Abstract 3960: Identification of osteosarcoma cancer stem cells using an imaging system for proteasome activity

Abstract

Abstract Osteosarcoma is the most common primary malignant bone tumor in the pediatric and adolescent age groups, comprising almost 60% of all bone sarcomas. Radioresistant cancers, including osteosarcoma, typically exhibit a considerable potential for recurrence and development of metastases following radiotherapy. Recurrence and metastatic potential can be due to a specific radioresistant subpopulation of cells with stem-like characteristics, cancer stem cells (often abbreviated CSCs) which maintain the capacity to regenerate entire tumors. Targeting of the CSCs in osteosarcoma is a promising avenue to explore to develop new therapies for this devastating cancer. CSC-enriched cancer cell populations exhibit the capacities for self-renewal, multilineage differentiation, tumorigenicity, chemo- and radioresistance. Also, CSCs usually are quiescent with low protein turnover, reduced metabolism, and a down-regulation of proteasome activity. The ubiquitin-proteasome system is the major non-lysosomal system for degradation of intracellular proteins. Recently, cancer cells with low proteasome activity have been identified as CSCs in breast cancer, glioma, and pancreatic cancer with a fluorescence marker system for level of proteasome activity. We stably infected two osteosarcoma cell lines, MG-63 and U2-OS, with an expression vector for a fusion protein between the green fluorescent protein, ZsGreen, and the C-terminal degron of murine ornithine decarboxylase to monitor 26S proteasome activity in living cells. We purified osteosarcoma cells with low proteasome activity by fluorescence activated cell sorting and verified whether these ZsGreen cells have CSC-like properties or not. ZsGreen cells showed enhanced sphere formation capacities, and underwent asymmetric divisions into ZsGreen cells and non-ZsGreen cells, showing self renewal and multipotency, while non-ZsGreen cells underwent only symmetric divisions into non-ZsGreen cells. We also performed clonogenic survival assays that showed ZsGreen cells were more radioresistant than non-ZsGreen cells. We conclude that established osteosarcoma cell lines are heterogeneous with regard to their intrinsic proteasome activity and that they contain osteosarcoma stem cells-enriched subpopulations that can be identified by low proteasome activity. Citation Format: Keisuke Tamari, Kazuhiko Hayashi, Yoshihiro Kano, Masamitsu Konno, Takahito Fukusumi, Shimpei Nishikawa, Shinichiro Hasegawa, Hisataka Ogawa, Atsushi Hamabe, Masaaki Miyo, Kozo Noguchi, Yuji Seo, Hideshi Ishii, Yuichiro Doki, Masaki Mori, Kazuhiko Ogawa. Identification of osteosarcoma cancer stem cells using an imaging system for proteasome activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3960. doi:10.1158/1538-7445.AM2014-3960