The proposal will elucidate critical regulators of the TRAIL signaling pathway that determine the exquisite sensitivity of 4/7 pediatric rhabdomyosarcomas (RMS) to TRAIL-induced cytotoxicity and apoptosis at concentrations < 1 ng/ml. All cultured cell lines express high levels of receptor DR5, but not DR4 or the decoy receptors DcR1 or DcR2, and express FADD and procaspase-8, with the exception of Rh36. Expression of c-FLIP is high in TRAIL-sensitive and -resistant lines, thereby not correlating directly with TRAIL sensitivity. Expression of procaspases-8 and -10 is highest in TRAIL-sensitive RD, Rh1 and Rh30. TRAIL-sensitive Rh18 expresses procaspase-8 in the absence of procaspase-10 and c-FLIP, and procaspase-10 is not expressed in TRAIL-resistant Rh41. Based upon these cellular characteristics the following hypotheses will be tested: 1) Following ligation of TRAIL to DR5, a DISC is formed among DR5, FADD, procaspase-8 and c-FLIP in RMS cell lines resulting in type I (direct) or type II (mitochondria- and procaspase-10- dependent) cell death, and 2) TRAIL resistance in Rh41 is due to deficiency in caspase-10. The second focus is to elucidate agents that can sensitize TRAIL- resistant RMS to TRAIL, or that can elicit synergistic interactions with TRAIL. Combination of TRAIL with actinomycin-D or doxorubicin has demonstrated > additive effects in TRAIL-sensitive RMS lines, and with recombinant human interferon-gamma (IFN-gamma) has induced cytotoxic response in TRAIL-resistant HT29 human .colon carcinoma cells. We will therefore test the following hypotheses: 1) TRAIL- resistant Rh28 and Rh41 can be sensitized to TRAIL by cycloheximide, actinomycin-D or IFN-gamma, and 2) specific chemotherapeutic agents can be identified that elicit synergistic interactions with TRAIL in TRAIL-sensitive RMS cell lines. Thirdly, we will test the hypothesis that TRAIL induces antitumor responses in human RMS xenografts when administered alone, or in combination with chemotherapeutic agents based upon data derived in tissue culture. The long-term objectives of the proposal are to develop highly effective therapy for metastatic disease in pediatric RMS from: 1) understanding specific signaling pathways involved in the regulation of cell death and apoptosis, 2) the identification of new molecular targets, and 3) developing new therapeutic strategies based upon specific molecular characteristics.
