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LMP1+SLAMF1high cells are associated with drug resistance in Epstein-Barr virus-positive Farage cells.

ABSTRACT: How Epstein-Barr virus (EBV) affects the clinical outcome of EBV-positive diffuse large B-cell lymphoma (DLBCL) remains largely unknown. The viral oncogene LMP1 is at the crux of tumorigenesis and cell survival. Therefore, we examined the association between LMP1high cells drug resistance. We first assessed SLAMF1 as a surrogate marker for LMP1high cells. LMP1 and its target gene CCL22 were highly expressed in SLAMF1high Farage cells. These cells survived longer following treatment with a combination of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP). Genes associated with interferon-alpha, allograft rejection, NF-?B and STAT3 were also overexpressed in the surviving Farage cells. Specifically, CHOP treatment increased IL10, LMP1 and pSTAT3 expression levels in a dose-dependent fashion. Addition of exogenous IL4 greatly increased the levels of LMP1 and pSTAT3, which rendered the Farage cells more resistant to CHOP by up-regulating the anti-apoptotic genes BCL-XL and MCL1. The Farage cells were sensitive to Velcade and STAT3, 5, and 6 inhibitors. Inhibition of NF-?B and STAT3, in combination with CHOP, decreased LMP1 levels and effectively induced cell death in the Farage cells. We suggest that LMP1high cells are responsible for the poor drug response of EBV+ DLBCL and that perturbation of the NF-?B and STAT signaling pathways increases toxicity in these cells.

SUBMITTER: Yoon H

PROVIDER: S-EPMC5421874 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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LMP1+SLAMF1high cells are associated with drug resistance in Epstein-Barr virus-positive Farage cells.

Yoon Heejei H Ko Young Hyeh YH

Oncotarget 20170401 15

How Epstein-Barr virus (EBV) affects the clinical outcome of EBV-positive diffuse large B-cell lymphoma (DLBCL) remains largely unknown. The viral oncogene LMP1 is at the crux of tumorigenesis and cell survival. Therefore, we examined the association between LMP1high cells drug resistance. We first assessed SLAMF1 as a surrogate marker for LMP1high cells. LMP1 and its target gene CCL22 were highly expressed in SLAMF1high Farage cells. These cells survived longer following treatment with a combin ...[more]

PMID: 28445949

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Project description:BackgroundEpstein-Barr virus (EBV)-encoded LMP1 protein is commonly expressed in nasopharyngeal carcinoma (NPC). LMP1 is a prime candidate for driving tumourigenesis given its ability to activate multiple signalling pathways and to alter the expression and activity of variety of downstream targets. Resistance to TGFbeta-mediated cytostasis is one of the growth transforming effects of LMP1. Of the downstream targets manipulated by LMP1, the induction of Id1 and inactivation of Foxo3a appear particularly relevant to LMP1-mediated effects. Id1, a HLH protein is implicated in cell transformation and plays a role in cell proliferation, whilst Foxo3a, a transcription factor controls cell integrity and homeostasis by regulating apoptosis. The mechanism(s) by which LMP1 induces these effects have not been fully characterised.ResultsIn this study, we demonstrate that the ability of LMP1 to induce the phosphorylation and inactivation of Foxo3a is linked to the upregulation of Id1. Furthermore, we show that the induction of Id1 is essential for the transforming function of LMP1 as over-expression of Id1 increases cell proliferation, attenuates TGFbeta-SMAD-mediated transcription and renders cells refractory to TGFbeta-mediated cytostasis. Id1 silencing in LMP1-expressing epithelial cells abolishes the inhibitory effect of LMP1 on TGFbeta-mediated cell growth arrest and reduces the ability of LMP1 to attenuate SMAD transcriptional activity. In response to TGFbeta stimulation, LMP1 does not abolish SMAD phosphorylation but inhibits p21 protein expression. In addition, we found the induction of Id1 in LMP1-expressing cells upon stimulation by TGFbeta. We provide evidence that LMP1 suppresses the transcriptional repressor ATF3, possibly leading to the TGFbeta-induced Id1 upregulation.ConclusionThe current data provide novel information regarding the mechanisms by which LMP1 suppresses TGFbeta-induced cytostasis, highlighting the importance of Id1 in LMP1 mediated cell transformation.

Project description:Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors that have latent infection, such as nasopharyngeal carcinoma (NPC), and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically infected cells, LMP1 expression is activated by the BRLF1 (R) immediate early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors KLF4 and BLIMP1 induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we found that differentiation of NOKs-Akata cells by either methylcellulose suspension or organotypic culture induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic infection-inducing effects of epithelial cell differentiation, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression and then cooperate with LMP1 to activate Z and R expression.IMPORTANCE The EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia) or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.

Project description:The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper, we characterize the EBV present in 21 pediatric and adult patients who were treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) were of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family in which several members have persistent T or NK cell infection by EBV indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient and not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children. IMPORTANCE EBV contributes to several types of human cancer. Some cancers and nonmalignant lymphoproliferative diseases involving T or NK cells contain EBV. These diseases are relatively frequent in Japan and China and have been shown sometimes to have deletions in the EBV genome in the disease cells. We identify further examples of deletions within the EBV genome associated with T or NK cell diseases, and we provide evidence that the virus genomes with these deletions are most likely selected in the individual cases, rather than being transmitted between people during infection. We demonstrate EBV infection of cord blood T cells by highly characterized, cloned EBV genomes and suggest that transient infection of T cells may be part of normal asymptomatic infection by EBV in young children.

Dataset Information

LMP1+SLAMF1high cells are associated with drug resistance in Epstein-Barr virus-positive Farage cells.

Publications

LMP1+SLAMF1high cells are associated with drug resistance in Epstein-Barr virus-positive Farage cells.

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