Tumor Suppressor Genes in Heritable Melanoma Models

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DESCRIPTION (provided by applicant): Interspecies hybrids from Xiphophorus fish have been used for decades to investigate genetic determinants of melanoma formation. Genetic and molecular analyses of inheritance of spontaneous and UV-induced melanomas in several Xiphophorus backcross hybrids have resulted in the discoveries of an EGFR-related oncogene - the Xiphophorus melanoma receptor kinase, Xmrk2 - and a melanoma susceptibility gene belonging to the CDKN2 family of cyclin-dependent kinase inhibitors, CDKN2X., which we discovered during the course of the funding of this grant. However, although the genetic evidence implicating this gene in melanoma susceptibility in Xiphophorus BC1 hybrids from particular crosses is strong and compelling, the specific role of this putative cell cycle regulator in tumorigenesis in Xiphophorus remains undefined. Our recent studies have shown that CDKN2X is overexpressed in primary melanomas, and that overexpression correlates with overexpression of other cell cycle regulating genes including cyclin D1 as well as overexpression of the upstream tyrosine receptor kinase, Xmrk2. In this project, we propose to investigate several cell cycle regulating genes in Xiphophorus organisms and cell culture models, including cyclin D1, Rb, and forkhead transcription factors. We will develop approaches and systems that will provide an experimental framework for investigating the cell physiology and biochemistry of melanoma formation in Xiphophorus, thereby providing greater scope for exploitation of this unique experimental melanoma model. We will characterize some of the early cellular and biochemical changes occurring in primary melanoma development. To accomplish this goal, we will (a) determine the relationship(s) between cell cycle regulating components and melanoma development in primary melanomas from Xiphophorus hybrids, (b) characterize the expression of specific cell cycle-regulating genes, at both the RNA and protein levels, in primary melanomas generated from Xiphophorus F1 and BC1 hybrids, at different temporal stages of tumor development; these results will be correlated with pathological descriptions of the tumors in situ, and spatial and temporal descriptions will be synthesized in an attempt to identify how primary melanomas manifest changes in cell cycle regulation as they develop. We will also (c) exploit cell culture models to investigate mechanisms by which Xmrk2 influences the expression of CDKN2X and other cell cycle regulators in cell culture models.