Late stage neuroblastoma tumors, particularly those with amplified MYCN genes, have a poor prognosis, primarily due to their ability to survive treatment with multiple chemotherapeutic agents/irradiation. The continuing goal of this research program is to understand how genetic alterations, such as MYCN gene amplification and chromosome lp36 loss-of-heterozygosity (LOH), contribute to this tumor phenotype. During the last funding period we found that a critical apoptotic signaling molecule, caspase-8, is preferentially silenced by methylation in >60% of the stage 4 neuroblastoma patient tumors with simplified MYCN, whereas <4% of those stage 1-4 tumors without amplified MYCN silence expression of the CASP8 gene. A similar observation has now been made in N-Myc-induced neuroblastoma tumors from mice. We also found that reprogramed expression of caspase-8 in human NB cells that are normally caspase-8 null resensitized them to apoptosis induced by the chemotherapeutic drugs doxorubicin and cisplatin. This was observed in both cell culture and in vivo xenograft mouse models. Finally, we have reported, as have others, that caspase-8 is capable of functioning as both an initiator and executioner caspase, allowing it to amplify certain mitochondrial-mediated cell death signals. This function is somewhat unique among the caspases identified thus far, and could be one reason it is selectively silenced in certain tumors. Based upon this data we hypothesize that the silencing of CASP8 by methylation may provide a more permissive cellular environment that can tolerate the overexpression of N-Myc without undergoing cell death, and perhaps contribute to the ability of these tumor cells to survive treatment with certain chemotherapeutic drugs. To test this hypothesis we propose to develop mouse models by gene knockout or transgenic expression of an inactive, dominant negative form of caspase-8 that either totally eliminate, or down-regulate enzyme activity, and determine whether it contributes to accelerated tumor cell growth in the presence of N-Myc overexpression or the response of these tumors to chemotherapeutic drugs. These experiments will include the use of complementary approaches; namely the induction of oncogenes such as MYCN in cultured neural crest cells isolated from these mice, as well as the response of the various cells, xenografts, and in vivo tumors to therapy. Finally, we will examine these different mouse NB tumors and normal adrenal gland tissue, as well as human patient samples of various stages and matched-treated/untreated samples by microarray analysis to identify possible genes, other than CASP8, whose expression is significantly altered by N-Myc overexpression and/or drug treatment. Such studies will provide significant insight into how these tumor cells circumvent apoptosis and prolong their life.
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