Abstract
Abstract The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation, and is a characteristic feature of a subset of rhabdomyosarcoma (RMS) with aggressive behavior and poor prognosis. This study utilizes an inducible expression system in human myoblasts to dissect the molecular mechanism of PAX3-FOXO1 action during RMS tumorigenesis and progression.Constitutive and doxycycline-inducible expression constructs were used to generate a human myoblast cell line that constitutively expresses MYCN and reversibly expresses PAX3-FOXO1, respectively. PAX3-FOXO1 expression was assessed by Western blotting, qRT-PCR and immunohistochemistry. Oncogenicity in these engineered myoblasts was studied in vitro by focus formation and in vivo by intramuscular injection of NOD-SCID mice. Cell proliferation, apoptosis and myogenic differentiation were assessed by western blot or immunohistochemical assays.In focus formation assays, doxycycline-treated myoblasts expressing constitutive MYCN and doxycycline inducible PAX3-FOXO1 showed a high level of oncogenic transformation. When doxycycline was removed during the course of this assay, smaller foci formed with prominent myogenic differentiation and cell death. Intramuscular injection of these engineered myoblasts resulted in RMS tumor formation when fusion protein expression was induced by feeding mice a doxycycline-supplemented diet. After small palpable tumors formed, doxycycline withdrawal resulted in decreased PAX3-FOXO1 expression and tumor regression. Microscopic examination of regressing tumors revealed widespread myogenic differentiation and cell death. In most cases, the tumors recurred several weeks later despite the absence of inducing agent. Analysis of recurrent tumor samples revealed that a subset emerged in the absence of PAX3-FOXO1 expression. A cell line generated from a PAX3-FOXO1-independent recurrence demonstrated transformation in vitro in the absence of doxycycline. Though cell lines derived from primary tumors were dependent on PAX3-FOXO1 and differentiated when doxycycline was removed, the recurrent tumor-derived cells did not differentiate under these conditions and instead proliferated continuously. Furthermore, reinjection of these recurrent tumor-derived cells resulted in tumor formation in mice in the absence of doxycycline, and even more rapid tumor formation in the presence of doxycycline.Our study provides evidence that the PAX3-FOXO1 fusion protein is necessary to develop primary tumors but recurrent tumors can develop by a PAX3-FOXO1-independent mechanism. The recurrent tumors are postulated to have acquired secondary oncogenic events that were activated or selected by initial PAX3-FOXO1 expression. These secondary events have an additive oncogenic effect with PAX3-FOXO1 expression, and then contribute to tumor recurrence in the absence of PAX3-FOXO1 expression. Citation Format: Puspa R. Pandey, Bishwanath Chatterjee, Javed Khan, Stephen M. Hewitt, Markku Martti Miettinen, Frederic G. Barr. Rhabdomyosarcoma tumors recur by a PAX3-FOXO1-independent mechanism in a human myoblast xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2461.
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