Linkage between STAT regulation and Epstein-Barr virus gene expression in tumors

Epstein-Barr virus (EBV) latency gene expression in lymphoblastoid cell lines is regulated by EBNA2. However, the factors regulating viral expression in EBV-associated tumors that do not express EBNA2 are poorly understood. In EBV-associated tumors, EBNA1 and frequently LMP1 are synthesized. We found that an alternative latent membrane protein 1 (LMP1) promoter, L1-TR, located within the terminal repeats is active in both nasopharyngeal carcinoma and Hodgkin's disease tissues. Examination of the L1-TR and the standard ED-L1 LMP1 promoters in electrophoretic mobility shift assays revealed that both promoters contain functional STAT binding sites. Further, both LMP1 promoters responded in reporter assays to activation of JAK-STAT signaling. Cotransfection of JAK1 or v-Src or treatment of cells with the cytokine interleukin-6 upregulated expression from ED-L1 and L1-TR reporter plasmids. Cotransfection of a dominant negative STAT3 beta revealed that STAT3 is likely to be the biologically relevant STAT for EBNA1 Qp and LMP1 L1-TR promoter regulation. In contrast, LMP1 expression from ED-L1 was not abrogated by STAT3 beta, indicating that the two LMP1 promoters are regulated by different STAT family members. Taken together with the previous demonstration of JAK-STAT activation of Qp driven EBNA1 expression, this places two of the EBV genes most commonly expressed in tumors under the control of the same signal transduction pathway. Immunohistochemical analyses of nasopharyngeal carcinoma tumors revealed that STAT3, STAT5, and STAT1 are constitutively activated in these tumors while STAT3 is constitutively activated in the malignant cells of Hodgkin's disease. We hypothesize that chronic or aberrant STAT activation may be both a necessary and predisposing event for EBV-driven tumorigenesis in immunocompetent individuals.     

Src Kinase and Syk Activation Initiate PI3K Signaling by a Chimeric Latent Membrane Protein 1 in Epstein-Barr Virus (EBV)+ B Cell Lymphomas

The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.     

Mechanism of STAT3 activation by insulin-like growth factor I receptor

Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation.     

STAT3 is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rbeta2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.     

A mutation in Epstein-Barr virus LMP2A reveals a role for phospholipase D in B-Cell antigen receptor trafficking

Epstein-Barr virus (EBV) latent infection of B cells blocks the interrelated signaling and antigen-trafficking functions of the BCR through the activity of its latent membrane protein 2A (LMP2A). At present, the molecular mechanisms by which LMP2A exerts its control of BCR functions are only poorly understood. Earlier studies showed that in B cells expressing LMP2A containing a tyrosine mutation at position 112 in its cytoplasmic domain (Y112-LMP2A), the BCR could initiate signaling but could not properly traffic antigen for processing. Here, we show that BCR signaling in Y112-LMP2A-expressing cells is attenuated with a reduction in both the degree and duration of phosphorylation of key components of the BCR signaling cascade including Syk, BLNK, PI3K, and Btk. Notably, Y112-LMP2A expression completely blocked the BCR-induced activation of phospholipase D (PLD), a lipase implicated in the intracellular trafficking of a variety of surface receptors. We show that blocking PLD activity, by expressing Y112-LMP2A, treating cells with the PLD inhibitor 1-butanol or reducing PLD expression by siRNA, blocked BCR trafficking to class II-containing compartments. Moreover, Y112-LMP2A expression blocked the recruitment of phosphorylated forms of the downstream BCR signaling components, Erk and JNK, through both PLD-dependent and PLD-independent mechanisms. Thus, the investigation of the mechanism by which Y112-LMP2A blocks BCR function revealed an essential role for PLD in BCR trafficking for antigen processing.     

Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) and LMP2A function cooperatively to promote carcinoma development in a mouse carcinogenesis model

The Epstein-Barr virus (EBV) proteins latent membrane proteins 1 and 2 (LMP1 and LMP2) are frequently expressed in EBV-associated lymphoid and epithelial cancers and have complex effects on cell signaling and growth. The effects of these proteins on epithelial cell growth were assessed in vivo using transgenic mice driven by the keratin 14 promoter (K14). The development of papillomas and carcinomas was determined in the tumor initiator and promoter model using dimethyl benzanthracene (DMBA), followed by repeated treatments of 12-O-tetradecanoyl phorbol 13-acetate (TPA). In these assays, LMP1 functioned as a weak tumor promoter and increased papilloma formation. In contrast, mice expressing LMP2A did not induce or promote papilloma formation. Transgenic LMP1 mice had slightly increased development of squamous cell carcinoma; however, the development of carcinoma was significantly increased in the doubly transgenic mice expressing both LMP1 and LMP2A. DMBA treatment induces an activating mutation in the Harvey-ras (H-ras(61)) oncogene, and this mutation was identified in most papillomas and carcinomas although several papillomas and carcinomas in K14-LMP1 and K14-LMP1/LMP2A mice lacked the mutation. Analysis of signaling pathways that are known to be activated by LMP1 and/or LMP2 indicated that all genotypes had high levels of activated extracellular signal-regulated kinase (ERK) and Stat3 in carcinomas with significantly higher activation in the doubly transgenic carcinomas. These findings suggest that, in combination, LMP1 and LMP2 contribute to carcinoma progression and that this may reflect the combined effects of the proteins on activation of multiple signaling pathways. This study is the first to characterize the effects of LMP2 on tumor initiation and promotion and to identify an effect of the combined expression of LMP1 and LMP2 on the increase of carcinoma development.     

Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor through effects on Bcl-3 and STAT3

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates multiple signaling pathways. Two regions, C-terminal-activating region 1 (CTAR1) and CTAR2, have been identified within the cytoplasmic carboxy terminal domain that activates NF-kappaB. CTAR2 activates the canonical NF-kappaB pathway, which includes p50/p65 complexes. CTAR1 can activate both the canonical and noncanonical pathways to produce multiple distinct NF-kappaB dimers, including p52/p50, p52/p65, and p50/p50. CTAR1 also uniquely upregulates the epidermal growth factor receptor (EGFR) in epithelial cells. Increased p50-Bcl-3 complexes have been detected by chromatin precipitation on the NF-kappaB consensus motifs within the egfr promoter in CTAR1-expressing epithelial cells and nasopharyngeal carcinoma cells. In this study, the mechanism responsible for the increase in Bcl-3 has been further investigated. The data indicate that LMP1-CTAR1 induces Bcl-3 mRNA and increases the nuclear translocation of both Bcl-3 and p50. LMP1-CTAR1 constitutively activates STAT3, and this activation was not due to the induction of interleukin 6 (IL-6). In LMP1-CTAR1-expressing cells, increased levels of activated STAT3 were detected by chromatin immunoprecipitation on STAT-binding sites located within both the promoter and the second intron of Bcl-3. A STAT3 inhibitor significantly reduced the activation of STAT3, as well as the CTAR1-mediated upregulation of Bcl-3 and EGFR. These data suggest that LMP1 activates distinct forms of NF-kappaB through multiple pathways. In addition to activating the canonical and noncanonical pathways, LMP1-CTAR1 constitutively activates STAT3 and increases Bcl-3. The increased nuclear Bcl-3 and p50 homodimer complexes positively regulate EGFR expression. These results indicate that LMP1 likely regulates distinct cellular genes by activating specific NF-kappaB pathways.     

Reactive oxygen species generated by hematopoietic cytokines play roles in activation of receptor-mediated signaling and in cell cycle progression

Hematopoietic cytokines, including interleukin (IL)-3 and erythropoietin (Epo), regulate hematopoiesis by stimulating their receptors coupled with the Jak2 tyrosine kinase to induce receptor tyrosine phosphorylation and activate mainly the STAT5, PI3K/Akt, and Ras/MEK/ERK signaling pathways. Here we demonstrate that IL-3 or Epo induces a rapid and transient (peaking at 30 min) as well as late progressive increase in reactive oxygen species (ROS) in a hematopoietic progenitor model cell line, 32Dcl3, and its subclone expressing the Epo receptor (EpoR), 32D/EpoR-Wt. The cytokine-induced ROS generation was not affected in 32Dcl3 cells depleted of mitochondrial DNA. The antioxidant N-acetyl-L-cysteine (NAC) inhibited IL-3-induced tyrosine phosphorylation of Jak2, IL-3 receptor betac subunit (IL-3Rbetac), and STAT5 as well as activation-specific phosphorylation of Akt, MEK, and ERK, while treatment of cells with H2O2 activated these signaling events. NAC also inhibited the EpoR-induced transphosphorylation of IL-3Rbetac. Moreover, NAC treatment reduced the expression levels of c-Myc, Cyclin D2, and Cyclin E, and induced expression of p27, thus inhibiting the G1 to S phase transition of cells cultured with IL-3. Further studies have shown that the degradation of c-Myc was facilitated or inhibited by treatment of cells with NAC or H2O2, respectively. These data indicate that the rapid generation of ROS by cytokine stimulation, which is at least partly independent of mitochondria, may play a role in activation of Jak2 and the STAT5, PI3K/Akt, and Ras/MEK/ERK signaling pathways as well as in transactivation of cytokine receptors. The cytokine-induced ROS generation was also implicated in G1 to S progression, possibly through stabilization of c-Myc and induction of G1 phase Cyclin expression leading to suppression of p27.     

鼻咽癌中EB病毒LMP1激活TRAFs的初步研究

在EB病毒潜伏膜蛋白LMP1介导的信号传导通路中,TRAFs作为LMP1活化的第一位信号分子,可能扮演着重要的分子开关角色。令人关注的是,在上皮性肿瘤NPC的发生中,EB病毒LMP1能否激活重要的TRAFs信号分子?究竟激活何种TRAFs信号分子,激活的机制何在?将LMP1cDNA导入LMP1表达阴性的HNE2中,建立稳定表达LMP1的鼻咽癌细胞系HNE2-LMP1。以此为材料,应用差异RT-PCR和Western blotting法证实,无论在RNA水平,还是蛋白水平上,TRAF1在HNE2-LMP1中表达较HNE2强,而TRAF2及TRAF3在HNE2-LMP1与HNE2细胞中表达无明显差异;进一步用免疫共沉淀-Western blotting证实LMP1可使TRAF1、TRAF2、TRAF3磷酸化而被活化。这些结果提示在鼻咽癌中,LMP1可能诱导TRAF1表达,而对TRAF2及TRAF3并不影响,但LMP1可磷酸化TRAF1、TRAF2、TRAF3而使其功能性活化。q     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.