Activating mutations in the BRAF kinase have been identified in most of the human melanomas and nevi screened for mutations in the kinase domain of this gene, of which >90% are BRAFV599E. Mutated BRAF is essential for growth in melanoma cells, as shown by a recent study using siRNA. The mutant form of the molecule is responsible for the constitutive activation of the Ras/MAPK pathway, resulting in high levels of phosphorylated ERK that promote cell growth and proliferation. In the present study, we use oligonucleotide microarrays to screen for gene expression changes between normal melanocytes without the BRaf mutation and melanoma cell lines with the V599E BRAF mutation. Our focus is to identify changes in the genes of the Ras/BRAF/MAPK pathway as well as other pathways, that may be a result of, or act in parallel with the BRAF mutant. Initial analysis and validation of the array results revealed changes of gene groups that were suggested by previous studies to be important for the development and progression of melanoma. The groups of genes induced in melanoma cells include matrix metalloproteinases, integrin subunits and growth factors such as VEGF, HGF, bFGF, PDGF-b and their receptors. E- and P-cadherins were found to be down-regulated in melanoma cells. Microarray meta-analysis was also performed to identify changes in expression of genes belonging to the Ras/BRAF/MAPK pathway. Using gene ontology keywords for the Ras/MAPK pathway, we retrieved gene expression data from ∼150 probe sets of the U133 Affymetrix array, corresponding to genes of this pathway. This screening process revealed up-regulation of MKP and sprouty genes in the melanoma cell lines. Both genes are known to act as inhibitors of the pathway, in response to increased levels of phosphorylated MAPK (ERK), therefore participating in a negative feedback mechanism. Our gene expression profiling suggests that there are changes in factors upsteam of BRAF that may act in parallel with the mutation, as well as changes in inhibitors of the BRAF/MAPK pathway, that may be a result of the BRAF-mediated ERK phosphorylation.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]