Oncostatin M Induces Bone Loss and Sensitizes Rat Osteosarcoma to the Antitumor Effect of MidostaurinIn vivo

Abstract

Purpose: In cultures, the cytokine oncostatin M (OSM) reduces the growth and induces differentiation of osteoblasts and osteosarcoma cells into glial/osteocytic cells. Moreover, OSM sensitizes these cells to apoptosis driven by various death inducers such as the kinase inhibitor staurosporine. Here, we asked whether OSM would have similar effects in vivo. Experimental Design: Adenoviral gene transfer of OSM (AdOSM) was done in naive and osteosarcoma-bearing rats, alone or in combination with Midostaurin (PKC412), a derivative of staurosporine currently used in cancer clinical trials. Bone variables were analyzed by microcomputed tomography scanner, by histology, and by the levels of various serum bone markers. Osteosarcoma progression was analyzed by the development of the primary bone tumor, evolution of pulmonary metastasis, histology (necrosis and fibrosis), and animal survival. Results: In naive rats, AdOSM reduced serum osteoblastic and osteoclastic markers in correlation with a reduced trabecular bone volume. In an osteosarcoma rat model, the combination of AdOSM with PKC412 reduced the progression of the primary bone tumor, pulmonary metastatic dissemination, and increased overall survival, whereas these agents alone had no antitumor effect. Increased tumor necrosis and tissue repair (fibrosis) were observed with this combination. Conclusion: These in vivo experiments confirm that systemic OSM overexpression alters osteoblast/osteosarcoma activity. Because OSM sensitizes rat osteosarcoma to apoptosis/necrosis, the use of kinase inhibitors such as Midostaurin in association with OSM could represent new adjuvant treatments for this aggressive malignancy.Osteosarcomas are rare bone-forming tumors that affect primarily young adults. These cancer cells arise from osteoblasts, the cells responsible for bone apposition. Rather than a unique gene alteration, multiple deregulations of the proteins controlling the G 1 -S-phase cell cycle checkpoint (p53, Rb, p16INK4A , MDM2, etc.) are involved in the pathogenesis and chemoresistance of osteosarcomas (1, 2). Although the prognosis and chemotherapies of patients with osteosarcoma were improved significantly in the seventies, the survival rate after 5 years is only 60% to 70% and as low as 30% when pulmonary metastases are detected at diagnosis. These survival rates were not further ameliorated during the past decades (3, 4).Thus, new therapies that inhibit the growth or metastasis of these tumors will have a significant effect on patient survival. Cytokines of the interleukin-6 (IL-6) family, such as oncostatin M (OSM), are recognized as pleiotropic factors influencing many pathophysiologic events in several organs, including bone (5 -8). These cytokines have all been reported to stimulate osteoclastogenesis and, in some cases, to stimulate osteoblast differentiation in cell cultures. However, recent in vivo and genetic data have challenged these concepts (reviewed in refs. 5 -8). For example, transgenic mice overexpressing IL-6 showed a decrease in osteobla...

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“…Sections (3 m thick, Leica Microsystems) were analyzed by tartrate-resistant acid phosphatase (TRAP) staining as described previously [17,18].…”

“…Immunostaining for Osterix was performed as described [17] For undecalcified histology, tibias were methylmethacrylate-embedded for microtome sectioning (6 m) for von Kossa staining.…”

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“…The tumor volume was quantified by measuring two perpendicular diameters with a vernier caliper and calculated with the formula: (l 2 Â L)/2 (l, the smallest and L, the largest diameter). 29,34 In-vivo treatment with Adenovirus Replication-deficient adenovirus encoding mouse OSM (AdOSM) has been described previously 29,36 and was produced, together with adenovirus encoding green fluorescent protein (AdGFP), in the vector facility of the INSERM U649 Laboratory (Nantes, France). Approximately 20 days after transplantation when tumor volumes reached 1,000 mm 3 (considered as progressive tumors), rats were injected at three points in the tumor with either PBS, AdOSM or AdGFP at a dose of 1.10 9 pfu.…”

“…15,28 However, OSM or IL-6 block osteoblasts and osteosarcoma cells in the G0/G1 phase of the cell cycle by inducing expression of the cdk inhibitor p21 WAF1 . 17,29,30 These cytokines do not induce apoptosis, but wild-type p53 osteosarcoma cells treated with OSM are particularly sensitive to apoptosis induced by staurosporine (STS), UV, TNFa or chemotherapeutic agents. 29,31 Indeed OSM, through activation of STATs and p53, increases the Bax/Bcl2 ratio which controls the mitochondrial apoptotic pathway.…”

“…17,29,30 These cytokines do not induce apoptosis, but wild-type p53 osteosarcoma cells treated with OSM are particularly sensitive to apoptosis induced by staurosporine (STS), UV, TNFa or chemotherapeutic agents. 29,31 Indeed OSM, through activation of STATs and p53, increases the Bax/Bcl2 ratio which controls the mitochondrial apoptotic pathway. 31 These effects on cell proliferation and apoptosis are concomitant with an induced terminal differentiation of osteoblasts or osteosarcoma cells into osteocyte-like cells.…”