Article

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

Laboratory of Experimental Oncology, CRS Development of Biomolecular Therapies, SSN Emilia Romagna Istituti Ortopedici Rizzoli IRCCS, Bologna, Italy.
The Journal of clinical investigation (Impact Factor: 13.22). 02/2010; 120(3):668-80. DOI: 10.1172/JCI36667
Source: PubMed

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 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.

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Available from: Mario P Colombo, Jan 18, 2014
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    • "In physiological conditions, CD99 protein is implicated in thymocyte differentiation, apoptosis of double-positive T cells, leucocyte diapedesis, and regulation of cell-cell adhesion (Schenkel et al. 2002). Furthermore, endogenous CD99 expression in EWS modulates tumor differentiation and malignancy and represents a medically relevant target molecule for the diagnosis and therapy of EWS (Rocchi et al. 2010). To this regard, it has been previously described that engagement of CD99 by agonistic murine monoclonal antibodies (mAbs) can significantly inhibit the growth of Ewing tumor cells in vitro and in vivo models by apoptotic stimulus, thus reducing the malignant potential of these cancer cells (Scotlandi et al. 2006). "
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    ABSTRACT: Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60 % soluble fraction, we obtained a yield of 25 mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75 % soluble fraction 35 mg C7 dAbd per L of cell culture grown in a synthetic media containing 3 g/L of vegetable peptone and 1 g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells.
    Full-text · Article · Dec 2015 · Applied Microbiology and Biotechnology
    • "Recent data suggest that CD99 is not only a marker and therapeutic target for ES but may also contribute to the disease phenotype. Knockdown of CD99 in ES cell lines resulted in decreased growth in tissue culture, diminished colony formation in soft agar assays, reduced cell motility and smaller tumors with less metastasis in xenograft models.[61] This study also suggested that CD99 inhibits full neuronal differentiation by decreasing the activity of the MAP kinase pathway. "
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    ABSTRACT: Ewing's sarcoma (ES) is a highly malignant tumor of children and young adults. Modern therapy for Ewing's sarcoma combines high-dose chemotherapy for systemic control of disease, with advanced surgical and/or radiation therapeutic approaches for local control. Despite optimal management, the cure rate for localized disease is only approximately 70%, whereas the cure rate for metastatic disease at presentation is less than 30%. Patients who experience long-term disease-free survival are at risk for significant side-effects of therapy, including infertility, limb dysfunction and an increased risk for second malignancies. The identification of new targets for innovative therapeutic approaches is, therefore, strongly needed for its treatment. Many new pharmaceutical agents have been tested in early phases of clinical trials in ES patients who have recurrent disease. While some agents led to partial response or stable disease, the percentages of drugs eliciting responses or causing an overall effect have been minimal. Furthermore, of the new pharmaceuticals being introduced to clinical practice, the most effective agents also have dose-limiting toxicities. Novel approaches are needed to minimize non-specific toxicity, both for patients with recurrence and at diagnosis. This report presents an overview of the potential molecular targets in ES and highlights the possibility that they may serve as therapeutic targets for the disease. Although additional investigations are required before most of these approaches can be assessed in the clinic, they provide a great deal of hope for patients with Ewing's sarcoma.
    No preview · Article · Oct 2012 · Indian journal of medical and paediatric oncology
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    • "Recent data suggest that CD99 is not only a marker and therapeutic target for Ewing's sarcoma, but may also contribute to the disease phenotype. Knockdown of CD99 in Ewing's sarcoma cell lines resulted in decreased growth in tissue culture, diminished colony formation in soft agar assays, reduced cell motility, and smaller tumors with less metastasis in xenograft models (Rocchi et al., 2010). This study also suggested that CD99 inhibits full neuronal differentiation by decreasing the activity of the MAP kinase pathway. "
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    ABSTRACT: Tumor development is a complex process resulting from interplay between mutations in oncogenes and tumor suppressors, host susceptibility factors, and cellular context. Great advances have been made by studying rare tumors with unique clinical, genetic, or molecular features. Ewing's sarcoma serves as an excellent paradigm for understanding tumorigenesis because it exhibits some very useful and important characteristics. For example, nearly all cases of Ewing's sarcoma contain the (11;22)(q24;q12) chromosomal translocation that encodes the EWS/FLI oncoprotein. Besides the t(11;22), however, many cases have otherwise simple karyotypes with no other demonstrable abnormalities. Furthermore, it seems that an underlying genetic susceptibility to Ewing's sarcoma, if it exists, must be rare. These two features suggest that EWS/FLI is the primary mutation that drives the development of this tumor. Finally, Ewing's sarcoma is an aggressive tumor that requires aggressive treatment. Thus, improved understanding of the pathogenesis of this tumor will not only be of academic interest, but may also lead to new therapeutic approaches for individuals afflicted with this disease. The purpose of this review is to highlight recent advances in understanding the molecular pathogenesis of Ewing's sarcoma, while considering the questions surrounding this disease that still remain and how this knowledge may be applied to developing new treatments for patients with this highly aggressive disease.
    Preview · Article · Aug 2010 · Oncogene
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