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FAT1 is a novel upstream regulator of HIF1? and invasion of high grade glioma.

ABSTRACT: The hypoxic microenvironment is an important contributor of glioblastoma (GBM) aggressiveness via HIF1?, while tumour inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study was designed to explore the functional interaction and significance of FAT1 and HIF1? under severe hypoxia-mimicking tumour microenvironment in primary human tumours. We first identified a positive correlation of FAT1 with HIF1? and its target genes in GBM tumour specimens, revealing the significance of the FAT1-HIF1? axis in glioma cells. We found that severe hypoxia leads to an increased expression of FAT1 and HIF1? in U87MG and U373MG cells. To reveal the relevance of FAT1 under hypoxic conditions, we depleted endogenous FAT1 under hypoxia and found a substantial reduction in the expression of HIF1? and its downstream target genes like CA9, GLUT1, VEGFA, MCT4, HK2, BNIP3 and REDD1, as well as a significant reduction in the invasiveness in GBM cells. At the molecular level, under hypoxia the FAT1 depletion-associated reduction in HIF1? was due to compromised EGFR-Akt signaling as well as increased VHL-dependent proteasomal degradation of HIF1?. In brief, for the first time, these results reveal an upstream master regulatory role of FAT1 in the expression and role of HIF1? under hypoxic conditions and that FAT1-HIF1? axis controls the invasiveness of GBM. Hence, FAT1 represents a novel potential therapeutic target for GBM.

SUBMITTER: Madan E

PROVIDER: S-EPMC6585695 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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FAT1 is a novel upstream regulator of HIF1α and invasion of high grade glioma.

Madan Evanka E Dikshit Bhawana B Gowda Srinivas H SH Srivastava Chitrangda C Sarkar Chitra C Chattopadhyay Parthaprasad P Sinha Subrata S Chosdol Kunzang K

International journal of cancer 20160829 11

The hypoxic microenvironment is an important contributor of glioblastoma (GBM) aggressiveness via HIF1α, while tumour inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study was designed to explore the functional interaction and significance of FAT1 and HIF1α under severe hypoxia-mimicking tumour microenvironment in primary human tumours. We first identified a positive correlation of FAT1 with HIF1α and its target genes in GBM tumour specimens, revealing the significanc ...[more]

PMID: 27536856

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Project description: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-1α, stemness genes and EMT markers in glioma cells. Here, we reveal NFкB (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 NFкB (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 NFкB (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 NFкB (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-NFкB (RelA)-motifs] showed the highest promoter activity. Exposure of GBM cells to known NFкB (RelA)-activators [severe-hypoxia/TNF-α/ectopic-NFкB (RelA) + IKBK vectors] led to increased pGL3F1-promoter activity and increased endogenous-FAT1 expression. Conversely, siRNA-mediated NFкB (RelA) knockdown led to decreased pGL3F1-promoter activity and decreased endogenous-FAT1 expression. Deletion of NFкB (RelA)-motif at - 90 bp/- 80 bp [pGL3F1δ1-construct] showed significant decrease in promoter activity. Site directed mutagenesis at -90 bp/- 80 bp and ChIP assay for endogenous-NFкB (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 NFкB (RelA). CONCLUSION:Since FAT1 and NFкB (RelA) are independently known to promote pro-tumorigenic inflammation and upregulate the expression of HIF-1α/EMT/stemness in tumors, targeting the NFкB (RelA)-FAT1 axis may attenuate an important tumor-promoting pathway in GBM. This may also be applicable to other tumors.

Project description:BackgroundMalignant glioma is the second leading cause of cancer-related death worldwide, and is known to exhibit a high degree of heterogeneity in its deregulation of different oncogenic pathways. The molecular subclasses of human glioma are not well known. Thus, it is crucial to identify vital oncogenic pathways in glioma with significant relationships to patient survival.MethodsIn this study, we devised a bioinformatics strategy to map patterns of oncogenic pathway activation in glioma, from the Gene Expression Omnibus (GEO). Bioinformatics analysis revealed that 749 genes were differentially expressed and classified into different glioma grades.ResultsUsing gene expression signatures, we identified three oncogenic pathways (MAPK signaling pathway, Wnt signaling pathway, and ErbB signaling pathway) deregulated in the majority of human glioma. Following gene microarray analysis, the gene expression profile in the differential grade glioma was further validated by bioinformatic analyses, with coexpression network construction. Furthermore, we found that cytochrome c oxidase subunit Vb (COX5B), the terminal enzyme of the electron transport chain, was the central gene in a coexpression network that transfers electrons from reduced cytochrome c to oxygen and, in the process, generates an electrochemical gradient across the mitochondrial inner membrane. The expression level of COX5B was then detected in 87 glioma tissues as well as adjacent normal tissues using immunohistochemistry. We found that COX5B was significantly upregulated in 67 of 87 (77.0%) glioma and glioblastoma tissues, compared with adjacent tissue (p<0.01). Furthermore, statistical analysis showed the COX5B expression level was significantly associated with clinical stage and lymph node status, while there were no correlations between COX5B expression and age or tumor size.ConclusionThese data indicate that COX5B may be implicated in glioma pathogenesis and as a biomarker for identification of the pathological grade of glioma.

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FAT1 is a novel upstream regulator of HIF1? and invasion of high grade glioma.

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FAT1 is a novel upstream regulator of HIF1α and invasion of high grade glioma.

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