IGF1R Signaling in Ewing Sarcoma Is Shaped by Clathrin-/Caveolin-Dependent Endocytosis Ana Sofia Martins1, Jose´ Luis Ordo´ n˜ ez1, Ana Teresa Amaral1, Frans Prins2, Giuseppe Floris3, Maria Debiec-Rychter4, Pancras C. W. Hogendoorn2, Enrique de Alava1* 1 Laboratory 20- Molecular Pathology of Sarcomas Laboratory, Centro de Investigaci o´n del Ca´ncer-IBMCC, Universidad de Salamanca-CSIC, Salamanca, Spain, 2 Department of Pathology, Leiden University Medical Center, Leiden, Netherlands, 3 Laboratory of Experimental Oncology, Catholic University of Leuven, Leuven, Belgium, 4 Department of Human Genetics, Catholic University of Leuven, Leuven, Belgium Abstract Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling. Citation: Martins AS, Ordo´ n˜ez JL, Amaral AT, Prins F, Floris G, et al. (2011) IGF1R Signaling in Ewing Sarcoma Is Shaped by Clathrin-/Caveolin-Dependent Endocytosis. PLoS ONE 6(5): e19846. doi:10.1371/journal.pone.0019846 Editor: Rakesh K. Srivastava, The University of Kansas Medical Center, United States of America Received January 21, 2011; Accepted April 5, 2011; Published May 17, 2011 Copyright: 2011 Martins 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: Funding provided by EuroBoNet, the European research network on bone tumors (FP6-2004-Lifescihealth-5, proposal number 018814)-European Commission and the Instituto de Salud Carlos Tercero, FIS- project PI081828. J.L. Ordo´ n˜ ez is supported by the ISCIII (Contratos Perfeccionamiento CD6/00001). 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: edealava@usal.es Introduction Receptor tyrosine kinases (RTKs) are involved in countless signaling pathways and their deregulation is involved in several diseases, in particular in cancer. Despite extensive study of these signaling cascades, until recently RTK internalization was believed to determine a down-regulation of RTK activity, ultimately leading to receptor degradation. However, increasing evidence indicates that endocytosis modulates and also sustains signaling transduction throughout the downstream targets of RTKs [1,2,3]. In most cell types, RTKs mainly internalize through clathrin-dependent internalization, converging in the formation of clathrin-coated membrane invaginations, or clathrin- coated pits (CCPs), in a series of highly regulated steps. Regarding caveolin-dependent internalization, receptor stimulation with caveolar budding leads to the formation of enclosed intracellular vesicles called ‘‘cavicles’’ [4]. Recently, it has been reported that the phosphorylation of CAV1 and dynamin-2 leads to caveolar fission [5]. Depending on the cellular context, internalized RTKs may then recycle back to the plasma membrane, recruit signaling proteins, thereby increasing active signaling from endosomes, or simply be degraded [1,6,7]. It has recently been reported that in lung cancer IGF1R endocytosis is triggered by ligand binding, causing IGF1R ubiquitination and internalization via clathrin-coated vesicles and/or caveolae [8]. Also, using an osteosarcoma model Sehat et al., have shown that IGF1R internalization is influenced by the ligand concentration [9], and Romanelli et al., have demonstrated that IGF1R internalization and recycling mediates the phosphor- ylation of AKT in glial progenitors [10]. Moreover, IGF1R is directly involved in the growth and survival of ES cells [11,12], and given the relevance of RTK endocytosis in cell survival here we study the role of clathrin and/or CAV1 in ES IGF1R signaling. Results IGF1R is internalized by both clathrin- and CAV1- dependent mechanisms In keeping with recent publications indicating that RTKs endocytosis regulates signaling transduction, we analyzed the role of IGF1R internalization in ES. This study was performed before and after ligand binding (IGF1) with the aim of exploring the two most common RTKs internalization mechanisms: clathrin- and caveolin-dependent endocytosis. PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19846