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Studies have indicated that dysfunction of autophagy is involved in the initiation and progression of multiple tumors and their chemoradiotherapy. Epstein-Barr virus (EBV) is a lymphotropic human gamma herpes virus that has been implicated in the pathogenesis of nasopharyngeal carcinoma (NPC). EBV encoded latent membrane protein1 (LMP1) exhibits the properties of a classical oncoprotein. In previous studies, we experimentally demonstrated that LMP1 could increase the radioresistance of NPC. However, how LMP1 contributes to the radioresistance in NPC is still not clear. In the present study, we found that LMP1 could enhance autophagy by upregulating the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3). Knockdown of BNIP3 could increase the apoptosis and decrease the radioresistance mediated by protective autophagy in LMP1-positive NPC cells. The data showed that increased BNIP3 expression is mediated by LMP1 through the ERK/HIF1α signaling axis, and LMP1 promotes the binding of BNIP3 to Beclin1 and competitively reduces the binding of Bcl-2 to Beclin1, thus upregulating autophagy. Furthermore, knockdown of BNIP3 can reduce the radioresistance promoted by protective autophagy in vivo. These data clearly indicated that, through BNIP3, LMP1 induced autophagy, which has a crucial role in the protection of LMP1-positive NPC cells against irradiation. It provides a new basis and potential target for elucidating LMP1-mediated radioresistance.

Several reports have demonstrated that Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1), which is transferred by extracellular vesicles (EVs) or exosomes, can promote cancer progression. However, its mechanism is still not fully understood. In the present study, we demonstrated that EV packaged LMP1 can activate normal fibroblasts (NFs) into cancer-associated fibroblasts (CAFs). The NF-κB p65 pathway is the key signal that promotes the activation of NFs to CAFs in nasopharyngeal carcinoma (NPC). In activated CAFs, aerobic glycolysis and autophagy were increased. Moreover, glucose uptake and lactate production were decreased, and mitochondrial activity in tumor cells was enhanced, which supported the Reverse Warburg Effect (RWE). During this process, upregulation of MCT4 in CAFs and MCT1 in tumor cells was observed. The NF-κB p65 pathway also plays an important role in the regulation of MCT4. Furthermore, co-culture with CAFs promoted the proliferation, migration and radiation resistance of NPC cells. And EV packaged LMP1 promoted tumor proliferation and pre-metastatic niche formation by activating CAFs in vivo. Our findings indicate that EV packaged LMP1-activated CAFs promote tumor progression via autophagy and stroma-tumor metabolism coupling., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The Epstein-Barr virus latent membrane protein 1 (EBV‑LMP1) is an oncoviral protein that plays an important role in oncogenic transformation in EBV‑associated nasopharyngeal carcinoma (NPC). Our previous studies demonstrated that LMP1 increased VEGFA expression and upregulated angiogenesis in NPC. Vasculogenic mimicry (VM) is a mechanism by which tumor cells can obtain nutrients to survive, and VM has been observed in numerous types of tumors. However, the occurrence and significance of VM in NPC and the relationship between LMP1 and VM have not yet been evaluated. In the present study, we observed that it was almost impossible for LMP1-negative NPC cells to form tubular structures, whereas LMP1-positive NPC cells were able to form tubular structures. Moreover, VEGFA was found to be involved in VM formation in LMP1-positive NPC cells. Knockdown of LMP1 or VEGFR1 distinctly disrupted tubular structures, whereas inhibition of VEGFR2 did not affect the process, indicating that VEGFR1 not VEGFR2 signaling was involved in LMP1-mediated VM formation. Furthermore, the data of immunohistochemistry (IHC) and CD34/PAS double staining in a tumor tissue array showed that LMP1 was positively correlated with VEGFR1 and VM. Meanwhile, after analyzing the clinicopathological features, we found that VM formation was associated with a poor prognosis in NPC patients. These results suggest that VM formation is increased by EBV‑LMP1 via VEGF/VEGFR1 signaling and provide additional information to clarify the role of EBV‑LMP1 in NPC pathophysiology.

We conducted this research to explore the role of latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) in modulating the DNA damage response (DDR) and its regulatory mechanisms in radioresistance. Our results revealed that LMP1 repressed the repair of DNA double strand breaks (DSBs) by inhibiting DNA-dependent protein kinase (DNA-PK) phosphorylation and activity. Moreover, LMP1 reduced the phosphorylation of AMP-activated protein kinase (AMPK) and changed its subcellular location after irradiation, which appeared to occur through a disruption of the physical interaction between AMPK and DNA-PK. The decrease in AMPK activity was associated with LMP1-mediated glycolysis and resistance to apoptosis induced by irradiation. The reactivation of AMPK significantly promoted radiosensitivity both in vivo and in vitro. The AMPKα (Thr172) reduction was associated with a poorer clinical outcome of radiation therapy in NPC patients. Our data revealed a new mechanism of LMP1-mediated radioresistance and provided a mechanistic rationale in support of the use of AMPK activators for facilitating NPC radiotherapy., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Epstein-Barr virus (EBV) was first discovered 50 years ago as an oncogenic gamma-1 herpesvirus and infects more than 90% of the worldwide adult population. Nasopharyngeal carcinoma (NPC) poses a serious health problem in southern China and is one of the most common cancers among the Chinese. There is now strong evidence supporting a role for EBV in the pathogenesis of NPC. Latent membrane protein 1 (LMP1), a primary oncoprotein encoded by EBV, alters several functional and oncogenic properties, including transformation, cell death and survival in epithelial cells in NPC. LMP1 may increase protein modification, such as phosphorylation, and initiate aberrant signalling via derailed activation of host adaptor molecules and transcription factors. Here, we summarise the novel features of different domains of LMP1 and several new LMP1-mediated signalling pathways in NPC. When then focus on the potential roles of LMP1 in cancer stem cells, metabolism reprogramming, epigenetic modifications and therapy strategies in NPC.

LMP1, which is encoded by the Epstein-Barr virus, is proposed to be one of the major oncogenic factors involved in nasopharyngeal carcinoma (NPC). Previous studies demonstrated that down-regulation of LMP1 by LMP1-targeted DNAzyme (DZ1) increases the radiosensitivity of NPC. However, the mechanism by which DZ1 contributes to this radiosensitivity remains unclear. In this study, we determined whether a DZ1 blockade of LMP1 expression has an overall positive effect on the radiotherapy of NPCs by repressing HIF-1/VEGF activity and to investigate the mechanisms underlying LMP1-induced HIF-1 activation in NPC cells. The results showed that DZ1 inhibited the microtubule-forming ability of HUVECs co-cultured with NPC cells, which occurs with the down-regulation of VEGF expression and secretion. Moreover, LMP1 increases phosphorylated JNKs/c-Jun signaling, which is involved in the regulation of HIF-1/VEGF activity. After silencing LMP1 and decreasing phosphorylation of JNKs, NPC cells exhibited an enhanced radiosensitivity. Furthermore, in vivo experiments revealed a significant inhibition of tumor growth and a marked reduction of the Ktrans parameter, which reflects the condition of tumor micro-vascular permeability. Taken together, our data suggested that VEGF expression is increased by LMP1 through the JNKs/c-Jun signaling pathway and indicated that DZ1 enhances the radiosensitivity of NPC cells by inhibiting HIF-1/VEGF activity.

The ability of the 10-23 DNAzyme to specifically cleave RNA with high efficiency has fuelled expectation that this agent may have useful applications for targeted therapy. Here, we, for the first time, investigated the antitumor and radiosensitizing effects of a DNAzyme (DZ1) targeted to the Epstein-Barr virus (EBV)-LMP1 mRNA of nasopharyngeal carcinoma (NPC) in patients. Preclinical studies indicated that the DNAzyme was safe and well tolerated. A randomized and double-blind clinical study was conducted in 40 NPC patients who received DZ1 or saline intratumorally, in conjunction with radiation therapy. Tumor regression, patient survival, EBV DNA copy number and tumor microvascular permeability were assessed in a 3-month follow-up. The mean tumor regression rate at week 12 was significantly higher in DZ1 treated group than in the saline control group. Molecular imaging analysis showed that DZ1 impacted on tumor microvascular permeability as evidenced by a faster decline of the K(trans) in DZ1-treated patients. The percentage of the samples with undetectable level of EBV DNA copy in the DZ1 group was significantly higher than that in the control group. No adverse events that could be attributed to the DZ1 injection were observed in patients.

The latent membrane protein 1 (LMP1), which is encoded by the Epstein-Barr virus (EBV), has been suggested to be one of the major oncogenic factors in nasopharyngeal carcinoma (NPC). In previous studies, we experimentally demonstrated that downregulation of LMP1 expression by targeting EBV-LMP1 DNAzyme (Dz1) could increase the radiosensitivity of NPC. However, how Dz1 contributes to the radiosensitivity in NPC is still not clear. In the present study, we confirmed that Dz1 decreases LMP1 expression and downregulates the expression of the catalytic subunit of telomerase (hTERT), both at the protein and mRNA levels (P<0.01), and therefore, consequently inhibits telomerase activity (P<0.05) in LMP1-positive NPC cells. We also observed that LMP1 mediated Akt phosphorylation is involved in the regulation of hTERT expression and phosphorylation. After LMP1 and hTERT expression was silenced by Dz1 and hTERT-targeted siRNA, respectively, the radiosensitivity increased in CNE1-LMP1 cells (P<0.05). The inhibition was more significant after Dz1 treatment was combined with siRNA (P<0.01). We concluded that hTERT expression and phosphorylation could be regulated by LMP1 through the Akt pathway, and Dz1 enhances radiosensitivity of LMP1-positive NPC cells by inhibiting telomerase activity.

This study aimed to determine the molecular mechanisms underlying the effect of the LMP1-targeted DNAzyme 1 (DZ1) on cell cycle progression in nasopharyngeal carcinoma (NPC) cells. We showed that the active DZ1 inhibited the expression of latent membrane protein 1 (LMP1) and induced a G1 phase arrest. In addition, this cell cycle deregulation was shown to be accompanied by upregulation of the DNA damage marker γ-H2AX, downregulation of the DNA damage response factor p-p53-Ser15 and cell proliferation inhibition. To investigate what affected the cell cycle progression, we examined the expression of two checkpoint-related cyclins and cyclin-dependent kinases (CDKs). We found a decrease of cyclin D1 and cyclin E protein levels at 24 h from the DZ1 treatment. Moreover, we observed inhibition of CDK4 activity and decreased cyclin D1 expression in the complexes immunoprecipitated with CDK4 antibody. We also found a reduction in cdc2 phosphorylation at Thr161 which partially stands for the cdc2 kinase activity in DZ1-treated CNE1-LMP1 cells, although the downregulation of LMP1 expression had no effect on the cyclin B1 and cdc2 expression. Further, we analyzed changes in cdc2 kinase activity induced by DZ1 and found that the downregulation of the LMP1 expression resulted in a 5-fold reduction in cdc2 kinase activity in CNE1-LMP1. The data suggest that the downregulation of the LMP1 expression by DZ1 was able to induce DNA damage, which then further inhibited the cell proliferation and resulted in malfunction of cell cycle checkpoints that led to G1 phase arrest and the decrease in number of cells in G2/M phase.

The latent membrane protein 1 (LMP1), which is encoded by the Epstein-Barr virus (EBV), is an important oncogenic protein that is closely related to carcinogenesis and metastasis of nasopharyngeal carcinoma (NPC), a prevalent cancer in China. We previously reported that the expression of the functional chemokine receptor CXCR4 is associated with human NPC metastasis. In this study, we show that LMP1 induces tyrosine sulfation of CXCR4 through tyrosylprotein sulfotransferase-1 (TPST-1), an enzyme that is responsible for catalysis of tyrosine sulfation in vivo, which is likely to contribute to the highly metastatic character of NPC. LMP1 could induce tyrosine sulfation of CXCR4 and its associated cell motility and invasiveness in a NPC cell culture model. In contrast, the expression of TPST-1 small interfering RNA reversed LMP1-induced tyrosine sulfation of CXCR4. LMP1 conveys signals through the epidermal growth factor receptor (EGFR) pathway, and EGFR-targeted siRNA inhibited the induction of TPST-1 by LMP1. We used a ChIP assay to show that EGFR could bind to the TPST-1 promoter in vivo under the control of LMP1. A reporter gene assay indicated that the activity of the TPST-1 promoter could be suppressed by deleting the binding site between EGFR and TPST-1. Finally, in human NPC tissues, the expression of TPST-1 and LMP1 was directly correlated and clinically, the expression of TPST-1 was associated with metastasis. These results suggest the up-regulation of TPST-1 and tyrosine sulfation of CXCR4 by LMP1 might be a potential mechanism contributing to NPC metastasis.

Latent membrane protein 1 (LMP1) is an important oncogenic protein encoded by Epstein-Barr virus (EBV) and plays an important role in the development of nasopharyngeal carcinoma (NPC). Our previous study has shown that p53 protein was accumulated and phosphorylated in NPC, implying its transcription factor activity in NPC tumorigenesis. However, the biological function and potential downstream target of p53 mediated by LMP1 in NPC remain unknown. In this study, we found that LMP1 simultaneously induced upregulation of both p53 and survivin at the protein level, as well as their phosphorylation. Knockdown of p53 by siRNA revealed that LMP1 increased survivin expression by p53 directly. Furthermore, we found that LMP1 upregulated survivin by p53 at the transcriptional level by increasing p53-mediated survivin promoter activity and DNA binding activity. Moreover, LMP1 induced the co-localization of p53 and survivin in the nucleus, conferring to their related functions in NPC tumorigenesis. We further found that p53 promoted G1/S cell cycle progression, but did not induce apoptosis in LMP1-positive NPC cells. Collectively, these findings suggest that p53 acting as a transcription factor promotes the transcriptional activity of survivin, and further increases its protein expression and phosphorylation in the regulation of LMP1, thus, leading to G1/S cell cycle progression with no effect on apoptosis in NPC tumorigenesis.

Background: The latent membrane protein 1 (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. In previous studies we experimentally demonstrated that down-regulation of LMP1 expression by DNAzymes could increase radiosensitivity both in cells and in a xenograft NPC model in mice., Results: In this study we explored the molecular mechanisms underlying the radiosensitization caused by the down-regulation of LMP1 in nasopharyngeal carcinoma. It was confirmed that LMP1 could up-regulate ATM expression in NPCs. Bioinformatic analysis of the ATM ptomoter region revealed three tentative binding sites for NF-κB. By using a specific inhibitor of NF-κB signaling and the dominant negative mutant of IkappaB, it was shown that the ATM expression in CNE1-LMP1 cells could be efficiently suppressed. Inhibition of LMP1 expression by the DNAzyme led to attenuation of the NF-κB DNA binding activity. We further showed that the silence of ATM expression by ATM-targeted siRNA could enhance the radiosensitivity in LMP1 positive NPC cells., Conclusions: Together, our results indicate that ATM expression can be regulated by LMP1 via the NF-κB pathways through direct promoter binding, which resulted in the change of radiosensitivity in NPCs.

The principal Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), has been suggested to contribute to the highly invasive nature of nasopharyngeal carcinoma (NPC). Signal transducer and activator of transcription 3 (STAT3) is a master transcriptional regulator in proliferation and apoptosis and is newly implicated in angiogenesis and invasiveness, which, in turn, are likely to contribute to the highly invasive character of NPC. The fundamental molecular mechanisms of LMP1-regulated STAT3 activation in NPC cell invasion have not been completely explored. Here, we showed that LMP1 signals the Janus kinase 3 (JAK3) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways upon the activation of STAT3 as well as STAT transactivation activity. LMP1 induces vascular endothelial growth factor (VEGF) expression via the JAK/STAT and mitogen-activated protein kinase (MAPK)/ERK signalling pathways. Induction of STAT3 by the human viral oncoprotein LMP1 may contribute to the invasion of NPC., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been known to have oncogenic properties during latent infection in nasopharyngeal carcinoma (NPC). Genetic manipulation of LMP1 expression may provide a novel strategy for the treatment of NPC. DNAzymes are synthetic, single-stranded DNA catalysts that can be engineered to bind and cleave the target mRNA of a disease-causing gene. By targeting the LMP1 mRNA, we successfully obtained a phosphorothioate-modified ''10-23'' DNAzyme namely DZ1, through screening a series of DNAzymes. DZ1 could significantly down-regulate the expression of LMP1 in NPC cells, inhibit cell proliferation, metastasis, promote apoptosis and enhance radiosensitivity of NPC through interfering signal pathways which are abnormally activated by LMP1, including NF-κB, AP-1 and STAT3 signal pathways. Together, interfering LMP1 signaling pathway could be a promising strategy to target the malignant phenotypes of NPC.