GLI1 Is a Central Mediator of EWS/FLI1 Signaling in Ewing Tumors Jay Joo1, Laura Christensen1, Kegan Warner1, Leith States1, Hyung-Gyoo Kang 2 , Kieuhoa Vo 1 , Elizabeth R. Lawlor1,2, William A. May1* 1 Division of Hematology-Oncology, Department of Pediatrics, Childrens Hospital Los Angeles and the Saban Research Institute, University of Southern California Los Angeles, Los Angeles, California, United States of America, 2 Department of Pathology, Childrens Hospital Los Angeles and the Saban Research Institute, University of Southern California Los Angeles, Los Angeles, California, United States of America Abstract The Ewing Sarcoma Family Tumors (ESFT) consist of the classical pathologic entities of Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor. Occurring largely in the childhood through young adult years, these tumors have an unsurpassed propensity for metastasis and have no defined cell of origin. The biology of these aggressive malignancies centers around EWS/FLI1 and related EWS/ETS chimeric transcription factors, which are largely limited to this tumor class. Much progress has been made in the identification of a network of loci whose expression is modulated by EWS/FLI1 and its congeners. To date, little progress has been made in reconstructing the sequence of direct and indirect events that produce this network of modulated loci. The recent identification of GLI1 as an upregulated target of EWS/ETS transcription factors suggests a target which may be a more central mediator in the ESFT signaling network. In this paper, we further define the relationship of EWS/FLI1 expression and GLI1 upregulation in ESFT. This relationship is supported with data from primary tumor specimens. It is consistently observed across multiple ESFT cell lines and with multiple means of EWS/FLI1 inhibition. GLI1 inhibition affects tumor cell line phenotype whether shRNA or endogenous or pharmacologic inhibitors are employed. As is seen in model transformation systems, GLI1 upregulation by EWS/FLI1 appears to be independent of Hedgehog stimulation. Consistent with a more central role in ESFT pathogenesis, several known EWS/FLI1 targets appear to be targeted through GLI1. These findings further establish a central role for GLI1 in the pathogenesis of Ewing Tumors. Citation: Joo J, Christensen L, Warner K, States L, Kang H-G, et al. (2009) GLI1 Is a Central Mediator of EWS/FLI1 Signaling in Ewing Tumors. PLoS ONE 4(10): e7608. doi:10.1371/journal.pone.0007608 Editor: Chris Jones, Institute of Cancer Research, United Kingdom Received April 24, 2009; Accepted October 7, 2009; Published October 27, 2009 Copyright: 2009 Joo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: WAM is supported by funding from the NIH (CA90666), the Alex’s Lemonade Stand Foundation, the Margaret Early Medical Research Trust, and the John W. Sullivan Cancer Research Fund. ERL is supported by the Margaret Early Medical Research Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wmay@chla.usc.edu Introduction Much of the unique biology of the Ewing Sarcoma Family Tumors (ESFT) stems from the unique effects of EWS/FLI1. This fusion transcription factor, along with related EWS/ETS fusions, is virtually pathognomonic of these aggressive malignancies[1]. Given the nature of these chimeric proteins, considerable work has gone into the identification of the transcriptional targets of EWS/FLI1[2,3]. Despite this effort, no identified target has been clinically demonstrated to be of prognostic or therapeutic significance. Together, this diverse group of targets constitute a signaling network. Elements of this transcrip- tional network have been identified[3] but the relationship between these elements has not been well studied. In a sense, such relationships constitute the topology of this network. Based on the biology of this disease, one can presume that EWS/FLI1 will be central to this network. But targets of EWS/FLI1 will vary in importance from isolated clients on the network to more centrally situated hubs or routers which regulate a subdomain of this network in concert. Establishing the existence and nature of such relationships will be critical to prioritizing which transcriptional targets are most likely to have maximal impact as targets for translational therapeutics. The recent finding that EWS/FLI1 enhances expression of GLI1 presents a potential clue to the interpretation of this network[4,5]. GLI1 is the principal transcriptional effector of the Hedgehog-GLI (HH-GLI) signaling pathway[6]. This pathway is of critical importance in many developmental processes and is important in the maintenance of stem cell compartments in both developing and mature tissues[7]. Furthermore, HH-GLI has been found to be involved in many human cancers from prostate cancer in adults to childhood medulloblastoma[8]. Translational efforts to target this pathway are ongoing[9,10,11]. While it has been implicated in EWS/FLI1 biology, much of this data comes from a murine model system for EWS/FLI1 transformation[4]. The establishment of the significance of GLI1 upregulation to ESFT biology remains to be more firmly established. Beyond this, if GLI1 is more than a peripheral event in the EWS/FLI1 signaling network, it can be expected to to leave an identifiable trans- criptional footprint which may encompass some previously identified EWS/FLI1 targets. Here we demonstrate that ESFT primary tumors express HH- GLI pathway members in a manner consistent with that seen in model transformation systems. The EWS/FLI1 dependence of GLI1 expression and signaling in multiple ESFT cell lines is clearly demonstrated. Using multiple means of GLI1 inhibition, we demonstrate the importance of GLI1 to the ESFT tumorigenic phenotype. Intriguingly, we show that GLI1 upregulation in ESFT PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7608