Chromatin , Gene , and RNA Regulation PhosphoproteomicPro fi lingReveals IL 6-MediatedParacrine Signaling within the Ewing Sarcoma Family of Tumors

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. Implications: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.Mol Cancer Res; 12(12); 1740–54. 2014 AACR. Introduction Advancements in the understanding of the molecular mechanisms of oncogenesis have led to the development of targeted therapeutics. For example, activating mutations in kinases such as EGFR in lung cancer or B-RAF in melanoma have been inhibited by specific small molecules to increase therapeutic efficacy. However, striking initial responses are rarely sustained due to innate and acquired resistance mechanisms (1, 2). In the case of melanoma, initial suppression of the MAPK pathway by B-RAF inhibitors is followed by reactivation that occurs through relief of a negative feedback loop (3). In other systems, activation of redundant pathways can occur through cellautonomous mechanisms or be mediated by stromal secretion of growth factors into the tumor microenvironment (4). These adaptive responses by tumor cells to evade the effects of targeted therapeutics present a challenge to single-agent therapy. Targeted therapy has also been used for the treatment of the Ewing sarcoma family of tumors (ESFT). As opposed to activating kinase mutations, ESFT pathogenesis is primarily driven by what appears to be an aberrant transcription factor generated by a chromosomal translocation. In most tumors, this translocation fuses the EWS gene to the ETS transcription factor FLI1 (5). The fusion protein EWS/FLI1 retains domains that facilitate interaction with transcriptional regulators and DNA binding, which provides the ability to alter gene expression (6). EWS/FLI1 is capable of oncogenic transformation, and maintenance of expression is required for ESFT cell 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. 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. SarcomaMedical 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. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California. UCLA Metabolomics Center, University of California, Los Angeles, Los Angeles, California. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Christopher T. Denny, University of California, 650 Charles E. Young Drive South, Factor 10-240, Los Angeles, CA 90095. Phone: 310-825-0704; Fax: 310-267-2848; E-mail: cdenny@ucla.edu doi: 10.1158/1541-7786.MCR-14-0159 2014 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 12(12) December 2014 1740 on July 5, 2017. © 2014 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst August 4, 2014; DOI: 10.1158/1541-7786.MCR-14-0159