Cutting Edge: The RNA-Binding Protein Ewing Sarcoma Is a Novel Modulator of Lymphotoxin ß Receptor Signaling.

Lymphotoxin ß receptor (LTßR) signaling is crucial for lymphoid tissue organogenesis and immune homeostasis. To identify novel regulatory mechanisms for signaling, we implemented a two-step screen that uses coexpression analysis of human fibroblasts undergoing LTßR stimulation and affinity-purification mass spectrometry for the LTßR signaling protein TNFR-associated factor 3 (TRAF3). We identify Ewing sarcoma (EWS) protein as a novel LTßR signaling component that associates with TRAF3 but not with TNFR-associated factor 2 (TRAF2). The EWS:TRAF3 complex forms under unligated conditions that are disrupted following activation of the LTßR. We conclude that EWS limits expression of proinflammatory molecules, GM-CSF, and ERK-2, promoting immune homeostasis.

New Insights about the Wnt/ß-Catenin Signaling Pathway in Primary Bone Tumors and Their Microenvironment: A Promising Target to Develop Therapeutic Strategies?

Osteosarcoma and Ewing sarcoma are the most common malignant primary bone tumors mainly occurring in children, adolescents and young adults. Current standard therapy includes multidrug chemotherapy and/or radiation specifically for Ewing sarcoma, associated with tumor resection. However, patient survival has not evolved for the past decade and remains closely related to the response of tumor cells to chemotherapy, reaching around 75% at 5 years for patients with localized forms of osteosarcoma or Ewing sarcoma but less than 30% in metastatic diseases and patients resistant to initial chemotherapy. Despite Ewing sarcoma being characterized by specific EWSR1-ETS gene fusions resulting in oncogenic transcription factors, currently, no targeted therapy could be implemented. It seems even more difficult to develop a targeted therapeutic strategy in osteosarcoma which is characterized by high complexity and heterogeneity in genomic alterations. Nevertheless, the common point between these different bone tumors is their ability to deregulate bone homeostasis and remodeling and divert them to their benefit. Therefore, targeting different actors of the bone tumor microenvironment has been hypothesized to develop new therapeutic strategies. In this context, it is well known that the Wnt/ß-catenin signaling pathway plays a key role in cancer development, including osteosarcoma and Ewing sarcoma as well as in bone remodeling. Moreover, recent studies highlight the implication of the Wnt/ß-catenin pathway in angiogenesis and immuno-surveillance, two key mechanisms involved in metastatic dissemination. This review focuses on the role played by this signaling pathway in the development of primary bone tumors and the modulation of their specific microenvironment.

EWS/FLI-l peptide-pulsed dendritic cells induces the antitumor immunity in a murine Ewing's sarcoma cell model.

An increasing number of T-cell epitopes derived from various tumor-associated antigens have been reported, and they proved to play significant roles for tumor rejection both in vivo and in vitro. Over 85% of Ewing's sarcoma family of tumors (ESFTs) express tumor-specific chimeric protein EWS/FLI-1, making it an attractive target for therapeutic cytotoxic T-lymphocyte responses. Here, we identified a novel peptide epitope derived from the EWS/FLI-1 protein and demonstrated that effectors induced by the peptide could specifically secrete IFN-γ and lyse the tumor cell line of EWS/FLI-1-positive and HLA-matched cells. In addition, mice treated with dendritic cells pulsed with the EWS/FLI-1 epitope were able to reject a lethal tumor inoculation of the Ewing's sarcoma A673 cells. Therefore, these data provide evidence for the use of the EWS/FLI-l peptide epitope in T cell-based immunotherapeutic concepts against Ewing's sarcoma cell in vitro and in vivo.

Prediction and identification of B cell epitopes derived from EWS/FLI-l fusion protein of Ewing's sarcoma.

To predict B cell epitope of Ewing's sarcoma EWS/FLI-l fusion protein and to analyze its antigenicity and immunogenicity. Comprehensive algorithms were applied to predict the possible B cell epitopes of EWS/FLI-l fusion protein. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis were performed to identify the synthesized epitope peptides, ELISA assays and Western blot to detect the antigenicity, and the immunogenicity of epitope peptides. Three B cell epitopes were screened out, and HPLC and MS analysis confirmed all three synthesized epitope peptides were demandable. ELISA assays verified all three epitope peptides could prime intense antigen-antibody reaction and induce ideal antibody titers after immunization to the New Zealand white rabbit. However, Western blot confirmed that antiserum of one of these epitope peptides could not recognize EWS/FLI-1 protein. Two B cell epitopes, PQDGNKPTETSQPQ and DPDEVARRWGQRKS, derived from EWS/FLI-l protein, are identified to have potential antigenicity and immunogenicity.

[Application of the in situ hybridization with EWS dual-color break-apart fluorescence probe and anti-CD99 and anti-FLI-1 antibodies in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor].

OBJECTIVE: To investigate the value of using EWS dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique, CD99 and FLI-1 antibodies immunohistochemistry in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor (EWS/PNET). METHODS: Thirty-five cases of EWS/PNET and 24 cases of non-EWS/PNET small round cell tumor were analyzed by FISH and immunohistochemically detected with FLI-1 and CD99 antibodies. Comparison between FISH and immunohistochemical results was carried out and their diagnostic value was evaluated. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of FISH in EWS/PNET were 93.8%(30/32), 81.8%(18/22), 88.2%(30/34) and 90%(18/20); those of CD99 were 100%(35/35), 58.3% (14/24), 77.8%(35/45) and 100%(14/14); those of FLI-1 were 71.4%(25/35), 62.5%(15/24), 73.5%(25/34), 60%(15/25), respectively. The sensitivity, specificity, positive predictive value and negative predictive value of combined use of CD99 and FLI-1 were 71.4%(25/35), 75%(18/24), 80.6%(25/31) and 64.3%(18/28), those of combined use of FLI-1 and FISH were 68.7%(22/32), 86.4%(19/22), 88%(22/25) and 65.5%(19/29), those of combined use of CD99 and FISH were 93.7%(30/32), 95.5%(21/22), 96.8%(30/31) and 91.3%(21/23), respectively. CONCLUSION: EWS dual-color, break-apart rearrangement probe FISH is a highly sensitive and specific technique in the diagnosis of EWS/PNET. The combination of CD99 and FISH is the method of choice for the diagnosis of EWS/PNET. The combination of CD99 and FLI-1 can improve the specificity in EWS/PNET without the data of FISH.

Generation of the novel monoclonal antibody against TLS/EWS-CHOP chimeric oncoproteins that is applicable to one of the most sensitive assays for myxoid and round cell liposarcomas.

The fusion oncoproteins, TLS-CHOP and EWS-CHOP, are characteristic markers for myxoid and round cell liposarcomas (MLS/RCLS). Especially, the peptide sequence of 26 amino acids corresponding to the normally untranslated CHOP exon 2 and parts of exon 3 (5'-UTR) is a unique structure for these chimeric proteins. In this report, we have generated monoclonal antibodies against the unique peptide sequence of TLS/EWS-CHOP oncoproteins. These antibodies reacted with TLS-CHOP fusion protein, but not reacted with normal TLS and CHOP proteins by Western blot analysis. In addition, one of the antibodies also recognized the chimeric oncoprotein in archival paraffin-embedded tissue samples of MLS/RCLS. The oncoprotein was detectable by the antibody even in the paraffin-embedded tissue samples whose mRNAs were too degraded to be detected by a nested reverse transcription-polymerase chain reaction-based assay. Thus, the molecular assay using the novel antibody is expected to be one of the most sensitive diagnostic assays for MLS/RCLS.

Identification of Ewing's sarcoma gene product as a glycoprotein using a monoclonal antibody that recognizes an immunodeterminant containing O-linked N-acetylglucosamine moiety.

The Ewing's sarcoma (EWS) oncogene is fused to a variety of cellular transcription factors in various forms of human cancers. Although EWS fusion proteins have been extensively studied, the normal function of EWS remains poorly characterized. We previously reported that a monoclonal antibody, referred to as MY95, recognized nucleoporins such as p62, Nup98, and CAN/Nup214 and an uncharacterized polypeptide with an apparent molecular mass of 83 kDa. In the present study, an amino acid sequence analysis of this 83-kDa protein revealed that it is, in fact, EWS, which is not known to belong to the nucleoporins. We further demonstrated that the immunodeterminant of MY95 contains an N-acetylglucosamine moiety, indicating that EWS is a glycoprotein. Interestingly, the glycosylation level of EWS changes during the neural differentiation of P19 cells. MY95 will be quite useful in further studies of the glycosylated form of EWS in terms of understanding the normal cellular function of this oncogene product.