The Ca2+/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
The Ca2+/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. 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 BRAFV600E was explored by BRAFV600E 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 Methylprednisolone hemisuccinate wild-type cells, overexpression of BRAFV600E 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. Conclusion: NFAT2 and 4 are expressed in human metastatic melanoma cell lines and are activated by oncogenic BRAFV600E 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 Methylprednisolone hemisuccinate 30% of all human cancers, the potential to exploit NFAT signalling for therapeutic benefit warrants further investigation. gene, occurring in 50C70% of all melanomas (Davies mutations are also common in melanoma occurring in up to 30% of Methylprednisolone hemisuccinate cases, and as the occurrence of or mutation in melanoma is mutually exclusive, up to 90% of melanomas harbour a mutated, hyperactive RasCRAF signalling pathway (Davies activating mutation accounts for approximately 90% of mutations in melanoma and BRAFV600E drives melanomagenesis in mice (Dhomen and Marais, 2007; Dankort is also mutated in up to 80% of benign melanocytic naevi (Pollock and mutations respectively (Dhomen and Marais, 2007). NFAT transcriptional activity was approximately 10-fold higher in A375 cells and approximately 70-fold higher in WM266-4 cells compared with wild-type CHL-1 cells (Figure 1A), consistent with increased B-RAF activity of BRAFV600D compared with BRAFV600E (Wan vehicle control. (C) Schematic of BRAFV600E-induced NFAT activation. NFAT regulates COX-2 protein induction in metastatic melanoma cell lines The importance of NFAT signalling in regulating COX-2 promoter activation and protein production was evaluated in BRAF-mutated melanoma cells using COX-2 luciferase reporter vectors that selectively lacked functional NFAT binding sites, calcineurin inhibitors and siRNA-mediated knockdown of NFAT2 and 4. Kl Treating A375 and WM266-4 cells with the classical NFAT activators TPA and ionomycin (TPA/iono) (Hogan cells treated with TPA/iono+Vh. Open in a separate window Figure 4 Small interfering RNA (siRNA)-mediated knockdown of nuclear factor of activated T-cells (NFAT) 2 or NFAT4 reduces cyclooxygenase-2 (COX-2) protein induction. WM266-4 and A375 cells were transfected with siRNA targeting either NFAT2 (A) or NFAT4 (B). 48?h post-transfection, cells were treated with TPA (50?nM) and ionomycin (1?control vector containing all functional NFAT binding sites ($). Discussion Previous studies have shown that NFAT signalling is important in haematological Methylprednisolone hemisuccinate malignancies and solid tumours (Buchholz and Ellenrieder, 2007; Medyouf and Ghysdael, 2008) and that NFAT is activated by environmental carcinogens such as ultraviolet radiation (Flockhart mutations are present in 29C69% of papillary thyroid carcinomas (Wojciechowska and Lewinski, 2006), are also common in colorectal cancers (Ogino (active in approximately 30% of all human cancers) signals partly via MEK/ERK (Schubbert may be limited as long term, Methylprednisolone hemisuccinate systemic application in transplant patients causes renal toxicity and increases cancer risk because of reduced immunosurveillance (Botti in the absence of significant toxicity (Yu et al, 2007) and may prove to be valuable as therapeutic NFAT antagonists. In summary, NFAT is expressed and is transcriptionally active in human metastatic melanoma cell lines and is activated by oncogenic BRAFV600E via canonical MEK/ERK signalling. Although NFAT is overexpressed in other cancers, activation has never previously been linked to the mutation of a specific oncogene. Data indicating that COX-2 expression in melanoma is regulated by NFAT further suggest that NFAT merits additional investigation as a transcription factor important in melanoma.