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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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 2

In vitro growth features of EWS cells silenced for CD99.

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In vitro growth features of EWS cells silenced for CD99. (A) CD99-silenc...
(A) CD99-silenced cells showed reduced growth in monolayer conditions compared with controls. Reexpression of CD99 rescued the growth inhibition caused by CD99 knockdown in TC-71 and IOR/BRZ cells. Data are presented as mean ± SEM of experiments performed in triplicate. Absorbance was measured at a wavelength of 550 nm. *P < 0.05, Student’s t test with respect to parental and CD99-reexpressing cells. (B) CD99-silenced cells showed reduced growth in anchorage-independent conditions. Reexpression of CD99 rescued the growth inhibition. Data are presented as mean ± SEM of experiments performed in triplicate. *P < 0.05, **P < 0.001, Student’s t test versus parental and CD99-reexpressing cells. Representative photomicrographs are shown for TC-71–derived cells. Digital images were taken under identical conditions at the same time. Scale bars: 600 μm. (C) Migratory ability of CD99-silenced cells was significantly reduced compared with that of controls. Reexpression of CD99 rescued the migration deficit caused by CD99 knockdown in TC-71 and IOR/BRZ cells. Data are presented as mean ± SEM of experiments performed in triplicate and indicate the number of cells that migrated 12 hours after cell seeding. **P < 0.001, Student’s t test versus parental and CD99-reexpressing cells. (D) Cell adhesion tests to extracellular matrix components demonstrate that CD99-deprived cells adhere faster to collagen I and IV. Data are presented as mean ± SEM of experiments performed in triplicate. *P < 0.05 versus parental and CD9-reexpressing cells, Student’s t test.