Expression of therapeutic targets in Ewing sarcoma family tumors
ABSTRACT Ewing sarcoma family tumor is an aggressive malignant tumor of bone and soft tissue in children and adolescents. Despite advances in modern therapy, metastasis occurs in 20% to 25% of cases and results in mortality in 80% of patients. Intracellular molecules mammalian target of rapamycin, Akt, vascular endothelial growth factor, nuclear factor κB, and BRAF are important kinases and transcription factors that regulate the proliferation of tumor cells. We studied the expression of these proteins in 72 Ewing sarcoma family tumors. Patients' survival data were available in 55 cases. Formalin-fixed, paraffin-embedded tumor sections were stained with antibodies against phosphorylated mammalian target of rapamycin, Akt, BRAF, vascular endothelial growth factor, and nuclear factor κB proteins. Stained sections were analyzed for percentage and strength of staining, and a composite score (0-200) was subsequently generated. Although most tumors expressed mammalian target of rapamycin, Akt, nuclear factor κB, and vascular endothelial growth factor, only 37%, 86%, 55%, and 12%, respectively, showed high expression (staining score ≥ 100). There was no significant correlation between mammalian target of rapamycin and Akt expression and clinical outcome. High nuclear factor κB expression was significantly associated with tumors in pelvic locations. Decreased vascular endothelial growth factor expression (score <100) was significantly associated with better prognosis (P < .05). BRAF was not expressed in most cases and showed negative or weak staining (score <100) in 97% of cases. Thus, except for BRAF, Ewing sarcoma family tumors may be amenable to treatment that targets the expressed proteins. High Akt expression suggests potential universal response to Akt-targeted therapy. BRAF kinase inhibitors are unlikely to be effective in the treatment of Ewing sarcoma family tumors.
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ABSTRACT: Background Hypoxia inducible factor-1 α (HIF-1α) has been identified as an important novel target in apoptosis resistance of pediatric tumors such as Rhabdomyosarcoma (RMS) and Ewing’s sarcoma (ES). Evidence suggests that PI3K/Akt signaling plays a role in regulation of HIF-1α activation as well as apoptosis resistance in various adult tumors. However the relevance of PI3K/Akt signaling in HIF-1bα activation and apoptosis resistance in childhood tumors has not been addressed yet. Thus, this study was to investigate whether PI3K/Akt signaling is involved in hypoxia induced activation of HIF-1α as well as in resistance to hypoxia-induced apoptosis in childhood tumors such as RMS and ES. Methods Constitutive activation of PI3K/Akt signaling was analyzed by Western blotting. Hypoxic activation of HIF-1α was determined by Western Blot analysis and electrophoretic mobility shift assay. Apoptosis was determined by flow cytometric analysis of the propidium iodine stained nuclei of cells treated with PI3K inhibitor LY294002 in combination with either TNF-related apoptosis-inducing ligand (TRAIL) or doxorubicin. Results This study demonstrated that PI3K/Akt signaling was constitutively activated in RMS and ES cell lines, A204 and A673, respectively. Targeting PI3K/Akt signaling by the inhibitor LY294002 (30 μM) significantly decreased the protein expression as well as DNA binding activity of HIF-1α and restored the apoptosis-inducing ability of cells in hypoxia Additionally, pretreatment with LY294002 sensitized A204 and A673 cells to TRAIL or doxorubicin induced apoptosis under hypoxia. Conclusion These results suggest that the constitutively active PI3K/Akt signaling contributes to hypoxic activation of HIF-1α as well as HIF1α-mediated apoptosis resistance in RMS and ES cells under hypoxia.Cancer Cell International 04/2013; 13(1). DOI:10.1186/1475-2867-13-36 · 1.99 Impact Factor
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ABSTRACT: Ewing's sarcoma family tumors are aggressive small round cell malignancies that arise in bone or soft tissues in adolescents and young adults. The addition of chemotherapy to local control measures has remarkably improved the survival of patients with localized disease. However, metastatic tumors are often refractory to conventional chemotherapy and irradiation, and the outcome of patients with metastatic or recurrent disease remains dismal. Despite growing understanding of the molecular biology of this tumor and the discovery of new therapeutic targets such as the insulin growth factor-1 receptor, tumor resistance continues to be a formidable challenge. Numerous adaptive mechanisms have been identified which allow tumor cells to escape the cytotoxic effect of chemotherapeutic agents. This review focuses on these mechanisms in an effort to highlight opportunities for more effective disease control.Cancer Chemotherapy and Pharmacology 01/2014; 73(4). DOI:10.1007/s00280-014-2392-1 · 2.57 Impact Factor
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ABSTRACT: Targeted therapies are increasingly being evaluated for patients with Ewing sarcoma (EWS). Optimal strategies for quantifying key signaling proteins in EWS remain unclear. We sought to quantify tumor expression of signaling pathway proteins in EWS using 3 methodologies. A total of 46 blocks of formalin-fixed paraffin-embedded tissue were obtained from 40 patients with EWS. Tumor was evaluated for the expression of proteins in the insulin-like growth factor type 1 receptor (IGF-1R), epithelial growth factor receptor (EGFR), and mTOR pathways using standard immunohistochemical analysis (IHC), automated quantitative analysis (AQUA) immunohistochemical analysis, and mass spectrometry quantification. The mean age at diagnosis was 14 years (range, 1 to 49 y). About 67.5% were male and 57.5% had localized disease. Samples displayed a wide range of expression by AQUA: mean (range) IGF-1R=10,702 (393 to 14,424); EGFR=2750 (672 to 9798); and phosphatase and tensin homolog (PTEN)=2250 (251 to 6557). Mean IGF-1R expression by AQUA did not differ between standard IHC expression categories (low IHC=11,255; medium IHC=11,070; high IHC=11,023; P=0.98). Mean PTEN expression by AQUA was higher in the medium and high IHC categories (low IHC=1229; medium IHC=2715; high IHC=2940; P=0.064). Only 2 samples expressed EGFR by standard IHC. Mass spectrometry trended toward correlation with standard IHC but did not yield interpretable results in the majority of samples. This study demonstrates that the relative quantification of signaling protein expression in EWS is dependent on the methodology used. Optimization and validation of these tools are necessary before clinical application for risk stratification of patients or measurement of biomarker expression.Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 02/2014; DOI:10.1097/PAI.0b013e3182a8d4bb · 2.06 Impact Factor