MITF creates a selective pressure to inactivate INK4A in melanoma

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PURPOSE: Why tumor suppressor genes tend to be targeted for inactivation in specific types of tumors remains unclear. We investigated whether a relationship between the differentiation factor MITF and the cell cycle inhibitor INK4A may explain the selective pressure to inactivate INK4A in melanoma. METHODS: Cells included wildtype and INK4A-null mouse embryo fibroblasts and human melanocytes and melanoma cells. Expression vectors included MITF, MITF mutant, empty vector, MITF siRNA, and control siRNA. Cells were analyzed for morphology, BrdU incorporation, expression of melanocyte markers (S100, DCT and TRP1), and mRNA and protein expression of MITF, 16Ink4a, and Rb. Luciferase promoter-reporter assays and chromatin IP were used to analyze MITF interaction with the INK4A promoter. RESULTS: MITF bound the INK4A promoter and induced expression of p16Ink4a mRNA and protein, causing hypophosphorylation of Rb and consequent cell cycle arrest. This relationship between MITF and INK4A was required for efficient melanocyte differentiation and for maintaining mature melanocytes, and it created a selective pressure to escape growth inhibition by inactivating INK4A. CONCLUSIONS: MITF regulates cell cycle exit by activating the cell cycle inhibitor INK4A, a tumor suppressor that frequently is mutated in melanomas. These findings establish a mechanistic link between melanocyte differentiation and cell cycle exit, and potentially explain the tissue-specific tendency for INK4A mutations to occur in melanoma.