ABSTRACT
During embryonic development the placental vasculature acts as a major hematopoietic niche, where endothelial to hematopoietic transition ensures emergence of hematopoietic stem cells (HSCs). However, the molecular mechanisms that regulate the placental hematoendothelial niche are poorly understood. Using a parietal trophoblast giant cell (TGC)-specific knockout mouse model and single-cell RNA-sequencing, we show that the paracrine factors secreted by the TGCs are critical in the development of this niche. Disruptions in the TGC-specific paracrine signaling leads to the loss of HSC population and the concomitant expansion of a KDR+/DLL4+/PROM1+ hematoendothelial cell-population in the placenta. Combining single-cell transcriptomics and receptor-ligand pair analyses, we also define the parietal TGC-dependent paracrine signaling network and identify Integrin signaling as a fundamental regulator of this process. Our study elucidates novel mechanisms by which non-autonomous signaling from the primary parietal TGCs maintain the delicate placental hematopoietic-angiogenic balance and ensures embryonic and extraembryonic development.
ABSTRACT
Methyltransferase-like 3 (METTL3), the catalytic enzyme of methyltransferase complex for m6A methylation of RNA, is essential for mammalian development. However, the importance of METTL3 in human placentation remains largely unexplored. Here, we show that a fine balance of METTL3 function in trophoblast cells is essential for successful human placentation. Both loss-of and gain-in METTL3 functions are associated with adverse human pregnancies. A subset of recurrent pregnancy losses and preterm pregnancies are often associated with loss of METTL3 expression in trophoblast progenitors. In contrast, METTL3 is induced in pregnancies associated with fetal growth restriction (FGR). Our loss of function analyses showed that METTL3 is essential for the maintenance of human TSC self-renewal and their differentiation to extravillous trophoblast cells (EVTs). In contrast, loss of METTL3 in human TSCs promotes syncytiotrophoblast (STB) development. Global analyses of RNA m6A modification and METTL3-RNA interaction in human TSCs showed that METTL3 regulates m6A modifications on the mRNA molecules of critical trophoblast regulators, including GATA2, GATA3, TEAD1, TEAD4, WWTR1, YAP1, TFAP2C and ASCL2, and loss of METTL3 leads to depletion of mRNA molecules of these critical regulators. Importantly, conditional deletion of Mettl3 in trophoblast progenitors of an early post-implantation mouse embryo also leads to arrested self-renewal. Hence, our findings indicate that METLL3 is a conserved epitranscriptomic governor in trophoblast progenitors and ensures successful placentation by regulating their self-renewal and dictating their differentiation fate.
ABSTRACT
Epstein-Barr virus (EBV) contributes to ~1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naïve B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus, ectopic expression of EBNA2 and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.
Subject(s)
Epstein-Barr Virus Infections , Herpesvirus 4, Human , Humans , Herpesvirus 4, Human/physiology , Epstein-Barr Virus Infections/genetics , B-Lymphocytes , Virus Latency , Trans-Activators/genetics , Virus Activation , Gene Expression Regulation, ViralABSTRACT
The placenta establishes a maternal-fetal exchange interface to transport nutrients and gases between the mother and the fetus. Establishment of this exchange interface relies on the development of multinucleated syncytiotrophoblasts (SynT) from trophoblast progenitors, and defect in SynT development often leads to pregnancy failure and impaired embryonic development. Here, we show that mouse embryos with conditional deletion of transcription factors GATA2 and GATA3 in labyrinth trophoblast progenitors (LaTPs) have underdeveloped placenta and die by ~embryonic day 9.5. Single-cell RNA sequencing analysis revealed excessive accumulation of multipotent LaTPs upon conditional deletion of GATA factors. The GATA factor-deleted multipotent progenitors were unable to differentiate into matured SynTs. We also show that the GATA factor-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. Loss of either GATA2 or GATA3 in cytotrophoblast-derived human trophoblast stem cells (human TSCs) drastically inhibits SynT differentiation potential. Identification of GATA2 and GATA3 target genes along with comparative bioinformatics analyses revealed that GATA factors directly regulate hundreds of common genes in human TSCs, including genes that are essential for SynT development and implicated in preeclampsia and fetal growth retardation. Thus, our study uncovers a conserved molecular mechanism, in which coordinated function of GATA2 and GATA3 promotes trophoblast progenitor-to-SynT commitment, ensuring establishment of the maternal-fetal exchange interface.
Subject(s)
Gene Expression Regulation, Developmental , Maternal-Fetal Exchange , Pregnancy , Female , Humans , Animals , Mice , Placenta , Trophoblasts , Cell Differentiation/physiology , Fetal Development , GATA Transcription FactorsABSTRACT
Landslides are a natural threat that poses a severe risk to human life and the environment. In the Kumaon mountains region in Uttarakhand (India), Nainital is among the most vulnerable areas prone to landslides inflicting harm to livelihood and civilization due to frequent landslides. Developing a landslide susceptibility map (LSM) in this Nainital area will help alleviate the probability of landslide occurrence. GIS and statistical-based approaches like the certainty factor (CF), information value (IV), frequency ratio (FR) and logistic regression (LR) are used for the assessment of LSM. The landslide inventories were prepared using topography, satellite imagery, lithology, slope, aspect, curvature, soil, land use and land cover, geomorphology, drainage density and lineament density to construct the geodatabase of the elements affecting landslides. Furthermore, the receiver operating characteristic (ROC) curve was used to check the accuracy of the predicting model. The results for the area under the curves (AUCs) were 87.8% for logistic regression, 87.6% for certainty factor, 87.4% for information value and 84.8% for frequency ratio, which indicates satisfactory accuracy in landslide susceptibility mapping. The present study perfectly combines GIS and statistical approaches for mapping landslide susceptibility zonation. Regional land use planners and natural disaster management will benefit from the proposed framework for landslide susceptibility maps.
Subject(s)
Landslides , Humans , Geographic Information Systems , Satellite Imagery , Machine Learning , TechnologyABSTRACT
The role of epigenetic alteration in prostate cancer pathogenesis was reported. We aimed to analyze dysregulation of DNA methylase (DNA methyl transferase/DNMT) and demethylase (ten eleven translocase/TET) and the associated interplay between them during prostate tumorigenesis. Promoter methylation and RNA/protein expression of selected DNMT and TETs were analysed in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer (PCa). Genomic 5-hydroxymethylcytosine (5hmC) level was detected and correlated with DNMT and TET proteins. Clinicopathological association of molecular data was done. Our data revealed a very low frequency of promoter methylation for DNMT1 (5-3% and high frequency for TET1 (22-38%), TET2 (68-90 %), and TET3 (43-32 %) in BPH and PCa. The promoter methylation of DNMT1 (p = 0.019) showed a significantly decreasing trend, while that of TET1 (p = 0.0005) and TET2 (p < 0.0001) showed an increasing trend from normal prostate to BPH to PCa, indicating their epigenetic dysregulation during prostate tumorigenesis. RNA/protein overexpression of DNMT1 and reduced expression of TET1 and TET2 in PCa compared to BPH were associated with the promoter methylation status of genes. The 5hmC level was significantly lower in PCa than in BPH and correlated negatively with DNMT1 but positively with TET1 and TET2 proteins, suggesting dysregulation of DNA methylase and de-methylase activities during prostate tumorigenesis. Lastly, tumors having methylated TET1 and TET2 promoters showed advanced clinicopathological features (a higher PSA level/Gleason score) and increased risk of bone metastasis. In conclusion, DNMT1 upregulation and epigenetic silencing of TET1 and TET2 was seen during PCa development. TET1 and TET2 promoter methylation has prognostic importance.
Subject(s)
Prostatic Hyperplasia , Prostatic Neoplasms , Male , Humans , Prostate/metabolism , Prognosis , Prostatic Hyperplasia/genetics , Prostatic Hyperplasia/metabolism , Proto-Oncogene Proteins/metabolism , DNA Methylation/genetics , Epigenesis, Genetic , Carcinogenesis/genetics , Cell Transformation, Neoplastic/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , DNA , RNA/metabolism , Mixed Function Oxygenases/metabolismABSTRACT
Actively treadmilling FtsZ acts as the pivotal scaffold for bacterial cell divisome components providing them with a circumferential ride along the site of future division. FtsZ from slow growing Helicobacter pylori (HpFtsZ), a class I carcinogen which thrives abundantly in the acidic environment is poorly understood. We studied HpFtsZ as a function of pH, cations and time and compared it with well-studied E. coli FtsZ (EcFtsZ). HpFtsZ shows pH dependent GTPase activity which is inhibited under acidic conditions. Mg+2 ions play an indispensable role in its GTPase activity, however, higher Mg+2 levels negatively affect its activity. As compared to EcFtsZ, HpFtsZ exhibits lower and slower nucleotide hydrolyzing activity. Molecular Dynamics Simulation studies of FtsZ reveal that GTP binding induces a rewiring of the hydrogen bond network which results in reduction of the binding cleft volume leading to the spontaneous release of GTP. The GTPase activity is linked to the extent of reduction in the binding cleft volume, which is also supported by the binding free energy analysis. Evidently, HpFtsZ is a pH sensitive GTPase with low efficiency that may reflect on the overall slow growth rate of H. pylori.
ABSTRACT
We analyze the phase diffusion, quantum fluctuations and their spectral features of a one-dimensional Bose-Josephson junction (BJJ) nonlinearly coupled to a bosonic heat bath. The phase diffusion is considered by taking into account of random modulations of the BJJ modes causing a phase loss of initial coherence between the ground and excited states, whereby the frequency modulation is incorporated in the system-reservoir Hamiltonian by an interaction term linear in bath operators but nonlinear in system (BJJ) operators. We examine the dependence of the phase diffusion coefficient on the on-site interaction and temperature in the zero- and π-phase modes and demonstrate its phase transition-like behavior between the Josephson oscillation and the macroscopic quantum self-trapping (MQST) regimes in the π-phase mode. Based on the thermal canonical Wigner distribution, which is the equilibrium solution of the associated quantum Langevin equation for phase, coherence factor is calculated to study phase diffusion for the zero- and π-phase modes. We investigate the quantum fluctuations of the relative phase and population imbalance in terms of fluctuation spectra which capture an interesting shift in Josephson frequency induced by frequency fluctuation due to nonlinear system-reservoir coupling, as well as the on-site interaction-induced splitting in the weak dissipative regime.
ABSTRACT
Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.
Subject(s)
Placenta , Placentation , Transcriptional Coactivator with PDZ-Binding Motif Proteins , Trophoblasts , Cell Differentiation , Female , Hippo Signaling Pathway , Humans , Infant, Newborn , Placenta/metabolism , Placentation/physiology , Pregnancy , Premature Birth/physiopathology , Transcriptional Coactivator with PDZ-Binding Motif Proteins/genetics , Transcriptional Coactivator with PDZ-Binding Motif Proteins/metabolism , Trophoblasts/cytology , Trophoblasts/metabolismABSTRACT
Apart from other risk factors, chronic inflammation is also associated with the onset of Prostate Cancer (PCa), wherein pathogen infection and tissue microbiome dysbiosis are known to play a major role in both inflammatory response and cancer development. However, except for a few studies, the link between microbes and PCa remained poorly understood. To explore the potential microbiome signature associated with PCa in Indian patients, we investigated differential compositions of commensal bacteria among patients with benign prostatic hyperplasia (BPH) and PCa using 16S rRNA amplicon sequencing followed by qPCR analyses using two distinct primer sets. Using two independent cohorts, we show that Prevotella copri, Cupriavidus campinensis, and Propionibacterium acnes represent the three most abundant bacteria in diseased prostate lesions. LEfSe analyses identified that while Cupriavidus taiwanensis and Methylobacterium organophilum are distinctly elevated in PCa samples, Kocuria palustris and Cellvibrio mixtus are significantly enriched in BPH samples. Furthermore, we identify that a number of human tumor viruses, including Epstein-Barr virus (EBV) and hepatitis B virus (HBV), along with two high-risk human papillomaviruses - HPV-16 and HPV-18, are significantly associated with the PCa development and strongly correlated with PCa bacterial signature. The study may thus offer to develop a framework for exploiting this microbial signature for early diagnosis and prognosis of PCa development.
Subject(s)
Epstein-Barr Virus Infections , Prostatic Hyperplasia , Prostatic Neoplasms , Herpesvirus 4, Human/genetics , Humans , Male , Prostatic Hyperplasia/diagnosis , Prostatic Hyperplasia/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
Since its emergence in December 2019 in Wuhan, China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created a worldwide pandemic of coronavirus disease (COVID-19) with nearly 136 million cases and approximately 3 million deaths. Recent studies indicate that like other coronaviruses, SARS-CoV-2 also hijacks or usurps various host cell machineries including autophagy for its replication and disease pathogenesis. Double membrane vesicles generated during initiation of autophagy cascade act as a scaffold for the assembly of viral replication complexes and facilitate RNA synthesis. The use of autophagy inhibitors - chloroquine and hydroxychloroquine initially appeared to be as a potential treatment strategy of COVID-19 patients but later remained at the center of debate due to high cytotoxic effects. In the absence of a specific drug or vaccine, there is an urgent need for a safe, potent as well as affordable drug to control the disease spread. Given the intricate connection between autophagy machinery and viral pathogenesis, the question arises whether targeting autophagy pathway might show a path to fight against SARS-CoV-2 infection. In this review we will discuss about our current knowledge linking autophagy to coronaviruses and how that is being utilized to repurpose autophagy modulators as potential COVID-19 treatment.
ABSTRACT
Infection with specific pathogens and alterations in tissue commensal microbial composition are intricately associated with the development of many human cancers. Likewise, dysbiosis of oral microbiome was also shown to play critical role in the initiation as well as progression of oral cancer. However, there are no reports portraying changes in oral microbial community in the patients of Indian subcontinent, which has the highest incidence of oral cancer per year, globally. To establish the association of bacterial dysbiosis and oral squamous cell carcinoma (OSCC) among the Indian population, malignant lesions and anatomically matched adjacent normal tissues were obtained from fifty well-differentiated OSCC patients and analyzed using 16S rRNA V3-V4 amplicon based sequencing on the MiSeq platform. Interestingly, in contrast to the previous studies, a significantly lower bacterial diversity was observed in the malignant samples as compared to the normal counterpart. Overall our study identified Prevotella, Corynebacterium, Pseudomonas, Deinococcus and Noviherbaspirillum as significantly enriched genera, whereas genera including Actinomyces, Sutterella, Stenotrophomonas, Anoxybacillus, and Serratia were notably decreased in the OSCC lesions. Moreover, we demonstrated HPV-16 but not HPV-18 was significantly associated with the OSCC development. In future, with additional validation, this panel could directly be applied into clinical diagnostic and prognostic workflows for OSCC in Indian scenario.
ABSTRACT
Natural compounds isolated from different medicinal plants remain one of the major resources of anticancer drugs due to their enormous chemical diversity. Studies suggested therapeutic potential for various tanshinones, key bioactive lipophilic compounds from the root extracts of Salvia miltiorrhiza Bunge, against multiple cancers including breast carcinoma. We designed, synthesized and evaluated anti-cancer properties of a series of condensed and doubly condensed furophenanthraquinones of tanshinone derivatives on two breast cancer lines - MCF7 and MDA-MB-231. We identified two thiophene analogues - compounds 48 and 52 with greater anti-proliferative efficiency (~4 fold) as compared to the natural tanshinones. Mechanistically, we showed that both compounds induced autophagy mediated cell death and partial but significant restoration of cell death in the presence of autophagy inhibitor further supported this notion. Both compounds transcriptionally activated several autophagy genes responsible for autophagosome formation along with two death regulators - GADD34 and CHOP for inducing cell death. Altogether, our studies provide strong evidence to support compounds 48 and 52 as promising leads for further development as anticancer agents through modulating autophagy mechanism.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Breast Neoplasms/drug therapy , Thiophenes/pharmacology , Antineoplastic Agents/chemical synthesis , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Thiophenes/chemical synthesis , Unfolded Protein Response/drug effectsABSTRACT
Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins-EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.
Subject(s)
Autophagic Cell Death/drug effects , Epstein-Barr Virus Nuclear Antigens/metabolism , Herpesvirus 4, Human/metabolism , Leupeptins/pharmacology , Lysosomes/metabolism , Oncogene Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Proteolysis/drug effects , Animals , Epstein-Barr Virus Nuclear Antigens/genetics , HEK293 Cells , Herpesvirus 4, Human/genetics , Humans , Lysosomes/genetics , Mice , Oncogene Proteins/genetics , Proteasome Endopeptidase Complex/geneticsABSTRACT
Helicase loaders are required for the loading of helicases at the vicinity of replication origins. In Helicobacter pylori, Hp0897 has been shown to be a potential helicase loader for replicative helicase (HpDnaB) although it does not show any sequence homology with conventional DnaC like helicase loader proteins. Therefore, it is important to investigate the in vivo role of Hp0897 and structure-function analysis with respect to domain mapping of Hp0897 and HpDnaB. Although HporiC is divided into oriC1 and oriC2, the latter has been assigned as functional origin based on loading of initiator protein HpDnaA. Using chromatin immunoprecipitation (ChIP) experiment, we show preferential binding of Hp0897 at oriC2 over oriC1 like HpDnaA highlighting its helicase loader function in vivo. Furthermore, we generated series of deletion mutants for HpDnaB and Hp0897 that enabled us to map the domains of interaction between these two proteins. Interestingly, the C-terminal domain of Hp0897 (Hp0897CTD) shows stronger interaction with HpDnaB over the N-terminal region of Hp0897 (Hp0897NTD). Similar to the full-length protein, Hp0897CTD also stimulates the DNA binding activity of HpDnaB. Furthermore, overexpression of Hp0897 full-length protein in H. pylori leads to an elongated cell phenotype. While the overexpression of Hp0897CTD does not show a phenotype of cell elongation, overexpression of Hp0897NTD shows extensive cell elongation. These results highlight the possible role of Hp0897CTD in helicase loading and Hp0897NTD's unique function linked to cell division that make Hp0897 as a potential drug target against H. pylori.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , DNA Helicases/chemistry , DNA Helicases/metabolism , Helicobacter pylori/enzymology , Bacterial Proteins/genetics , DNA Helicases/genetics , DnaB Helicases/chemistry , DnaB Helicases/genetics , DnaB Helicases/metabolism , Helicobacter pylori/chemistry , Helicobacter pylori/genetics , Protein Binding , Protein DomainsABSTRACT
Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus which asymptomatically infects the majority of the world population. Under immunocompromised conditions, EBV can trigger human cancers of epithelial and lymphoid origin. The oncogenic potential of EBV is demonstrated by in vitro infection and transformation of quiescent B cells into lymphoblastoid cell lines (LCLs). These cell lines, along with primary infection using genetically engineered viral particles coupled with recent technological advancements, have elucidated the underlying mechanisms of EBV-induced B-cell lymphomagenesis.
Subject(s)
B-Lymphocytes/virology , Carcinogenesis , Herpesvirus 4, Human/genetics , Lymphoma, B-Cell/virology , Cell Line , Cell Transformation, Viral , Epstein-Barr Virus Infections/virology , Epstein-Barr Virus Nuclear Antigens/genetics , Gene Expression Regulation, Viral , Humans , Immunocompromised Host , Lymphoma, B-Cell/genetics , Neoplasms , RNA, Untranslated , Virus LatencyABSTRACT
EBV latent antigen 3C (EBNA3C) is essential for EBV-induced primary B-cell transformation. Infection by EBV induces hypermethylation of a number of tumor suppressor genes, which contributes to the development of human cancers. The Ras association domain family isoform 1A (RASSF1A) is a cellular tumor suppressor, which regulates a broad range of cellular functions, including apoptosis, cell-cycle arrest, mitotic arrest, and migration. However, the expression of RASSF1A is lost in many human cancers by epigenetic silencing. In the present study, we showed that EBNA3C promoted B-cell transformation by specifically suppressing the expression of RASSF1A. EBNA3C directly interacted with RASSF1A and induced RASSF1A degradation via the ubiquitin-proteasome-dependent pathway. SCFSkp2, an E3-ubiquitin ligase, was recruited by EBNA3C to enhance RASSF1A degradation. Moreover, EBNA3C decreased the transcriptional activity of RASSF1A promoter by enhancing its methylation through EBNA3C-mediated modulation of DNMTs expression. EBNA3C also inhibited RASSF1A-mediated cell apoptosis, disrupted RASSF1A-mediated microtubule and chromosomal stability, and promoted cell proliferation by upregulating Cyclin D1 and Cyclin E expression. Our data provides new details, which sheds light on additional mechanisms by which EBNA3C can induce B-cell transformation. This will also facilitate the development of novel therapeutic approaches through targeting of the RASSF1A pathway.
Subject(s)
Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Nuclear Antigens/metabolism , Tumor Suppressor Proteins/genetics , Antigens, Viral/genetics , Apoptosis , B-Lymphocytes/metabolism , B-Lymphocytes/virology , Cell Line, Tumor , Cell Proliferation/genetics , Cell Transformation, Viral/genetics , DNA Methylation/genetics , Down-Regulation , Epigenesis, Genetic/genetics , Epstein-Barr Virus Infections/genetics , Epstein-Barr Virus Nuclear Antigens/genetics , Gene Expression Regulation/genetics , HEK293 Cells , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/metabolism , Humans , Lymphocyte Activation/genetics , Promoter Regions, Genetic/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Epstein-Barr virus (EBV) oncoprotein EBNA3C is indispensable for primary B-cell transformation and maintenance of lymphoblastoid cells outgrowth. EBNA3C usurps two putative cellular pathways-cell-cycle and apoptosis, essentially through modulating ubiquitin-mediated protein-degradation or gene transcription. In cancer cells, these two pathways are interconnected with autophagy,-a survival-promoting catabolic network in which cytoplasmic material including mis/un-folded protein aggregates and damaged organelles along with intracellular pathogens are degraded and recycled in lysosomal compartments. Studies have shown that tumor viruses including EBV can manipulate autophagy as a survival strategy. Here, we demonstrate that EBNA3C elevates autophagy, which serves as a prerequisite for apoptotic inhibition and maintenance of cell growth. Using PCR based micro-array we show that EBNA3C globally accelerates autophagy gene transcription under growth limiting conditions. Reanalyzing the ENCODE ChIP-sequencing data (GSE52632 and GSE26386) followed by ChIP-PCR demonstrate that EBNA3C recruits several histone activation epigenetic marks (H3K4me1, H3K4me3, H3K9ac, and H3K27ac) for transcriptional activation of autophagy genes, notably ATG3, ATG5, and ATG7 responsible for autophagosome formation. Moreover, under growth limiting conditions EBNA3C further stimulates the autophagic response through upregulation of a number of tumor suppressor genes, notably cyclin-dependent kinase inhibitors-CDKN1B (p27Kip1) and CDKN2A (p16INK4a) and autophagy mediated cell-death modulators-DRAM1 and DAPK1. Together our data highlight a new role of an essential EBV oncoprotein in regulating autophagy cascade as a survival mechanism and offer novel-targets for potential therapeutic expansion against EBV induced B-cell lymphomas.
Subject(s)
Autophagy/genetics , B-Lymphocytes/pathology , Epigenesis, Genetic , Herpesvirus 4, Human/metabolism , Transcription, Genetic , Viral Proteins/metabolism , Apoptosis/genetics , Autophagy-Related Protein 5/metabolism , B-Lymphocytes/metabolism , Cell Cycle/genetics , Cell Proliferation/genetics , Cell Survival/genetics , Cytoprotection , HEK293 Cells , Histones/metabolism , Humans , Models, BiologicalABSTRACT
Epigenetic modifications leading to either transcriptional repression or activation, play an indispensable role in the development of human cancers. Epidemiological study revealed that approximately 20% of all human cancers are associated with tumor viruses. Epstein-Barr virus (EBV), the first human tumor virus, demonstrates frequent epigenetic alterations on both viral and host genomes in associated cancers-both of epithelial and lymphoid origin. The cell type-dependent different EBV latent gene expression patterns appear to be determined by the cellular epigenetic machinery and similarly viral oncoproteins recruit epigenetic regulators in order to deregulate the cellular gene expression profile resulting in several human cancers. This review elucidates the epigenetic consequences of EBV-host interactions during development of multiple EBV-induced B-cell lymphomas, which may lead to the discovery of novel therapeutic interventions against EBV-associated B-cell lymphomas by alteration of reversible patho-epigenetic markings.