Constitutive activation of distinct NF-κB signals in EBV-associated nasopharyngeal carcinoma.

As a distinct type of head and neck cancer, non-keratinizing nasopharyngeal carcinoma (NPC) is closely associated with EBV infection and massive lymphoid infiltration. The unique histological features suggest that local inflammation plays an important role in NPC tumourigenesis. We comprehensively characterized NF-κB signalling, a key inflammatory pathway which might contribute to the tumourigenesis of this EBV-associated cancer. By EMSA, western blotting, and immunohistochemical staining, constitutive activation of distinct NF-κB complexes, either p50/p50/Bcl3 or p50/RelB, was found in almost all EBV-positive NPC tumours. siRNA or chemical inhibition of NF-κB signalling significantly inhibited the growth of EBV-positive NPC cells C666-1. Gene expression profiling identified a number of NF-κB target genes involved in cell proliferation, apoptosis, immune response, and transcription. We further confirmed that p50 signals modulate the expression of multiple oncogenes (MYB, BCL2), chemokines, and chemokine receptors (CXCL9, CXCL10, CX3CL1, and CCL20). The findings support a crucial role of these constitutively activated NF-κB signals in NPC tumourigenesis and local inflammation. In addition to expression of the viral oncoprotein LMP1, genetic alteration of several NF-κB regulators (eg TRAF3, TRAF2, NFKBIA, A20) also contributes to the aberrant NF-κB activation in EBV-associated NPC. Except for LMP1-expressing C15 cells, all NPC tumour lines harbour at least one of these genetic alterations. Importantly, missense mutations of TRAF3, TRAF2, and A20 were also detected in 3/33 (9.1%) primary tumours. Taken together with the reported LTBR amplification in 7.3% of primary NPCs, genetic alterations in NF-κB pathways occurred in at least 16% of cases of this cancer. The findings indicate that distinct NF-κB signals are constitutively activated in EBV-positive NPC cells by either multiple genetic changes or EBV latent genes.

Chung GT ，Lou WP ，Chow C ，To KF ，Choy KW ，Leung AW ，Tong CY ，Yuen JW ，Ko CW ，Yip TT ，Busson P ，Lo KW ... -  《-》

NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

Verhoeven RJ ，Tong S ，Zhang G ，Zong J ，Chen Y ，Jin DY ，Chen MR ，Pan J ，Chen H ... -  《-》

Insufficient PINX1 expression stimulates telomerase activation by direct inhibition of EBV LMP1-NF-κB axis during nasopharyngeal carcinoma development.

Early malignant transformation of nasopharyngeal carcinoma(NPC) is associated with Epstein-Barr virus(EBV) infection and telomerase activation. The EBV latent membrane protein 1(LMP1) regulates expression of various genes by triggering NF-κB signaling pathway. PINX1 is a well-identified tumor suppressor gene by inhibiting telomerase activity and cancer cell growth. However, whether and how EBV inhibit PINX1 expression and activate telomerase in NPC is still incompletely elucidated. Immunohistochemistry, real-time PCR and Western blotting were utilized to explore the expression of PINX1. Chromatin immunoprecipitation(ChIP) and Dual-luciferase reporter assay were used to elucidate the regulatory mechanism between NF-κB and PINX1. TRAP-SYBR Green assay and Southern blotting were utilized to detect telomerase activity and telomere length. CCK8 and EdU tests were conducted to measure proliferation ability. We demonstrated that PINX1 is down-regulated in NPC for the first time. Mechanistically, we found that LMP1 could inhibit the transcriptional activity of PINX1 by promoting the binding of p65 to three specific sites in PINX1 promoter, significantly, two(-1698/-1689, tgcaatttcc; -206/-197, cgggctttac) of which have not been reported. In addition, we also observed that LMP1 overexpression resulted in increased telomerase activity, prolonged telomere length and enhanced proliferation. We first discovered EBV led to reduced PINX1 expression through LMP1-NF-κB-PINX1 axis, which up-regulated telomerase activity in NPC. And hence, the tumor cells acquired the ability to proliferate more exuberantly. This signaling pathway illustrates the relationship between EBV latent infection and telomerase activation, and further provides new thinking for early diagnosis and treatment in NPC.

Liu Y ，Gong P ，Zhou N ，Zhang J ，He C ，Wang S ，Peng H ... -  《-》

Positive regulation of HIF-1A expression by EBV oncoprotein LMP1 in nasopharyngeal carcinoma cells.

Latent membrane protein 1 (LMP1) is a pivotal viral oncoprotein that contributes to the carcinogenesis of Epstein-Barr virus (EBV)-associated malignancies, including nasopharyngeal carcinoma (NPC). We investigated the regulation of hypoxia-inducible factor 1-α (HIF-1α) by LMP1. In NPC cells, we found that LMP1 significantly enhanced the HIF-1α mRNA level, and not only the protein amount as described previously. Mechanistically, the stability of the HIF-1α transcript was remarkably prolonged by LMP1 via reduced expressions of RNA-destabilizing proteins tristetraprolin (TTP) and pumilio RNA-binding family member 2 (PUM2) through C-terminal activation region 1 (CTAR1) and CTAR3 interaction with the ERK1/2 and STAT3 signaling pathways, respectively, in parallel with hindrance of PUM2 binding to the HIF-1α mRNA 3'-untranslated region (3'-UTR). On the other hand, HIF-1A promoter activity was also obviously facilitated by the LMP1 CTAR1-recruited ERK1/2/NF-κB pathway. Intriguingly, in this scenario, augmented HIF-1α further exhibited positive auto-regulation of its own gene transcription. Our results showed the first time that LMP1 directly up-regulates HIF-1A transcription and post-transcription in NPC cells, in addition to providing evidence of an increase in the HIF-1α mRNA level caused by a tumor-associated virus under normoxic conditions.

Sung WW ，Chu YC ，Chen PR ，Liao MH ，Lee JW ... -  《-》

SPLUNC1 reduces the inflammatory response of nasopharyngeal carcinoma cells infected with the EB virus by inhibiting the TLR9/NF-κB pathway.

Ou C ，Sun Z ，Zhang H ，Xiong W ，Ma J ，Zhou M ，Lu J ，Zeng Z ，Bo X ，Chen P ，Li G ，Li X ，Li X ... -  《-》