RESUMEN
BACKGROUND: Osteosarcoma is the most common primary bone cancer in children and young adults. It is highly aggressive and patients that present with metastasis have a poor prognosis. Angiopoietin-like 4 (ANGPTL4) drives the progression and metastasis of many solid tumours, but has not been described in osteosarcoma tissue. ANGPTL4 also enhances osteoclast activity, which is required for osteosarcoma growth in bone. We therefore investigated the expression and function of ANGPTL4 in human osteosarcoma tissue and cell lines. METHODS: Expression of ANGPTL4 in osteosarcoma tissue microarrays was determined by immunohistochemistry. Hypoxic secretion of ANGPTL4 was tested by ELISA and Western blot. Regulation of ANGPTL4 by hypoxia-inducible factor (HIF) was investigated using isoform specific HIF siRNA (HIF-1α, HIF-2α). Effects of ANGPTL4 on cell proliferation, migration (scratch wound assay), colony formation and osteoblastogenesis were assessed using exogenous ANGPTL4 or cells stably transfected with ANGPTL4. Osteoclastogenic differentiation of CD14+ monocytes was assessed by staining for tartrate-resistant acid phosphatase (TRAP), bone resorption was assessed by lacunar resorption of dentine. RESULTS: ANGPTL4 was immunohistochemically detectable in 76/109 cases. ANGPTL4 was induced by hypoxia in 6 osteosarcoma cell lines, under the control of the HIF-1α transcription factor. MG-63 cells transfected with an ANGPTL4 over-expression plasmid exhibited increased proliferation and migration capacity and promoted osteoclastogenesis and osteoclast-mediated bone resorption. Individually the full-length form of ANGPTL4 could increase MG-63 cell proliferation, whereas N-terminal ANGPTL4 mediated the other pro-tumourigenic phenotypes. CONCLUSIONS: This study describes a role(s) for ANGPTL4 in osteosarcoma and identifies ANGPTL4 as a treatment target that could potentially reduce tumour progression, inhibit angiogenesis, reduce bone destruction and prevent metastatic events.
Asunto(s)
Proteína 4 Similar a la Angiopoyetina/metabolismo , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Osteogénesis/genética , Osteosarcoma/patología , Proteína 4 Similar a la Angiopoyetina/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Resorción Ósea/genética , Resorción Ósea/patología , Carcinogénesis/genética , Movimiento Celular/genética , Proliferación Celular/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Osteoclastos/fisiología , Osteosarcoma/irrigación sanguínea , Osteosarcoma/genética , ARN Interferente Pequeño/metabolismo , Análisis de Matrices TisularesRESUMEN
Mononuclear precursors of human osteoclasts are found in the CD14(+) monocyte fraction of circulating peripheral blood mononuclear cells (PBMCs). It is possible to generate osteoclasts in vitro from PBMCs cultured with macrophage colony-stimulating factor and receptor activator for nuclear factor κB ligand. In these cultures, however, it is not possible to distinguish the effect of a specific agent on osteoclast resorption activity as opposed to osteoclast differentiation. To produce a population of mature human osteoclasts to study osteoclast lacunar resorption specifically, we cultured CD14(+) human monocytes on hydrophobic dishes in order to generate and maintain osteoclasts in suspension prior to culturing them on coverslips and dentine slices. Multinucleated cells formed in these cultures expressed vitronectin receptor, tartrate-resistant acid phosphatase, and cathepsin K. These cells also produced F-actin rings and were capable of extensive lacunar resorption on dentine slices after 24 h in culture. Lacunar resorption was inhibited by calcitonin and zoledronate but not by osteoprotegerin. This method of generating a highly enriched population of mature human osteoclasts should provide a valuable means of specifically assessing the effect of molecular factors (e.g., cytokines, growth factors, hormones) and therapeutic agents on osteoclast resorption activity.