P01.05 Overexpression of FAT1 gene in GBM is regulated by transcription factor NFkB (RelA)

Abstract

Introduction: Glioblastoma (GBM) is the most common and aggressive form of primary brain cancer with high tendency for local invasiveness. Hypoxia is a critical feature of glioblastoma, promoting tumor invasiveness via HIF1α. In our laboratory, we have identified the oncogenic role of ‘FAT1’ in glioma. FAT1 is a human homolog of Drosophila tumor suppressor gene ‘fat’. Increased FAT1 expression in human GBM tissue and in GBM cell lines have been observed, augmenting tumor migration/invasiveness; upregulating proinflammatory microenvironment by regulating AP1 transcriptional ­activity as well as found to be modulating HIF1a expression and activity in GBM cells. Since high expression of FAT1 has been observed in GBM and other tumors, here, we have analyzed how FAT1 expression is being regulated in glioma. Materials and Methods: Online tools like transfac, DBD etc. were used to identify potential transcription factors binding sites at the FAT1 promoter. The TCGA online dataset for GBM was analyzed for gene expression study. The expression of genes in tumor samples and GBM cell lines (U87MG and A172) was done by qPCR and Western blot analysis. The transcription regulation of FAT1 gene by NFkB (RelA) was confirmed by in vitro luciferase assay of the FAT1 promoter (5kb), 5’ deletion constructs and site directed mutagenesis of transcription factor binding site at the FAT1 promoter in U87MG cells. Chromatin-immunoprecipitation (ChIP) assay was done for analyzing the binding of endogenous RelA to FAT1 promoter. Results:In silico analysis showed multiple binding sites with high matrix score for RelA on FAT1 promoter. TCGA dataset (n=430) showed significantly higher mRNA expression of FAT1, RelA, IKBKB and RelA target genes (BCL2, MMP9 and Serpine) in GBM. A positive correlation (p<0.01) of FAT1 and BCL2 mRNA was observed in GBM tumors analyzed (n=16). We further observed increased expression of FAT1 on RelA (RelApBABE vector) overexpression and downregulation of FAT1 expression upon PDTC (NFκB inhibitor) treatment in U87MG cells, suggesting RelA having a role in regulation of endogenous FAT1 expression. Furthermore, to confirm the regulatory effect of RelA on FAT1 expression, FAT1 promoter (5kb) and multiple sequential 5’deletion constructs were analyzed by in-vitro luciferase assay. Among all constructs, 5’deletion construct containing single consensus binding site for RelA showed highest luciferase activity endogenously and on exogenous RelA overexpression in U87MG cells. Site directed mutagenesis of RelA consensus binding site decreased luciferase activities by three-fold. ChIP assay confirmed the binding of endogenous RelA on FAT1 promoter. CONCLUSION: RelA transcription factor potentially regulates FAT1 expression in GBM, which is a novel finding. This finding may be clinically relevant, helping in identifying a subset of GBMs with high FAT1 and RelA expression for molecular targeting.