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American Journal of Cancer Research logoLink to American Journal of Cancer Research
. 2023 Apr 15;13(4):1363–1376.

EBNA2 mediates lipid metabolism and tumorigenesis through activation of ATF4 pathway

Jia Feng 1, Ping Zhang 1, Paul Yao 1, Hongyu Zhang 1
PMCID: PMC10164800  PMID: 37168348

Abstract

Epstein-Barr virus (EBV) can infect the majority of the human population with no obvious symptoms and is associated with tumor development, although the mechanism is still largely unknown. In this study, we investigated the role and the underlying mechanism of EBV nuclear antigen 2 (EBNA2) in tumorigenesis. We found that the infection of EBNA2 in human B lymphocytes (HBL) upregulated the expression of activating transcription factor 4 (ATF4). Furthermore, we used gene expression or knockdown approach to demonstrate the effect of EBNA2 on redox balance, mitochondrial function, lipid metabolism, and cell proliferation in both HBL and EBV-transformed lymphocyte cell line (LCL). More importantly, we applied in vivo xenograft tumor mouse model to explore the contribution of EBNA2 and ATF4 in tumor growth and mouse survival. Mechanistically, we revealed that EBNA2 exposure caused persistent expression of ATF4 via EBNA2-mediated epigenetic changes, which increased the binding ability of upstream stimulating factor 1 (USF1) on the ATF4 promoter. ATF4 activation in HBL cells modulated the expression of lipid metabolism-related genes and potentiated fatty acid oxidation and lipogenesis. Conversely, knockdown of either EBNA2 or ATF4 in LCL suppressed lipid metabolism, modulated redox balance and mitochondrial function, as well as inhibited tumor cell proliferation. In consistent with these findings from in vitro study, an in vivo xenograft model confirmed that knockdown of either EBNA2 or ATF4 inhibited the gene expression of SREBP1, ChREBP, and FAS, as well as suppressed tumor growth and prolonged animal survival. Collectively, this study demonstrates that EBNA2 mediates tumorigenesis through ATF4 activation and the modulation of lipid metabolism; therefore, our findings provide a novel avenue for the clinical treatment of EBV-mediated cancer.

Keywords: EBV, EBNA2, lipid metabolism, ATF4 and USF1

Introduction

Epstein-Barr virus (EBV) has been reported to be associated with a variety of tumors, including nasopharyngeal carcinoma and nasal Nature/Killer/T-cell lymphoma NKTCL [1,2]. Most of the human population has latent EBV infection but usually shows no obvious symptoms [3]. It has been reported that EBV infection may cause tumorigenesis through EBV-encoded oncogenic proteins such as EBV nuclear antigen 2 (EBNA2) 1 [4,5]. As a transcription factor, EBNA2 plays a critical role in EBV-mediated cell transformation and tumorigenesis [6-8] by activating multiple latent viral and cellular genes, including Latent Membrane Protein 1 (LMP1) and Myc [9-11]. Intriguingly, recent studies have also shown that EBNA2 regulates gene expression through epigenetic changes [12-15] with metabolic rearrangement toward aerobic glycolysis, thereby contributing to tumorigenesis [16], although the detailed mechanism is largely unknown.

Activating transcription factor 4 (ATF4) is a stress-mediated transcription factor that is highly expressed in tumors [17-19]. ATF4 upregulation results in a poor prognosis [20-22], and ATF4 is involved in many metabolic processes during tumor development, including glucose metabolism, protein synthase, redox balance, and mitochondrial modulation [23-25]. In addition, ATF4 is involved in mitochondrial fat oxidation [26] and lipid metabolism through the upregulation of fatty acid synthase (FAS), carbohydrate-responsive element-binding protein (ChREBP), and sterol regulatory element-binding protein 1 (SREBP1) [27], although its regulator mechanism remains to be determined.

It has been known that LMP1 plays a central role in EBV modulated lipid metabolism and the subsequent tumorigenesis [16,28]. LMP1 regulates lipogenesis through the upregulation of FAS [29] and SREBP1, contributing to cell proliferation and nasopharyngeal carcinoma progression [30]. In addition, EBV and LMP2A affect lipid accumulation and increase the migration of cancer cells [28,31]. Similarly, EBNA2 and SREBP1 play important roles in the fatty acid pathway to promote B-cell proliferation and survival [32]. However, there are few studies regarding EBV-mediated lipogenesis through ChREBP [33,34]. Hence, the mechanism of EBV-mediated lipogenesis and its contribution to tumorigenesis require further characterization.

In this study, we investigated the effects as well as the underlying mechanism of EBNA2-mediated lipid metabolism in tumorigenesis. Specifically, we explored the mechanism of transient EBV/EBNA2 exposure-mediated persistent ATF4 activation through in vitro study in human B lymphocytes (HBL) via luciferase reporter assay and chromatin immunoprecipitation techniques. Furthermore, the mechanism of ATF4 activation-induced cell growth was investigated through analyzing fatty acid oxidation (FAO) and lipogenesis as well as its target genes such as SREBP1, ChREBP, and FAS. Moreover, the potential modulatory effects on redox balance and mitochondrial function were also determined. Simultaneously, EBV-transformed lymphoblastoid cell lines were employed to evaluate the influence of EBNA2/ATF4 on cancer cell proliferation in vitro and xenograft tumor growth in vivo as well as animal survival in a mouse model.

Materials and methods

The full section of the Experimental Procedures can be found in Supplementary Materials, and all primers used for gene knockdown and quantitative PCR were summarized in Table S1.

Reagents and cell lines

Antibodies against ATF4 (sc-390063), ChREBP (sc-515922), FAS (sc-48357), Smad4 (sc-7966), Sp1 (sc-17824), SREBP1 (sc-13551), USF1 (sc-390027), Zic3 (sc-101201) were purchased from SCBT (Beijing, China). Antibodies against EBNA2 (ab90543), HNF3 (ab108422), and SPZ1 (ab235302) were purchased from Abcam. The EBNA2 adenovirus and shEBNA2 lentivirus, as well as the corresponding empty controls, were kindly provided by Dr. Haimou Zhang (Hubei University). shRNA lentivirus particles targeting human ATF4 (sc-35112-V) and USF1 (sc-36783-V) were purchased from SCBT. HBL cells were isolated from healthy human peripheral blood using the EasySep™ Human B Cell Isolation Kit (#17954), while lymphoblastoid cell line 1 (LCL1) was generated by transforming HBL cells with EBV virus infection as described previously [35].

Construction of ATF4 luciferase reporter plasmids

The human ATF4 promoter was identified by Ensembl #: ENST00000337304.2 ATF4-201, amplified (2 kb upstream + 1st exon) by PCR using human B lymphocytes genomic DNA as template, and inserted into pGL3-basic plasmid. The PCR primer sequences were: ATF4 forward primer: 5’-atat-ggtacc-ggg ctc aag caa tcc tcc cac-3’ (KpnI) and ATF4 reverse primer: 5’-atat-agatct-cct tgc tgt tgt tgg agg gac-3’ (Bgl II). ATF4 promoter deletion/mutation plasmids were also produced by PCR and confirmed by DNA sequencing.

Construction of ATF4/USF1 lentivirus plasmids

The cDNA plasmids expressing human ATF4 and USF1 were obtained from Open Biosystems. Then, the coding sequences of ATF4 and USF1 were amplified using the following primers: ATF4 forward: 5’-atat-gaattc (EcoR1) - agc cat ttc tac ttt gcc cgc-3’, ATF4 reverse: 5’-atat-ggatcc (BamH1) - ttt ttt ttt ttt ttt act ttc cct aca-3’; USF1 forward: 5’-atat-gaattc (EcoR1) - gcc cct cac caa cat ggc cgc-3’, USF1 forward: 5’-atat-ggatcc (BamH1) - aga taa aat tgt tca aag cca-3’. The amplified cDNA fragments were cloned into the pLVX-Puro vector for lentivirus production.

Methods

Total RNA was extracted by Trizol reagent; mRNA expression was determined by quantitative PCR, and protein levels were determined by western blotting following standard protocols. To investigate EBAN2-regulated ATF4 transcriptional activation, the activity of wild type and mutant ATF4 promoter was measured by luciferase reporter assay. The binding ability of EBAN2 to ATF4 promoter was determined by chromatin immunoprecipitation. To evaluate the redox balance, reactive oxygen species (ROS) formation was determined using CM-H2DCFDA-based fluorescence. Mitochondrial function was determined through mitochondrial replication, ATP generation, mitochondrial membrane potential (ΔΨm) and apoptosis rate. EBV DNA copy numbers were determined by qPCR of the extracted genomic DNA [36]. The colony formation assay and Ki-67 immunostaining were performed to measure cell growth in vitro using standard protocol. Fatty acid oxidation (FAO) was determined by calculating the oxidation of [14C] palmitate using a previously described method [37]. The redox balance of the tumor tissues was evaluated using the superoxide anion (O2.-) release through the DMSO/TBAC-based chemiluminescence method, as described previously [38].

De novo lipogenesis

Briefly, cells were incubated in serum-free medium with 100 nM insulin at 37°C overnight followed by incubatiion in lipogenesis medium (DMEM medium with 100 nM insulin, 10 μM cold acetate and 0.5 μCi 3H-acetate) at 37°C for 2 h. Cells were then lysed in 120 μl of 0.1 N HCl, and 100 µl of lysates were transferred to a 1.5 ml centrifuge tube for further analysis. An aliquot of 10 μl lysates was used for protein quantification with the Pierce™ BCA Protein Assay Kit according to the manufacturer’s instruction. The lipids were extracted by adding 500 μl of 2:1 chloroform-methanol (v/v) followed by 5 min incubation at room temperature (RT). After a brief vortex, 250 μl of water was added to the cell lysates, which was mixed again by vortexing and was incubated at RT for another 5 min. After centrifugation at 3000×g for 10 min at RT, the lower phase was transferred to vial containing 4 ml of liquid scintillation fluid, and 3H activity was determined by the scintillation counter and expressed as counts per minute (cpm) [39].

In vivo mouse experiments

Balb/c athymic nude male mice (6 weeks old) were purchased from Guangdong Medical Animal Center. To generate xenograft tumors, mice were injected via tail vein with 2×106 (in 0.1 ml PBS) of 5 different treated LCL1 cells (n=9) as 5 treatment groups: Group 1 (CTL): Cells infected by control (CTL) lentivirus; Group 2 (shEBNA2): Cells infected by shEBNA2 lentivirus; Group 3 (shATF4): Cells infected by shATF4 lentivirus; Group 4 (shUSF1): Cells infected with shUSF1 lentivirus; and Group 5 (shEBNA2/↑ATF4): Cells infected with shEBNA2 and ATF4 lentivirus in the presence of 10 µg/ml puromycin. The experimental mice were monitored daily and euthanized by cervical dislocation when their body weight was reduced by 20% of their initial weight. Tumor tissues were dissected from lung and used for gene expression analysis and in vivo superoxide anions (O2.-) release assay [38], as well as were fixed and examined for the number of metastases per lung under a microscope. The tumor spots were imaged by H&E staining and quantified using the 3DHISTECH Pannoramic Viewer.

Results

Overexpression of EBNA2 induced ATF4 upregulation and modulated lipid metabolism

To investigate the effects of EBV/EBNA2 on cellular functions, we first infected hTERT-immortalized HBL cells with EBV or EBNA2 adenovirus and found that EBNA2 mRNA expression was induced from passage 0 and diminished quickly following continuous culture and completely disappeared at passage 6 (Figure 1A). Notably, ATF4 mRNA level was significantly increased upon EBV/EBNA2 exposure and remained high until passage 6 (Figure 1B). In addition, western blot analysis indicated the ATF4 protein level was consistent with its mRNA level (Figure 1C, 1D). Furthermore, we determined EBV genomic copy number following EBV infection and found that EBV virus diminished quickly following cell culture and disappeared completely at passage 6 (Figure S1A, S1B). After confirming the expression of EBV/EBNA2 in HBL cells, we analyzed the effect of EBV/EBNA2 on lipogenesis-related genes and found that EBV/EBNA2 overexpression significantly increased the protein levels of SREBP1, ChREBP, and FAS (Figures 1E, 1F, S2A) compared with that in the vector infected cells. We also evaluated the effect of EBV/EBNA2 on lipid metabolism and found a significantly increased palmitate oxidation (Figure 1G) and de novo lipogenesis (Figure 1H). Together, these data suggested that EBNA2 exposure induced ATF4 expression and modulated lipid metabolism in HBL cells.

Figure 1.

Figure 1

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EBNA2 expression induces persistent ATF4 upregulation and modulates lipid metabolism. HBL cells were infected with control (CTL), EBV virus (↑EBV) or EBNA2 adenovirus (↑EBNA2) and analyzed. (A) mRNA levels of EBNA2, n=4. (B) mRNA levels of ATF4, n=4. (C-H) EBV/EBNA2-infected HBL cells were analyzed in passage 6. (C) Quantification of ATF4 protein levels, n=5. (D) Representative western blot of ATF4 for (C). (E) Representative western blots. (F) Quantification of protein levels for (E), n=5. (G) Palmitate oxidation, n=5. (H) De novo lipogenesis, n=5. *, P<0.05, vs CTL group; ¶, P<0.05, vs EBV group.

EBNA2 activated ATF4 through epigenetic modification and subsequently increased USF1 binding on ATF4 promoter

We further explored how EBNA2 activated ATF4 transcription in HBL cells by using luciferase reporter assay. We generated a series of ATF4 reporter plasmids with 5’-promoter deletion and infected these mutant constructs individually in HBL cells in the presence of EBNA2 adenovirus infection. Our results showed that ATF4 promoter deletion at -2000, -1600, -1200, -800, -600, -400, -300, -200, and -100 had no effect on EBNA2 activity. However, the reporter activation was completely diminished in the p-ATF4-0 construct containing -100~0 deletion, suggesting that the EBNA2-responsive element on the ATF4 promoter is located in the region of -100~0 (Figure 2A). Furthermore, to reveal the binding motif of EBNA2 in ATF4, we performed sequence analysis and identified the following potential binding motifs in this region: USF1 (-89), SPZ1 (-76), Zic3 (-53), HNF3 (-36), Sp1 (-26) and Smad4 (-18) (Figure 2B). Then, we generated mutation constructs for each binding motif in the ATF4 full-length reporter plasmid and used luciferase activity assay to examine the activity of these mutants. The results showed that only mutation in USF1 motif (from red to green, Figure 2B) completely diminished EBNA2-mediated ATF4 activation, suggesting that EBNA2 activated ATF4 expression through the USF1 binding element (Figure 2C). Moreover, we assessed the potential epigenetic modifications in HBL cells and found that EBV and EBNA2 infection significantly decreased H3K9me2 and H3K27me3 modifications compared with that in the control cells, but had no effect on H3K9me3 and H3K27me2 modification (Figure 2D). Interestingly, exposure to EBV and EBNA2 did not affect DNA methylation (Figure S3). Importantly, we performed ChIP analysis using the corresponding antibodies to confirm the binding abilities of these motifs and found that treatment with EBV and EBNA2 infection significantly increased USF1 binding, but had no effect on other motifs (Figure 2E), consistent with the results of luciferase reporter assay. To determine the functional significance of USF1 on EBNA2 activation of ATF4, we knocked down EBNA2 and USF1 by shEBNA2 and shUSF1, respectively, and found that shEBNA2 and shUSF1 in LCL1 cells significantly decreased ATF4 protein levels compared to those in the CTL cells (Figures 2F, 2G, S2B), suggesting that ATF4 expression was activated by EBNA2-mediated H3 demethylation and subsequently increased USF1 binding to the ATF4 promoter.

Figure 2.

Figure 2

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EBNA2 activates ATF4 through epigenetic modification and subsequently increased USF1 binding to the ATF4 promoter. (A-E) The hTERT-immortalized HBL cells were used for promoter mapping and activity assays. (A) Luciferase reporter activities of ATF4 promoter deletion mutants in hTERT-immortalized HBL cells, n=5. *, P<0.05, vs pATF4-2000 group. (B) A schematic diagram of binding motifs in the range of -100~0 on the ATF4 promoter. (C) Luciferase activity of ATF4 promoter mutation reporters, n=5. *, P<0.05, vs pATF4-2000 group. (D, E) ChIP analysis on ATF4 promoter for H3 methylation (D) and different transcription factors (E), n=4. *, P<0.05, vs CTL group. (F, G) EBNA2 or USF1 knockdown in LCL1 cells by either shEBNA2 or shUSF1, as determined by western blot analysis. (F) Representative blots. (G) Quantification for (F), n=5. *, P<0.05, vs CTL group.

EBNA2 regulated lipid metabolism through ATF4 activation

Since ATF4 is a central regulator of cellular stress response, we investigated the effects of EBNA2 on lipid metabolism. LCL1 cells were infected with control (CTL), shEBNA2, shATF4, shUSF1, or shEBNA2 plus ATF4 expressing lentivirus (shEBNA2/↑ATF4). The efficient knockdown of gene expression by shRNA and ATF overexpression were confirmed by qPCR. We found that shEBNA2 reduced ATF4 mRNA level but had no effect on USF1 mRNA level, while shATF4 had no effect on the expression of USF1. Meanwhile, shUSF1 significantly reduced ATF4 levels (Figure 3A). We further examined ATF4 protein levels by western blot, and the results were consistent with the mRNA levels (Figure 3B, 3C). We then determined the effect of these shRNAs on the expression of genes involved in lipid metabolism and found that treatment with shEBNA2, shATF4, and shUSF1 significantly reduced the protein levels of SREBP1, ChREBP, and FAS, but shEBNA2/↑ATF4 completely reversed the shEBNA2-induced effect (Figures 3D, 3E, S2C). Consistently, treatment with shEBNA2, shATF4, and shUSF1 affected lipid metabolism in these cells as palmitate oxidation (Figure 3F) and de novo lipogenesis was significantly reduced (Figure 3G), while shEBNA2/↑ATF4 reversed this effect. Taken together, these results suggested that EBNA2 regulated lipid metabolism by activating the ATF4 pathway.

Figure 3.

Figure 3

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EBNA2 regulates lipid metabolism through ATF4 activation. LCL1 cells were infected with control (CTL), shEBNA2, shATF4, shUSF1 or shEBNA2 plus ATF4 expression (↑ATF4) lentivirus (shEBNA2/↑ATF4) and were examined. (A) mRNA level, n=4. (B) ATF4 quantitation, n=5. (C) Representative immunostaining (green) for ATF4 with nuclei staining by DAPI (blue) for (B). (D) Protein quantitation, n=5. (E) Representative western blots for (D). (F) Palmitate oxidation, n=5. (G) De novo lipogenesis, n=5. *, P<0.05, vs CTL group.

EBNA2 modulated redox balance and mitochondrial function through ATF4 activation

We also examined the effect of EBNA2 on redox balance and revealed that shEBNA2 treatment significantly increased both ROS (Figure 4A) and 8-oxo-dG generation (Figure 4B, 4C). Treatment with shATF4 and shUSF1 partly, while shEBNA2/↑ATF4 completely reversed these effects. Furthermore, treatment with shEBNA2, shATF4, and shUSF1 significantly reduced mitochondrial replication (Figure 4D), ATP generation (Figure 4E), and mitochondrial membrane potential (ΔΨm, Figure 4F), but significantly increased the apoptosis rate (Figure 4G). In contrast, treatment with shEBNA2/↑ATF4 showed little effect (except for ATP generation) compared with the shEBNA2 group. Collectively, these results suggested that EBNA2 modulated redox balance and mitochondrial function partly through ATF4 activation.

Figure 4.

Figure 4

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EBNA2 modulates redox balance and mitochondrial function through ATF4 activation. LCL1 cells were infected with control (CTL), shEBNA2, shATF4, shUSF1 or shEBNA2 plus ATF4 expression (↑ATF4) lentivirus (shEBNA2/↑ATF4) and examined. (A) ROS generation, n=5. (B) Quantitation of 8-oxo-dG staining, n=5. (C) Representative immunostaining (green) with nuclei staining by DAPI (blue) for (B). (D) Mitochondrial DNA copy number, n=4. (E) Intracellular ATP generation, n=5. (F) Mitochondrial membrane potential (ΔΨm), n=5. (G) Apoptosis, n=5. *, P<0.05, vs CTL group.

EBNA2 regulated cell proliferation in vitro through ATF4 activation

The effect of EBNA2 expression on cell transformation and cell proliferation was investigated, and our results showed that shEBNA2 significantly reduced colony formation (Figure 5A, 5B) and Ki-67 positive cell rate (Figure 5C, 5D), compared with those in the CTL group, while shATF4, shUSF1, and shEBNA2/↑ATF4 partly reversed this effect, suggesting that EBNA2 promoted cell proliferation partly through ATF4 activation.

Figure 5.

Figure 5

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EBNA2 regulates cell proliferation in vitro through ATF4 activation. LCL1 cells were infected with control (CTL), shEBNA2, shATF4, shUSF1 or shEBNA2 plus ATF4 lentivirus (shEBNA2/↑ATF4) and examined. (A) Colony formation, n=5. (B) Representative images for (A). (C) Quantitation of Ki-67 staining, n=5. (D) Representative pictures for (C). *, P<0.05, vs CTL group; ¶, P<0.05, vs shEBNA2 group.

EBNA2 regulated tumor growth in vivo through ATF4-mediated lipogenesis

More importantly, to confirm our in vitro cell line-based findings, we investigated the role of EBNA2 expression in tumor growth by using mouse xenograft tumor model. LCL1 cells were injected into mice through tail vein, and the tumor growth as well as the survival of the injected mice were monitored. We first measured the mRNA levels of EBNA2, shATF4, and shUSF1 in tumors and confirmed that shRNA lentivirus significantly knocked down the corresponding genes, while ATF4 lentivirus (↑ATF4) infection significantly increased ATF4 mRNA expression, indicating a successful lentivirus infection-mediated gene manipulation (Figure 6A). We then determined the expression of genes involved in lipid metabolism in tumors and found that treatment with shEBNA2, shATF4, and shUSF1 significantly reduced the protein levels of SREBP1, CHREBP, and FAS compared with the CTL group; shEBNA2/↑ATF4 treatment partly reversed this effect (Figures 6B, 6C, S2D). In addition, the redox balance of tumor tissues was determined, and the data showed that shEBNA2 treatment significantly potentiated O2.- release compared with the CTL group and that treatment with shATF4, shUSF1 and shEBNA2/↑ATF4 ameliorated this effect (Figure 6D). When examining the tumor growth in lung, we found that shEBNA2 treatment significantly increased lung metastatic nodules (Figure 6E) and lung tumor spots (Figure 6F, 6G) compared with the CTL group, and similarly, treatments with shATF4, shUSF1, and shEBNA2/↑ATF4 partly reversed this effect. Finally, mouse survival was evaluated, and the data indicated that shEBNA2 treatment significantly increased mouse survival compared with the CTL group, whereas treatment with shATF4, shUSF1 and shEBNA2/↑ATF4 had smaller effects compared to shEBNA2 (Figure 6H). These results suggest that EBNA2 modulates tumor growth in vivo partly through ATF4 activation and the subsequently enhanced lipogenesis.

Figure 6.

Figure 6

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EBNA2 regulates tumor growth in vivo through ATF4-mediated effect. Treated LCL1 cells were injected through tail vein, and the tumor growth and the survival of mice were monitored. (A-C) Tumor tissues were isolated from the lung and analyzed. (A) mRNA levels, n=4. (B) Protein quantitation, n=5. (C) Representative western blots for (B). (D) Superoxide anion release from tissues, n=5. (E-G) Tumor colonies from lung were counted and used for H&E staining, n=9. (E) Metastatic nodules. (F) Quantitation of tumor spots. (G) Representative H&E staining for (F). (H) Kaplan-Meier analysis for mouse survival, P value represents log-rank Mantel-Cox test result, n=9. *, P<0.05, vs CTL group; ¶, P<0.05, vs shEBNA2 group.

Schematic model for EBNA2-mediated ATF4 activation and tumorigenesis

Based on the results above, we proposed a working model for the mechanism underlying the regulation of ATF4 by EBNA2 and the subsequent increased tumorigenesis. In normal situations, ATF4 expression is suppressed by H3 methylation of the ATF4 promoter (Figure 7A); however, in the presence of EBV/EBNA2 exposure, ATF4 expression is upregulated by EBNA2-mediated demethylation of the ATF4 promoter (Figure 7B). ATF4 activation then modulates the expression of SREBP1, ChREBP, FAS, FAO, and lipogenesis (Figure 7C), and together with ATF4 activation-mediated modulation of oxidative stress and mitochondrial function, EBNA2-mediated ATF4 activation potentiates tumorigenesis (Figure 7D).

Figure 7.

Figure 7

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Schematic model for EBNA2-mediated ATF4 activation and tumorigenesis. A. ATF4 expression is suppressed by H3 methylation on the ATF4 promoter. B. ATF4 expression is up-regulated by EBNA2-mediated demethylation on the ATF4 promoter in the presence of EBV or EBNA2. C. ATF4 activation modulates the expression of SREBP1, ChREBP and FAS as well as FAO and lipogenesis. D. ATF4 activation-mediated modulation of lipid metabolism, and ROS generation and mitochondrial function promotes tumorigenesis. Abbreviations: ATF4: activates transcription factor-4; ChREBP: carbohydrate-responsive element-binding protein; EBNA2: Epstein-Barr virus nuclear antigen 2; EBV: Epstein-Barr virus; FAS: fatty acid synthase; FAO: fatty acid oxidation; H3: histone 3; ROS: reactive oxygen species; SREBP1: sterol regulatory element-binding protein 1; USF1: upstream stimulating factor 1.

Discussion

In this study, we demonstrate that EBNA2 exposure causes persistent ATF4 expression and that ATF4 is regulated through EBNA2-mediated epigenetic changes, which subsequently increases the USF1 binding ability to the ATF4 promoter. Furthermore, EBNA2-mediated ATF4 activation modulates lipid metabolism and the expression of SREBP1, ChREBP and FAS, by which, together with ATF4-mediated redox balance and modulation of mitochondrial function, EBNA2 potentiates tumorigenesis through ATF4 activation.

Our findings are novel. First, we revealed that transient EBV/EBNA2 exposure induced persistent ATF4 expression through decreased H3K9me2 and H3K27me3 levels on the ATF4 promoter. In consistent with our findings, it has been reported that EBNA2 mediates epigenetic changes through DNA methylation [13], chromatin rearrangement [12] and phase separation [10], indicating that EBNA2 expression triggers epigenetic changes. Nevertheless, whether the epigenetic modifications on the ATF4 promoter is a direct effect of EBNA2 needs further investigation. EBNA2-mediated epigenetic changes and persistent gene regulation of ATF4 may play important roles in cell transformation and tumorigenesis [6]. Although ATF4 activation is usually regulated at the translational level due to the phosphorylation of eIF2α as part of the integrated stress response [40], our study showed ATF4 regulation at the transcriptional level by epigenetic changes, indicating that EBNA2-mediated gene expression is relatively persistent, which is favorable for tumorigenesis. Interestingly, our study demonstrated that EBNA2-mediated epigenetic changes increased USF1 binding to the ATF4 promoter and subsequently activated ATF4 expression. Furthermore, EBNA2 expression increased USF1 binding to the ATF4 promoter with ATF4 upregulation, whereas USF1 knockdown suppressed ATF4 expression. Hence, our data indicated that EBNA2 regulated ATF4 activation and lipid metabolism through USF1, which subsequently modulated lipogenesis via SREBP1, ChREBP, and FAS [34,41,42].

Our data also showed that ATF4 activation increased the expression of SREBP1, ChREBP, and FAS as well as potentiated FAO and lipogenesis, while ATF4 knockdown suppressed the gene expression and lipid metabolism, indicating that ATF4 may modulate lipid metabolism [27,43]. It has been reported that ATF4 does not directly upregulate mitochondrial FAO but may modulate mitochondrial function and upregulate antioxidant machinery to mitigate the presence of defective mitochondria [44,45]. As a transcription factor, ATF4 may regulate the expression of SREBP1, ChREBP, and FAS either directly or indirectly and subsequently modulate lipid metabolism, although the mechanism remains unclear.

Moreover, we observed that knockdown of either EBNA2 or ATF4 increased oxidative stress, while ATF4 overexpression partly reverses this effect, indicating that ATF4 is involved in the redox balance in tumor cells [17,24]. In addition, knockdown of either EBNA2 or ATF4 modulated mitochondrial function [25], including decreased mitochondrial DNA copies, intracellular ATP formation, ΔΨm, and potentiated apoptosis rate, indicating that EBNA2-mediated ATF4 activation plays an important role in sustaining mitochondrial function in tumor cells. It has been proposed that ATF4 may modulate redox balance and mitochondrial function through mitochondrial fat oxidation and lipogenesis [26,27,46]. In this study, we showed that ATF4 overexpression could rescue ROS generation, mitochondrial function, and lipid metabolism in the absence of EBNA2, and EBNA2 triggers this effect in an ATF4-dependent manner, indicating that EBNA2 transient exposure may cause persistent changes during tumorigenesis, which can explain why many forms of lymphoma show the absence of EBV.

More significantly, using in vivo mouse model, we confirmed that EBNA2-mediated ATF4 activation increased the gene expression of SREBP1, ChREBP, and FAS [16], subsequently potentiating FAO and lipogenesis as well as modulating redox balance and mitochondrial function. In addition, knockdown of EBNA2/ATF4 significantly increased mouse survival in the in vivo mouse model. Therefore, we speculate that the major function of ATF4 in promoting tumorigenesis is to increase the antioxidant activity and regulate ROS production; however, the precise mechanisms of ATF4 function remain to be fully elucidated [44,45]. Although our data suggest that EBNA2 mediates tumorigenesis through ATF4 activation and altered lipid metabolism [19], EBNA2 may also contribute to tumorigenesis through other pathways, such as Myc activation, Notch signaling, and PD-L1 expression [6-8,28].

Conclusions

In summary, EBNA2 expression causes persistent ATF4 activation through EBNA2-mediated epigenetic changes and enhances USF1 binding to the ATF4 promoter. Furthermore, EBNA2-mediated ATF4 activation increases the expression of SREBP1, ChREBP, and FAS, as well as modulates lipid metabolism, redox balance, and mitochondrial function, which subsequently contributes to tumorigenesis. Given our findings that EBNA2 mediates tumorigenesis through ATF4 activation and modulation of lipid metabolism, our study may reveal novel potential targets for the clinical treatment of EBV-mediated tumors.

Acknowledgements

EBNA2 shRNA lentivirus, EBNA2 adenovirus, and related empty controls were kindly provided by Dr. Haimou Zhang (Hubei University, China). This study was financially supported by the Shenzhen Science and Technology Innovation Project #: JCYJ20210324105802007; Shenzhen Health System Research Project #: SZXJ2018081; Research Foundation of Peking University Shenzhen Hospital Project #: JCYJ2020017; and National Natural Science Foundation of China, Project #: 82170191 & 82100210.

The protocols for animal (#2021-092) and human subjects (#2020-050) were approved by the Peking University Shenzhen Hospital Ethical Committee and written consent was obtained from all participants.

Disclosure of conflict of interest

None.

Abbreviations

ATF4

activating transcription factor-4

ChREBP

carbohydrate-responsive element-binding protein

EBNA2

Epstein-Barr virus nuclear antigen 2

EBV

Epstein-Barr virus

FAS

fatty acid synthase

MMP

mitochondrial membrane potential (ΔΨm)

SREBP1

sterol regulatory element-binding protein 1

USF1

upstream stimulating factor 1

Supporting Information

ajcr0013-1363-f8.pdf (557.5KB, pdf)

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