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 6

Insights into the mechanisms of CD99-silenced cells.

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Insights into the mechanisms of CD99-silenced cells. (A) Increased expre...
(A) Increased expression of activated p-ERK in cells deprived of CD99 as shown by cytofluorometric analysis. (B) Cell-based ELISA evaluation of activated p-ERK. Once normalized to cell number, the phosphorylation level is directly related to the extent of activation. Cells deprived of CD99 showed a higher levels of p-ERK. Wavelength, 550 nm. Data are presented as mean ± SEM of experiments performed in triplicate; *P < 0.05 versus controls, Student’s t test. (C) Western blot analysis of ERK and p-ERK confirmed the higher levels of activation in CD99-silenced cells. (D) Confocal microscopy further confirmed higher expression of the activated ERK in CD99-silenced cells compared with controls. No remarkable differences were observed between parental and transfected cells versus total ERK. Scale bars: 120 μm. (E) Forty-eight hours of treatment of TC-71 and derived cells with the ERK inhibitor PD98059 (50 μM) resulted in growth inhibition of the parental cells but not of the CD99-silenced cells. Data are presented as mean ± SEM of experiments performed in triplicate. (F) H-NF and β-III tubulin staining of CD99-silenced cells after 48-hour treatment with the ERK inhibitor PD98059. ERK inactivation inhibited expression of H-NF in CD99-silenced cells. Nuclei are counterstained with bisbenzimide Hoechst 33258. Digital images were taken under identical conditions, at the same time, and using the same image analysis software (Quips-XL genetic workstation). Scale bars: 120 μm.