Melanoma is a common human malignancy of increasing incidence and mortality in people over the age of 45. It is also increasing rapidly in the population under age 35. Overexpression and mislocalization of beta-catenin, a cell adhesion and signaling molecule, has been shown to correlate with severity of melanoma and other malignancies. This occurs through down-regulation of its adhesive function and an increase in its transcriptional activation function, leading to unregulated proliferation. Retinoic acid (RA) is known to inhibit the growth rate and invasiveness of melanoma cell lines. Retinoids have been shown to regulate beta-catenin signaling during development and can alter its function in breast cancer cell lines to produce a less malignant phenotype. The interaction of RA and beta-catenin in melanoma has not been investigated. Our hypothesis is that beta-catenin transcriptional activity is enhanced in melanoma and that RA will inhibit this activity. The hypothesis will be tested by characterizing beta-catenin expression and function in mouse B16 melanoma cells, nonmalignant mouse melanocytes and in human melanoma cell lines derived from progressively more malignant tumors. The effect of RA on beta-catenin expression/function will be determined, including beta-catenin RNA and protein expression, cytoskeletal attachment, intracellular localization and reporter gene transactivation. It is expected that even if retinoic acid does not directly affect these measures, there will be important basic information derived on the status of beta-catenin in melanoma compared to non-malignant melanocytes. To examine a functional link between beta-catenin action and the melanoma phenotype, constructs expressing constitutively active or dominant negative beta-catenin signaling proteins will be used. Non-malignant cells will be transfected with active beta-catenin and tested for malignant transformation. Melanoma cells will be transfected with beta-catenin inhibitory proteins and examined for loss of the malignant phenotype. If it is determined that gene transactivation by beta-catenin drives a malignant phenotype in melanocytes, analysis by cDNA microarrays will be used to determine the population and pattern of genes induced. The physical interaction of beta-catenin and retinoic acid receptors will be examined. These studies will provide the basis for future investigations of mechanisms by which beta-catenin function affects the development and progression of melanoma.
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