Melanoma is a serious form of skin cancer which is rising in the United States. Our studies plus others demonstrated that interspecies Xiphophorus hybrids exhibit spontaneous and environmentally induced tumors of several types, including melanoma and other cancers. Biochemical and histological similarities between Xiphophorus and human melanoma have been revealed making these hybrids an attractive model for melanoma research. We will test the hypothesis that each of these different melanoma models share a common set of modulated genetic factors (due to the interspecies cross) that predispose interspecies hybrid individuals to induced melanoma development. These genetic factors and pathways they represent may be identified by study of melanoma paradigms in these Xiphophorus hybrid models and may be translated to melanoma study in other organisms, including humans. We have employed traditional cloning as well as next generation 454 titanium (454-Ti) analyses to examine the Xiphophorus transcriptome. We have identified 10,679 unique EST sequences encoding proteins from seven different organs of adult male and female X. maculatus Jp 163 A. We propose to investigate two inter-species crosses Xiphophorus that produce animals representing a spontaneous melanoma model and an induced model whereby melanoma forms only after treatment with MNU or UVB light. In aim 1, RNA from melanoma and normal tissues will be isolated then used to synthesis normalized full-length cDNAs. 454-Ti will be employed to develop deep transcript sequence coverage of these samples. ESTs obtained from the 454-Ti from melanoma and control tissues will be added to our EST database and used to data mine those genes showing specific expression in the melanoma. In aim 2, each of the RNAs collected in aim 1 will be used to construct SAGE libraries. 454-Ti will be used for deep transcriptome sequence coverage of these SAGE libraries to develop transcript profiles of each sample. SAGE tags will be mapped to the ESTs obtained in aim 1 to identify genes that are specifically expressed in melanoma and those subsets of genes that appear to be expressed in each distinct melanoma model (spontaneous, UVB-induced, and MNU-induced). For aim 3, real time PCR and in situ hybridization will be used to validate and confirm our results. Results from these studies will further illuminate the range of genetic factors that may lead to genetic predisposition to induced melanoma. PUBLIC HEALTH RELEVANCE: Melanoma is a serious form of skin cancer. This proposal focuses on understanding the various genetic routes to melanoma, among varied Xiphophorus interspecies hybrid models will further illuminate the range of genetic factors that may be shared in humans that may lead to genetic predisposition to induced melanoma. Robust melanoma biomarker candidates can be identified in this investigation.