Issn Print: 0960-8931

Publication Date: 2006/09/01


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Deregulation of critical cell cycle regulatory genes in melanocytic nevi with the B-RAF V600E mutation

S. Bloethner; J. Lorenzo Bermejo; E. Snellman; K. Hemminki; R. Kumar

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Author Information: Division of Molecular Genetic Epidemiology, German Cancer Research Center, Heidelberg, Germany

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Purpose of the Study Human melanomas are characterized by a constitutively activated RAS/RAF/MAP-kinase signaling pathway through autocrine growth factor stimulation or oncogenic mutations in the B-RAF or N-RAS genes. Somatic mutations in the B-RAF gene occur in a majority of melanomas, with the V600E alteration in the kinetic activation domain of the gene being the most common. Mutations in the B-RAF, owing to presence in melanocytic nevi at a high frequency, are considered early but insufficient genetic events in melanoma development. Nevertheless, recent evidence has suggested an active role for B-RAF mutations at the nevus stage. In this study, we investigated the differential global gene expression in melanocytic nevi with and without the V600E B-RAF mutation.
Description We determined the mutational status of the B-RAF gene in 30 nevi excised from 10 individuals. Global gene expression using microarray technology was determined and compared in 18 nevi with mutations and four without mutations. Statistical significance of the difference in gene expression between the two groups was assessed by Wilcoxon tests. Principal component analysis was used to investigate the relationship between global gene expression and the mutational status. Finally, pathway analysis and geneontology using the differentially expressed genes were utilized to identify networks of interacting genes, and to map differentially expressed genes to known signaling pathways. Microarray data for selected genes were validated by quantitative real-time PCR.
Results and Conclusions Our data showed significant upregulation of 93 genes and downregulation of 105 genes in nevi with the V600E B-RAF mutation compared with those without mutation. In the principal component analysis, a relationship between mutational status and gene expression was clearly observed. Pathway analysis and geneontology revealed a significant deregulation of genes associated with cell death, cellular growth and proliferation, and cell cycle in nevi with B-RAF mutations. Important pathways affected by the V600E B-RAF mutation included the integrin, ERK/MAPK, and G-protein coupled receptor signaling pathways, as well as G2/M DNA damage checkpoint regulation. The tumor suppressor gene CDKN2A with a known major role in cellular senescence was found to be significantly overexpressed in nevi with B-RAF mutations. In addition, another significantly upregulated gene identified was CDKN1C.
Thus, our results support the hypothesis that the B-RAF mutation per se does not transform melanocytes, but rather induces senescence through induction of CDKN2A. In addition, overexpression of CDKN1C points to its possible role in melanocyte senescence. Melanocytic nevi probably require a second genetic ‘hit’ in the form of loss of CDKN2A and probably also CDKN1C to overcome oncogene-induced senescence and to undergo transformation.

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