CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis.

Ewing sarcoma (EWS) is an aggressive bone tumor of uncertain cellular origin. CD99 is a membrane protein that is expressed in most cases of EWS, although its function in the disease is unknown. Here we have shown that endogenous CD99 expression modulates EWS tumor differentiation and malignancy. We determined that knocking down CD99 expression in human EWS cell lines reduced their ability to form tumors and bone metastases when xenografted into immunodeficient mice and diminished their tumorigenic characteristics in vitro. Further, reduction of CD99 expression resulted in neurite outgrowth and increased expression of beta-III tubulin and markers of neural differentiation. Analysis of a panel of human EWS cells revealed an inverse correlation between CD99 and H-neurofilament expression, as well as an inverse correlation between neural differentiation and oncogenic transformation. As knockdown of CD99 also led to an increase in phosphorylation of ERK1/2, we suggest that the CD99-mediated prevention of neural differentiation of EWS occurs through MAPK pathway modulation. Together, these data indicate a new role for CD99 in preventing neural differentiation of EWS cells and suggest that blockade of CD99 or its downstream molecular pathway may be a new therapeutic approach for EWS.

[1]  C. Pagès,et al.  A TAT–DEF–Elk-1 Peptide Regulates the Cytonuclear Trafficking of Elk-1 and Controls Cytoskeleton Dynamics , 2007, The Journal of Neuroscience.

[2]  P. Cohen,et al.  Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. , 1992, The Biochemical journal.

[3]  S. Maeda,et al.  PKC pathway and ERK/MAPK pathway are required for induction of cyclin D1 and p21Waf1 during 12-o-tetradecanoylphorbol 13-acetate-induced differentiation of myeloleukemia cells. , 2006, The Kobe journal of medical sciences.

[4]  H Kovar,et al.  MIC2 is a specific marker for ewing's sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of ewing's sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration , 1991, Cancer.

[5]  D Rampling,et al.  Immunocytochemical study of 12E7 in small round‐cell tumours of childhood: an assessment of its sensitivity and specificity , 1993, Histopathology.

[6]  M. Suvà,et al.  EWS-FLI-1 expression triggers a Ewing's sarcoma initiation program in primary human mesenchymal stem cells. , 2008, Cancer research.

[7]  M. Ticchioni,et al.  CD99 (E2) up‐regulates α 4β 1‐dependent T cell adhesion to inflamed vascular endothelium under flow conditions , 2000 .

[8]  M. Toyoda,et al.  Inducible Expression of Chimeric EWS/ETS Proteins Confers Ewing's Family Tumor-Like Phenotypes to Human Mesenchymal Progenitor Cells , 2008, Molecular and Cellular Biology.

[9]  J. Sweatt,et al.  Mitogen-activated protein kinases in synaptic plasticity and memory , 2004, Current Opinion in Neurobiology.

[10]  S. Jhanwar,et al.  Neural differentiation in small round cell tumors of bone and soft tissue with the translocation t(11;22)(q24;q12): an immunohistochemical study of 11 cases. , 1990, Human pathology.

[11]  P. Lollini,et al.  CD99 engagement: an effective therapeutic strategy for Ewing tumors. , 2000, Cancer research.

[12]  C. Turc‐Carel,et al.  Immunologic characterization of Ewing's sarcoma using mesenchymal and neural markers. , 1989, The American journal of pathology.

[13]  O. Delattre,et al.  Mesenchymal stem cell features of Ewing tumors. , 2007, Cancer cell.

[14]  M. Lipinski,et al.  Neuroectoderm-associated antigens on Ewing's sarcoma cell lines. , 1987, Cancer research.

[15]  S. Lessnick,et al.  NR0B1 Is Required for the Oncogenic Phenotype Mediated by EWS/FLI in Ewing's Sarcoma , 2006, Molecular Cancer Research.

[16]  T. Golub,et al.  Supplemental Information for , 2002 .

[17]  Robert E. Brown,et al.  Mesenchymal chondrosarcoma: molecular characterization by a proteomic approach, with morphogenic and therapeutic implications. , 2003, Annals of clinical and laboratory science.

[18]  S. Knuutila,et al.  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 , 2004, Oncogene.

[19]  H. Kovar,et al.  Ewing's sarcoma family of tumors: current management. , 2006, The oncologist.

[20]  F. Barr,et al.  Molecular diagnosis of ewing family tumors: too many fusions... ? , 2007, The Journal of molecular diagnostics : JMD.

[21]  S. Grewal,et al.  Extracellular-signal-regulated kinase signalling in neurons , 1999, Current Opinion in Neurobiology.

[22]  Agata K. Zupanska,et al.  Alternative pathway of transcriptional induction of p21WAF1/Cip1 by cyclosporine A in p53-deficient human glioblastoma cells. , 2007, Cellular signalling.

[23]  G. Perini,et al.  Functional cooperation between TrkA and p75(NTR) accelerates neuronal differentiation by increased transcription of GAP-43 and p21(CIP/WAF) genes via ERK1/2 and AP-1 activities. , 2007, Experimental cell research.

[24]  C. Turc‐Carel,et al.  Abnormal expression of neurofilament proteins in Ewing's sarcoma cell cultures. , 1992, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine.

[25]  P. Lollini,et al.  Targeting CD99 in association with doxorubicin: an effective combined treatment for Ewing's sarcoma. , 2006, European journal of cancer.

[26]  C. Denny,et al.  Loss of p16 pathways stabilizes EWS/FLI1 expression and complements EWS/FLI1 mediated transformation , 2001, Oncogene.

[27]  K. Unsicker,et al.  Extracellular signal-regulated kinase as an inducer of non-apoptotic neuronal death , 2006, Neuroscience.

[28]  S. Park,et al.  Differential activation of MAP kinase family members triggered by CD99 engagement , 2000, FEBS letters.

[29]  D. Jeoung,et al.  A Splice Variant of CD99 Increases Motility and MMP-9 Expression of Human Breast Cancer Cells through the AKT-, ERK-, and JNK-dependent AP-1 Activation Signaling Pathways* , 2006, Journal of Biological Chemistry.

[30]  Nicolò Riggi,et al.  Identification of cancer stem cells in Ewing's sarcoma. , 2009, Cancer research.

[31]  M. Ticchioni,et al.  CD99 (E2) up-regulates alpha4beta1-dependent T cell adhesion to inflamed vascular endothelium under flow conditions. , 2000, European journal of immunology.

[32]  S. Baker,et al.  Ewing tumor fusion proteins block the differentiation of pluripotent marrow stromal cells. , 2003, Cancer research.

[33]  J. Slingerland,et al.  Multiple Roles of the PI3K/PKB (Akt) Pathway in Cell Cycle Progression , 2003, Cell cycle.

[34]  D. Leroith,et al.  The Insulin-like Growth Factor-I Receptor Is Required for EWS/FLI-1 Transformation of Fibroblasts* , 1997, The Journal of Biological Chemistry.

[35]  P. Sorensen,et al.  EWS/ETS fusion genes induce epithelial and neuroectodermal differentiation in NIH 3T3 fibroblasts. , 1999, Laboratory investigation; a journal of technical methods and pathology.

[36]  J. Trojanowski,et al.  Expression of neurofilament subunits in neurons of the central and peripheral nervous system: an immunohistochemical study with monoclonal antibodies , 1986, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[37]  Y. Iwamoto,et al.  Identification of p21 WAF1/CIP1 as a Direct Target of EWS-Fli1 Oncogenic Fusion Protein* , 2003, The Journal of Biological Chemistry.

[38]  G. Thomas,et al.  Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours , 1992, Nature.

[39]  E. Álava,et al.  Stable interference of EWS–FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target , 2009, British Journal of Cancer.

[40]  R. Pochampally,et al.  Isolation of a Highly Clonogenic and Multipotential Subfraction of Adult Stem Cells from Bone Marrow Stroma , 2004, Stem cells.

[41]  Naomi J Balamuth,et al.  Ewing's sarcoma. , 2010, The Lancet. Oncology.

[42]  H. Kovar.  Context matters: the hen or egg problem in Ewing's sarcoma. , 2005, Seminars in cancer biology.

[43]  C. Denny,et al.  The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1 , 1993, Molecular and cellular biology.

[44]  V. Thomas,et al.  The Ews/Fli-1 Fusion Gene Switches the Differentiation Program of Neuroblastomas to Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumors , 2004, Cancer Research.

[45]  A. Bernard,et al.  Apoptosis of immature thymocytes mediated by E2/CD99. , 1997, Journal of immunology.

[46]  I. Black,et al.  Marrow Stromal Cells, Mitosis, and Neuronal Differentiation: Stem Cell and Precursor Functions , 2003, Stem cells.

[47]  P. Crespo,et al.  Ras proteins in the control of the cell cycle and cell differentiation , 2000, Cellular and Molecular Life Sciences CMLS.

[48]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[49]  E. Melamed,et al.  Human mesenchymal stem cells express neural genes, suggesting a neural predisposition. , 2006, Stem cells and development.

[50]  R. Steinman.  Cell cycle regulators and hematopoiesis , 2002, Oncogene.

[51]  P. Lollini,et al.  CD99 acts as an oncosuppressor in osteosarcoma. , 2006, Molecular biology of the cell.

[52]  H. Kovar,et al.  Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor. , 1990, Oncogene.

[53]  A. Trumpp,et al.  Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells. , 2005, Cancer research.

[54]  R. Ilaria,et al.  Interference with the constitutive activation of ERK1 and ERK2 impairs EWS/FLI-1-dependent transformation , 2000, Oncogene.

[55]  T. Golub,et al.  Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma. , 2006, Cancer cell.

[56]  W. Muller,et al.  CD99 plays a major role in the migration of monocytes through endothelial junctions , 2002, Nature Immunology.

[57]  P. Picci,et al.  Prognostic Value of CCN3 in Osteosarcoma , 2008, Clinical Cancer Research.

[58]  A. Benabid,et al.  Functional Neuronal Differentiation of Bone Marrow‐Derived Mesenchymal Stem Cells , 2006, Stem cells.

[59]  T. Triche,et al.  EWS-FLI1 fusion protein up-regulates critical genes in neural crest development and is responsible for the observed phenotype of Ewing's family of tumors. , 2005, Cancer research.

[60]  E. Dufour,et al.  The Murine CD99-Related Molecule CD99-Like 2 (CD99L2) Is an Adhesion Molecule Involved in the Inflammatory Response , 2007, Cell communication & adhesion.

[61]  Giselle Chamberlain,et al.  Concise Review: Mesenchymal Stem Cells: Their Phenotype, Differentiation Capacity, Immunological Features, and Potential for Homing , 2007, Stem cells.

[62]  E. Álava,et al.  EWS/FLI-1 oncoprotein subtypes impose different requirements for transformation and metastatic activity in a murine model , 2007, Journal of Molecular Medicine.

[63]  Yinyan Xia,et al.  Basic Fibroblast Growth Factor-induced Neuronal Differentiation of Mouse Bone Marrow Stromal Cells Requires FGFR-1, MAPK/ERK, and Transcription Factor AP-1* , 2008, Journal of Biological Chemistry.

[64]  H. Kovar,et al.  CD99-positive "Ewing's sarcoma" from mouse-bone marrow-derived mesenchymal progenitor cells? , 2006, Cancer research.

[65]  K. Fukunaga,et al.  Role of MAP kinase in neurons , 1998, Molecular Neurobiology.

[66]  Julie H. Campbell,et al.  J. Submicrosc. Cytol. Pathol. , 2000 .

[67]  G. Basso,et al.  CD99 expression in T-lineage ALL: implications for flow cytometric detection of minimal residual disease , 2004, Leukemia.

[68]  P. Pelicci,et al.  A function of p21 during promyelocytic leukemia cell differentiation independent of CDK inhibition and cell cycle arrest , 1999, Oncogene.

[69]  Y. Iwamoto,et al.  The Prognostic and Therapeutic Relevance of p27kip1 in Ewing’s Family Tumors , 2004, Clinical Cancer Research.

[70]  T. Triche,et al.  Immunohistochemical analysis of Ewing's sarcoma cell surface antigen p30/32MIC2. , 1991, The American journal of pathology.

[71]  O. Delattre,et al.  EWS/FLI-1 Silencing and Gene Profiling of Ewing Cells Reveal Downstream Oncogenic Pathways and a Crucial Role for Repression of Insulin-Like Growth Factor Binding Protein 3 , 2004, Molecular and Cellular Biology.

[72]  C. Denny,et al.  Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[73]  M. Deckert,et al.  The E2 molecule (CD99) specifically triggers homotypic aggregation of CD4+ CD8+ thymocytes. , 1995, Journal of immunology.

[74]  P. Picci,et al.  CD99 isoforms dictate opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase activity , 2007, Oncogene.