CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling

// Maria Cristina Manara 1 , Mario Terracciano 1, 6 , Caterina Mancarella 1 , Marika Sciandra 1, 2 , Clara Guerzoni 1, 2 , Michela Pasello 1, 2 , Andrea Grilli 1 , Nicoletta Zini 3, 4 , Piero Picci 1, 2 , Mario P. Colombo 5 , Andrea Morrione 6 , Katia Scotlandi 1, 2 1 CRS Development of Biomolecular Therapies, Experimental Oncology Laboratory, Istituto Ortopedico Rizzoli, Bologna 40136, Italy 2 PROMETEO Laboratory, STB, RIT Department, Istituto Ortopedico Rizzoli, Bologna 40136, Italy 3 CNR, National Research Council of Italy, Institute of Molecular Genetics, Bologna 40136, Italy 4 SC Laboratory of Musculoskeletal Cell Biology, Istituto Ortopedico Rizzoli, Bologna 40136, Italy 5 Molecular Immunology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan 20133, Italy 6 Department of Urology and Biology of Prostate Cancer Program, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA Correspondence to: Katia Scotlandi, email: katia.scotlandi@ior.it Andrea Morrione, email: Andrea.Morrione@jefferson.edu Keywords: antibody, CD99, cell death, Ewing sarcoma, RAS Received: August 12, 2016      Accepted: October 14, 2016      Published: November 07, 2016 ABSTRACT CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.