Effect of common B-RAF and N-RAS mutations on global gene expression in melanoma cell lines

Abstract

We studied global gene expression in three melanoma cell lines with the most common and potent V600E mutation in the B-RAF gene-four cell lines with a common Q61R mutation in the N-RAS gene and three cell lines with no mutations using human HG-U133A 2.0 micro-arrays with 22 277 transcripts. Data analysis using stringent criteria revealed several upregulated and downregulated genes in cell lines with B-RAF and N-RAS mutations compared with cell lines without mutations. We found 29 genes specifically upregulated and 32 genes downregulated in cell lines with B-RAF mutations, whereas 70 genes were upregulated and 39 downregulated in cell lines with N-RAS mutations; 11 genes showed overlapping upregulation and 45 downregulation. The micro-array data for nine selected genes were validated by the real-time PCR technique. Expression of a large number of genes, that encode members or regulators of the RAS/RAF/MEK/ERK pathways or are involved in metastasis or invasion, was affected in cell lines with mutations in B-RAF and N-RAS. Upregulated genes in cell lines with mutations included dual-specificity phosphatase 6 (DUSP6), sprouty 2 (SPRY2), v-akt murine thymoma viral oncogene homolog 3 (AKT3) and matrix metalloproteinase 14 (MMP14); downregulated genes included interleukin 18 (IL18), Krüppel-like factor 5 (KLF5) and inhibitor of DNA binding 2 (ID2). Our results, though carried on cell lines, provide a novel insight into the effect of mutations in the B-RAF and N-RAS genes on global gene expression in melanoma and highlight the complexity of mechanisms involved in tumour initiation and maintenance.

Activating K-Ras mutations outwith ‘hotspot’ codons in sporadic colorectal tumours – implications for personalised cancer medicine

“…DUSPs are key regulators of the balance between kinase pathway activation and inactivation, and have previously been reported to be upregulated in an adaptive response, creating a negative feedback loop following MAPK pathway activation (Keyse, 2008). Consistent with our own data, DUSP4 expression has previously been shown to be increased in pancreatic tumours with K-Ras mutations (Yip-Schneider et al, 2001) and DUSP6 expression increased in a variety of tumour types with mutations in Ras or Raf pathway genes (Croonquist et al, 2003;Warmka et al, 2004;Bloethner et al, 2005). Of particular interest, however, was our observation that certain signalling cascades, including those mediated by Igf1 and Vegf, were differentially activated by cluster 1 and cluster 2 K-Ras mutants, suggesting that not only the presence but the specific molecular characteristics of individual K-Ras mutations may be important determinants of both tumour progression and treatment response.…”

V600E BRAF is associated with disabled feedback inhibition of RAF–MEK signaling and elevated transcriptional output of the pathway

“…Other oncogenic mutants may require loss of these proteins to effect transformation. In fact, DUSP and SPRY gene expression have been reported to be elevated in V600E BRAF melanomas (36,37), but significantly reduced in a variety of other tumors (38)(39)(40)(41). Evasion of pathway feedback may be a fundamental requirement for oncogenic transformation.…”

NRAS mutation causes a human autoimmune lymphoproliferative syndrome

“…3B). This included up-regulation of two dual-specificity phosphatases (DUSP4 and -6) that are inhibitors of ERK signaling and were previously shown to be overexpressed in tumor cell lines containing somatic activating mutations in NRAS (20).…”