Abstract 3534: FAT1: A potential target of NFkB (RelA) in GBM

Abstract

Abstract FAT1 is a large transmembrane protein (502 kD), reportedly having dual role in different human cancers. Our lab has reported oncogenic role of FAT1 overexpression in glioblastoma multiforme (GBM, grade IV glioma) increasing migration-invasion as well as tumor inflammation and HIF1α activity. Morris et al. (2013), have reported that glioma patients with low FAT1-expressing tumors have significantly (p=0.037) longer survival (Rembrandt dataset), suggesting high FAT1 expression to have adverse effect on GBM patient survival. Since NFκB is a major transcription factor overexpressed in GBM and involve in regulating many oncognes at transcriptional level. There are no reports available on FAT1 upstream regulation. Therefore in this study we investigated the role of NFkB (RelA) in regulating FAT1 expression in GBM. In-silico analysis of FAT1 promoter predicted binding sites for several transcription factors including NFkB (RelA) having multiple binding motifs with high matrix score. We assessed the mRNA expression correlation of FAT1 and NFκB target gene (BCL2) in small cohort of GBM tumors (n=16). A significant positive correlation (0.647, p<0.01) was established between FAT1 and BCL2 in GBM tumors compared to normal brain (Clontech). In a sequential bioinformatics study, we analyzed TCGA GBM dataset and found significantly elevated expression of FAT1, RelA, IKBKB and RelA target genes (BCL2, MMP9 and Serpine) in GBM tumors (n=430) as compared to normal brain (n=10). In survival analysis of TCGA dataset {Grouped into high (n=100) and low FAT1 expression (n=100)}, low FAT1 expressing patients showed higher survival, corroborating the observation of Morris et al 2013. The role of NFkB (RelA) in FAT1 expression regulation was further analyzed in in-vitro in U87MG (GBM) cell line. We overexpressed and chemically inhibited NFkB using RelApBABE expression vector and PDTC respectively. There was increased FAT1expression in cells overexpressing NFkB and this effect could be reversed on NFkB inhibition, suggesting that RelA plays an important role in regulation of endogenous FAT1 expression. Furthermore, to confirm the regulatory role of NFκB (RelA) on FAT1 expression, 2-kb FAT1 promoter (pGLF2) and sequential 5’ deletion constructs (pGLF2A, pGLF2B, pGLF2C and pGLF2D) with different RelA binding sites were cloned into luciferase basic vector (pGL3B) and analyzed for their activity by in vitro luciferase assay. Among all constructs, 5’ deletion construct (pGLF2C) containing single consensus binding site for RelA and least no. of repressor sites, showed highest luciferase activity in U87MG. Furthermore, site directed mutagenesis of RelA consensus binding site decreased luciferase activities by three-fold as compared to the wild type construct, pGLF2C. The endogenous binding of RelA on FAT1 promoter was confirmed by ChIP (chromatin-immunoprecipitation) assay. Conclusively, our study for the first time identified NFkB (RelA) transcription factor as a potential regulator of FAT1. Citation Format: Chitrangda Srivastava, Khushboo Irshad, Parthaprasad Chattopadhyay, Chitra Sarkar, Ashish Suri, Subrata Sinha, Kunzang Chosdol. FAT1: A potential target of NFkB (RelA) in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3534. doi:10.1158/1538-7445.AM2017-3534