CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis
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
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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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 7

Relationship of CD99 expression and EWS/FLI expression.

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Relationship of CD99 expression and EWS/FLI expression. (A) Hierarchical...
(A) Hierarchical clustering of CD99-associated signature based on TC-71 and IOR/BRZ silenced cells. The signature included 106 genes: 76 upregulated and 30 downregulated (Supplemental Table 1; t test, P < 0.05). The top 10 CD99 up- (red) or downmodulated genes (blue) are shown. (B) We applied PCA using the CD99-associated signature (Supplemental Table 1) on EWS/FLI1-silenced EWS cells derived from SK-M-NC and EW24 (dataset from ref. 10) or from EWS502 and TC-71 cell lines (dataset from ref. 55). CD99 signature allowed for a clear separation of EWS/FLI knockdown cells from controls. Circles indicate EWS/FLI1 silenced cells; triangles indicate controls. (C) Interaction between EWS/FLI1 and CD99 promoter (prom). ChIP was carried out in TC71 and IOR/BRZ cells as described in Supplemental Methods by using anti-Fli1 antibody. The CD99 promoter region containing the ets sequence was detected by PCR with specific primers listed in Supplemental Methods. One microliter of initial preparations of soluble chromatin (Input) was amplified to control input DNA. In control samples (N), normal rabbit IgG was used instead of the primary Ab as control of Ab specificity. The occupancy of EWS/FLI1 on the TGFβR2 promoter was tested with specific primers as a control for ChIP reaction. The negative control promoter primers used for ChIP were previously described (72).