Upregulation of Atypical Cadherin FAT1 Promotes an Immunosuppressive Tumor Microenvironment via TGF-ß.

FAT atypical cadherin 1 (FAT1) promotes glioblastoma (GBM) by promoting protumorigenic inflammatory cytokine expression in tumor cells. However, tumors also have an immunosuppressive microenvironment maintained by mediators such as transforming growth factor (TGF)-ß cytokines. Here, we have studied the role of FAT1 in tumor immune suppression. Our preliminary TIMER2.0 analysis of The Cancer Genome Atlas (TCGA) database revealed an inverse correlation of FAT1 expression with infiltration of tumor-inhibiting immune cells (such as monocytes and T cells) and a positive correlation with tumor-promoting immune cells [such as myeloid-derived suppressor cells (MDSCs)] in various cancers. We have analyzed the role of FAT1 in modulating the expression of TGF-ß1/2 in resected human gliomas, primary glioma cultures, and other cancer cell lines (U87MG, HepG2, Panc-1, and HeLa). Positive correlations of gene expression of FAT1 and TGF-ß1/2 were observed in various cancers in TCGA, Glioma Longitudinal Analysis Consortium (GLASS), and Chinese Glioma Genome Atlas (CGGA) databases. Positive expression correlations of FAT1 were also found with TGF-ß1/2 and Serpine1 (downstream target) in fresh-frozen GBM samples using q-PCR. siRNA-mediated FAT1 knockdown in cancer cell lines and in primary cultures led to decreased TGF-ß1/2 expression/secretion as assessed by q-PCR, Western blotting, and ELISA. There was increased chemotaxis (transmigration) of THP-1 monocytes toward siFAT1-transfected tumor cell supernatant as a consequence of decreased TGF-ß1/2 secretion. Reduced TGF-ß1 expression was also observed in THP-1 cultured in conditioned media from FAT1-depleted glioma cells, thus contributing to immune suppression. In U87MG cells, decreased TGF-ß1 upon FAT1 knockdown was mediated by miR-663a, a known modulator. FAT1 expression was also observed to correlate positively with the expression of surrogate markers of MDSCs [programmed death ligand-1 (PD-L1), PD-L2, and interleukin (IL)-10] in glioma tumors, suggesting a potential role of FAT1 in MDSC-mediated immunosuppression. Hence, our findings elaborate contributions of FAT1 to immune evasion, where FAT1 enables an immunosuppressive microenvironment in GBM and other cancers via TGF-ß1/2.

GLIS1 regulates trabecular meshwork function and intraocular pressure and is associated with glaucoma in humans.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 10-6), further supporting a role for GLIS1 into glaucoma etiology. Our study identifies GLIS1 as a critical regulator of TM function and maintenance, AqH dynamics, and IOP.

NFкB is a critical transcriptional regulator of atypical cadherin FAT1 in glioma.

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.

FAT1 modulates EMT and stemness genes expression in hypoxic glioblastoma.

Glioblastoma (GBM) is characterized by the presence of hypoxia, stemness and local invasiveness. We have earlier demonstrated that FAT1 promotes invasiveness, inflammation and upregulates HIF-1α expression and its signaling in hypoxic GBM. Here, we have identified the role of FAT1 in regulating EMT (epithelial-mesenchymal transition) and stemness characteristics in GBM. The expression of FAT1, EMT (Snail/LOX/Vimentin/N-cad), stemness (SOX2/OCT4/Nestin/REST) and hypoxia markers (HIF-1α/VEGF/PGK1/CA9) was upregulated in ≥39% of GBM tumors (n = 31) with significant positive correlation (p ≤ 0.05) of the expression of FAT1 with LOX/Vimentin/SOX2/HIF-1α/PGK1/VEGF/CA9. Furthermore, positive correlation (p ≤ 0.01) of FAT1 with Vimentin/N-cad/SOX2/REST/HIF-1α has been observed in TCGA GBM-dataset (n = 430). Analysis of cells (U87MG/A172) exposed to severe hypoxia (0.2%O2 ) revealed elevated mRNA expression of FAT1, EMT (Snail/LOX/Vimentin/N-cad), stemness (SOX2/OCT4/Nestin/REST) and hypoxia markers (HIF-1α/PGK1/VEGF/CA9) as compared to their normoxic (20%O2 ) counterparts. FAT1 knockdown in U87MG/A172 maintained in severe hypoxia and in normoxic primary glioma cultures led to significant reduction of EMT/stemness markers as compared to controls. HIF-1α knockdown in U87MG cells markedly reduced the expression of all the EMT/stemness markers studied except for Nestin and SOX2 which were more under the influence of FAT1. This indicates FAT1 has a novel regulatory effect on EMT/stemness markers both via or independent of HIF-1α. The functional relevance of our study was corroborated by significant reduction in the number of soft-agar colonies formed in hypoxic-siFAT1 treated U87MG cells. Hence, our study for the first time reveals FAT1 as a novel regulator of EMT/stemness in hypoxic GBM and suggests FAT1 as a potential therapeutic candidate.

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

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 significance of the FAT1-HIF1α axis in glioma cells. We found that severe hypoxia leads to an increased expression of FAT1 and HIF1α 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 HIF1α 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 HIF1α was due to compromised EGFR-Akt signaling as well as increased VHL-dependent proteasomal degradation of HIF1α. In brief, for the first time, these results reveal an upstream master regulatory role of FAT1 in the expression and role of HIF1α under hypoxic conditions and that FAT1-HIF1α axis controls the invasiveness of GBM. Hence, FAT1 represents a novel potential therapeutic target for GBM.

A combined gene signature of hypoxia and notch pathway in human glioblastoma and its prognostic relevance.

Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor-clusters representing high and low HIF-1α/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O2). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures.