling pathway, plays a central role in bone homeostasis by controlling both angiogenesis and osteoclastogenesis, and therapeutic targeting of PHD enzymes can potentially be used for the treatment of bone disorders.

Cancer-associated muscle weakness is a poorly understood phenomenon, and there is no effective treatment. Here we find that seven different mouse models of human osteolytic bone metastases-representing breast, lung and prostate cancers, as well as multiple myeloma-exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that transforming growth factor (TGF)-β, released from the bone surface as a result of metastasis-induced bone destruction, upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor and calcium (Ca(2+)) release channel (RyR1). The oxidized RyR1 channels leaked Ca(2+), resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-β signaling, TGF-β release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-β activity. Thus, pathological TGF-β release from bone contributes to muscle weakness by decreasing Ca(2+)-induced muscle force production.

Bone is a common site of cancer metastasis. Breast, prostate, and lung cancers show a predilection to metastasize to bone. Recently, we reported that the chemokine interleukin 8 (IL-8) stimulates both human osteoclast formation and bone resorption. IL-8 mRNA expression was surveyed in a panel of human breast cancer lines MDA-MET, MDA-MB-231, MDA-MB-435, MCF-7, T47D, and ZR-75, and the human lung adenocarcinoma cell line A549. IL-8 mRNA expression was higher in cell lines with higher osteolytic potential in vivo. Human osteoclast formation was increased by MDA-MET or A549 cell-conditioned medium, but not by MDA-MB-231. Pharmacologic doses of receptor activator of nuclear factor-κB (RANK)-Fc or osteoprotogerin had no effect on the pro-osteoclastogenic activity of the conditioned medium; however, osteoclast formation stimulated by conditioned medium was inhibited 60% by an IL-8-specific neutralizing antibody. The data support a model in which tumor cells cause osteolytic bone destruction independently of the RANK ligand (RANKL) pathway. Tumor-produced IL-8 is a major contributor to this process. The role of secreted IL-8 isoforms was examined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, which detected distinct IL-8 isoforms secreted by MDA-MET and MDA-231 cells, suggesting different pro-osteoclastogenic activities of the two IL-8-derived peptides. These data indicate that (a) osteoclast formation induced by MDA-MET breast cancer cells and A549 adenocarcinoma cells is primarily mediated by IL-8, (b) cell-specific isoforms of IL-8 with distinct osteoclastogenic activities are produced by tumor cells, and (c) tumor cells that support osteoclast formation independent of RANKL secrete other pro-osteoclastogenic factors in addition to IL-8.

Understanding the mechanism by which tumor cells influence osteoclast differentiation is crucial for improving treatment of osteolytic metastasis. Here, we report broad microRNA (miRNA) expression changes in differentiating osteoclasts after exposure to tumor-conditioned media, in part through activation of NFκB signaling by soluble intracellular adhesion molecule (sICAM1) secreted from bone-metastatic cancer cells. Ectopic expression of multiple miRNAs downregulated during osteoclastogenesis suppresses osteoclast differentiation by targeting important osteoclast genes. Intravenous delivery of these miRNAs in vivo inhibits osteoclast activity and reduces osteolytic bone metastasis. Importantly, serum levels of sICAM1 and two osteoclast miRNAs, miR-16 and miR-378, which are elevated in osteoclast differentiation, correlate with bone metastasis burden. These findings establish miRNAs as potential therapeutic targets and clinical biomarkers of bone metastasis.

Overexpression of the short isoform of p53 (p44) has unexpectedly uncovered a role for p53 in the regulation of size and life span in the mouse. Hyperactivation of the insulin-like growth factor (IGF) signaling axis by p44 sets in motion a kinase cascade that clamps potentially unimpeded growth through p21Cip1. This suggests that pathways of gene activity known to regulate longevity in lower organisms are linked in mammals via p53 to mechanisms for controlling cell proliferation. Thus, appropriate expression of the short and long p53 isoforms might maintain a balance between tumor suppression and tissue regeneration, a major requisite for long mammalian life span.

The extracellular calcium-sensing receptor (CaR; alternate gene names, CaR or Casr) is a membrane-spanning G protein-coupled receptor. CaR is highly expressed in the parathyroid gland, and is activated by extracellular calcium (Ca(2+)(o)). Mice homozygous for null mutations in the CaR gene (CaR(-/-)) die shortly after birth because of the effects of severe hyperparathyroidism and hypercalcemia. A wide variety of functions have been attributed to CaR. However, the lethal CaR-deficient phenotype has made it difficult to dissect the direct effect of CaR deficiency from the secondary effects of hyperparathyroidism and hypercalcemia. We therefore generated parathyroid hormone-deficient (PTH-deficient) CaR(-/-) mice (Pth(-/-)CaR(-/-)) by intercrossing mice heterozygous for the null CaR allele with mice heterozygous for a null Pth allele. We show that genetic ablation of PTH is sufficient to rescue the lethal CaR(-/-) phenotype. Pth(-/-)CaR(-/-) mice survive to adulthood with no obvious difference in size or appearance relative to control Pth(-/-) littermates. Histologic examination of most organs did not reveal abnormalities. These Pth(-/-)CaR(-/-) mice exhibit a much wider range of values for serum calcium and renal excretion of calcium than we observe in control littermates, despite the absence of any circulating PTH. Thus, CaR is necessary for the fine regulation of serum calcium levels and renal calcium excretion independent of its effect on PTH secretion.

BACKGROUND Certain solid tumors metastasize to bone and cause an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not completely understood. METHODS The authors identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provided evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response. RESULTS Tumor conditioned media, as well as exogenous ET-1, stimulated osteoblast proliferation and new bone formation in cultures of mouse calvariae. These effects were blocked by antagonists of the endothelin A (ETA), but not ETB, receptors. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ETA receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on growth in vitro or at the orthotopic site of ZR-75-1 or MDA-MB-231 cells. CONCLUSIONS Collectively, the data suggested that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. ETA receptor blockade may be useful for prevention and the treatment of osteoblastic bone metastases due to breast or prostate cancer. Cancer 2003;97(3 Suppl):779–84. © 2003 American Cancer Society. DOI 10.1002/cncr.11129

Hypoxia often occurs under various physiological and pathophysiological conditions, including solid tumors; it is linked to malignant transformation, metastatic progression, and treatment failure or resistance. Tip110 protein plays important roles in several known physiological and pathophysiological processes, including cancers. Thus, in the present study we investigated the regulation of Tip110 expression under hypoxia. Hypoxia led to Tip110 protein degradation through the ubiquitin-proteasome system. Under hypoxia, Tip110 stabilized p53, which in return destabilized Tip110. In addition, Tip110 regulated hypoxia-inducible factor 1α (HIF-1α), likely through enhancement of its protein stability. Furthermore, Tip110 upregulated p300, a known coactivator for both p53 and HIF-1α. Expression of a p53(22/23) mutant deficient in p300 binding accelerated Tip110 degradation under hypoxia. Tip110 knockdown resulted in the inhibition of cell proliferation and cell death in the presence of p53. Finally, significantly less Tip110, p53, and HIF-1α was detected in the hypoxic region of bone metastasis tumors in a mouse model of human melanoma cells. Taken together, these results suggest Tip110 is an important mediator in the cross talk between p53 and HIF-1α in response to hypoxic stress.

Despite the high prevalence and significant morbidity of spinal anomalies in neurofibromatosis type 1 (NF1), the pathogenesis of these defects remains largely unknown. Here, we present two murine models: Nf1flox/-;PeriCre and Nf1flox/-;Col.2.3Cre mice, which recapitulate spinal deformities seen in the human disease. Dynamic histomorphometry and microtomographic studies show recalcitrant bone remodeling and distorted bone microarchitecture within the vertebral spine of Nf1flox/-;PeriCre and Nf1flox/-;Col2.3Cre mice, with analogous histological features present in a human patient with dystrophic scoliosis. Intriguingly, 36-60% of Nf1flox/-;PeriCre and Nf1flox/-;Col2.3Cre mice exhibit segmental vertebral fusion anomalies with boney obliteration of the intervertebral disc (IVD). While analogous findings have not yet been reported in the NF1 patient population, we herein present two case reports of IVD defects and interarticular vertebral fusion in patients with NF1. Collectively, these data provide novel insights regarding the pathophysiology of dystrophic spinal anomalies in NF1, and provide impetus for future radiographic analyses of larger patient cohorts to determine whether IVD and vertebral fusion defects may have been previously overlooked or underreported in the NF1 patient population.

Increased fracture risk is commonly reported in cancer patients receiving radiotherapy, particularly at sites within the field of treatment. The direct and systemic effects of ionizing radiation on bone at a therapeutic dose are not well-characterized in clinically relevant animal models. Using 20-week-old male C57Bl/6 mice, effects of irradiation (right hindlimb; 2 Gy) on bone volume and microarchitecture were evaluated prospectively by microcomputed tomography and histomorphometry and compared to contralateral-shielded bone (left hindlimb) and non-irradiated control bone. One week postirradiation, trabecular bone volume declined in irradiated tibias (-22%; p 0.0001) and femurs (-14%; p = 0.0586) and microarchitectural parameters were compromised. Trabecular bone volume declined in contralateral tibias (-17%; p = 0.003), and no loss was detected at the femur. Osteoclast number, apoptotic osteocyte number, and marrow adiposity were increased in irradiated bone relative to contralateral and non-irradiated bone, whereas osteoblast number was unchanged. Despite no change in osteoblast number 1 week postirradiation, dynamic bone formation indices revealed a reduction in mineralized bone surface and a concomitant increase in unmineralized osteoid surface area in irradiated bone relative to contralateral and non-irradiated control bone. Further, dose-dependent and time-dependent calvarial culture and in vitro assays confirmed that calvarial osteoblasts and osteoblast-like MC3T3 cells were relatively radioresistant, whereas calvarial osteocyte and osteocyte-like MLO-Y4 cell apoptosis was induced as early as 48 hours postirradiation (4 Gy). In osteoclastogenesis assays, radiation exposure (8 Gy) stimulated murine macrophage RAW264.7 cell differentiation, and coculture of irradiated RAW264.7 cells with MLO-Y4 or murine bone marrow cells enhanced this effect. These studies highlight the multifaceted nature of radiation-induced bone loss by demonstrating direct and systemic effects on bone and its many cell types using clinically relevant doses; they have important implications for bone health in patients treated with radiation therapy.

Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl(-)/1H(+) antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene.

Dysregulated transforming growth factor beta (TGF-β) signaling is associated with a spectrum of osseous defects as seen in Loeys-Dietz syndrome, Marfan syndrome, and Camurati-Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF-β1 signaling pivotally underpins osseous defects in Nf1(flox/-) ;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF-β1 levels are fivefold to sixfold increased both in Nf1(flox/-) ;Col2.3Cre mice and in a cohort of NF1 patients. Nf1-deficient osteoblasts, the principal source of TGF-β1 in bone, overexpress TGF-β1 in a gene dosage-dependent fashion. Moreover, Nf1-deficient osteoblasts and osteoclasts are hyperresponsive to TGF-β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21-Ras-dependent hyperactivation of the canonical TGF-β1-Smad pathway. Reexpression of the human, full-length neurofibromin guanosine triphosphatase (GTPase)-activating protein (GAP)-related domain (NF1 GRD) in primary Nf1-deficient osteoblast progenitors, attenuated TGF-β1 expression levels and reduced Smad phosphorylation in response to TGF-β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF-β receptor 1 (TβRI) kinase inhibitor, SD-208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1(flox/-) ;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF-β1 signaling in the pathogenesis of NF1-associated osteoporosis and pseudarthrosis, thus implicating the TGF-β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies.

Copyright: © 2013 Casimiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., The osteolytic nature of bone metastasis results from a tumor-driven increased bone resorption. Bone remodeling is orchestrated by the molecular triad RANK-RANKL-OPG. This process is dysregulated in bone metastases, mostly via induction of RANKL by tumor-derived factors. These factors increase expression of RANKL, which induce osteoclast formation, function, and survival, thereby increasing bone resorption. RANK is unexpectedly expressed by cancer cells, and the activation of RANKL-RANK pathway correlates with an increased invasive phenotype. To investigate the interaction between RANK expression in human breast and prostate cancer cells and their pro-metastatic phenotype we analyzed the activation of RANKL-RANK pathway and its effects on cell migration, invasion, gene expression in vitro, and osteolysis-inducing ability in vivo. RANKL activates kinase signaling pathways, stimulates cell migration, increases cell invasion, and up-regulates MMP-1 expression. In vivo, MMP-1 knockdown resulted in smaller x-ray osteolytic lesions and osteoclastogenesis, and decreased tumor burden. Therefore, RANKL inhibition in bone metastatic disease may decrease the levels of the osteoclastogenesis inducer MMP-1, contributing to a better clinical outcome., The work was supported in part by grants from the National Institutes of Health (National Cancer Institute) CA69158 and CA143057 (to TAG and KM); and FCT Fellowships SFRH/BPD/34801/2007, SFRH/BD/45219/2008 and SFRH/BD/44716/2008 (to SC, JT-C and IA).

Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR) and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231), and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p

Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1) patients. NF1 (Nf1) haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1(+/-) mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21(Ras) in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF). Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1(+/) (-) osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1(+/-) osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1(+/-) osteoporotic mice induced by ovariectomy (OVX) and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1(+/-)-OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.

Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a complex genetic disorder with a high predisposition of numerous skeletal dysplasias including short stature, osteoporosis, kyphoscoliosis, and fracture non-union (pseudoarthrosis). We have developed murine models that phenocopy many of the skeletal dysplasias observed in NF1 patients, including reduced bone mass and fracture non-union. We also show that the development of these skeletal manifestations requires an Nf1 haploinsufficient background in addition to nullizygous loss of Nf1 in mesenchymal stem/progenitor cells (MSCs) and/or their progenies. This is replicated in two animal models of NF1, PeriCre(+);Nf1(flox/-) and Col2.3Cre(+);Nf1(flox/-) mice. Adoptive transfer experiments demonstrate a critical role of the Nf1+/- marrow microenvironment in the impaired fracture healing in both models and adoptive transfer of WT bone marrow cells improves fracture healing in these mice. To our knowledge, this is the first demonstration of a non-cell autonomous mechanism in non-malignant NF1 manifestations. Collectively, these data provide evidence of a combinatory effect between nullizygous loss of Nf1 in osteoblast progenitors and haploinsufficiency in hematopoietic cells in the development of non-malignant NF1 manifestations.

Extensive data suggest that estradiol contributes to the development of breast cancer by acting as a mitogen and exerting direct genotoxic effects after enzymatic conversion to 4-hydroxyestradiol (4-OHE2) via cytochrome P450 1B1 (CYP1B1). The mammary gland, ovary, and uterus all express CYP1B1. Overexpression of this enzyme has been associated with an increased risk of breast cancer and blockade might reduce this carcinogenic effect. For this reason, we conducted systematic in vitro and in vivo studies of a CYP1B1 inhibitor, TMS (2,3′,4,5′-tetramethoxystilbene). We found that TMS blocked the enzymatic conversion of radiolabeled estradiol to both 2-hydroxyestradiol (2-OHE2) and 4-OHE2, but did not inhibit Cyp1b1 message formation. In vivo studies using mass spectrometry showed that TMS inhibited formation of 2-OHE2 and 4-OHE2 and the resulting estrogen-DNA adducts. To examine its biologic actions in vivo, we investigated whether TMS could block the hyperplastic changes that occur in the developing breast of aromatase-transfected mice. We found that TMS induced a significant reduction of ductal structures in mice less than 6 months in age. In older mice, no reduction in breast morphology occurred. These latter studies uncovered unexpected estrogen agonistic actions of TMS at high doses, including a paradoxical stimulation of breast ductal structures and the endometrium. These studies suggest that the enzyme inhibitory properties of TMS, as well as the effects on developing breast, could implicate a role for TMS in breast cancer prevention, but only in low doses and on developing breast.

Contains fulltext : 88566.pdf (Publisher’s version ) (Closed access) BACKGROUND: The transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling. METHODS: We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. RESULTS: Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors. CONCLUSION: GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

Background Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- β. We asked whether hypoxia (via HIF-1α) and TGF-β signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model. Methodology/Principal Findings We analyzed interactions between HIF-1α and TGF-β pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-β and hypoxia, with effects on the proximal promoters. We inhibited HIF-1α and TGF-β pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells. Conclusions/Significance Hypoxia and TGF-β signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1α and TGF-β may improve treatment of bone metastases and increase survival.

During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-beta has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-beta signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-beta signaling on bone remain unclear. To examine the role of TGF-beta in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-beta type I receptor (TbetaRI) kinase on bone mass, architecture and material properties. Inhibition of TbetaRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TbetaRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TbetaRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TbetaRI inhibitors may be effective in treating conditions of skeletal fragility.

Bone formation is a complex process, and testing anabolic effects on the skeleton of agents is slow and expensive in animals. Neonatal mouse calvariae cultured ex vivo show strong anabolic or catabolic bone responses to 1-week treatments and can be analyzed by quantitative histomorphometry. Changes in new bone area and osteoblast number caused by added proteins, drugs, or transfected genes can be quantified and analyzed for statistical significance. The organ cultures preserve much of the skeletal architecture and cellular diversity present in vivo and offer greater physiological relevance than cell lines studied in vitro.

Melanoma has a propensity to metastasize to bone, where it is exposed to high concentrations of transforming growth factor-β (TGF-β). Because TGF-β promotes bone metastases from other solid tumors, such as breast cancer, we tested the role of TGF-β in melanoma metastases to bone. 1205Lu melanoma cells, stably transfected to overexpress the natural TGF-β/Smad signaling inhibitor Smad7, were studied in an experimental model of bone metastasis whereby tumor cells are inoculated into the left cardiac ventricle of nude mice. All mice bearing parental and mock-transfected 1205Lu cells developed osteolytic bone metastases 5 weeks post-tumor inoculation. Mice bearing 1205Lu-Smad7 tumors had significantly less osteolysis on radiographs and longer survival compared with parental and mock-transfected 1205Lu mice. To determine if the reduced bone metastases observed in mice bearing 1205Lu-Smad7 clones was due to reduced expression of TGF-β target genes known to enhance metastases to bone from breast cancer cells, we analyzed gene expression of osteolytic factors, parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), the chemotactic receptor CXCR4, and osteopontin in 1205Lu cells. Quantitative reverse transcription-PCR analysis indicated that PTHrP, IL-11, CXCR4, and osteopontin mRNA steady-state levels were robustly increased in response to TGF-β and that Smad7 and the TβRI small-molecule inhibitor, SB431542, prevented such induction. In addition, 1205Lu-Smad7 bone metastases expressed significantly lower levels of IL-11, connective tissue growth factor, and PTHrP. These data suggest that TGF-β promotes osteolytic bone metastases due to melanoma by stimulating the expression of prometastatic factors via the Smad pathway. Blockade of TGF-β signaling may be an effective treatment for melanoma metastasis to bone. [Cancer Res 2007;67(5):2317–24]

Tumor-produced endothelin-1 (ET-1) stimulates osteoblasts to form new bone and is an important mediator of osteoblastic bone metastasis. The anabolic actions of ET-1 in osteoblasts were investigated by gene microarray analyses of murine neonatal calvarial organ cultures. Targets of ET-1 action were validated by real-time RT-PCR in murine primary osteoblast cultures. IL-6, IL-11, the CCN (CYR61, CTGF, NOV) family members cysteine-rich protein 61 and connective tissue growth factor, inhibin beta-A, serum/glucocorticoid regulated kinase, receptor activator of nuclear factor kappaB ligand, snail homolog 1, tissue inhibitor of metalloproteinase 3, and TG-interacting factor transcripts were increased by ET-1. ET-1 decreased the transcript for the Wnt signaling pathway inhibitor, dickkopf homolog 1 (Dkk1). Calvarial organ cultures treated with ET-1 had lower concentrations of DKK1 protein in conditioned media than control cultures. High DKK1 concentrations in bone marrow suppress bone formation in multiple myeloma. We hypothesized that the converse occurs in osteoblastic bone metastasis, where ET-1 stimulates osteoblast activity by reducing autocrine production of DKK1. Recombinant DKK1 blocked ET-1-mediated osteoblast proliferation and new bone formation in calvarial organ cultures, whereas a DKK1-neutralizing antibody increased osteoblast numbers and new bone formation. ET-1 directed nuclear translocation of beta-catenin in osteoblasts, indicating activation of the Wnt signaling pathway. The data suggest that ET-1 increases osteoblast proliferation and new bone formation by activating the Wnt signaling pathway through suppression of the Wnt pathway inhibitor DKK1.

Although mammals do not regenerate most appendages, they are able to regenerate toetips if the amputation occurs through the nail bed. The reasons for different outcomes following amputation at different levels are not understood. It is possible that cells at regenerating and nonregenerating sites migrate from fundamentally different tissues. If so, different migratory pathways could be detected. To identify putative migrating cells, microscope slides were made from both regenerating and nonregenerating toes of rats and mice on successive days after amputation. Fluorescent-labeled phalloidin, which binds polymerized f-actin, was used to identify actin filaments and fibers. Cells containing prominent actin bundles were distinguishable from those containing diffuse fibrils and those in which visible fibers were absent. Phalloidin labeling was similar in regenerating and nonregenerating digits after amputation. As early as 2 days after amputation at either proximal or distal levels, many cells of the hypodermis adjacent to the wound became labeled with phalloidin. The number and intensity of labeled hypodermal cells containing stress fiber-like bundles increased rapidly with time, and at successive times cells were seen progressively further distally. By approximately 7 days, they occupied the wound site immediately distal to bone of both regenerating and nonregenerating digits. Most dermal cells were unlabeled and endosteal and marrow cells contained only fibrillar actin. Phalloidin labeling does not support the concept of migration from different tissues in regenerating and nonregenerating amputation sites.

Certain solid tumors metastasize to bone, causing an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not understood completely. We identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provide evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response. Tumor-conditioned media and exogenous ET-1 stimulated osteoblast proliferation and new bone formation in cultures of calvarias from mice. These effects were blocked by endothelin A (ETA) but not by ETB receptor antagonists. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ETA receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on cell growth in vitro or at the orthotopic site (mammary fat pad) of ZR-75-1, or MDA-MB-231 cells. Collectively, the data suggest that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. Endothelin A receptor blockade may be useful for the prevention and treatment of osteoblastic bone metastases attributable to breast or prostate cancer.

SummaryTransforming growth factor-β (TGF-β) regulates the expression of genes supporting breast cancer cells in bone, but little is known about prostate cancer bone metastases and TGF-β. Our study reveals that the TGFBR1 inhibitor SD208 effectively reduces prostate cancer bone metastases. TGF-β upregulates in prostate cancer cells a set of genes associated with cancer aggressiveness and bone metastases, and the most upregulated gene was PMEPA1. In patients, PMEPA1 expression decreased in metastatic prostate cancer and low Pmepa1 correlated with decreased metastasis-free survival. Only membrane-anchored isoforms of PMEPA1 interacted with R-SMADs and ubiquitin ligases, blocking TGF-β signaling independently of the proteasome. Interrupting this negative feedback loop by PMEPA1 knockdown increased prometastatic gene expression and bone metastases in a mouse prostate cancer model.

Introduction Adrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases—a major site of treatment-refractory tumor growth in patients with advanced disease. Methods The role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR. Results Overexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity. Conclusions The results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0458-y) contains supplementary material, which is available to authorized users.