EBV membrane protein LMP2A interactions with ubiquitin ligases and a signaling scaffold

Over 90% of the adult human population carries Epstein-Barr Virus (EBV). EBV stays for life, striking a balance with the immune system. The virus evades immune elimination by severely restricting its own gene expression. The latent infection is established with the resting memory type B-cell as the reservoir. In the majority of infected individuals worldwide the coexistence with EBV is harmless. The dependence on a functioning immune system is clearly borne out by the long list of EBV-associated diseases that also feature accompanying immune dysfunction. An RNA message for the Latent Membrane Protein 2A (LMP2A) of EBV is constantly detected both in peripheral B-cells of healthy EBV carriers and in EBVassociated tumors. Elucidation of LMP2A interactions with the host cell will therefore contribute both to a better understanding of cellular signaling pathways, the regulation of EBV latency and to treatment of EBV-associated malignancies. Years of LMP2A research lead to the conclusion that LMP2A serves as a safeguard for the latency of EBV in the resting memory type B cells by interfering with and intercepting B-Cell Receptor (BCR) functions. In the normal resting B cell, BCR provides tonic signals to promote cell survival until the B-cell encounters its cognate antigen, upon which antigen-induced BCR signals will initiate differentiation to antibody-producing plasma cells. LMP2A sends surrogate tonic survival signals but interferes with the antigen-induced signals from the BCR. It does so by activating a key survival molecule in the cell, the serine-threonine kinase Akt and by down-regulating the Src-family tyrosine kinase Lyn, which is essential for antigen driven B-cell activation and differentiation. This knowledge about the functions of LMP2A was gained by using in-vitro cultured cells, biochemical and reverse genetic methods, as were the present studies, since the in-vivo cells that harbor the latent EBV infection are so scarce that direct studies can not be performed. Studies of viral perturbation of cellular systems are relevant model systems for learning about the normal functions of the cell and for pointing to vulnerable functions that are targeted also in non-viral disease, as in cancer. In the present thesis I provide evidence that: 1. Activation of LMP2A by constitutive tyrosine phosphorylation requires clustering of LMP2A molecules in the raft compartments of cell membranes (Paper I). 2. Src-family kinases, normally associated with the BCR, are labeled for degradation by E3 ubiquitin ligases of the Nedd4/AIP4 HECT-domain family, which are bound to LMP2A (Paper II); 3. LMP2A interacts with the Shb signaling scaffold, which can mediate Akt activation (Paper III)

MTSS1 is downregulated in nasopharyngeal carcinoma (NPC) which disrupts adherens junctions leading to enhanced cell migration and invasion

Loss of cell-cell adhesions is the indispensable first step for cancer cells to depart from the primary tumor mass to metastasize. Metastasis suppressor 1 (MTSS1) is frequently lost in metastatic tissues, correlating to advanced tumor stages and poor prognosis across a variety of cancers. Here we explore the anti-metastatic mechanisms of MTSS1, which have not been well understood. We found that MTSS1 is downregulated in NPC tissues. Lower levels of MTSS1 expression correlate to worse prognosis. We show that MTSS1 suppresses NPC cell migration and invasion in vitro through cytoskeletal remodeling at cell-cell borders and assembly of E-cadherin/beta-catenin/F-actin in adherens junctions. The I-BAR domain of MTSS1 was both necessary and sufficient to restore this formation of E-cadherin/beta-catenin/F-actin-mediated cell adherens junctions

Epstein-Barr virus-encoded LMP2A stimulates migration of nasopharyngeal carcinoma cells via the EGFR/Ca2+/calpain/ITGβ4 axis

Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) promotes the motility of nasopharyngeal carcinoma (NPC) cells. Previously, we have shown that the localization of integrin β4 (ITGβ4) is regulated by LMP2A, with ITGβ4 concentrated at the cellular protrusions in LMP2A-expressing NPC cells. In the present study, we aim to further investigate mechanisms involved in this process and its contribution to cell motility. We show that expression of LMP2A was correlated with increased epidermal growth factor receptor (EGFR) activation, elevated levels of intracellular Ca2+, calpain activation and accelerated cleavage of ITGβ4. Activation of EGFR and calpain activity was responsible for a redistribution of ITGβ4 from the basal layer of NPC cells to peripheral membrane structures, which correlated with an increased migratory capacity of NPC cells. Furthermore, we demonstrated that the calpain inhibitor calpastatin was downregulated in NPC primary tumors. In conclusion, our results point to LMP2A-mediated targeting of the EGFR/Ca2+/calpain/ITGβ4 signaling system as a mechanism underlying the increased motility of NPC cells. We suggest that calpain-facilitated cleavage of ITGβ4 contributes to the malignant phenotype of NPC cells

Additional file 1: of Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution

Supplementary tables. (ZIP 20649 kb

Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

<div><p>The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype.</p></div

N-butyric acid induces PARP cleavage.

<p>Western blot analysis of cells treated with butyrate or TPA for 24hr, A-C) Raji cells, D-F) C666-1 cells. A,D) PonceauS staining. C,E) Actin. B,F) anti PARP Ab.</p

N-butyric acid induces EBV virus production.

<p>Flow cytometric analysis of GFP fluorescence in Raji cells upon superinfection with EBV-GFP recombinant virus produced by EBV-GFP carrying AGS epithelial cells, upon treatment with n-butyric acid as stated in the M&M.</p

Analysis of short chain fatty acids induced BZLF1 gene expression in C666-1 NPC cells.

<p>RT-PCR analysis upon overnight treatment with 10mM of short chain fatty acids as indicated. A. BZLF1 expression. B. BART A expression. C. GAPDH expression. Cells were treated with 10mM of SCFAs as indicated or were left untreated. D. Digital analysis of data from panels A-C, demonstrating BZLF1 expression in relation to GAPDH.</p