Curcumin downregulates FAT1 expression via NFkB in glioblastoma

Abstract

Background: Glioblastoma (GBM) is the most aggressive brain cancer. Curcumin, a natural dietary polyphenol extracted from Curcuma longa L, has a demonstrated promising outcome against the GBM. Many molecular targets of curcumin are still undefined. Our lab is focused on the functional role of FAT1 which is a transmembrane protein and found to have oncogenic role in GBM. This study is designed to analyze the regulation of FAT1 expression by curcumin. Methods: U87MG cells were treated with Curcumin (5uM, 10uM and 20uM) for 24hours, PDTC (Pyrrolidine dithiocarbamate) (5uM and 10uM) for1hour and TNFa (100ng/ml) for 24hours. FAT1 promoter pGL3F1 [1kb;-200bp/θ848bp from transcription start site (TSS) (θ1)] was cloned in luciferase vector (pGL3Basic) and promoter activity was analysed inU87MG, A172, and U373MG. Characterizationof NFkB binding sites was done by FAT1 deletion constructs (5' and 3' NFkB site deletion), site directed mutagenesis (SDM) and ChIP assay. FAT1 and NFkB expression were correlated inGBM tumors (n=16) and TCGA GBM data set. Results: FAT1 and NFkB was significantly downregulated in U87MG cells treated with curcumin. Also, PDTC (NFkB inhibitor) treatment showed significant decrease in FAT1 expression. But, TNFa (NFkB stimulator) treatment showed increased FAT1 expression in U87MG cells. Transfac (in-silico) analysis predicted three NFkB sites at-90bp/-80bp, θ347bp/θ356bp and θ360bp/θ368bp on1kb FAT1 promoter. Glioma cell lines were transfected with cloned FAT1 promoter constrcut (pGL3F1), showed significant increase in promoter activity. Interestingly, FAT1 promoter activity was down-regulated in curcumin treated cells and upregulated in TNFa treated cells. The most potential binding site for NFkB was found to be at-90bp/-80bp on FAT1 promoter upstream to TSS (θ1). Inour inhouse GBM tumors (n=16), increased expression and a positive correlation between FAT1 and NFkB (r = 0.70, p < 0.01) was observed, which was also corroborated in TCGA GBM data set. Conclusions: Our in-vitro and GBM tumor analysis for the first time confirms the regulation of FAT1 by curcumin via NFkB transcription factor in glioblastoma and also validating a novel link between NFkB and FAT1.