Pediatric rhabdomyosarcoma occurs as two biologically distinct histological variants, embryonal (ERMS) and alveolar (ARMS). To identify genomic changes that drive ERMS pathogenesis, we used a new array comparative genomic hybridization (aCGH) platform to examine a specific subset of ERMS tumors, those occurring in children with clinically defined intermediate-risk disease. The aCGH platform used has an average probe spacing ∼1 kb, and can identify genomic changes with single gene resolution. Our data suggest that these tumors share a common genomic program that includes inactivation of a master regulator of the p53 and Rb pathways, CDKN2A/B, and activation of FGFR4, Ras, and Hedgehog (Hh) signaling. The CDKN2A/B tumor suppressor is deleted in most patient samples. FGFR4, which encodes a receptor tyrosine kinase, is activated in 20% of tumors, predominantly by amplification of mutant, activating FGFR4 alleles. Over 50% of patients had low-level gains of a region containing the Hh-pathway transcription factor GLI1, and a gene expression pattern consistent with Hh-pathway activation. We also identified intragenic deletions affecting NF1, a tumor suppressor and inhibitor of Ras, in 15% of tumor samples. Deletion of NF1 and the presence of activating Ras mutations (in 42% of patients) were mutually exclusive, suggesting NF1 loss is an alternative and potentially common mechanism of Ras activation in ERMS. Our data suggest that intermediate-risk ERMS is driven by a common set of genomic defects, a finding that has important implications for the application of targeted therapies to improve the treatment of children diagnosed with this disease.

Germ-cell tumors (GCTs), which arise from pluripotent embryonic germ cells, exhibit a wide range of histologic differentiation states with varying clinical behaviors. Although testicular GCT is the most common cancer of young men, the genes controlling the development and differentiation of GCTs remain largely unknown. Through a forward genetic screen, we previously identified a zebrafish mutant line, tgct, which develops spontaneous GCTs consisting of undifferentiated germ cells [Neumann JC, et al. (2009) Zebrafish 6:319-327]. Using positional cloning we have identified an inactivating mutation in alk6b, a type IB bone morphogenetic protein (BMP) receptor, as the cause of the zebrafish GCT phenotype. Alk6b is expressed in spermatogonia and early oocytes, and alk6b mutant gonads display impaired BMP signal transduction, altered expression of BMP target genes, and abnormal germ-cell differentiation. We find a similar absence of BMP signaling in undifferentiated human GCTs, such as seminomas and embryonal carcinoma, but not in normal testis or in differentiated GCTs. These results indicate a germ-cell-autonomous role for BMP signal transduction in germ-cell differentiation, and highlight the importance of the BMP pathway in human GCTs.non-seminoma | SMAD | teleost | haplotype

Germ cell tumors (GCTs) affect infants, children and adults and are the most common cancer type in young men. Progress in understanding the molecular basis of germ cell tumors has been hampered by a lack of suitable animal models. Here we report the identification of a zebrafish model of highly penetrant, heritable testicular germ cell tumor isolated as part of a forward-genetic screen for cancer-susceptibility genes. The mutant line develops spontaneous testicular tumors at a median age of 7 months, and pedigree analysis indicates dominant inheritance of the germ cell tumor susceptibility trait. The zebrafish model exhibits disruption of testicular tissue architecture and the accumulation of primitive, spermatogonial-like cells with loss of spermatocytic differentiation. Radiation treatment leads to apoptosis of the tumor cells and tumor regression. The germ cell tumor-susceptible line can serve as a model for understanding the mechanisms regulating germ cells in normal development and disease, and as a platform investigating new therapeutic approaches for germ cell tumors.

SUMMARYEwing’s sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing’s sarcoma family tumors (ESFTs), which include peripheral primitive neuroectodermal tumors (PNETs), are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing’s sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing’s sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing’s sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

Ewing's sarcoma, a member of the small round blue cell family of tumors, is an aggressive and malignant bone tumor of children and young adults. The cells are generally poorly differentiated, although can also have characteristics of neuroectodermal cells. Ewing's tumors are characterized by a t(11:22) that generates a fusion of the EWS protein and the FLI1 transcription factor. This fusion oncoprotein, EWS-FLI1, represents a potential therapeutic target for treatment of Ewing's sarcoma. However, the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown. Few animal models exist in which to study the function of EWS-FLI1. Here, we report the use of zebrafish as a new vertebrate model of EWS-FLI1 function and tumorigenesis. Using the zebrafish Tol2 transposon system, we have developed several transgenic models to study the function of EWS-FLI1 in a vertebrate model organism. First, we have injected zebrafish with transposons containing human EWS-FLI1 driven by either the heat shock or the β-actin promoter. A subset of the injected fish developed tumors when raised to adulthood. These tumors strongly resemble Ewing's sarcoma. This finding demonstrates that the functional targets of EWS-FLI1 that mediate tumor development are conserved from zebrafish to human. We observe an increased incidence of tumors in a p53 mutant background, suggesting that the p53 pathway may suppress EWS-FLI1 driven tumorigenesis. We are performing microarray analysis to identify candidate downstream effector genes and to compare to previously identified EWS-FLI1 target genes. In addition to these tumors, we identified transgenic lines that transmit the EWS-FLI1 transgene to their progeny. The embryos that express EWS-FLI1 exhibit abnormal embryonic development. This result suggests that EWS-FLI1 may regulate conserved developmental pathways. These transgenic lines represent a novel physiological context in which to study the cellular function of EWS-FLI1. From these two models of EWS-FLI1 expression in the zebrafish, we conclude that human EWS-FLI1 functions in zebrafish and that these models provide a novel context in which to study the function of this fusion oncogene. Additionally, the rapid development and small size of the zebrafish will allow us to utilize these models for genetic and chemical screens for specific EWS-FLI1 inhibitors for the treatment of Ewing's sarcoma. Citation Information: Cancer Res 2009;69(23 Suppl):B36.