Cisplatin selects for stem-like cells in osteosarcoma by activating Notch signaling

// Ling Yu 1, * , Zhengfu Fan 2, * , Shuo Fang 1, * , Jian Yang 1 , Tian Gao 2 , Bruno M. Simões 3 , Rachel Eyre 3 , Weichun Guo 1 , Robert B. Clarke 3 1 Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei, China 2 Department of Orthopedic Oncology, Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Peking University Cancer Hospital & Institute, Beijing, China 3 Breast Cancer Now Research Unit, Institute of Cancer Sciences, University of Manchester, Manchester, UK * These authors have contributed equally to this work Correspondence to: Robert B. Clarke, email: rclarke@picr.man.ac.uk Weichun Guo, email: guoweichun@aliyun.com Keywords: osteosarcoma, cancer stem-like cells, chemo-resistance, Notch signaling pathway Received: February 11, 2016 Accepted: March 27, 2016 Published: April 20, 2016 ABSTRACT Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo , and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro , and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro , including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance.