NF?B is a critical transcriptional regulator of atypical cadherin FAT1 in glioma.

ABSTRACT: BACKGROUND:Overexpression of FAT1 gene and its oncogenic effects have been reported in several cancers. Previously, we have documented upregulation of FAT1 gene in glioblastoma (GBM) tumors which was found to increase the expression of proinflammatory markers, HIF-1?, stemness genes and EMT markers in glioma cells. Here, we reveal NF?B (RelA)/RelA/p65 as the transcriptional regulator of FAT1 gene in GBM cells. METHODS:In-silico analysis of FAT1 gene promoter was performed using online bioinformatics tool Promo alggen (Transfac 8.3) to identify putative transcription factor(s) binding motifs. A 4.0?kb FAT1 promoter (-?3220?bp to +?848?bp w.r.t. TSS?+?1) was cloned into promoter less pGL3Basic reporter vector. Characterization of FAT1 promoter for transcriptional regulation was performed by in-vitro functional assays using promoter deletion constructs, site directed mutagenesis and ChIP in GBM cells. RESULTS:Expression levels of NF?B (RelA) and FAT1 were found to be increased and positively correlated in GBM tumors (n?=?16), REMBRANDT GBM-database (n?=?214) and TCGA GBM-database (n?=?153). In addition to glioma, positive correlation between NF?B (RelA) and FAT1 expression was also observed in other tumors like pancreatic, hepatocellular, lung and stomach cancers (data extracted from TCGA tumor data). A 4.0?kb FAT1-promoter-construct [-?3220?bp/+?848?bp, transcription start site (TSS)?+?1, having 17 NF?B (RelA) motifs] showed high FAT1 promoter luciferase-activity in GBM cells (U87MG/A172/U373MG). FAT1 promoter deletion-construct pGL3F1 [-?200?bp/+?848?bp, with 3-NF?B (RelA)-motifs] showed the highest promoter activity. Exposure of GBM cells to known NF?B (RelA)-activators [severe-hypoxia/TNF-?/ectopic-NF?B (RelA)?+?IKBK vectors] led to increased pGL3F1-promoter activity and increased endogenous-FAT1 expression. Conversely, siRNA-mediated NF?B (RelA) knockdown led to decreased pGL3F1-promoter activity and decreased endogenous-FAT1 expression. Deletion of NF?B (RelA)-motif at -?90?bp/-?80?bp [pGL3F1?1-construct] showed significant decrease in promoter activity. Site directed mutagenesis at -90?bp/-?80?bp and ChIP assay for endogenous-NF?B (RelA) confirmed the importance of this motif in FAT1 expression regulation. Significant reduction in the migration, invasion as well as colony forming capacity of the U87MG glioma cells was observed on siRNA-mediated knockdown of NF?B (RelA). CONCLUSION:Since FAT1 and NF?B (RelA) are independently known to promote pro-tumorigenic inflammation and upregulate the expression of HIF-1?/EMT/stemness in tumors, targeting the NF?B (RelA)-FAT1 axis may attenuate an important tumor-promoting pathway in GBM. This may also be applicable to other tumors.

SUBMITTER: Srivastava C 

PROVIDER: S-EPMC6988320 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature