ABSTRACT
Ewing's sarcoma (ES) is a primary bone tumor characterized by a chromosomic translocation between the EWS gene and a member of the ETS gene family, mainly FLI1, which leads to an aberrant transcription factor EWS-FLI1 that promotes tumorigenicity. Gap junctions are intercellular channels composed of transmembrane proteins (connexin: Cx), that allow direct intercellular communication between adjacent cells. Numerous studies have shown that tumorigenesis may be associated with a loss of gap junctional intercellular communication (GJIC). Loss of Cx43 expression was observed at the protein and mRNA levels in ES cell lines compared to those measured in human mesenchymal stem cells. A673 ES cells stably transfected with an shRNA targeting EWS-FLI1 showed an increase in Cx43 expression (at the mRNA, protein and transcriptional levels) and GJIC. In an osteolytic murine model of ES, the overexpression of Cx43 in ES cells dramatically reduced tumor growth, leading to a significant increase in animal survival. In vitro assays showed that Cx43 overexpression increases the p27 level with an associated marked decrease of Rb phosphorylation, consistent with the observed blockade of the cell cycle in G0/G1 phase. In addition, the bone microarchitectural parameters, assessed by micro-CT analysis, showed an increased bone volume when Cx43 expression was enhanced. Histological analysis demonstrated that the overexpression of Cx43 in ES tumor cells inhibits osteoclast activity and therefore bone resorption. Our study demonstrated that the loss of Cx43 expression in ES cells plays a crucial role in the development of the primary tumor and the associated bone osteolysis.
Subject(s)
Bone Neoplasms , Cell Transformation, Neoplastic , Connexin 43 , Sarcoma, Ewing , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Bone Neoplasms/secondary , Cell Communication , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Connexin 43/genetics , Connexin 43/metabolism , Embryonic Stem Cells , Gap Junctions , Gene Expression Regulation, Neoplastic , Humans , Oncogene Proteins, Fusion/genetics , Osteolysis/genetics , Osteolysis/pathology , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathologyABSTRACT
Aseptic loosening as a result of wear debris is considered to be the main cause of long-term implant failure in orthopaedic surgery and improved biomaterials for bearing surfaces decreases significantly the release of micrometric wear particles. Increasingly, in-depth knowledge of osteoimmunology highlights the role of nanoparticles and ions released from some of these new bearing couples, opening up a new era in the comprehension of aseptic loosening. Mouse models have been essential in the progress made in the early comprehension of pathophysiology and in testing new therapeutic agents for particle-induced osteolysis. However, despite this encouraging progress, there is still no valid clinical alternative to revision surgery. The present review provides an update of the most commonly used bearing couples, the current concepts regarding particle-cell interactions and the approaches used to study the biology of periprosthetic osteolysis. It also discusses the contribution and future challenges of mouse models for successful translation of the preclinical progress into clinical applications.
Subject(s)
Arthroplasty, Replacement/adverse effects , Macrophages/physiology , Osteolysis/etiology , Animals , Biomechanical Phenomena , Ceramics/adverse effects , Clinical Trials as Topic , Disease Models, Animal , Equipment Failure , Humans , Inflammation/physiopathology , Mice , Nanoparticles/adverse effects , Osteolysis/physiopathology , Particle Size , Polyethylenes/adverse effects , Polyethylenes/chemistry , Polymethyl Methacrylate/adverse effects , Reoperation , Translational Research, BiomedicalABSTRACT
Genes that mediate breast cancer metastasis to lung are different from those which mediate bone metastasis. However, which markers accounts for the diversity of breast cancer metastasis remains unknown. The aim of this study was identify proteins associated with the soft-tissue metastatic ability of breast cancer tumors in metastases, coupling microarray data from clinical metastases and immunohistochemistry, for further screening for early detection at the first diagnosis in patients. We use a bioinformatic program to create and analyze protein interaction networks from protein experimental data, and to translate RNA expression analysis of breast cancer human metastases to protein, in a search for the phenotype associated with soft-tissue metastases. The pre-validated proteins constituted the protein signature for each metastasis: 37 (8.9%) from liver, 92 (8.5%) from lung and 167 (13%) from bone. Pleiotrophin, BAG 2, HSP 60 and vinculin were pre-validated in liver and lung metastases performing the soft-tissue phenotype. After IHC validation, we conclude that HSP 60, one of the best-known mitochondrial chaperone machines, is a key protein in soft-tissue metastases phenotype interacting with BAG 2, which competes for binding to GRP 75, the other mitochondrial chaperone. The relationship between HSP 60/GRP 75 and BAG 2 might result in the activation of several transcription pathways, different in liver from in lung metastases, as a nodal point coupling positive and negative actuators in the multiple survival-signal pathways and so achieving metastatic growth.
Subject(s)
Breast Neoplasms/pathology , Liver Neoplasms/secondary , Lung Neoplasms/secondary , Molecular Chaperones/physiology , Breast Neoplasms/physiopathology , Cell Line, Tumor , HumansABSTRACT
Bisphosphonates are the standard of care in the treatment of malignant bone diseases, because of their ability to inhibit osteoclast-mediated bone destruction. We review here preclinical evidence that bisphosphonates also exert direct antitumour effects and antiangiogenic properties. Furthermore, we describe new insights on how bisphosphonates may act synergistically in combination with antineoplastic drugs or gammadelta T cells to exhibit antitumour activity. These findings reveal new exciting possibilities to fully exploit the antitumour potential of bisphosphonates in the clinical practice.
Subject(s)
Diphosphonates/therapeutic use , Neoplasms/drug therapy , Angiogenesis Inhibitors/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Apoptosis , Bone and Bones/metabolism , Cell Adhesion , Cell Proliferation , Diphosphonates/chemistry , Disease Models, Animal , Humans , Models, Biological , Models, Chemical , Neoplasm Invasiveness , Neoplasm MetastasisABSTRACT
Receptor activator of nuclear factor-κB (RANK) and RANK ligand (RANKL) play a pivotal role in bone metabolism, and selective targeting of RANK signaling has become a promising therapeutic strategy in the management of resorptive bone diseases. Existing antibody-based therapies and novel inhibitors currently in development were designed to target the ligand, rather than the membrane receptor expressed on osteoclast precursors. We describe here an alternative approach to designing small peptides able to specifically bind to the hinge region of membrane RANK responsible for the conformational change upon RANKL association. A nonapeptide generated by this method was validated for its biological activity in vitro and in vivo and served as a lead compound for the generation of a series of peptide RANK antagonists derived from the original sequence. Our study presents a structure- and knowledge-based strategy for the design of novel effective and affordable small peptide inhibitors specifically targeting the receptor RANK and opens a new therapeutic opportunity for the treatment of resorptive bone disease.
Subject(s)
Bone Resorption/drug therapy , Models, Biological , Peptides/therapeutic use , Receptor Activator of Nuclear Factor-kappa B/antagonists & inhibitors , Amino Acid Motifs , Animals , Bone Resorption/diagnostic imaging , Bone Resorption/pathology , Bone Resorption/prevention & control , Computer Simulation , Drug Evaluation, Preclinical , Female , Humans , Mice, Inbred C57BL , Osteoclasts/drug effects , Osteoclasts/metabolism , Ovariectomy , Peptides/antagonists & inhibitors , Peptides/chemistry , Peptides/pharmacology , Protective Agents/pharmacology , Protective Agents/therapeutic use , RANK Ligand/pharmacology , Radiography , Receptor Activator of Nuclear Factor-kappa B/metabolism , Reproducibility of Results , Signal Transduction/drug effectsABSTRACT
PURPOSE: Osteosarcoma is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastasis is present at diagnosis. Because transforming growth factor-ß (TGFß) has been shown to promote metastasis in many solid tumors, we investigated the effect of the natural TGFß/Smad signaling inhibitor Smad7 and the TßRI inhibitor SD-208 on osteosarcoma behavior. EXPERIMENTAL DESIGN: By using a mouse model of osteosarcoma induced by paratibial injection of cells, we assessed the impact of Smad7 overexpression or SD-208 on tumor growth, tumor microenvironment, bone remodeling, and metastasis development. RESULTS: First, we demonstrated that TGFß levels are higher in serum samples from patients with osteosarcoma compared with healthy volunteers and that TGFß/Smad3 signaling pathway is activated in clinical samples. Second, we showed that Smad7 slows the growth of the primary tumor and increases mice survival. We furthermore demonstrated that Smad7 expression does not affect in vitro osteosarcoma cell proliferation but affects the microarchitectural parameters of bone. In addition, Smad7-osteosarcoma bone tumors expressed lower levels of osteolytic factors such as RANKL, suggesting that Smad7 overexpression affects the "vicious cycle" established between tumor cells and bone cells by its ability to decrease osteoclast activity. Finally, we showed that Smad7 overexpression in osteosarcoma cells and the treatment of mice with SD208 inhibit the development of lung metastasis. CONCLUSION: Taken together, these results demonstrate that the inhibition of the TGFß/Smad signaling pathway may be a promising therapeutic strategy against tumor progression of osteosarcoma, specifically against the development of lung metastasis.
Subject(s)
Bone Neoplasms/genetics , Bone Neoplasms/pathology , Gene Expression , Lung Neoplasms/secondary , Osteosarcoma/genetics , Osteosarcoma/pathology , Smad7 Protein/genetics , Animals , Bone Neoplasms/metabolism , Bone Neoplasms/mortality , Bone Resorption/genetics , Cell Line, Tumor , Disease Models, Animal , Female , Heterografts , Humans , Mice , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteosarcoma/metabolism , Osteosarcoma/mortality , Pteridines/pharmacology , Signal Transduction/drug effects , Smad3 Protein/metabolism , Smad7 Protein/metabolism , Transforming Growth Factor beta/blood , Tumor Burden/geneticsABSTRACT
Osteosarcoma is the most frequent malignant primary bone tumor and a main cause of cancer-related death in children and adolescents. Although long-term survival in localized osteosarcoma has improved to about 60% during the 1960s and 1970s, long-term survival in both localized and metastatic osteosarcoma has stagnated in the past several decades. Thus, current conventional therapy consists of multi-agent chemotherapy, surgery and radiation, which is not fully adequate for osteosarcoma treatment. Innovative drugs and approaches are needed to further improve outcome in osteosarcoma patients. This review describes the current management of osteosarcoma as well as potential new therapies.
ABSTRACT
The nitrogen-containing bisphosphonate zoledronic acid (ZOL), a potent inhibitor of farnesyl pyrophosphate synthase, blocks the mevalonate pathway, leading to intracellular accumulation of isopentenyl pyrophosphate/triphosphoric acid I-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI) mevalonate metabolites. IPP/ApppI accumulation in ZOL-treated cancer cells may be recognized by Vγ9Vδ2 T cells as tumor phosphoantigens in vitro. However, the significance of these findings in vivo remains largely unknown. In this study, we investigated the correlation between the anticancer activities of Vγ9Vδ2 T cells and the intracellular IPP/ApppI levels in ZOL-treated breast cancer cells in vitro and in vivo. We found marked differences in IPP/ApppI production among different human breast cancer cell lines post-ZOL treatment. Coculture with purified human Vγ9Vδ2 T cells led to IPP/ApppI-dependent near-complete killing of ZOL-treated breast cancer cells. In ZOL-treated mice bearing subcutaneous breast cancer xenografts, Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels, but not those expressing low IPP/ApppI levels. Moreover, IPP/ApppI not only accumulated in cancer cells but it was also secreted, promoting Vγ9Vδ2 T-cell chemotaxis to the tumor. Without Vγ9Vδ2 T-cell expansion, ZOL did not inhibit tumor growth. These findings suggest that cancers-producing high IPP/ApppI levels after ZOL treatment are most likely to benefit from Vγ9Vδ2 T-cell-mediated immunotherapy.
Subject(s)
Antigens, Neoplasm/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , Diphosphonates/pharmacology , Imidazoles/pharmacology , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocytes/immunology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Animals , Antigens, Neoplasm/immunology , Breast Neoplasms/metabolism , Cell Line, Tumor , Cytotoxicity, Immunologic , Female , Hemiterpenes/metabolism , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Organophosphorus Compounds/metabolism , Phosphorylation , T-Lymphocytes/drug effects , Zoledronic AcidABSTRACT
Nitrogen-containing bisphosphonates (N-BPs) are widely used to block bone destruction associated with bone metastasis because they are effective inhibitors of osteoclast-mediated bone resorption. More specifically, once internalized by osteoclasts, N-BPs block the activity of farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. In addition to their antiresorptive activity, preclinical evidence shows that N-BPs have antiangiogenic properties. However, the exact reasons for which N-BPs inhibit angiogenesis remain largely unknown. Using different angiogenesis models, we examined here the effects of zoledronate, risedronate and three structural analogs of risedronate (NE-58025, NE-58051 and NE-10790) with lower potencies to inhibit FPPS activity. Risedronate and zoledronate were much more potent than NE-compounds at inhibiting both endothelial cell proliferation in vitro and vessel sprouting in the chicken egg chorioallantoic membrane (CAM) assay. In addition, only risedronate and zoledronate inhibited the revascularization of the prostate gland in testosterone-stimulated castrated rats. Moreover, as opposed to NE-compounds, risedronate and zoledronate induced intracellular accumulation of isopentenyl pyrophosphate (IPP) in endothelial cells by blocking the activity of the IPP-consuming enzyme FPPS. Thus, these results indicated that N-BPs inhibited angiogenesis in a FPPS-dependent manner. However, drug concentrations used to inhibit angiogenesis, both in vitro and in the CAM and prostate gland assays, were high. In contrast, a low concentration of risedronate (1 µM) was sufficient to inhibit blood vessel formation in the ex vivo rat aortic ring assay. Moreover, NE-58025 (which had a 7-fold lower potency than risedronate to inhibit FPPS activity) was as effective as risedronate to reduce angiogenesis in the rat aortic ring assay. In conclusion, our results suggest that low concentrations of N-BPs inhibit angiogenesis in a FPPS-independent manner, whereas higher drug concentrations were required to inhibit FPPS activity in vivo.
Subject(s)
Diphosphonates/pharmacology , Geranyltranstransferase/metabolism , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Chick Embryo , Chorioallantoic Membrane , Diphosphonates/chemistry , Diphosphonates/therapeutic use , Endothelial Cells/cytology , Endothelial Cells/drug effects , Etidronic Acid/analogs & derivatives , Etidronic Acid/chemistry , Etidronic Acid/pharmacology , Etidronic Acid/therapeutic use , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Imidazoles/therapeutic use , Male , Neovascularization, Pathologic/drug therapy , Rats , Rats, Sprague-Dawley , Risedronic Acid , Zoledronic AcidABSTRACT
Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and have demonstrated clinical utility in the treatment of patients with osteolytic bone metastases. They also exhibit direct antitumor activity in vitro and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo. However, whether such effects are caused by a direct action of bisphosphonates on tumor cells or indirectly through inhibition of bone resorption remains unclear. To address this question, we used here a structural analog of the bisphosphonate risedronate, NE-58051, which has a bone mineral affinity similar to that of risedronate, but a 3000-fold lower bone antiresorptive activity. In vitro, risedronate and NE-58051 inhibited proliferation of breast cancer and melanoma cell lines. In vivo, risedronate and NE-58051 did not inhibit the growth of subcutaneous B02 breast tumor xenografts or the formation of B16F10 melanoma lung metastasis. In contrast to NE-58051, risedronate did inhibit B02 breast cancer bone metastasis formation by reducing both bone destruction and skeletal tumor burden, indicating that the antitumor effect of bisphosphonates is achieved mainly through inhibition of osteoclast-mediated bone resorption.
Subject(s)
Bone Neoplasms/prevention & control , Breast Neoplasms/drug therapy , Diphosphonates/therapeutic use , Melanoma, Experimental/drug therapy , Pyridines/therapeutic use , Animals , Bone Density Conservation Agents/pharmacology , Bone Density Conservation Agents/therapeutic use , Bone Neoplasms/secondary , Bone Resorption/drug therapy , Bone Resorption/pathology , Breast Neoplasms/pathology , Cell Line, Tumor , Diphosphonates/pharmacology , Etidronic Acid/analogs & derivatives , Etidronic Acid/pharmacology , Etidronic Acid/therapeutic use , Female , Humans , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Melanoma, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Pyridines/pharmacology , Risedronic Acid , Xenograft Model Antitumor AssaysABSTRACT
Metastatic breast cancer cells are characterized by their high propensity to colonize the skeleton and form bone metastases, causing major morbidity and mortality. Identifying key proteins involved in the osteotropic phenotype would represent a major step toward the development of both new prognostic markers and new effective therapies. Cell surface proteins differentially expressed in cancer cells are preferred potential targets for antibody-based targeted therapies. In this study, using cell surface biotinylation and a mass spectrometric approach, we have compared the profile of accessible cell surface proteins between the human breast cancer cell line MDA-MB-231 and its highly osteotropic B02 subclone. This strategy allowed the identification of several proteins either up- or downregulated in the osteotropic cell line, and differential protein expressions were validated using antibody-based techniques. Class I HLAs were down-regulated in the bone metastatic variant, whereas alpha(v)beta(3) integrins, among others, were consistently up-regulated in this latter cell line. These results show that comprehensive profiling of the cell surface proteome of mother cancerous cell lines and derived organ-specific metastatic cell lines provides an effective approach for the identification of potential accessible marker proteins for both prognosis and antibody-based targeted therapies.
Subject(s)
Bone Neoplasms/metabolism , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Proteome/metabolism , Biotinylation , Bone Neoplasms/secondary , Bone and Bones , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Membrane , Female , Gene Expression , Humans , Mass SpectrometryABSTRACT
Bcl-xL gene induces metastasis in the lung, lymph nodes and bone when breast cancer cells are inoculated in Nude Balb/c mice. In an attempt to identify the molecules required for diverse metastatic foci, we compared gene expression levels in tumor cells and metastatic variants with a cDNA GeneFilter containing 4000 known genes. The transcriptional regulators of alpha1-fetoprotein transcription factor, TBP-associated factor 172 (TAF-172) and the human zinc finger protein 5 (ZFP5) were downregulated. The expression of TAF-172 was inversely proportional to Bcl-xL expression (ANOVA P < 0.0001) and metastatic activity (ANOVA P < 0.0001). A protein interaction program allowed us to functionally associate Bcl-xL and TAF through TATA-binding protein (TBP), suggesting that Bcl-xL connects metabolic pathways with transcriptional machinery. The prediction included proteins involved in apoptosis, electron transfer, kinases and transcription factors. These results indicate that the selection of diverse metastatic cells from the broad spectrum of tumor cell leads to the underexpression of certain transcriptional regulators that might act as adaptor molecules to different microenvironments, and indicate that the synergistic activity of several genes is needed for the selection process in several metastatic foci.