Studies of gene fusions in rhabdomyosarcoma

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

We investigated the molecular changes occurring during P3F-induced tumorigenesis in the myoblast system that may ultimately allow a recurrent tumor to be independent of the fusion protein. Comparison of expression profiles of untreated parental myoblasts with recurrent tumor cells and MYCN/P3F-expressing primary tumor cells revealed numerous differences between primary and recurrent tumor cells. In particular, most direct targets up-regulated by P3F in primary tumor cells were not up-regulated in recurrent tumor cells in the absence of P3F. One of the few P3F targets up-regulated in a subset of recurrent tumors is FGF8, which encodes a member of the fibroblast growth factor family that functions upstream of several signaling pathways involved in growth and oncogenesis. FGF8 is up-regulated by P3F in MYCN/P3F-expressing parental and primary tumor cells, and is expressed at higher levels in recurrent tumor cells in a P3F-independent manner. We investigated whether FGF8 is involved in the oncogenic phenotype of recurrent tumor cells. CRISPR-Cas9 knockout of FGF8 in recurrent tumor cells impaired in vitro transforming activity and in vivo tumorigenesis whereas expression of an FGF8 cDNA in parental myoblasts (transduced with MYCN) stimulated in vitro transformation and vivo tumorigenesis. To determine if FGF8 is acting in an autocrine fashion, we studied conditioned medium from these cell lines. FGF8 protein is present in conditioned medium from recurrent tumor cells but not parental cells, and conditioned medium from recurrent tumor cells is able to transform parental myoblasts whereas medium from parental cells does not have this effect. In further tests of conditioned medium on transformation of parental cells, FGF8 overexpression in parental cells results in gain of transforming activity by its conditioned medium whereas FGF8 knockout in recurrent tumor cells or FGF8 immunodepletion of medium from recurrent tumor cells abrogates transforming activity of this medium. To study the role of FGF8 in primary tumors, we examined primary tumor cells from the myoblast system and human FP RMS lines, which both show P3F-dependent transformation. The primary tumor cells express FGF8 when P3F is induced and most human FP RMS cell lines express FGF8. Using a CRISPR-Cas9 approach, FGF8 knockout results in decreased proliferation and loss of transformation in primary tumor cells and FP RMS lines. We also identified a variant of the P3F-positive RH30 RMS cell line which spontaneously lost most P3F expression, resulting in cells which grow in culture but are not transformed. Transduction of a FGF8 expression construct resulted in recovery of in vitro transforming activity without any change in P3F expression. These findings show that FGF8 is necessary and sufficient for much of the oncogenic activity of P3F, and highlight the importance of FGF8 as a downstream P3F target involved in the mechanism of P3F-dependent tumorigenicity. Finally, these combined results indicate that deregulated FGF8 expression can sustain P3F-independent tumorigenicity, thus providing a mechanism for P3F-independent recurrence and resistance to targeted therapy directed against P3F.