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The EBV-Encoded Oncoprotein, LMP1, Induces an Epithelial-to-Mesenchymal Transition (EMT) via Its CTAR1 Domain through Integrin-Mediated ERK-MAPK Signalling.

ABSTRACT: The Epstein?Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) oncogene can induce profound effects on epithelial growth and differentiation including many of the features of the epithelial-to-mesenchymal transition (EMT). To better characterise these effects, we used the well-defined Madin Darby Canine Kidney (MDCK) epithelial cell model and found that LMP1 expression in these cells induces EMT as defined by characteristic morphological changes accompanied by loss of E-cadherin, desmosomal cadherin and tight junction protein expression. The induction of the EMT phenotype required a functional CTAR1 domain of LMP1 and studies using pharmacological inhibitors revealed contributions from signalling pathways commonly induced by integrin?ligand interactions: extracellular signal-regulated kinases/mitogen-activated protein kinases (ERK-MAPK), PI3-Kinase and tyrosine kinases, but not transforming growth factor beta (TGFβ). More detailed analysis implicated the CTAR1-mediated induction of Slug and Twist in LMP1-induced EMT. A key role for β1 integrin signalling in LMP1-mediated ERK-MAPK and focal adhesion kianse (FAK) phosphorylation was observed, and β1 integrin activation was found to enhance LMP1-induced cell viability and survival. These findings support an important role for LMP1 in disease pathogenesis through transcriptional reprogramming that enhances tumour cell survival and leads to a more invasive, metastatic phenotype.

SUBMITTER: Morris MA

PROVIDER: S-EPMC5977103 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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The EBV-Encoded Oncoprotein, LMP1, Induces an Epithelial-to-Mesenchymal Transition (EMT) via Its CTAR1 Domain through Integrin-Mediated ERK-MAPK Signalling.

Morris Mhairi A MA Laverick Louise L Wei Wenbin W Davis Alexandra M AM O'Neill Samantha S Wood Liam L Wright Jack J Dawson Christopher W CW Young Lawrence S LS

Cancers 20180501 5

The Epstein⁻Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) oncogene can induce profound effects on epithelial growth and differentiation including many of the features of the epithelial-to-mesenchymal transition (EMT). To better characterise these effects, we used the well-defined Madin Darby Canine Kidney (MDCK) epithelial cell model and found that LMP1 expression in these cells induces EMT as defined by characteristic morphological changes accompanied by loss of E-cadherin, desmosom ...[more]

PMID: 29723998

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Project description:The Epstein Barr virus (EBV) encoded latent membrane protein-1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of Nuclear Factor-kappaB (NF-kappaB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis, and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative RT-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-kappaB binding sites in the miR-146a promoter and identified a role for an OCT-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response. Keywords: microRNA expression modified by EBV encoded oncogene, LMP1 EBV negative Mutu E1dn Cl. 3 cells were transduced with either a control retrovirus (pEhyg) or an LMP1 containing retrovirus (pEhyg-FLAG-LMP1). Cells were selected with hygromycin for approximately 2 weeks afterwhich 5 pairs of RNA preps were carried out on 5 successive days. Arrays GSM255656 and GSM255659 are dye swaps in which LMP1 samples were labeled with Cy3 and control samples were labeled with Cy5.

Project description:The Epstein-Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) constitutively activates nuclear factor κB (NFκB) from intracellular membranes to promote cell growth and survival. LMP1 associates with CD63 in intracellular membranes and is released via exosomes. Whether tumour necrosis factor (TNF) receptor-associated factors (TRAFs) mediate LMP1 NFκB signalling from endosomes and modulate exosomal sorting is unknown. In this article, we show that LMP1-TRAF2 signalling complexes accumulate at endosomes in a palmitoylation-dependent manner, thereby driving LMP1-dependent oncogenicity. Palmitoylation is a reversible post-translational modification and is considered to function as a membrane anchor for proteins. Mutagenesis studies showed that LMP1-TRAF2 trafficking to endosomes is dependent on one single cysteine residue (C78), a known palmitoylation site of LMP1. Notably, growth assays in soft agar revealed that oncogenic properties of the palmitoylation-deficient LMP1 mutant C78A were diminished compared to wild-type LMP1. Since LMP1 recruitment of TRAF2 and downstream NFκB signalling were not affected by a disturbance in palmitoylation, the specific localization of LMP1 at endosomal membranes appears crucial for its transforming potential. The importance of palmitoylation for trafficking to and signalling from endosomal membranes was not restricted to LMP1, as similar observations were made for the cellular oncoproteins Src and Fyn. Despite abundant LMP1-TRAF2 association at endosomal membranes TRAF2 could not be detected in exosomes by Western blotting or proteomics. Interestingly, point mutations that prevented TRAF binding strongly promoted the sorting and release of LMP1 via exosomes. These observations reveal that LMP1-TRAF2 complexes at endosomes support oncogenic NFκB activation and suggest that LMP1 dissociates from the activated signalling complexes upon sorting into intraluminal vesicles. We propose that "signalling endosomes" in EBV-infected tumour cells can fuse with the plasma membrane, explaining LMP1 release via exosomes.

Dataset Information

The EBV-Encoded Oncoprotein, LMP1, Induces an Epithelial-to-Mesenchymal Transition (EMT) via Its CTAR1 Domain through Integrin-Mediated ERK-MAPK Signalling.

Publications

The EBV-Encoded Oncoprotein, LMP1, Induces an Epithelial-to-Mesenchymal Transition (EMT) via Its CTAR1 Domain through Integrin-Mediated ERK-MAPK Signalling.

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