Khushboo Irshad

An insight into the diagnosis and pathogenesis of hepatitis C virus infection.

This review focuses on research findings in the area of diagnosis and pathogenesis of hepatitis C virus (HCV) infection over the last few decades. The information based on published literature provides an update on these two aspects of HCV. HCV infection, previously called blood transmitted non-A, non-B infection, is prevalent globally and poses a serious public health problem worldwide. The diagnosis of HCV infection has evolved from serodetection of non-specific and low avidity anti-HCV antibodies to detection of viral nucleic acid in serum using the polymerase chain reaction (PCR) technique. Current PCR assays detect viral nucleic acid with high accuracy and the exact copy number of viral particles. Moreover, multiplex assays using real-time PCR are available for identification of HCV-genotypes and their isotypes. In contrast to previous methods, the newly developed assays are not only fast and economic, but also resolve the problem of the window period as well as differentiate present from past infection. HCV is a non-cytopathic virus, thus, its pathogenesis is regulated by host immunity and metabolic changes including oxidative stress, insulin resistance and hepatic steatosis. Both innate and adaptive immunity play an important role in HCV pathogenesis. Cytotoxic lymphocytes demonstrate crucial activity during viral eradication or viral persistence and are influenced by viral proteins, HCV-quasispecies and several metabolic factors regulating liver metabolism. HCV pathogenesis is a very complex phenomenon and requires further study to determine the other factors involved.

FAT1 acts as an upstream regulator of oncogenic and inflammatory pathways, via PDCD4, in glioma cells

Glioblastoma multiforme (GBM) is the most aggressive and the commonest primary brain tumor with a tendency for local invasiveness. The pathways of neoplasia, invasion and inflammation are inextricably linked in cancer and aberrations in several regulatory pathways for these processes have been identified. Here we have studied the FAT1 (Homo sapiens FAT tumor-suppressor homolog 1 (Drosophila)) gene to identify its role in the tumorigenecity of the gliomas. The expression of FAT1 was found to be high in grade IV glioma cell lines (U87MG, A172, U373MG and T98G) but low in grade III glioma cell lines (GOS3 and SW1088). Two cell lines (U87MG and A172) with high FAT1 expression were chosen for in vitro FAT1-knockdown studies. FAT1 knockdown by small interfering RNA resulted in decreased migration and invasion of both the cell lines along with increased expression of the tumor-suppressor gene programmed cell death 4 (PDCD4). Increased PDCD4 expression led to the attenuation of activator protein-1 (AP-1) transcription by inhibiting c-Jun phosphorylation and resulted in concomitant decrease in the expression of AP-1-target genes like MMP3, VEGF-C and PLAU, the pro-inflammatory regulator COX-2 and cytokines IL1β and IL-6. Conversely, simultaneous silencing of PDCD4 and FAT1 in these cells significantly enhanced AP-1 activity and expression of its target genes, resulting in increase in mediators of inflammation and in enhanced migratory and invasive properties of the cells. We also observed a negative correlation between the expression of FAT1 and PDCD4 (P=0.0145), a positive correlation between the expression of FAT1 and COX-2 (P=0.048) and a similar positive trend between FAT1 and IL-6 expression in 35 primary human GBM samples studied. Taken together, this study identifies a novel signaling mechanism mediated by FAT1 in regulating the activity of PDCD4 and thereby the key transcription factor AP-1, which then affects known mediators of neoplasia and inflammation.

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.

HIF-2α mediates a marked increase in migration and stemness characteristics in a subset of glioma cells under hypoxia by activating an Oct-4/Sox-2-Mena (INV) axis

Hypoxia is a salient feature of most solid tumors and plays a central role in tumor progression owing to its multiple contributions to therapeutic resistance, metastasis, angiogenesis and stemness properties. Reports exist in literature about hypoxia increasing stemness characteristics and invasiveness potential of malignant cells. In order to delineate molecular crosstalk among factors driving glioma progression, we used knockdown and overexpression strategies. We have demonstrated that U87MG and A172 glioma cells inherently have a subset of cells with high migratory potential due to migration-inducing Mena transcripts. These cells also have elevated stemness markers (Sox-2 and Oct-4). There was a significant increase of number in this subset of migratory cells on exposure to hypoxia with corresponding elevation (over 1000 fold) in migration-inducing Mena transcripts. We were able to demonstrate that a HIF-2α-Sox-2/Oct-4-Mena (INV) axis that is strongly activated in hypoxia and markedly increases the migratory potential of the cells. Such cells also formed tumor spheres with greater efficiency. We have correlated our in-vitro results with human glioblastoma samples and found that hypoxia, invasiveness and stemness markers correlated well in native tumor samples. This study identifies a novel signaling mechanism mediated by HIF-2α in regulating invasiveness and stemness characteristics, suggesting that under hypoxic conditions, some tumor cells acquire more migratory potential by increased Pan Mena and Mena INV expression as a consequence of this HIF-2α mediated increase in Oct-4 and Sox-2. These properties would help the cells to form a new nidus after local invasion or metastasis.

Novel single-step multiplex real-time polymerase chain reaction assay for simultaneous quantification of hepatitis virus A, B, C, and E in serum.

Viral hepatitis needs an earliest diagnosis for its proper and timely treatment. Although serodiagnosis of viral hepatitis is in regular practice, however, it has certain limitations and points to alternate procedures of diagnosis. Present study was designed to develop a single‐step multiplex real‐time polymerase chain reaction (PCR) assay for detection of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis E virus (HEV) related nucleic acids in sera from infected patients.

Molecular basis of hepatocellular carcinoma induced by hepatitis C virus infection

Present study outlines a comprehensive view of published information about the underlying mechanisms operational for progression of chronic hepatitis C virus (HCV) infection to development of hepatocellular carcinoma (HCC). These reports are based on the results of animal experiments and human based studies. Although, the exact delineated mechanism is not yet established, there are evidences available to emphasize the involvement of HCV induced chronic inflammation, oxidative stress, insulin resistance, endoplasmic reticulum stress, hepato steatosis and liver fibrosis in the progression of HCV chronic disease to HCC. Persistent infection with replicating HCV not only initiates several liver alterations but also creates an environment for development of liver cancer. Various studies have reported that HCV acts both directly as well as indirectly in promoting this process. Whereas HCV related proteins, like HCV core, E1, E2, NS3 and NS5A, modulate signal pathways dysregulating cell cycle and cell metabolism, the chronic infection produces similar changes in an indirect way. HCV is an RNA virus and does not integrate with host genome and therefore, HCV induced hepatocarcinogenesis pursues a totally different mechanism causing imbalance between suppressors and proto-oncogenes and genomic integrity. However, the exact mechanism of HCC inducement still needs a full understanding of various steps involved in this process.

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.

T372R Mutation Status in Yin Yang 1 Gene in Insulinoma Patients

Insulinomas are rare pancreatic neuroendocrine tumors. The genetic causes underlying insulinoma are still being investigated. Recently, 3 independent studies reported a recurrent somatic mutation in YY1 gene (C>G; Thr372Arg) among insulinoma patients belonging to Chinese and Western Caucasian populations, which was found to increase insulin secretion by β-cells. However, the status of this key gene variation remains unknown in patients of other ethnicities. We, therefore, screened Indian sporadic insulinoma patients for YY1 T372R mutation in the present study. Seventeen patients diagnosed with insulinoma were recruited retrospectively and their records of family history and clinical parameters were collected. Formalin-fixed paraffin-embedded tumor tissues were used to extract genomic DNA, which was subjected to PCR amplification of YY1 exon 5, followed by Sanger sequencing. Nucleotide sequences thus obtained were aligned against the documented sequence of YY1 exon 5. We found absence of C to G mutation at YY1 codon 372 in all 17 (100%) insulinoma tissues analyzed. On comparison with the mutation frequency observed in the Chinese patients, our results point to genetic heterogeneity in the pathogenesis of insulinoma. This is the first report on the status of YY1 T372R in insulinoma cases of Indian origin. This also warrants analysis of other documented as well as novel mutations in genes in insulinoma tumorigenesis.

PO-125 FAT1 on salvador-warts-hippo (SWH) pathway in human glioblastoma

Introduction Glioblastoma(GBM) is an aggressive brain tumour arising from glial cells. Our lab has identified the oncogenic role of FAT1 gene in GBM, regulating inflammatory and hypoxic microenvironment of the tumour as well as migratory/invasive properties of the tumour cells. In Drosophila, fat, the ortholog of FAT1, is known to regulate the Salvador-Warts-Hippo (SWH) pathway, but its role in human is not clear. Here, we have analysed the effect of FAT1 on SWH pathway in glioma. Material and methods Glioma cell lines (U87MG, U373, A172, GOS3 and SW1088) were transfected with FAT1 specific siRNA/control siRNA and analysed the expression of SWH pathway molecules by qPCR/Western blot. Protein-protein interactions were analysed by Co-immunoprecipitation (Co-IP) after overexpression of YAP1 (wild-type and mutated) and TEAD1 with and without FAT1 knockdown. Sub-cellular localization of proteins was analysed by Confocal microscopy. Results and discussions The mRNA expression of FAT1 and SWH pathway molecules (MST1, LATS1, LATS2, YAP1 and TEAD1) was highest in U87MG cells followed by A172, U373MG and GOS3. After FAT1 knockdown, the mRNA expression of MST1 and BIRC2 were significantly decreased with no change in the levels of LATS1, LATS2, YAP1, TEAD1 and BIRC5. At protein level, increased YAP1 and phospho-YAP1 was observed after FAT1 knockdown with increased total as well as phospho-YAP1 in the cytoplasmic extract as compared to the nuclear extract. There was significant reduction in the interaction between YAP1 and TEAD1 in siFAT1 treated cells as compared to siControl treated cells. Conclusion Knockdown of FAT1 (i) increases the YAP1 protein level, could be by increasing the protein stability as no change was observed at the mRNA level (ii) it relieves the inhibitory effect on YAP1 phosphorylation, thereby increasing the phospho-YAP1 level (iii) it affects the sub-cellular localization of YAP1 by retaining YAP1 in the cytosol and thereby (iv) decrease in the YAP1:TEAD1 interaction with decreased expression of their target gene Birc2. This finding of the effect of FAT1 on YAP1 in GBM is novel with features pointing towards the oncogenic role of FAT1 by regulating YAP1 sub-cellular localization and co-transcriptional activity independent of SWH pathway.

Molecular targeting of antiviral drugs used against hepatitis C virus infection

Present study reports an update on the molecular interaction of antiviral drugs with viral and host cell components during hepatitis C virus (HCV) infection. In addition to the traditional therapeutic drug regimen, termed as standard of care, some recent drugs have been added in the existing regimen used for HCV infection. These drugs were categorized as direct-acting antivirals (DAAs) agents and “other agents”, with their efficacious impact in the control of HCV infection. They target both viral proteases and host cell receptor proteins/enzymes involved in HCV entry into the cell, replication, and assembly to check their propagation both in situ as well as in cell to cell transmission. Recent studies have reported a significant rise in sustained virological response after the use of these drugs both alone and in combination with pegylated interferon-α (PegIFN-α) plus ribavirin. Recently, DAAs have been reported to be highly effective in eradication of HCV infection, especially liver cirrhosis, reducing but not avoiding the occurrence of liver cancer. Some studies have demonstrated that the presence of resistant HCV variants, arising during viral replication, may be controlled by the new drug regimen. It is important to note here that all these drugs are influenced by viral as well as host factors including basic viral load, HCV genotypes, IFN action, interleukin 28B polymorphism and some liver and metabolic diseases, etc . This is an area with on-going investigations to explore more antiviral agents that may address new challenges in HCV therapy.