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 understanding 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. Current therapeutic regimens cure only 50% to 70% of patients with localized ESFT and less than 30% of patients with metastatic disease. Intensified chemotherapy has not improved the patients survival. Also, due to absence of biologic indicators of aggressiveness, stratification of patients for more aggressive treatments is difficult. Because chemotherapeutic agents kill tumor cells through the mitochondrial apoptotic pathway which is inhibited by survivin, we studied survivin expression in ESFT tissues from 35 patients with localized disease and correlated its expression with patient survival. We found a statistically significant poor outcome of patients with survivin-positive vs. survivin-negative tumors. This suggests that survivin may be used as a prognostic indicator and a possible target in ESFT patients with drug-resistant tumors.We also explored the possibility of engaging alternative pathways to kill ESFT cells, such the receptor-mediated apoptotic pathway, using TRAIL as a ligand. TRAIL binds with two death receptors (DR) 4 and 5 and induces apoptosis through activation of caspase 8. Because it kills only tumor and not normal cells, TRAIL is a desirable alternative agent for drug-resistant tumors. We showed that ESFT cells are TRAIL sensitive in vitro, except when they express low caspase 8. Furthermore, we found that in 25% of ESFT specimens caspase 8 is expressed in less than 50% of tumor cells in a given tumor sample. These data suggested that a significant number of ESFT patients may have TRAIL-resistant tumors due to low caspase 8 expression and may benefit from treatments that upregulate caspase 8, such as interferon. Furthermore, we noticed that in addition to low caspase 8 expression, TRAIL-resistant ESFT have high expression of survivin. Because low caspase 8-expressing cells cannot activate the apoptotic pathway directly, but depend on the mitochondria to amplify the apoptotic signal, we hypothesized that in TRAIL-resistant ESFT cells with low caspase 8 expression, the mitochondrial pathway is blocked by a survivin-dependent mechanism. Indeed, we showed that survivin functions through binding and neutralization of the mitochondrial protein Smac, and that survivin silencing releases Smac from the survivin/Smac complex and reverses TRAIL resistance. Our data indicate that survivin also contributes to TRAIL resistance in ESFT cells and can serve as a therapeutic target in ESFT treated with TRAIL. More recently, we found that survivin is transcriptionally upregulated by EWS/FLI-1 through activation of the NF-kB pathway and that stably EWS/FLI-1 silenced ESFT clones express low survivin and are extemely sensitive to TRAIL. Because it has been postulated that ESFT patients in clinical remission may have circulating undetectable tumor cells which temporarily do not express EWS/FLI-1, but are capable of recurrences, we hypothesized that the EWS/FLI-1 silenced clones may express the phenotype of these cells and are in the process of further evaluating their biology. We are also interested in exploring the relevance of apoptosis protein expression in ESFT specimens from patients treated with TRAIL receptor agonistic antibodies, such as the HGS-ETR2 monoclocal antibody, which is currently in a phase 1 clinical trial at the Pediatric Oncology Branch, National Cancer Institute. 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.
