Molecular mechanisms of CD99-induced caspase-independent cell death and cell–cell adhesion in Ewing’s sarcoma cells: actin and zyxin as key intracellular mediators Vanessa Cerisano 1 , Yan Aalto 2 , Stefania Perdichizzi 1 , Ghislaine Bernard 3 , Maria Cristina Manara 1 , Stefania Benini 1 , Giovanna Cenacchi 4 , Paola Preda 4 , Giovanna Lattanzi 5 , Ba´ lint Nagy 2 , Sakari Knuutila 2 , Mario Paolo Colombo 6 , Alain Bernard 3 , Piero Picci 1 and Katia Scotlandi* ,1 1 Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna 40136, Italy; 2 Departments of Pathology and Medical Genetics, Haartman Institute and University of Helsinki, Helsinki FIN-00014, Finland; 3 Unite´INSERM UMR 576, Hoˆspital de l’Archet, Nice Cedex 3, France; 4 Dipartimento Clinico di Scienze Radiologiche e Istocitopatologiche, Universita`di Bologna, Bologna, Italy; 5 ITOI-CNR, Unit of Bologna, c/o Istituti Ortopedici Rizzoli, Bologna 40136, Italy; 6 Immunotherapy and Gene Therapy Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan 20133, Italy CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing’s sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase- independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell–cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement. Oncogene (2004) 23, 5664–5674. doi:10.1038/sj.onc.1207741 Published online 7 June 2004 Keywords: CD99; Ewing’s sarcoma; homotypic aggre- gation; apoptosis; zyxin; actin Introduction CD99 is a ubiquitous 32-kDa transmembrane protein encoded by the MIC2 gene, which is located to the pseudoautosomal regions of both X and Y human chromosomes, and shares no homology with any known family of proteins (Goodfellow et al., 1986; Banting et al., 1989; Gelin et al., 1989). Broadly distributed on normal cells, CD99 is highly expressed on T- and B- lymphocytes (Bernard et al., 1988; Dworzak et al., 1999). Immunohistochemistry detected CD99 in several types of cancer, including lymphoblastic lymphoma/ leukemia (Dworzak et al., 1999), some rhabdomyosar- comas (Ramani et al., 1993) and prostate cancer (Shen etal., 1998). Sporadic expression has also been found in synovial sarcoma (Fisher, 1998) and mesenchymal chondrosarcoma (Granter et al., 1996; Scotlandi et al., 1996), and antigen retrieval technology extended the number of tumors in which detection of the antigen was found (Jung et al., 2002). However, Ewing’s sarcoma (ES) remains the tumor that, without exception, most consistently expresses CD99, a feature that allows differential diagnosis to distinguish ES from other types of small round cell tumors (Kovar et al., 1990; Ambros et al., 1991). The functional role of CD99 is not well known. The ligand (s) for CD99 has not been identified, and most of our knowledge about its functions derives from trigger- ing CD99 on hematopoietic cells by agonistic mono- clonal antibodies (MAb). In normal cells, ligation of CD99 has been functionally implicated in cell adhesion and homotypic aggregation, apoptosis, cell migration, Th1 cell differentiation, activation and proliferation of mature T cells and upregulation and transport of several transmembrane proteins (Bernard et al., 1995, 1997, 2000; Hahn et al., 1997; Choi et al., 1998; Kim et al., 1998, 2000; Waclavicek etal., 1998; Wingett etal., 1999; Pettersen et al., 2001; Sohn et al., 2001; Schenkel et al., 2002), but the molecular mechanisms of CD99-mediated signal transduction remains largely unknown. In tumors, the functional role of CD99 is even less understood. In ES, the engagement of CD99 induces massive apoptosis (Sohn et al., 1998; Scotlandi et al., 2000), increases sensitivity to conventional chemother- apeutic agents (Scotlandi et al., 2000) and inhibits the Received 27 January 2004; revised 17 March 2004; accepted 19 March 2004; published online 7 June 2004 *Correspondence: K Scotlandi; E-mail: katia.scotlandi@ior.it Oncogene (2004) 23, 5664–5674 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc