ORIGINAL ARTICLE Efficacy of and resistance to anti-IGF-1R therapies in Ewing’s sarcoma is dependent on insulin receptor signaling C Garofalo 1,9 , MC Manara 1,9 , G Nicoletti 1 , MT Marino 1 , P-L Lollini 2 , A Astolfi 3 , G Pandini 4 , JA Lo´pez-Guerrero 5 , K-L Schaefer 6 , A Belfiore 7 , P Picci 1 and K Scotlandi 1,8 1 Laboratory of Experimental Oncology, Istituto Ortopedico Rizzoli, Bologna, Italy; 2 Department of Hematology and Oncology Sciences L. A. Sera`gnoli, Sant’Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy; 3 G.Prodi Cancer Research Center, University of Bologna, Bologna, Italy; 4 Department of Internal Medicine and Medical Specialties, Endocrinology, University of Catania, Catania, Italy; 5 Laboratory of Molecular Biology, Fundacio´n Instituto Valenciano de Oncologı´a, Valencia, Spain; 6 University Medical Center, Inst. f. Pathology, Dusseldorf, Germany; 7 Department of Clinical and Experimental Medicine, University of Catanzaro, Catanzaro, Italy and 8 CRS Development of Biomolecular Therapies, Istituto Ortopedico Rizzoli, Bologna, Italy Identification of patient selection criteria and under- standing of the potential mechanisms involved in the development of resistance are crucial for an appropriate and successful design of clinical trials with anti-insulin- like growth factor (IGF)-1R therapies. Few Ewing’s sarcomas are highly sensitive to IGF-1R targeting and understanding the reason why, may hold the secret to improve successful treatments. In this paper, we show that a major mechanism of resistance to highly specific inhibitors of IGF-1R, either antibodies or tyrosine kinase inhibitors may involve enhanced insulin receptor (IR)-A homodimer formation and IGF-2 production. Resistant cells are able to switch from IGF-1/IGF-1R to IGF-2/IR- A dependency to maintain sustained activation of AKT and ERK1/2, proliferation, migration and metastasis. These cells also showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF-1R therapies. Thus, we indicate that tumors with low IGF- 1R:IR ratio are unlikely to greatly benefit from anti-IGF- 1R therapies and that the efficacy of anti-IGF-1R therapies should be evaluated in relationship to the IR- A:IGF-1R ratio in cancer cells. Moreover, we provide evidences supporting IR-A as an important target in sarcoma therapy. Oncogene (2011) 30, 2730–2740; doi:10.1038/onc.2010.640; published online 31 January 2011 Keywords: sarcomas; IGF-1R; insulin signaling; drug resistance Introduction Insulin-like growth factor (IGF) signaling has been implicated as a critical contributor to malignant transformation and tumor resistance to cytotoxic chemotherapy, targeted therapies, hormonal therapy and radiation (for reviews see Casa et al., 2008; Chitnis et al., 2008). As a result, IGF signaling has become an attractive target for development of novel anticancer agents, and a large number of compounds, including blocking antibodies and tyrosine kinase inhibitors (TKIs) disrupting IGF-I receptor (IGF-1R) functions are in preclinical and clinical development. In general, phase I and II studies in different tumors have provided evidences suggesting safety of anti-IGF-1R antibodies and a few anecdotes of impressive single-agent activity particularly in Ewing’s sarcoma (Pollak, 2008; Wahner Hendrickson et al., 2009; Buck et al., 2010) indicated anti-IGF-1R drugs as an effective clinical possibility. However, these early studies also clearly pointed out that only a minority of patients really benefit from IGF- 1R targeting. Evidences so far accumulated have in fact revealed that, although most tumors express the IGF-1R, expression alone is unlikely to be sufficient for sensitivity to IGF-targeted treatments. Deeper under- standing of the IGF signaling system and its down- stream effectors is thus necessary to identify patients that may really benefit from this new targeted therapy. Ewing’s sarcoma is one of the few tumors in which the efficacy of IGF-1R targeting in preclinical models has been partly predictive of clinical activity (Scotlandi and Picci, 2008). However, we still do not know exactly the mechanisms responsible of the exquisite efficacy of anti- IGF-1R therapy in this tumor. It is possible that the particular sensitivity of Ewing’s sarcoma cells to anti- IGF-1R therapies derives from the direct connection established between the genetic hallmark of Ewing’s sarcoma, the oncogene fusion EWS/ETS transcripts (Delattre et al., 1994) and the IGF system (Toretsky et al., 1997; Prieur et al., 2004; Cironi et al., 2008). Autocrine production of IGF-1 (Scotlandi et al., 1996) is in fact sustained by abnormal transcription activity of EWS/ETS hybrid products (Cironi et al., 2008) and Received 24 October 2010; revised 22 December 2010; accepted 23 December 2010; published online 31 January 2011 Correspondence: Dr K Scotlandi, CRS Development of Biomolecular Therapies, Istituto Ortopedico Rizzoli, Via di Barbiano 1/10, Bologna 40136, Italy. E-mail: katia.scotlandi@ior.it 9 These authors contributed equally to this work. Oncogene (2011) 30, 2730–2740 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc