CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis
Anna Rocchi, … , Piero Picci, Katia Scotlandi
Anna Rocchi, … , Piero Picci, Katia Scotlandi
Published February 8, 2010
Citation Information: J Clin Invest. 2010;120(3):668-680. https://doi.org/10.1172/JCI36667.
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Research Article Oncology

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

Abstract

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 β-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.

Authors

Anna Rocchi, Maria Cristina Manara, Marika Sciandra, Diana Zambelli, Filippo Nardi, Giordano Nicoletti, Cecilia Garofalo, Stefania Meschini, Annalisa Astolfi, Mario P. Colombo, Stephen L. Lessnick, Piero Picci, Katia Scotlandi

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Figure 3

CD99 influences cell proliferation and apoptosis of EWS cells.

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CD99 influences cell proliferation and apoptosis of EWS cells. (A) Cell ...
(A) Cell cycle analysis of TC-71–derived cells. Cells were stained with propidium iodide (a DNA stain) and DNA content analyzed by ModFit LT software. Data are from an experiment representative of 2 independent experiments. Percentages of cells in G2/M phase were significantly different in CD99-silenced cells compared with controls; P < 0.05, Student’s t test. (B) Detection of annexin V–FITC–labeled apoptotic cells by flow cytometric analysis in TC-71–derived cells. The simultaneous application of propidium iodide as a DNA stain allows the discrimination of necrotic cells (upper right) from the apoptotic cells positively stained for annexin V (lower right). Data are from an experiment representative of 2 independent experiments. Percentages of apoptotic cells (annexin V–positive and propidium iodide–negative) were significantly different in CD99-silenced cells compared with controls; P < 0.05, Student’s t test. (C) Western blot analysis of a series of cell cycle and apoptosis regulators. Induction of the cell cycle inhibitors p21 or p27 was observed together with repression of the antiapoptotic protein Bcl-2. The phosphorylation of Akt was also inhibited in CD99-silenced cells.