Abstract 1747: The Pax3:Foxo1a translocation product is a cell cycle-specific modifier of the alveolar rhabdomyosarcoma tumor phenotype

• Published in: Cancer research (Chicago, Ill.) Ill.), 2013-04, Vol.73 (8_Supplement), p.1747-1747
• Main Authors: Kikuchi, Ken, Hettmer, Simone, Aslam, M. Imran, Michalek, Joel E., Laub, Wolfram, Rubin, Brian P., Wagers, Amy J., Keller, Charles
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2013-1747

BACKGROUND: Rhabdomyosarcoma is the most common soft-tissue sarcoma in childhood, which falls into one of two biologically distinct subtypes, alveolar (aRMS) or embryonal rhabdomyosarcoma. aRMS harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. MATERIALS AND METHODS: Primary murine aRMS cultures were obtained from the Myf6Cre, Pax3:Foxo1a, p53 conditional mouse aRMS model, in which eYFP is expressed as a second cistron in the targeted Pax3:Foxo1a-ires-eYFP allele. Limited dilution transplantation experiments were performed using Pax3:Foxo1a knockdown by siRNA of eYFP or control aRMS primary cultures. To elucidate the dynamical function of Pax3:Foxo1a, time-lapse confocal microscopy experiments, cell cycle analysis, quantitative RT-PCR, western blotting, immunohistochemistry and mRNA array were performed using murine aRMS primary cell cultures with or without Pax3:Foxo1a knockdown treated by 6 Gy irradiation, or selected for ploidy using hoechst33342 sorted cell cycle-specific cells. RESULTS: Pax3:Foxo1a is not required for tumor repopulating ability in mouse limiting-dilution transplantation experiments. The expression level of Pax3:Foxo1a was discovered to be dynamic and varied during the cell cycle in murine aRMS primary cell cultures and human aRMS cell lines. Pax3:Foxo1a is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation in genome-wide expression analysis and quantitative RT-PCR. Radiation resulted in a higher fraction of DNA breaks amongst mitotic cells (as represented by dual pHH3 positive, H2AX positive cells) under conditions of Pax3:Foxo1a expression compared with knockdown, suggesting that Pax3:Foxo1a facilitates G2/M transit, consistent with checkpoint adaptation. CONCLUSION: We demonstrated that Pax3:Foxo1a does not function in tumor cell maintenance, the expression level of Pax3:Foxo1a is dynamic and varies during the cell cycle, and Pax3:Foxo1a facilitates checkpoint adaptation under stress conditions such as irradiation. Furthermore, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer biology. Citation Format: Ken Kikuchi, Simone Hettmer, M. Imran Aslam, Joel E. Michalek, Wolfram Laub, Brian P. Rubin, Amy J. Wagers, Charles Keller. The Pax3:Foxo1a translocation product is a cell cycle-specific modifier of the alveolar rhabdomyosarcoma tumor phenotype. [