Proteomic Analysis of Chemonaı¨ve Pediatric Osteosarcomas and Corresponding Normal Bone Reveals Multiple Altered Molecular Targets Cecilia Folio, †,# Marı ´a I. Mora, ‡,# Marta Zalacain, † Fernando J. Corrales, ‡ Victor Segura, ‡ Luis Sierrasesu´maga, † Gemma Toledo, § Mikel San-Julia´n, | and Ana Patin˜o-Garcı ´a* ,† Laboratory and Department of Pediatrics, University Clinic of Navarra, Pamplona, Spain, Division of Hepatology and Gene Therapy, Proteomics and Bioinformatics Unit, Centre for Applied Medical Research (CIMA), Pamplona, Spain, Department of Pathology, M.D. Anderson International Espan˜ a, Madrid, Spain, and Department of Traumatology and Orthopaedic Surgery, University Clinic of Navarra, Pamplona, Spain Received February 10, 2009 With a view to identify the proteins involved in transformation, metastasis or chemoresistance in pediatric osteosarcoma,we carried out a new experimentalapproach based on comparison of the proteomic profile of paired samples of osteosarcoma and normal bone tissues from the same patient. The proteomic profiles of five pairs of cell lines (normal vs tumoral) were obtained by two-dimensional difference gel electrophoresis. We detected 56 differential protein spots ( t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify some of these proteins, 16 of which were chosen on the basis of the change of their relative abundance between osteosarcomas and paired normal bones and also because their involvement was supported by the genomic analysis. Two of the 16 proteins, Alpha-crystallin B chain (CRYAB) and ezrin (EZR1), were selected for further studies: an immunohistochemical analysis of a TMA (tissue microarray) and real-time PCR for a set of 14 osteosarcoma/normal-bone pairs. The results of this second tier of studies confirmed that there were significant increases in the amounts of CRYAB and ezrin, especially in advanced stages of the disease. Our overall conclusion is that proteomic profiling of paired samples of osteosarcoma and normal bone tissues from the same patient is a practicable and potentially powerful way of initiating and proceeding with a search for proteins and genes involved in pediatric osteosarcoma. Keywords: pediatric osteosarcoma • proteomics• CRYAB • ezrin Introduction Osteosarcoma is the most common primary malignant tumor of bone in children and adolescents and is characterized by production of osteoid. It most frequently occurs in the second decade of life; about 60% of patients are under 25 years- old, whereas only 30% are over 40. 1,2 At present, the standard treatment for high-grade osteosarcoma includes neoadjuvant chemotherapy followed by surgical resection and postoperative chemotherapy. Several clinical factors have been found to be related to survival, and these include the presence of metastatic disease and the histological response to preoperative chemo- therapy. Despite new therapeutic modalities, the survival rate is around 65-70%. 3,4 In studies of classical high-grade osteosarcomas, few, if any, genetic alterationshave been found to be common to a substantial proportion of tumors. For alterations which have been identified, different studies usually find different frequen- cies depending on the study design and the criteria used to determine which specimens to include. About 70% of osteosa- rcomas have altered karyotypes: the most frequently encoun- tered chromosomal aberrations are an extra chromosome 1; losses of chromosomes 9, 13 (including the RB1 gene), and 17 (including the TP53 gene); and structural alterations to chro- mosomes 11, 19, and 20. 3,5-7 At present,globalproteomic profiling of human osteosar- comas has scarcely begun. To our knowledge,there are only two papers in the literature about proteomic profiling of primary osteosarcoma samples. One is by Li and co-workers, 8 who use SELDI-TOF-MS to identify secreted proteins in 29 osteosarcoma specimensand thereby establish a way to differentiate between osteosarcoma and benign osteochon- droma. The other paper is by Kawai and co-workers, 9 who use proteomic profiling of 23 osteosarcoma biopsies to identify 10 electrophoretic protein spots potentially involved in osteosa- rcoma chemosensitivity.Neither of these studiesincluded profiling the biomarkers in a matched control group. In the * Corresponding author: Ana Patin˜ o-Garcı ´a,Ph.D., Laboratory of Pedi- atrics, University of Navarra/University Clinic, Irunlarrea SN, Los Castan˜ os Building, 31080 Pamplona, Spain. T. +34948425600/6304/F. +34948425649. E-mail: apatigar@unav.es. † Laboratory and Department of Pediatrics, University Clinic of Navarra. # These authors have contributed equally to the work. ‡ Centre for Applied Medical Research (CIMA). § M.D. Anderson International Espan˜ a. | Department of Traumatology and Orthopaedic Surgery, University Clinic of Navarra. 3882Journal of Proteome Research 2009, 8, 3882–3888 10.1021/pr900113w CCC: $40.75  2009 American Chemical Society Published on Web 06/03/2009