BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.

Inherited polymorphisms in immuno-modulatory genes may contribute to variations in immune function and genetic susceptibility for complex diseases, including cancer. We report results from a comprehensive study to discover novel single nucleotide polymorphisms (SNPs) and to estimate allelic frequency for both novel and known coding and regulatory region SNPs in genes encoding proteins that have been implicated in the immune response to tumors. We identified 12 novel nucleotide substitution variants and one deletion variant in 17 genes analyzed (TGFβR, β2M, IFNγ, TNFα, TNFαR, LTα, IL-6, IL-12, IL-2, IL-1α, IL-1β, IL-1RN, IL-10, CTLA4, CD40L, Fas and FasL). We determined the frequency of these novel polymorphisms, as well as 17 previously identified polymorphisms, in a control sample of 158 individuals, approximately half of which were Caucasian (n = 74) and half of which were African American (n = 84). Significant differences in allele frequencies were observed between the two racial groups for 13/17 genes tested. These allelic variations maybe associated with alterations in immune function and thus susceptibility to a number of complex disease states such as cancer.