Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

// Shingo Shimozaki 1, 4, * , Norio Yamamoto 1 , Takahiro Domoto 4, * , Hideji Nishida 1 , Katsuhiro Hayashi 1 , Hiroaki Kimura 1 , Akihiko Takeuchi 1 , Shinji Miwa 1, 3, 4 , Kentaro Igarashi 1 , Takashi Kato 1 , Yu Aoki 1 , Takashi Higuchi 1 , Mayumi Hirose 4 , Robert M Hoffman 2, 3 , Toshinari Minamoto 4 , Hiroyuki Tsuchiya 1 1 Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan 2 Department of Surgery, University of California, San Diego, CA, U.S.A 3 AntiCancer Incorporated, San Diego, CA, U.S.A 4 Division of Translational and Clinical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan * These authors contributed equally to this work Correspondence to: Robert M. Hoffman, email: all@anticancer.com Norio Yamamoto, email: norinori@med.kanazawa-u.ac.jp Keywords: glycogen synthase kinase-3β, molecular target, treatment, osteosarcoma, orthotopic nude mice Received: September 09, 2016 Accepted: October 05, 2016 Published: October 20, 2016 ABSTRACT Development of innovative more effective therapy is required for refractory osteosarcoma patients. We previously established that glycogen synthase kinase-3β (GSK- 3β) is a therapeutic target in various cancer types. In the present study, we explored the therapeutic efficacy of GSK-3β inhibition against osteosarcoma and the underlying molecular mechanisms in an orthotopic mouse model. Expression and phosphorylation of GSK-3β in osteosarcoma and normal osteoblast cell lines was examined, together with efficacy of GSK-3β inhibition on cell survival, proliferation and apoptosis and on the growth of orthotopically-transplanted human osteosarcoma in nude mice. We also investigated changes in expression, phosphorylation and co-transcriptional activity of β-catenin in osteosarcoma cells following GSK-3β inhibition. Expression of the active form of GSK- 3β (tyrosine 216-phosphorylated) was higher in osteosarcoma than osteoblast cells. Inhibition of GSK-3β activity by pharmacological inhibitors or of its expression by RNA interference suppressed proliferation of osteosarcoma cells and induced apoptosis. Treatment with GSK-3β-specific inhibitors attenuated the growth of orthotopic osteosaroma in mice. Inhibition of GSK-3β reduced phosphorylation at GSK- 3β-phospho-acceptor sites in β-catenin and increased β-catenin expression, nuclear localization and co-transcriptional activity. These results suggest the efficacy of GSK-3β inhibitors is associated with activation of β-catenin, a putative tumor suppressor in bone and soft tissue sarcoma and an important component of osteogenesis. Our study thereby demonstrates a critical role for GSK-3β in sustaining survival and proliferation of osteosarcoma cells, and identifies this kinase as a potential therapeutic target against osteosarcoma.