title: NFAT signalling is a novel target of oncogenic BRAF in metastatic melanoma.
abstract: BACKGROUND
  Metastatic melanoma is the most deadly form of skin cancer and with an overall 5-year survival rate of <11%, there is an acute need for novel therapeutic strategies. Activating mutations in the BRAF oncogene are present in 50-70% of cases and contribute to tumourigenesis, thus, defining downstream targets of oncogenic BRAF may help define novel targets for therapeutic intervention. The Ca(2+)/calcineurin-regulated transcription factor, Nuclear factor of activated T-cells (NFAT), is important in the pathogenesis of several human cancers, target genes of which are also known to contribute to melanoma progression. One such NFAT target gene is COX-2, increased expression of which correlates with poor prognosis; however, upstream regulators of COX-2 in melanoma remain undefined. Therefore, the aim of this study was to evaluate NFAT expression and activity in metastatic melanoma and establish whether or not oncogenic BRAF signalling modulates NFAT activity and determine if NFAT is a key upstream regulator of COX-2 in melanoma.
  
  
  METHODS
  Nuclear factor of activated T-cells transcriptional activity and protein expression were determined in three human metastatic melanoma cell lines with differing B-RAF mutational status. NFAT activation by oncogenic BRAF(V600E) was explored by BRAF(V600E) overexpression and application of the specific MEK inhibitor PD98059. Regulation of COX-2 expression by NFAT was investigated using NFAT-targeted siRNA, calcineurin inhibitors cyclosporin A and FK506, in addition to COX-2 luciferase reporter vectors that selectively lacked NFAT binding sites.
  
  
  RESULTS
  NFAT transcriptional activity was increased in BRAF-mutated melanoma cells compared with wild-type cells. Furthermore, in wild-type cells, overexpression of BRAF(V600E) increased NFAT activity, which was blocked by the MEK inhibitor PD98059. Using calcineurin inhibitors and siRNA-mediated knockdown of NFAT2 and 4, we show NFAT is required for COX-2 promoter activation and protein induction in metastatic melanoma cells.
  
  
  CONCLUSIONS
  NFAT2 and 4 are expressed in human metastatic melanoma cell lines and are activated by oncogenic BRAF(V600E) via MEK/ERK signalling. NFAT is an important upstream regulator of COX-2 in metastatic melanoma. Furthermore, as the BRAF/MEK/ERK pathway is hyperactive in other malignancies and MEK/ERK are also activated by oncogenic RAS in 30% of all human cancers, the potential to exploit NFAT signalling for therapeutic benefit warrants further investigation.
authors.affiliation: Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: R J
authors.initials: RJ
authors.lastname: Flockhart
authors.forename: J L
authors.initials: JL
authors.lastname: Armstrong
authors.forename: N J
authors.initials: NJ
authors.lastname: Reynolds
authors.forename: P E
authors.initials: PE
authors.lastname: Lovat
mesh_terms: D065095:Calcineurin Inhibitors; D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000556:secondary* / Q000628:therapy; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors / Q000502:physiology*; D011401:Promoter Regions, Genetic; D048493:Proto-Oncogene Proteins B-raf / Q000502:physiology*; D015398:Signal Transduction
chemical_list: D065095:Calcineurin Inhibitors; D050778:NFATC Transcription Factors; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf