STAT1 mediated downregulation of the tumor suppressor gene PDCD4, is driven by the atypical cadherin FAT1, in glioblastoma.

STAT1 (Signal Transducer and Activator of Transcription 1), belongs to the STAT protein family, essential for cytokine signaling. It has been reported to have either context dependent oncogenic or tumor suppressor roles in different tumors. Earlier, we demonstrated that Glioblastoma multiforme (GBMs) overexpressing FAT1, an atypical cadherin, had poorer outcomes. Overexpressed FAT1 promotes pro-tumorigenic inflammation, migration/invasion by downregulating tumor suppressor gene, PDCD4. Here, we demonstrate that STAT1 is a novel mediator downstream to FAT1, in downregulating PDCD4 in GBMs. In-silico analysis of GBM databases as well as q-PCR analysis in resected GBM tumors showed positive correlation between STAT1 and FAT1 mRNA levels. Kaplan-Meier analysis showed poorer survival of GBM patients having high FAT1 and STAT1 expression. SiRNA-mediated knockdown of FAT1 decreased STAT1 and increased PDCD4 expression in glioblastoma cells (LN229 and U87MG). Knockdown of STAT1 alone resulted in increased PDCD4 expression. In silico analysis of the PDCD4 promoter revealed four putative STAT1 binding sites (Site1-Site4). ChIP assay confirmed the binding of STAT1 to site1. ChIP-PCR revealed decrease in the binding of STAT1 on the PDCD4 promoter after FAT1 knockdown. Site directed mutagenesis of Site1 resulted in increased PDCD4 luciferase activity, substantiating STAT1 mediated PDCD4 inhibition. EMSA confirmed STAT1 binding to the Site 1 sequence. STAT1 knockdown led to decreased expression of pro-inflammatory cytokines and EMT markers, and reduced migration/invasion of GBM cells. This study therefore identifies STAT1 as a novel downstream mediator of FAT1, promoting pro-tumorigenic activity in GBM, by suppressing PDCD4 expression.

Homeodomain Transcription Factors Nkx2.2 and Pax6 as Novel Biomarkers for Meningioma Tumor Treatment.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01085-1.

Elucidating the Role of miRNA-326 Modulating Hedgehog Signaling in Pancreatic Carcinoma.

BACKGROUND AND AIM: Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies worldwide characterized by poor prognosis. MicroRNAs (miRNAs) function as the key regulators in carcinogenesis and may act as noninvasive biomarkers in various malignancies including PDAC. The present study aimed to elucidate the role of miR-326, a known modulator of hedgehog (Hh) pathway in PDAC. MATERIALS AND METHODS: miR-326 circulating levels were assessed in 105 PDAC patients, 31 with chronic pancreatitis (CP) and 36 healthy controls by quantitative Polymerase chain reaction. The expression of miR-326 and smoothened (SMO) was checked in surgical PDAC tissue. SMO protein expression was analyzed by immunohistochemistry in different groups. Finally, the role of miR-326 as a modulator of Hh pathway was assessed in vitro. RESULTS: Our results demonstrate that miR-326 is downregulated in both blood and tissue of PDAC patients as compared with controls. In contrast, the target gene/protein expression of SMO is upregulated in PDAC. Moreover, the tumor stromal expression of SMO was found to be clinically associated with lymph-node metastasis and vascular encasement in PDAC. Overexpression of miR-326 in Panc1 cell line was found to induce downregulation of SMO suggesting the tumor suppressor role of miR-326 in PDAC. CONCLUSIONS: Taken together, miR-326 acts as a tumor suppressor in PDAC by modulating Hh pathway. It may be a promising target for the development of efficient drug therapies for the treatment of PDAC.

Poly(lactic-co-glycolic) acid nanoparticles localize in vesicles after diffusing into cells and are retained by intracellular traffic modulators.

Aim: We investigated our previous finding of increased retention of poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs) with metabolic inhibitors (MI) and studied the effect of some small molecule inhibitors on PLGA-NP assimilation. Materials & methods: Intracellular PLGA-NP colocalization in the presence of MI was investigated by confocal microscopy. Intracellular retention of PLGA-NPs by some small molecules was estimated by fluorescence microscopy and flow cytometry after Pulse/Chase experiments. Results: MI caused PLGA-NP colocalization in intracellular membranous structures, mainly endosomes and lysosomes. Some small molecule inhibitors demonstrated increased intracellular PLGA-NP accumulation. Conclusion: This study elucidates the movement of PLGA-NP in cells and suggests that clinically used small molecules can reduce their extrusion by enhancing their stay within intracellular vesicles, with possible clinically beneficial consequences.

Nanoparticles are increasingly being used to carry drugs for treatment of cancer. We wish to decrease their movement out of the cells. This may give time for them to unload their drugs. Cells were treated with nanoparticles for 30 min and observed. Then the nanoparticles were washed off. Cells were again observed after 30 min. Various intracellular trafficking inhibitors were also added. Nanoparticle retention and subcellular localization were measured. We found that nanoparticles are trapped in some membranous compartments within the cells after energy depletion. We also discovered some commonly used clinical molecules that can decrease the excretion of nanoparticles from the cells. These inhibitors can be utilized for increasing the intracellular stay of the drug-loaded nanoparticles.

Prognostic significance of melanogenesis pathway and its association with the ultrastructural characterisation of melanosomes in uveal melanoma.

BACKGROUND: Pigmentation could be a relevant prognostic factor in uveal melanoma (UM) development. Microphthalmia-associated transcription factor (MITF) regulates melanin synthesis by activating tyrosinase-related protein 2 (TYRP2) and silver protein (SILV) that induce the melanogenesis pathway. Although their oncogenic potential has been observed in various malignancies but has not been investigated in UM Asian population. Our aim is to study the ultrastructure of melanosomes and the prognostic significance of pigmentation markers such as TYRP2, MITF and SILV in UM. METHODS: Transmission electron microscopy was performed to compare the ultrastructure of melanosomes in the normal choroid and UM cases. Immunoexpression of TYRP2, SILV and MITF was analysed in 82 UM samples. The mRNA expression level of all genes was measured in 70 UM cases. A statistical correlation was performed to determine the prognostic significance of all markers. RESULTS: Premelanosomes and mature melanosomes undergoing dedifferentiation were observed in high-pigmented UM cases as compared with low-pigmented UM cases. Seventy per cent of UM cases showed high SILV expression while TYRP2 and MITF expression was present in 58% and 56% of cases, respectively. At the mRNA level, upregulation of TYRP2, SILV and MITF markers was seen in around 50% of UM cases, which was statistically significant with high pigmentation. Reduced metastatic-free survival was statistically significant with the MITF protein expression. CONCLUSION: Our results demonstrated that ultrastructural changes in melanosomes and high expression of TYRP2, MITF and SILV could dysregulate the melanogenesis pathway and might be responsible for the aggressive behaviour of UM.

Protumorigenic role of the atypical cadherin FAT1 by the suppression of PDCD10 via RelA/miR221-3p/222-3p axis in glioblastoma.

The atypical cadherin FAT1 function either as a pro or antitumorigenic in tumors of different tissue origins. Our group previously demonstrated the protumorigenic nature of FAT1 signaling in glioblastoma (GBM). In this study, we investigated how FAT1 influences the expression of clustered oncomiRs (miR-221-3p/miR-222-3p) and their downstream effects in GBM. Through several experiments involving the measurement of specific gene/microRNA expression, gene knockdowns, protein and cellular assays, we have demonstrated a novel oncogenic signaling pathway mediated by FAT1 in glioma. These results have been verified using antimiRs and miR-mimic assays. Initially, in glioma-derived cell lines (U87MG and LN229), we observed FAT1 as a novel up-regulator of the transcription factor NFκB-RelA. RelA then promotes the expression of the clustered-oncomiRs, miR-221-3p/miR-222-3p, which in turn suppresses the expression of the tumor suppressor gene (TSG), PDCD10 (Programmed cell death protein10). The suppression of PDCD10, and other known TSG targets (PTEN/PUMA), by miR-221-3p/miR-222-3p, leads to increased clonogenicity, migration, and invasion of glioma cells. Consistent with our in-vitro findings, we observed a positive expression correlation of FAT1 and miR-221-3p, and an inverse correlation of FAT1 and the miR-targets (PDCD10/PTEN/PUMA), in GBM tissue-samples. These findings were also supported by publicly available GBM databases (The Cancer Genome Atlas [TCGA] and The Repository of Molecular Brain Neoplasia Data [Rembrandt]). Patients with tumors displaying high levels of FAT1 and miR-221-3p expression (50% and 65% respectively) experienced shorter overall survival. Similar results were observed in the TCGA-GBM database. Thus, our findings show a novel FAT1/RelA/miR-221/miR-222 oncogenic-effector pathway that downregulates the TSG, PDCD10, in GBM, which could be targeted therapeutically in a specific manner.

A Combination of Inflammatory and Hematological Markers is Strongly Associated with the Risk of Death in Both Mild and Severe Initial Disease in Unvaccinated Individuals with COVID-19 Infection.

Background: Inflammatory and hematological markers are used extensively for early prognostication and monitoring in COVID-19.We aimed to determine whether routinely prescribed laboratory markers can predict adverse outcome at presentation in COVID-19. Methods: This retrospective observational study was performed on 401 samples collected between July to December 2020 from COVID-19 positive subjects, admitted at All India Institute of Medical Sciences, Delhi, India. Clinical details and laboratory investigations within 3 days of COVID-19 positivity were obtained. Clinical outcomes were noted from patient medical records, till discharge or death. Laboratory parameters, with individually defined cut-offs, were used, either singly or in combination to distinguish survival and death for those having severe and non-severe disease at initial presentation. Findings: Total Leukocyte count, Absolute neutrophil count, Neutrophil to Lymphocyte ratio, C-Reactive Protein (CRP), Interleukin-6 (IL-6), Lactate Dehydrogenase, Ferritin and Lymphocyte to CRP ratio (LCR) were significantly altered at presentation in severe COVID-19 as compared to non-severe cases; and, also in those who died due to COVID-19 compared to those who survived. A combination of four markers, CRP (≥3.9mg/dL); IL-6 (≥45.37pg/ml); Ferritin (≥373ng/mL); 1/LCR ≥0.405 was found to strongly predict mortality in cases with non-severe presentation as also in severe cases. Conclusion and Interpretation: The combination of routinely used markers, CRP, IL-6, Ferritin and 1/LCR can be used to predict adverse outcomes, even in those presenting with mild to moderate disease. This would identify subset of patients who would benefit from closer monitoring than usual for non-severe disease.

Differential role of Pax6 and its interaction with Shh-Gli1-IDH2 axis in regulation of glioma growth and chemoresistance.

Glioma is a major brain tumor, and the associated mortality rate is very high. Contemporary therapies provide a chance of survival for 9-12 months. Therefore, a novel approach is essential to improve the survival rate. Sonic hedgehog (Shh) cell signaling is critical for early development in various tumors. This investigation attempted to explore the potential interaction and regulation of Shh-Gli1 cell signaling in association with paired box 6 (Pax6) and isocitrate dehydrogenase 2 (IDH2). The expression pattern of Shh, Gli1, Pax6, and IDH2 was examined by transcriptome analysis, immunohistochemistry, and confocal images. The results suggest the interaction of Shh-Gli1 cell signaling pathway with Pax6 and IDH2 and potential regulation. Thereafter, we performed protein-protein docking and molecular dynamic simulations (MDS) of Gli1 with Pax6 and IDH2. The results suggest differential dynamic interactions of Gli1-IDH2 and Gli1-Pax6. Gli1 knockdown downregulated the expression of Pax6 and upregulated the expression of IDH2. Moreover, Gli1 knockdown decreased the expression of the drug resistance gene MRP1. The knockdown of Pax6 gene in glioma cells downregulated the expression of Gli1 and IDH2 and promoted cell proliferation. Moreover, the efficacy of the treatment of glioma cells with temozolomide (TMZ) and Gli1 inhibitor GANT61 was higher than that of TMZ alone. MDS results revealed that the interactions of Gli1 with IDH2 were stronger and more stable than those with Pax6. Intriguingly, inhibition of Pax6 promoted glioma growth even in the presence of TMZ. However, the tumor-suppressive nature of Pax6 was altered when Gli1 was inhibited by GANT61, and it showed potential oncogenic character, as observed in other cancers. Therefore, we conclude that Pax6 interacted with IDH2 and Gli1 in glioma. Moreover, the Shh-Gli1-IDH2/Pax6 cell signaling axis provides a new therapeutic approach for inhibiting the progression of the disease and mitigating drug resistance in glioma.

microRNAs in exhaled breath condensate for diagnosis of lung cancer in a resource-limited setting: a concise review.

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.

Molecular Characterization of IDH Wild-type Diffuse Astrocytomas: The Potential of cIMPACT-NOW Guidelines.

IDH wild-type (wt) grade 2/3 astrocytomas are a heterogenous group of tumors with disparate clinical and molecular profiles. cIMPACT-NOW recommendations incorporated in the new 2021 World Health Organization (WHO) Classification of Central Nervous System (CNS) Tumors urge minimal molecular criteria to identify a subset that has an aggressive clinical course similar to IDH -wt glioblastomas (GBMs). This paper describes the use of a panel of molecular markers to reclassify IDH -wt grade 2/3 diffuse astrocytic gliomas (DAGs) and study median overall survival concerning for to IDH -wt GBMs in the Indian cohort. IDH -wt astrocytic gliomas (grades 2, 3, and 4) confirmed by IDHR132H immunohistochemistry and IDH1/2 gene sequencing, 1p/19q non-codeleted with no H3F3A mutations were included. TERT promoter mutation by Sanger sequencing, epidermal growth factor receptor amplification, and whole chromosome 7 gain and chromosome 10 loss by fluorescence in situ hybridization was assessed and findings correlated with clinical and demographic profiles. The molecular profile of 53 IDH -wt DAGs (grade 2: 31, grade 3: 22) was analyzed. Eleven cases (grade 2: 8, grade 3: 3) (20.75%) were reclassified as IDH -wt GBMs, WHO grade 4 ( TERT promoter mutation in 17%, epidermal growth factor receptor amplification in 5.5%, and whole chromosome 7 gain and chromosome 10 loss in 2%). Molecular GBMs were predominantly frontal (54.5%) with a mean age of 36 years and median overall survival equivalent to IDH -wt GBMs (18 vs. 19 mo; P =0.235). Among grade 2/3 DAGs not harboring these alterations, significantly better survival was observed for grade 2 versus grade 3 DAGs (25 vs. 16 mo; P =0.002). Through the incorporation of a panel of molecular markers, a subset of IDH -wt grade 2 DAGs can be stratified into molecular grade 4 tumors with prognostic and therapeutic implications. However, IDH -wt grade 3 DAGs behave like GBMs irrespective of molecular profile.

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.

Vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1ɑ) in lacrimal gland Adenoid cystic carcinoma: Correlation with clinical outcome.

PURPOSE: VEGF and HIF-1α are important regulators of angiogenesis, overexpressed in various tumors. Lacrimal gland Adenoid cystic carcinoma (ACC) is a malignant tumor whose angiogenic properties remain unexplored. This study was designed to evaluate the expression of HIF-1α and VEGF in lacrimal gland ACC. METHODS: VEGF and HIF-1α immunoexpression was undertaken in 30 lacrimal gland ACC cases. mRNA expression of VEGF and HIF-1α was analysed in 17 cases by quantitative real time PCR. The results obtained were correlated with clinicopathological features and survival of the patients to determine the prognostic significance. RESULTS: Immunoexpression of HIF-1α and VEGF was seen in 36.6% and 46.6% ACC cases. HIF-1α expression showed significant association with advanced T-stage (P = 0.001) and VEGF with intracranial extension (P = 0.014) and solid histological pattern (P = 0.045). HIF-1α mRNA expression was seen in 29.4% cases and showed significant association with perineural invasion (P = 0.027). Recurrence occurred in 60%, distant metastasis in 20% and death in 20% cases. Survival analysis revealed that patients with HIF-1α, VEGF immunoexpression, solid histological pattern, perineural invasion, bone erosion, intracranial extension, metastasis, advanced T-stage, and exenteration had poor survival. On multivariate analysis VEGF immunoexpression (hazard ratio, 16.785; 95% confidence interval, 1.872-150.495; P = 0.012) was the most significant poor prognostic factor. CONCLUSIONS: This study demonstrates that VEGF is a potential predictor for poor clinical outcome in lacrimal gland Adenoid cystic carcinoma.

Identification of Gender-Specific Molecular Differences in Glioblastoma (GBM) and Low-Grade Glioma (LGG) by the Analysis of Large Transcriptomic and Epigenomic Datasets.

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.

The quest for ligands and binding partners of atypical cadherin FAT1.

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.

Prognostic impact of Notch1 receptor and clinicopathological High-Risk Predictors in lacrimal gland adenoid cystic carcinoma.

PURPOSE: Adenoid Cystic Carcinoma (ACC) is an aggressive malignant lacrimal gland tumour associated with poor prognosis. Aberrant Notch signalling has been investigated in various tumours. However, very few studies on Notch signalling in lacrimal gland ACC are reported. The aim of the present study was to evaluate the status of Notch1 receptor and activated Notch1 (NICD1) in lacrimal gland ACC and to correlate it with high-risk clinicopathological features. METHODS: A total of 23 cases of histopathologically proven lacrimal gland ACC, who underwent surgical treatment, were included in this study. Expression of Notch1 receptor and NICD1 was evaluated by immunohistochemistry on formalin fixed paraffin embedded tissues. The results obtained were correlated with clinicopathological high-risk features and survival of the patients. Kaplan-Meier survival and multivariate analysis was performed to determine the prognostic significance. RESULTS: Overexpression of Notch1 receptor and NICD1 was observed in 65% and 39% of lacrimal gland ACC cases, respectively. On Kaplan-Meier survival analysis, patients with Notch1 receptor overexpression had reduced disease free survival. On univariate analysis, male gender, bone erosion, perineural invasion, solid histologic pattern, intracranial extension and advanced tumour stage were also indicators of poor prognosis. On multivariate analysis bone erosion was the most significant poor prognostic indicator. CONCLUSION: Our study demonstrates that overexpression of Notch1 receptor plays a critical role in the biology and aggressive behaviour of lacrimal gland ACC. Bone erosion, solid histologic pattern, advanced T stage, perineural invasion and intracranial extension are other high-risk clinicopathological predictors of lacrimal gland ACC.

Passive internalization and active extrusion determines PLGA-nanoparticle concentration in cancer cell lines.

Background: Poly(lactic-co-glycolic) acid nanoparticle (PLGA-NP) trafficking across cell membranes was investigated to confirm preliminary results that contradicted existing studies. Materials & methods: Uptake and retention of PLGA-NPs at 37 and 4°C in the presence and absence of metabolic inhibitors in various cell lines was estimated. Results: Pulse experiments with metabolic inhibitors and culturing at 4°C demonstrated the predominantly passive nature of PLGA-NP uptake. Chase experiments with metabolic inhibitors indicated the role of active exocytosis in the extrusion of these NPs. PLGA-NPs with ionic or nonionic hydrophilic coats with highly positive or negative ζ-potential also showed similar results. Conclusion: Our study opens up the possibility of modulation of active exocytosis to increase intracellular retention of NPs for an extended period of drug delivery.

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.

Serum Angiopoietin-1 and -2 and VEGF are associated with severe disease in vivax malaria.

BACKGROUND & OBJECTIVES: Malaria continues to be a significant public health problem in tropical countries including India; however, there are limited tools to predict occurrence of severe disease due to malaria. This study was designed to evaluate the role of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Vascular endothelial growth factor (VEGF) and Asymmetric Dimethylarginine (ADMA)as disease biomarkers in uncomplicated malaria (UM) and severe malaria (SM). METHODS: This is a prospective observational study carried out at All India Institute of Medical Sciences (AIIMS), tertiary referral hospital in New Delhi, India. The study population included patients diagnosed with malaria (Plasmodium falciparum or Plasmodium vivax) either by rapid diagnostic kit test or positive peripheral smear and age more than 12 years. Forty-nine patients (25 with SM, 24 with UM) and 22 controls were recruited. In addition to routine investigations, serum concentrations of Ang-1, Ang-2, VEGF and ADMA were measured using ELISA technique. RESULTS: We observed Ang-1 serum levels to be significantly lower in patients with severe malaria (7775 pg/ml) compared to uncomplicated malaria (17629 pg/ml) and healthy controls (43472 pg/ml) [p <0.001]. Ang-2 levels were significantly higher in severe malaria (11100 pg/ml) compared to uncomplicated malaria (7315 pg/ml) and healthy controls (3679 pg/ml) (p <0.001). The ratio of Ang-2/Ang-1 was significantly higher in patients with severe malaria. VEGF serum levels was significantly lower in severe malaria (130.36 pg/ml) compared to uncomplicated malaria (317.3 pg/ml). The Ang-1, Ang-2 and VEGF levels were able to differentiate severe malaria from uncomplicated malaria caused by P. vivax but not with P. falciparum. INTERPRETATION & CONCLUSION: We conclude that Ang-1, Ang-2 and VEGF are markers of disease severity in vivax malaria.

Vimentin overexpression as a novel poor prognostic biomarker in eyelid sebaceous gland carcinoma.

BACKGROUND: Vimentin is an intermediate-sized filament which is highly expressed in mesenchymal cells and is associated with epithelial-mesenchymal transition (EMT). EMT markers ZEB2 and Slug lead to Vimentin overexpression and E-cadherin loss, resulting in invasion and metastasis. However, the status of Vimentin remains unexplored in eyelid sebaceous gland carcinoma (SGC). The study aims to determine status of Vimentin in SGC and its association with EMT markers E-cadherin, ZEB2 and Slug. METHODS: Vimentin protein expression was undertaken in 66 cases with SGC by immunohistochemistry (IHC). Messenger RNA (mRNA) expression was determined in 42 fresh tissues by quantitative real-time PCR. Association of Vimentin with E-cadherin, ZEB2 and Slug was also analysed. Patients were followed up for 17-69 months (mean 34.02 ± 14.73 months). RESULTS: IHC revealed Vimentin overexpression in 37/66 (56%) cases. This overexpression showed significant association with lymph node metastasis (p=0.004) and pagetoid spread (p=0.05). Patients with high Vimentin expression also had poor disease-free survival (p=0.033). Univariate Cox regression model indicated that high Vimentin expression (p=0.043) and advanced tumour stage (p=0.002) were independent adverse prognostic factors. High Vimentin mRNA expression was seen in 16/42 (38%) cases and correlated significantly with lymph node metastasis (p=0.027), advanced tumour stage (p=0.002) and large tumour size (p=0.023). Vimentin expression overall showed a significant inverse association with E-cadherin and direct association with ZEB2 expression. CONCLUSIONS: Vimentin overexpression in SGC is associated with EMT and leads to poor clinical outcome. It also emerged as a novel predictor for lymph node metastasis and poor survival.

Differential expression of p52 and RelB proteins in the metastatic and non-metastatic groups of uveal melanoma with patient outcome.

PURPOSE: Non-canonical NFκB (NC-NFκB) pathway plays an influential role in metastasis, which promotes cancer proliferation and progression. The aim of the study was to examine the expression of NC-NFκB proteins and their correlation with clinicopathological factors associated with metastatic cases of uveal melanoma (UM) and with the patient outcome. METHOD: Expression of NC-NFκB proteins (p52, RelB, and co-expression of p52/RelB) was evaluated in 75 formalin-fixed cases of uveal melanoma by immunohistochemistry. Validation of nuclear immunoreactivity was done by western blotting. Transcriptional status of NC-NFκB genes was assessed in 60 fresh tumor tissues by quantitative real-time PCR. Co-immunoprecipitation was performed to determine the presence of native p52/RelB heterodimer in UM. Prognostic relevance was determined using Cox proportional hazard and Kaplan-Meier methods. RESULTS: Immunohistochemical expression of p52, RelB, and their co-expression was observed in 81%, 68.7%, 56.2% of metastatic cases, respectively, while their expression was seen only in 38%, 33% and 30% of non-metastatic cases. Loss of BAP-1 was correlated with expression of p52 and RelB proteins. Co-immunoprecipitation assay confirmed the putative interaction of p52 with RelB protein in metastatic cases of uveal melanoma. Co-expression of p52/RelB and expression of p52 protein was significantly correlated with decreased metastasis-free survival (MFS) (p = 0.004; p = 0.002) and overall survival (OS) (p = 0.004; p = 0.032), while the RelB expression only correlated with reduced MFS (p = 0.003). CONCLUSION: Our data showed that non-canonical NFκB proteins were significantly higher in metastatic cases and associated with poor outcome of the patients. Furthermore, the p52 protein could be used as a potential therapeutic biomarker for metastatic cases in uveal melanoma.