PAX3-FOXO1 is essential for initiation but not for recurrence during rhabdomyosarcoma tumorigenesis

Puspa R. Pandey, Stephen M. Hewitt, Markku M. Miettinen, and Frederic G. Barr

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA See Original Here

The PAX3-FOXO1 fusion gene, which is generated by a 2; 13 chromosomal translocation, is a characteristic feature of fusion-positive rhabdomyosarcoma (RMS), a major RMS subtype associated with aggressive behavior and poor prognosis. This study utilizes a novel inducible expression system in human myoblasts to dissect the molecular mechanism and contribution of PAX3-FOXO1 in RMS tumorigenesis.A human myoblast cell line (immortalized by BMI1 and TERT expression) was transduced with a retroviral construct that constitutively expresses MYCN and a lentiviral based- doxycycline inducible construct that variably and reversibly expresses PAX3-FOXO1. Focus formation and animal xenograft experiments were performed to study transformation in vitro and tumorigenesis in vivo, respectively. Myogenic differentiation was assessed by light microscopy and by western blot or immunohistochemical assays of muscle-specific protein expression.PAX3-FOXO1-transduced myoblasts treated with doxycycline demonstrated a time- and dose-dependent increase in expression of PAX3-FOXO1 mRNA and protein and its downstream targets genes; doxycycline withdrawal led to cessation of fusion protein expression. Though myoblasts expressing PAX3-FOXO1 or MYCN alone did not show any evidence of transformation in culture, combined PAX3-FOXO1 and MYCN expression resulted in myoblast transformation. Under differentiation-promoting culture conditions, combined PAX3-FOXO1 and MYCN expression inhibited myogenic differentiation. Intramuscular injection of myoblasts with MYCN and PAX3-FOXO1 resulted in rapid RMS tumor formation in NOD-SCID mice when fusion protein expression was induced by feeding mice a doxycycline-containing diet. Myoblasts with MYCN expression alone did not form any tumors while PAX3-FOXO1 induction without MYCN expression resulted in RMS tumors only after a much longer latency period. After tumors formed from myoblasts expressing PAX3-FOXO1 with or without MYCN, down-regulation of PAX3-FOXO1 expression by doxycycline withdrawal resulted in tumor regression associated with widespread myogenic differentiation. However, the regressed tumors slowly grew back in the absence of doxycycline induction demonstrating a PAX3-FOXO1-independent oncogenic mechanism for recurrence.The PAX3-FOXO1 fusion protein collaborated with MYCN in the initial stage of RMS tumorigenesis to promote dysregulated cell proliferation and inhibit myogenic differentiation. Though most cells in the initial tumor were dependent on the fusion protein, recurrent tumors formed in which the fusion protein was not required to maintain the tumorigenic phenotype.

Citation Format: Puspa R. Pandey, Stephen M. Hewitt, Markku M. Miettinen, Frederic G. Barr. PAX3-FOXO1 is essential for initiation but not for recurrence during rhabdomyosarcoma tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3273. doi:10.1158/1538-7445.AM2015-3273

©2015 American Association for Cancer Research.