Glycogen Synthase Kinase-3β, NF-κB Signaling, and Tumorigenesis of Human Osteosarcoma

Background Glycogen synthase kinase-3 beta (GSK-3 beta), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3 beta in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent. Methods We used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3 beta expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5-8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3 beta in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3 beta inhibition on the nuclear factor-kappa B (NF-kappa B) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3 beta activity with overall survival. Results Osteosarcoma cells with low levels of inactive p-Ser9-GSK-3 beta formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3 beta had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3 beta resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3 beta resulted in inhibition of the NF-kappa B pathway and reduction of NF-kappa B-mediated transcription. Combination treatments with GSK-3 beta inhibitors, NF-kappa B inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3 beta, and nuclear NF-kappa B had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3 beta and NF-kappa B (109.2 months). Conclusion GSK-3 beta activity may promote osteosarcoma tumor growth, and therapeutic targeting of the GSK-3 beta and/or NF-kappa B pathways may be an effective way to enhance the therapeutic activity of anticancer drugs against osteosarcoma.