In the initial phase of these studies, we introduced constitutive MYCN and doxycycline-inducible PAX3-FOXO1 expression constructs into immortalized human myoblasts. Treatment with doxycycline induces increased PAX3-FOXO1 expression and doxycycline removal leads to decreased PAX3-FOXO1 expression. In cell culture experiments, treatment of these engineered myoblasts with doxycycline induces oncogenic transformation in focus formation assays. In subsequent animal studies, mice were injected with these cells in an intramuscular site, and when fed a doxycycline-supplemented diet, tumors that resemble human ARMS formed within 3-4 weeks. To determine if continued PAX3-FOXO1 expression is required for tumor maintenance, doxycycline supplementation was discontinued after small tumors formed, and this discontinuation resulted in overall tumor regression associated with decreased cellular proliferation and increased myogenic differentiation and cell death. Based on these findings, this inducible system is proposed to be a model of targeted therapy against the fusion protein. In the animal studies, tumors generally recur several weeks after doxycycline discontinuation. In most of these recurrent tumors, the fusion protein was not detectable, suggesting that these tumors are recurring by a fusion protein-independent mechanism. We generated cell lines from the primary and recurrent tumors and confirmed the expression of PAX3-FOXO1 in the primary tumor-derived cells and its absence in the recurrent tumor-derived cells. Though these recurrent tumor-derived cell lines did not express the fusion protein when grown without doxycycline, these cells demonstrate transforming activity in culture and tumorigenesis in vivo under these conditions. Based on these findings, we propose that rare cells with additional oncogenic events were selected during recurrence, and the action of these new oncogenic events allow the tumor cells to become independent of the fusion protein. These oncogenic events thus provide a mechanism for resistance to the targeted therapy directed against the fusion protein. We then further investigated the molecular changes occurring during PAX3-FOXO1-induced tumorigenesis that may ultimately allow a recurrent tumor to be independent of the fusion protein. Using a microarray platform, our comparison of the expression profiles of primary and recurrent tumor-derived cells with the expression profile of parental myoblasts revealed multiple deregulated genes that show significant overlap with growth factor-, adhesion-PI3K and KRAS-related transcriptional signatures. Further comparison revealed substantial differences between the gene expression profiles of the recurrent tumor-derived and the primary tumor-derived cell lines. In particular, most PAX3-FOXO1 transcriptional targets that are upregulated in primary tumor-derived cells were not upregulated in recurrent tumor-derived cells. One of the few PAX3-FOXO1 transcriptional targets upregulated in recurrent tumor-derived cells encodes a growth factor, which is an upstream component of the PI3K/Akt and MAPK pathways. Though this growth factor is normally upregulated in primary tumor-derived cells, it is expressed at even higher levels in recurrent tumor-derived cells. We investigated whether this growth factor is involved in the oncogenic phenotype of the primary and recurrent tumor-derived cells. Using an RNA interference approach, knockdown of this growth factor in recurrent and primary tumor-derived cells reduced their transforming activity in focus formation assays, with a higher impact on recurrent tumor-derived cells than primary tumor-derived cells. Furthermore, overexpression of the growth factor in parental myoblasts stimulated their proliferation and transformation capability in vitro compared to cells transfected with an empty vector. These results are consistent with the hypothesis that this growth factor is normally involved in the mechanism of PAX3-FOXO1-dependent tumorigenicity and can be deregulated in recurrent tumors to sustain PAX3-FOXO1-independent tumorigenicity.
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