Ewing sarcoma family tumors (ESFT) and neuroblastoma (NB) are two of the most frequently encountered extracranial solid tumors in children and adolescents. Despite major advances in the understanding of the biology of these tumors, the prognosis of patients with recurrent and metastatic disease remains dismal. Therefore, it is important to identify novel agents or strategies, which will eradicate chemotherapy-resistant tumor cells. Chemotherapeutic agents, irrespective of their initial targets, exert their action by converging into a common apoptotic pathway. Our studies seek to identify mechanisms that lead to dysregulation of apoptosis in ESFT and NB in order to understand the reasons for therapy failures, and discover alternative pathways to overcome drug resistance in these tumors. Two main signaling pathways initiate apoptosis: (a) the mitochondrial pathway, triggered by cell distress and DNA damage and (b) the death receptor-mediated pathway, triggered by the interaction of the death receptors with their ligands. Both pathways proceed through activation of caspases. Among the death receptor-mediated pathways, the Tumor Necrosis Factor Apoptosis-Inducing Ligand (TRAIL) pathway has generated a great deal of interest, because of the efficacy of TRAIL and TRAIL receptor agonists as tumoricidal agents and their lack of toxicity to normal cells, which provide exciting opportunities for development of novel therapeutic strategies in cancer. We have previously shown that TRAIL is a potent inducer of apoptosis in ESFT cells, and that ESFT cells express the TRAIL receptors DR4 and DR5. However, in our in vitro studies, we observed that some ESFT cells exhibit resistance to TRAIL and in collaborative studies with Dr. Mackalls laboratory using ESFT xenografts, we found that ESFT cell resistance to TRAIL is associated with downregulation of the TRAIL receptors, and can be overcome with interferon treatment. We also found that low caspase 8 expression contributes to TRAIL resistance in ESFT and is present in 24% of the ESFT tissues. Recent data in our laboratory have shown that drug-resistant ESFT cells have high levels of the antiapoptotic protein survivin. By downregulating survivin, we were able to restore the sensitivity of ESFT cells to chemotherapeutic agents in vitro. Furthermore, we found that patients with localized ESFT expressing high levels of survivin had an overall poor prognosis when compared to those with no survivin expression and this was statistically significant. We are currently exploring the role of survivin in TRAIL-resistant ESFT cells and its regulation by the ESFT-specific fusion gene EWS/FLI-1. With regard to NB cells, we have shown that they have several regulatory apoptotic defects, which lead to the inactivation of the mitochondrial and the death receptor pathways. NB cells have been reported to express low levels of caspase 8, but this may not be the only defect, because as we have shown in collaboration with Dr. Thiele's laboratory, even after upregulation of caspase 8 with interferon, TRAIL-induced apoptosis is defective due to low or absent levels of TRAIL receptors. Our laboratory has also shown a novel mechanism of caspase 8 inactivation in NB cells through direct binding with the antiapoptotic protein Bcl-2. More recent data from our laboratory have shown that a newly reported protein under the name of Bifunctional Apoptosis Regulator (BAR) is expressed at high levels in TRAIL-resistant NB cells and forms complexes with Bccl-2 and caspase 8. Its downregulation results in dissociation of caspase 8 from bcl-2 and reverses TRAIL resistance in NB cells. These data support that association of Bcl-2 and caspase 8 is accomplished through BAR, which is an important inhibitor of TRAIL-induced apoptosis in NB cells and therefore, a possible therapeutic target.
