New therapies to eliminate melanoma must be identified both for the high-risk patient after surgery and for metastatic disease. Interferons (IFNs) have therapeutic activity in both clinical settings but little expansion has occurred in understanding of mechanisms underlying clinical effectiveness in melanoma -- or indeed other malignancies. Underlying clinical antitumor effects must be the potent and pleiotropic gene modulatory effects that occur at a transcriptional level. Utilizing oligonucleotide array analysis, over 100 genes that are induced by IFNs in a melanoma cell line have been identified. On an equimolar basis these genes are induced to a substantially greater extent by IFN-beta than by IFN-alpha2. Based upon the hypothesis that the antitumor effects of IFNs in melanoma may result from direct effects on tumor cells, apoptosis, an understudied effect of IFNs, is a focus of our studies. Induction of apoptosis, coupled with array studies, led to the protein TRAIL (TNF-Related Apoptosis Inducing Ligand) as a mechanism that in part accounts for melanoma cell death. In sensitive melanoma cell lines, IFN-beta, but not IFN-alpha2, induces caspase dependent apoptosis that was associated with TRAIL. Resistant melanoma cell lines are characterized by lack of proliferative inhibition or apoptosis induction by either IFN- alpha2 or IFN-beta and lack of TRAIL induction. The data further suggest that apoptosis induction is negatively influenced by NFkappaB (Nuclear Factor kappa B) activation by TRAIL. With a goal of understanding of IFNs as antitumor cytokines and using melanoma as a model, our working hypothesis is that genes and functional mechanisms, yet to be identified, contribute substantially to the antitumor effects of IFNs. Our estimates based upon genome size suggest that greater than 500 interferon- stimulated genes (ISGs) remain to be identified. The goals of the proposal are: 1) By assessment of sensitive and resistant melanomas to identify new genes that may contribute to mediating apoptosis response and resistance in response to IFNs, 2) To define and confirm functional effects of the newly identified ISG, TRAIL, on IFN-induced apoptosis, 3) To determine how NF- kappaB or other induced anti-apoptotic pathways influence actions of IFNs and TRAIL, and 4) As an effective inducer of of apoptosis in mice and of TRAIL, to conduct a Phase II trial of IFN-beta to identify disease response in metastatic melanoma. The data should contribute to understanding of IFNs effects in melanoma but also other neoplasms and to a beginning understanding of the therapeutic potential of TRAIL.
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