Abstract
Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin + methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically improved overall survival of OS patients. However, further research is needed to identify new therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme, nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin, doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of NMNAT1−/− cells showed features of both apoptosis and necroptosis, as indicated by the protective effect of the caspase-3 inhibitor z-DEVD-FMK and the necroptosis inhibitor necrostatin-1. Activation of the DNA damage sensor enzyme poly(ADP-ribose) polymerase 1 (PARP1), a major consumer of NAD+ in the nucleus, was fully blocked by NMNAT1 inactivation, leading to increased DNA damage (phospho-H2AX foci). The PARP inhibitor, olaparib, sensitized wild type but not NMNAT1−/− cells to cisplatin-induced anti-clonogenic effects, suggesting that impaired PARP1 activity is important for chemosensitization. Cisplatin-induced cell death of NMNAT1−/− cells was also characterized by a marked drop in cellular ATP levels and impaired mitochondrial respiratory reserve capacity, highlighting the central role of compromised cellular bioenergetics in chemosensitization by NMNAT1 inactivation. Moreover, NMNAT1 cells also displayed markedly higher sensitivity to cisplatin when grown as spheroids in 3D culture. In summary, our work provides the first evidence that NMNAT1 is a promising therapeutic target for osteosarcoma and possibly other tumors as well.
Highlights
Bone tumors constitute 4–7% of all cancers in children aged 0–14 years and 7–8% in children aged15–19 years [1]
We found that the absence of nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) sensitized cells to the toxic effects of cisplatin (Figure 3A)
To demonstrate the role of NMNAT1 in cellular models that more closely resemble in vivo conditions or clinical settings, we investigated a) chemosensitization to cisplatin by NMNAT1 deficiency in a 3D model and b) a treatment protocol based on a combination of chemotherapeutic drugs
Summary
Bone tumors constitute 4–7% of all cancers in children aged 0–14 years and 7–8% in children aged. Osteosarcoma is the most prevalent neoplasm of the bone in children and adolescents, with approximately 850,000 cases reported each year in the US. Tumors are typically diagnosed between 10 and 30 years of age and mostly arise in the femur, tibia, and humerus. Most osteosarcoma cases are sporadic, a higher incidence of osteosarcoma has been reported in certain hereditary cancer syndromes. Mutations of the tumor suppressor retinoblastoma protein (Rb) in retinoblastoma [2] or p53 in Li-Fraumeni syndrome [3], as well as the DNA repair helicase. Werner protein are associated with osteosarcoma [4]. Previous radiotherapy of other tumors may lead, in the long term, to osteosarcoma development
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