title: Phosphoproteomic profiling reveals IL6-mediated paracrine signaling within the Ewing sarcoma family of tumors.
abstract: Members of the Ewing sarcoma family of tumors (ESFT) contain tumor-associated translocations that give rise to oncogenic transcription factors, most commonly EWS/FLI1. EWS/FLI1 plays a dominant role in tumor progression by modulating the expression of hundreds of target genes. Here, the impact of EWS/FLI1 inhibition, by RNAi-mediated knockdown, on cellular signaling was investigated using mass spectrometry-based phosphoproteomics to quantify global changes in phosphorylation. This unbiased approach identified hundreds of unique phosphopeptides enriched in processes such as regulation of cell cycle and cytoskeleton organization. In particular, phosphotyrosine profiling revealed a large upregulation of STAT3 phosphorylation upon EWS/FLI1 knockdown. However, single-cell analysis demonstrated that this was not a cell-autonomous effect of EWS/FLI1 deficiency, but rather a signaling effect occurring in cells in which knockdown does not occur. Conditioned media from knockdown cells were sufficient to induce STAT3 phosphorylation in control cells, verifying the presence of a soluble factor that can activate STAT3. Cytokine analysis and ligand/receptor inhibition experiments determined that this activation occurred, in part, through an IL6-dependent mechanism. Taken together, the data support a model in which EWS/FLI1 deficiency results in the secretion of soluble factors, such as IL6, which activate STAT signaling in bystander cells that maintain EWS/FLI1 expression. Furthermore, these soluble factors were shown to protect against apoptosis.
  
  
  CONCLUSIONS
  EWS/FLI1 inhibition results in a novel adaptive response and suggests that targeting the IL6/STAT3 signaling pathway may increase the efficacy of ESFT therapies.
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California. Division of Hematology/Oncology, Department of Pediatrics, Gwynne Hazen Cherry Memorial Laboratories, University of California, Los Angeles, Los Angeles, California.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Anderson
authors.affiliation: Crump Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, California. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California.
authors.forename: Björn
authors.initials: B
authors.lastname: Titz
authors.affiliation: Division of Hematology/Oncology, Department of Pediatrics, Gwynne Hazen Cherry Memorial Laboratories, University of California, Los Angeles, Los Angeles, California.
authors.forename: Ryan
authors.initials: R
authors.lastname: Akiyama
authors.affiliation: Crump Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, California. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California.
authors.forename: Evangelia
authors.initials: E
authors.lastname: Komisopoulou
authors.affiliation: Division of Hematology/Oncology, Department of Pediatrics, Gwynne Hazen Cherry Memorial Laboratories, University of California, Los Angeles, Los Angeles, California.
authors.forename: Ann
authors.initials: A
authors.lastname: Park
authors.affiliation: Sarcoma Medical Oncology Service, Division of Solid Tumors, Department of Medicine, Memorial Sloan Kettering Cancer Center and Department of Medicine, Weill Cornell Medical College, New York, New York.
authors.forename: William D
authors.initials: WD
authors.lastname: Tap
authors.affiliation: Crump Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, California. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California. UCLA Metabolomics Center, University of California, Los Angeles, Los Angeles, California.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Graeber
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California. Division of Hematology/Oncology, Department of Pediatrics, Gwynne Hazen Cherry Memorial Laboratories, University of California, Los Angeles, Los Angeles, California. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California. cdenny@ucla.edu.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Denny
mesh_terms: D017209:Apoptosis; D001859:Bone Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D019899:Paracrine Communication; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics*; D034802:RNA-Binding Protein EWS / Q000235:genetics*; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D012512:Sarcoma, Ewing / Q000378:metabolism*
chemical_list: C106031:EWS-FLI fusion protein; C508600:IL6 protein, human; D015850:Interleukin-6; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human