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)-β 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-β1/2 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-β1/2 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-β1/2 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-β1/2 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-β1/2 secretion. Reduced TGF-β1 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-β1 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-β1/2.
Differences in the incidence and outcome of glioma between males and females are well known, being more striking for glioblastoma (GB) than low-grade glioma (LGG). The extensive and well-annotated ...data in publicly available databases enable us to analyze the molecular basis of these differences at a global level. Here, we have analyzed The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases to identify molecular indicators for these gender-based differences by different methods. Based on the nature of data available/accessible, the transcriptomic profile was studied in TCGA by using DeSeq2 and in CGGA by T-test, after correction based. Only IDH1 wild-type tumors were studied in CGGA. Using weighted gene co-expression network analysis (WGCNA), network analysis was done, followed by the assessment of modular differential connectivity. Differentially affected signaling pathways were identified. The gender-based effects of differentially expressed genes on survival were determined. DNA methylation was studied as an indicator of gender-based epigenetic differences. The results clearly showed gender-based differences in both GB and LGG, whatever method or database was used. While there were differences in the results obtained between databases and methods used, some major signaling pathways such as Wnt signaling and pathways involved in immune processes and the adaptive immune response were common to different assessments. There was also a differential gender-based influence of several genes on survival. Also, the autosomal genes NOX, FRG1BP, and AL354714.2 and X-linked genes such as PUDP, KDM6A, DDX3X, and SYAP1 had differential DNA methylation and expression profile in male and female GB, while for LGG, these included autosomal genes such as CNIH3 and ANKRD11 and X-linked genes such as KDM6A, MAOB, and EIF2S3. Some, such as FGF13 and DDX3X, have earlier been shown to have a role in tumor behavior, though their dimorphic effects in males and females have not been identified. Our study thus identifies several crucial differences between male and female glioma, which could be validated further. It also highlights that molecular studies without consideration of gender can obscure critical elements of biology and emphasizes the importance of parallel but separate analyses of male and female glioma.
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.
A recent study in Scientific Reports identified glypican-3 (GPC3) as a novel extracellular interacting protein for FAT1 in hepato-cellular carcinoma (HCC) cells. FAT1 is a large transmembrane ...atypical cadherin with limited knowledge existing about its binding partners. While in Drosophila, dachsous (ds), another transmembrane member of the cadherin superfamily, is known to function as FAT1 ligand, no ligand is known in mammals so far. The revelation of GPC3 as a potential binding partner of FAT1 extracellular domain unfolds an opportunity to study potential triggers of FAT1 signaling in cancers. Available inhibitors of GPC3 in various phases of clinical trials also present an attractive option to curb GPC3-FAT1 signaling in tumors that overexpress these proteins.
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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.
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.
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).
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.
Meningioma is a common brain tumour which has neither a specific detection nor treatment method. The Sonic hedgehog (Shh) cell signaling pathway is a crucial regulatory pathway of mammalian ...organogenesis and tumorigenesis including meningioma. Shh cell signalling pathway cascade function by main transcription factor Gli1 and which further regulates in its downstream to Pax6 and Nkx2.2. This current study is aimed to explore the regulation of the Sonic hedgehog-Gli1 cell signaling pathway and its potential downstream targets in meningioma samples. A total of 24 surgically resected meningioma samples were used in this current study.Cytological changes were assessed using electron microscopic techniques as well as hematoxylin & eosin and DAPI staining. The expression pattern of Gli1, Nkx2.2 and Pax6 transcription factors were determined by using immunohistochemistry. The mRNA expression was assessed using RT-qPCR assays. Later, the whole transcriptome analysis of samples was performed with the amploseq technique. Results were compared with those obtained in normal human brain tissue (or normal meninges). Compared to the normal human brain tissue, meningioma samples showed crowded nuclei with morphological changes. Transcription factor Nkx2.2 expressed highly in all samples (24/24, 100%). Twenty-one of the 24 meningiomas (88%) showed high Gli1 and Pax6 expression. Whole transcriptome analysis of two meningioma samples also exhibited a very high increase in Gli1 expression signal in meningioma samples as compare to normal control. Hence, we may conclude that the Shh–Gli1 pathway is aberrantly activated in meningioma cells and is canonically upregulating the expression of transcription factors Pax6 and Nkx2.2.
Summary Epithelial-mesenchymal transition (EMT) plays a pivotal role in tumor invasion and metastasis in various malignancies. ZEB2/SIP1 is an important EMT regulator and down-regulates E-cadherin ...expression. The present study was planned to explore status of EMT-associated markers ZEB2/SIP1 and E-cadherin in eyelid sebaceous gland carcinoma (SGC) and to correlate with clinicopathological high-risk features. Expressions of ZEB2 and E-cadherin were evaluated by immunohistochemistry in 65 cases of histopathologically proven eyelid SGC. The results were correlated with clinicopathological high-risk features and survival of the patients to determine the prognostic significance of ZEB2, E-cadherin, and various high-risk features. Cytoplasmic overexpression of ZEB2 and membranous loss of E-cadherin were seen in 68% and 66% of cases of eyelid SGC, respectively. ZEB2 overexpression was significantly associated with E-cadherin loss ( P = .002). Overexpression of ZEB2 also showed significant association with lymph node metastasis ( P = .046), orbital invasion ( P = .049), large tumor size ( P = .018), and advanced tumor stages ( P = .036). Survival analysis revealed that patients with ZEB2 overexpression had poor survival. ZEB2 overexpression and orbital invasion were found to be independent prognostic indicators (univariate analysis). However, multivariate analysis showed that ZEB2 (hazard ratio, 0.094; 95% confidence interval, 00.012-0.709; P = .022) was the best poor prognostic indicator of eyelid SGC. Our study demonstrates the role of both ZEB2 and E-cadherin in the promotion of EMT in eyelid SGC. The outcome of this study also points toward ZEB2 as an independent prognostic marker as well as a potential therapeutic target in eyelid SGC.
Hypoxia is a characteristic of solid tumors especially Glioblastoma and is critical to chemoresistance. Cancer stem cells present in hypoxic niches are known to be a major cause of the progression, ...metastasis and relapse. We tried to identify synergistic combinations of drugs effective in both hypoxia and normoxia in tumor cells as well as in cancer stem cells. Since COX-2 is over-expressed in subset of glioblastoma and is also induced in hypoxia, we studied combinations of a prototype Cyclooxygenase (COX-2) inhibitor, NS-398 with various drugs (BCNU, Temozolomide, 2-Deoxy-D-glucose and Cisplatin) for their ability to abrogate chemoresistance under both severe hypoxia (0.2% O
) and normoxia (20% O
) in glioma cells. The only effective combination was of NS-398 and BCNU which showed a synergistic effect in both hypoxia and normoxia. This synergism was evident at sub-lethal doses for either of the single agent. The effectiveness of the combination resulted from increased pro- apoptotic and decreased anti-apoptotic molecules and increased caspase activity. PGE
levels, a manifestation of COX-2 activity were increased during hypoxia, but were reduced by the combination during both hypoxia and normoxia. The combination reduced the levels of epithelial-mesenchymal transition (EMT) markers. It also resulted in a greater reduction of cell migration. While single drugs could reduce the number of gliomaspheres, the combination successfully abrogated their formation. The combination also resulted in a greater reduction of the cancer stem cell marker CD133. This combination could be a prototype of possible therapy in a tumor with a high degree of hypoxia like glioma.
Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 ...region (21-47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21-47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants.
Lung cancer is one of the common cancers globally with high mortality and poor prognosis. Most cases of lung cancer are diagnosed at an advanced stage due to limited diagnostic resources. Screening ...modalities, such as sputum cytology and annual chest radiographs, have not proved sensitive enough to impact mortality. In recent years, annual low-dose computed tomography has emerged as a potential screening tool for early lung cancer detection, but it may not be a feasible option for developing countries. In this context, exhaled breath condensate (EBC) analysis has been evaluated recently as a noninvasive tool for lung cancer diagnosis. The breath biomarkers also have the advantage of differentiating various types and stages of lung cancer. Recent studies have focused more on microRNAs (miRNAs) as they play a key role in tumourigenesis by regulating the cell cycle, metastasis and angiogenesis. In this review, we have consolidated the current published literature suggesting the utility of miRNAs in EBC for the detection of lung cancer.
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