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Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy.

ABSTRACT: Generating oxidative stress is a critical mechanism by which host cells defend against infection by pathogenic microorganisms. Radiation resistance is a critical problem in radiotherapy against cancer. Epstein-Barr virus (EBV) is a cancer-causing virus and its reactivation plays an important role in the development of EBV-related tumors. This study aimed to explore the inner relationship and regulatory mechanism among oxidative stress, EBV reactivation, and radioresistance and to identify new molecular subtyping models and treatment strategies to improve the therapeutic effects of radiotherapy. Methods: ROS, NADP⁺/NADPH, and GSSG/GSH were detected to evaluate the oxidative stress of cells. 8-OHdG is a reliable oxidative stress marker to evaluate the oxidative stress in patients. Its concentration in serum was detected using an ELISA method and in biopsies was detected using IHC. qPCR array was performed to evaluate the expression of essential oxidative stress genes. qPCR, Western blot, and IHC were used to measure the level of EBV reactivation in vitro and in vivo. A Rta-IgG ELISA kit and EBV DNA detection kit were used to analyze the reactivation of EBV in serum from NPC patients. NPC tumor tissue microarrays was used to investigate the prognostic role of oxidative stress and EBV reactivation. Radiation resistance was evaluated by a colony formation assay. Xenografts were treated with NAC, radiation, or a combination of NAC and radiation. EBV DNA load of tumor tissue was evaluated using an EBV DNA detection kit. Oxidative stress, EBV reactivation, and the apoptosis rate in tumor tissues were detected by using 8-OHdG, EAD, and TUNEL assays, respectively. Results: We found that EBV can induce high oxidative stress, which promotes its reactivation and thus leads to radioresistance. Basically, EBV caused NPC cells to undergo a process of 'Redox Resetting' to acquire a new redox status with higher levels of ROS accumulation and stronger antioxidant systems by increasing the expression of the ROS-producing enzyme, NOX2, and the cellular master antioxidant regulator, Nrf2. Also, EBV encoded driving protein LMP1 promotes EBV reactivation through production of ROS. Furthermore, high oxidative stress and EBV reactivation were positively associated with poor overall survival of patients following radiation therapy and were significant related to NPC patients' recurrence and clinical stage. By decreasing oxidative stress using an FDA approved antioxidant drug, NAC, sensitivity of tumors to radiation was increased. Additionally, 8-OHdG and EBV DNA could be dual prognostic markers for NPC patients. Conclusions: Oxidative stress mediates EBV reactivation and leads to radioresistance. Targeting oxidative stress can provide therapeutic benefits to cancer patients with radiation resistance. Clinically, we, for the first time, generated a molecular subtyping model for NPC relying on 8-OHdG and EBV DNA level. These dual markers could identify patients who are at a high risk of poor outcomes but who might benefit from the sequential therapy of reactive oxygen blockade followed by radiation therapy, which provides novel perspectives for the precise treatment of NPC.

SUBMITTER: Hu J

PROVIDER: S-EPMC7667690 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy.

Hu Jianmin J Li Yueshuo Y Li Hongde H Shi Feng F Xie Longlong L Zhao Lin L Tang Min M Luo Xiangjian X Jia Weihua W Fan Jia J Zhou Jian J Gao Qiang Q Qiu Shuangjian S Wu Weizhong W Zhang Xin X Liao Weihua W Bode Ann M AM Cao Ya Y

Theranostics 20201025 26

Generating oxidative stress is a critical mechanism by which host cells defend against infection by pathogenic microorganisms. Radiation resistance is a critical problem in radiotherapy against cancer. Epstein-Barr virus (EBV) is a cancer-causing virus and its reactivation plays an important role in the development of EBV-related tumors. This study aimed to explore the inner relationship and regulatory mechanism among oxidative stress, EBV reactivation, and radioresistance and to identify new mo ...[more]

PMID: 33204320

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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:Interferon alpha (IFN-α) and IFN-β are type I IFNs that are induced by virus infection and are important in the host's innate antiviral response. EBV infection activates multiple cell signaling pathways, resulting in the production of type I IFN which inhibits EBV infection and virus-induced B-cell transformation. We reported previously that EBV tegument protein BGLF2 activates p38 and enhances EBV reactivation. To further understand the role of BGLF2 in EBV infection, we used mass spectrometry to identify cellular proteins that interact with BGLF2. We found that BGLF2 binds to Tyk2 and confirmed this interaction by coimmunoprecipitation. BGLF2 blocked type I IFN-induced Tyk2, STAT1, and STAT3 phosphorylation and the expression of IFN-stimulated genes (ISGs) IRF1, IRF7, and MxA. In contrast, BGLF2 did not inhibit STAT1 phosphorylation induced by IFN-γ. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of the protein to repress type I IFN signaling. Treatment of gastric carcinoma and Raji cells with IFN-α blocked BZLF1 expression and EBV reactivation; however, expression of BGLF2 reduced the ability of IFN-α to inhibit BZLF1 expression and enhanced EBV reactivation. In summary, EBV BGLF2 interacts with Tyk2, inhibiting Tyk2, STAT1, and STAT3 phosphorylation and impairs type I IFN signaling; BGLF2 also counteracts the ability of IFN-α to suppress EBV reactivation.IMPORTANCE Type I interferons are important for controlling virus infection. We have found that the Epstein-Barr virus (EBV) BGLF2 tegument protein binds to a protein in the type I interferon signaling pathway Tyk2 and inhibits the expression of genes induced by type I interferons. Treatment of EBV-infected cells with type I interferon inhibits reactivation of the virus, while expression of EBV BGLF2 reduces the ability of type I interferon to inhibit virus reactivation. Thus, a tegument protein delivered to cells during virus infection inhibits the host's antiviral response and promotes virus reactivation of latently infected cells. Therefore, EBV BGLF2 might protect virus-infected cells from the type I interferon response in cells undergoing lytic virus replication.

Project description:UnlabelledThe latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediate EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels than EBV-negative B cells. Moreover, cellular PAR levels were up to 2-fold greater in type III than type I latently infected EBV B cells. We identified a positive association between expression of the EBV genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation.ImportanceEBV is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

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Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy.

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Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy.

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