Abstract 5346: Dual role of <i>PAX3-NCOA2</i> in tumorigenesis of rhabdomyosarcoma
Authors: Mitsuru Miyachi, Hideki Yoshida, Daisuke Asai, Atsushi Fujiki, Shigeki Yagyu, Yasumichi Kuwahara, Kunihiko Tsuchiya, Toshihiko Imamura, Tomoko Iehara, Hajime Hosoi
Journal: Cancer Research (2011)

Abstract Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults, and can be classified into two major histological subtypes, embryonal RMS and alveolar RMS. Recently, two novel fusion genes, PAX3-NCOA1 and PAX3-NCOA2, were identified in the embryonal and alveolar subtypes. We previously analyzed the complex chromosomal translocation in one case with embryonal RMS by means of spectral karyotyping (SKY) and identified a translocation involving chromosome band 2q35, which is the locus of the PAX3 gene (Hosoi H, Kakazu N, et al. Cancer Genetics and Cytogenetics 189 (2009) 98-104). The patient is alive and in remission at nine years after treatment. We later identified a PAX3 partner gene as NCOA2 using fluorescence in situ hybridization (FISH) and cDNA sequence analysis. Because the role of PAX3-NCOA2 in rhabdomyosarcoma tumorigenesis is unknown, we investigated the biological function of PAX3-NCOA2 in this study. Methods: Mouse myoblast C2C12 cells and human embryonic kidney HEK293 cells were cultured in DMEM containing 10% fetal bovine serum. HEK293 cells were transfected with GFP-PAX3-NCOA2 expression vector and the fusion protein was localized by confocal microscopy. C2C12 cell lines expressing wild type PAX3, PAX3-FOXO1, PAX3-NCOA2 and a C-terminal activation domain deletion mutant of PAX3 were established using a murine stem cell virus (MSCV) retrovirus expression system. Anchorage-independent growth was assessed using soft agar colony formation. Myosin Heavy Chain (MHC), a marker of fully differentiated myocytes, was identified immunohistochemically. Myogenic differentiation was induced by switching the medium to DMEM containing 2% horse serum. Results and Discussion: In transfected HEK293 cells, GFP-PAX3-NCOA2 was observed in the nucleus, consistent with the hypothesis that PAX3-NCOA2 is involved in transcriptional regulation. Expression of PAX3-NCOA2 protein promoted anchorage-independent growth in C2C12 myoblasts. The number of colonies of the PAX3-NCOA2 stable cell line was half the number of colonies of the PAX3-FOXO1 stable cell line. Negligible colony formation was observed in wild-type PAX3 and PAX3 deletion mutant stable cell lines. Expression of PAX3-NCOA2, wild-type PAX3 and PAX3-FOXO1 each blocked the fusion of myoblasts to myotubes in differentiation medium. MHC was not detected in any of the PAX3-NCOA2, wild-type PAX3 or PAX3-FOXO1 stable cell lines. The PAX3-NCOA2 and PAX3-FOXO1 stable cell lines had the comparable phenotype. However, the finding that the PAX3-NCOA2 stable cell line produced fewer colonies than the PAX3-FOXO1 stable cell line may indicate that RMS with PAX3-NCOA2 fusion gene has a less aggressive phenotype and a better prognosis. Conclusions: The PAX3-NCOA2 fusion gene promoted anchorage-independent growth and inhibited myogenic differentiation in mouse myoblasts. These data suggest that PAX3-NCOA2 has a dual role in the tumorigenesis of RMS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2011-5346