One of the best defined apoptotic pathways is mediated by the surface receptor Fas, which upon ligation with the Fas ligand (FasL) induces the formation of a death-inducing signaling complex (DISC). The DISC consists of the Fas receptor, the adaptor molecule FADD and FLICE/caspase 8. Caspase 8 is activated at the DISC and triggers the proteolytic activation of downstream caspases, either directly (type I cells), or indirectly through release of cytochrome c from the mitochondria and activation of caspase 9 (type II cells). In type II cells, the bcl-2 family of proteins are important inhibitors of apoptosis inhibiting the release of cytochrome c from the mitochondria. Various tumor cell types express Fas and FasL, yet not all of them have a functional Fas/FasL pathway. Because cytotoxic drugs induce apoptosis, irrespective of their intracellular target, and Fas- resistant tumors show cross resistance to drug-induced apoptosis in vitro, we made the hypothesis that tumors with a defective Fas/FasL signaling pathway are refractory to treatment and exhibit a more aggressive behavior. We have investigated the signaling components of the Fas/FasL pathway in tumors of the Ewings sarcoma family (ESF) and in neuroblastoma (NB), in order to understand the molecular events that lead to their impaired apoptosis and hence treatment failures. This approach is expected to lead to the discovery of novel therapeutic agents in ESF tumors and NB.We have found that many ESF tumors are Fas- sensitive in vitro, whereas NB cell lines with high N-myc copy number are Fas-resistant. We also demonstrated that in NB cells, the Fas signaling pathway requires participation of mitochondria (type II cells) and by downregulating bcl-2 with antisense oligonucleotides we can reverse their resistance to Fas-mediated apoptosis. We are investigating the therapeutic efficacy of combined antisense bcl-2 treatment and chemotherapy in a NB model in SCID mice. Because high N- myc copy numbers have been associated with aggressive NB biology in vivo, and aggressive NB is refractory to existing therapeutic modalities, a combination treatment with chemotherapeutic agents and antisense bcl2 oligonucleotides may prove to be beneficial in vivo. Our data have also shown that NB cells with a defective Fas signaling pathway in vitro show expression of a protein that has been recently shown to inhibit the activation of FLICE/caspase 8 at the DISC and known as FLICE-inhibitory protein (FLIP). We have shown FLIP to be present not only in NB cell lines but also in tumor tissues from patients with aggressive NB. In the Fas-responsive ESFT cell lines, we found that we can induce apoptosis not only by ligation with a Fas activating antibody, but also by inducing enhanced expression of Fas and FasL in tumor cells. This can be accomplished either post- translationally after treatment with synthetic metalloproteinase inhibitors (MMPIs) which inhibit the cleavage of the full length FasL molecule to its soluble form, or transcriptionally by upregulation of the synthesis of the the Fas and FasL molecules by chemotherapeutic agents. We confirmed the induction of FasL by chemotherapeutic agents in ESF tumor cells by FasL promoter activation experiments. Furthermore, we found that the chemotherapeutic agents can induce FasL expression through activation of the NF-kB transcription factor. We further confirmed the requirement of the FasL molecule for the induction of apoptosis by chemotherapeutic agents by generating stable transfectants with antisense FasL sequences which blocked FasL synthesis and inhibited the drug-induced apoptosis in ESF tumor cells. These findings support that chemotherapeutic agents use the Fas/FasL pathway to induce apoptosis in ESF tumors. We also showed that MMPI- treatment had a synergistic effect with chemotherapeutic agents in vitro, thus supporting a role for synthetic MMPIs in the treatment of ESF tumors. - apoptosis, Ewing's sarcoma family of tumors (ESFT), neuroblastoma, Fas/FasL, MMPIs, metalloproteinase inhibitors, - Human Tissues, Fluids, Cells, etc.
