Abstract
SUMMARYWe report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants.
Highlights
Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation generating a fusion between EWS and an Ets family transcription factor, most commonly FLI1 (Jedlicka, 2010; Lawlor and Sorensen, 2015; Lessnick and Ladanyi, 2012; Mackintosh et al, 2010; Toomey et al, 2010)
Secretome Proteomics Identifies growth and differentiation factor 6 (GDF6) as a Target of EWS-FLI1 To dissect the cytokine signaling in Ewing sarcoma, we analyzed the proteins secreted from A673 and TC71 Ewing sarcoma cells by mass spectrometry
By using chromatin immunoprecipitation (ChIP), we detected the binding of EWS-FLI1 to the GDF6 gene promoter in A673 cells, which was abolished by EWS-FLI1 silencing (Figure 1D)
Summary
Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation generating a fusion between EWS and an Ets family transcription factor, most commonly FLI1 (Jedlicka, 2010; Lawlor and Sorensen, 2015; Lessnick and Ladanyi, 2012; Mackintosh et al, 2010; Toomey et al, 2010). EWS-FLI1 regulates the expression of a number of genes important for cell proliferation and tumor progression (Hancock and Lessnick, 2008), can transform mouse cells (Gonzalez et al, 2007; May et al, 1993), and is required for proliferation and tumorigenicity of Ewing sarcoma cells. EWSFLI1 is considered a driver for Ewing sarcoma. Previous research on Ewing sarcoma largely focused on intracellular pathways. Little is known about the extracellular signaling that regulates Ewing sarcoma
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