Downregulation of KLF5 by EBER1 via the ERK signaling pathway in EBV-positive nasopharyngeal carcinoma cells: implications for latent EBV infection.

Nasopharyngeal carcinoma (NPC) carcinogenesis and malignant transformation are intimately associated with Epstein-Barr virus (EBV) infection. A zinc-fingered transcription factor known as Krüppel-like factor 5 (KLF5) has been shown to be aberrantly expressed in a number of cancer types. However, little is known about the regulatory pathways and roles of KLF5 in EBV-positive NPC. Our study found that KLF5 expression was significantly lower in EBV-positive NPC than in EBV-negative NPC. Further investigation revealed that EBER1, which is encoded by EBV, down-regulates KLF5 via the extracellular signal-regulated kinase (ERK) signalling pathway. This down-regulation of KLF5 by EBER1 contributes to maintaining latent EBV infection in NPC. Furthermore, we uncovered the biological roles of KLF5 in NPC cells. Specifically, KLF5 may influence the cell cycle, prevent apoptosis, and encourage cell migration and proliferation - all of which have a generally pro-cancer impact. In conclusion, these findings offer novel strategies for EBV-positive NPC patients' antitumour treatment.

Noncanonical functions of telomerase and telomeres in viruses-associated cancer.

The cause of cancer is attributed to the uncontrolled growth and proliferation of cells resulting from genetic changes and alterations in cell behavior, a phenomenon known as epigenetics. Telomeres, protective caps on the ends of chromosomes, regulate both cellular aging and cancer formation. In most cancers, telomerase is upregulated, with the telomerase reverse transcriptase (TERT) enzyme and telomerase RNA component (TERC) RNA element contributing to the maintenance of telomere length. Additionally, it is noteworthy that two viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), utilize telomerase for their replication or persistence in infected cells. Also, TERT and TERC may play major roles in cancer not related to telomere biology. They are involved in the regulation of gene expression, signal transduction pathways, cellular metabolism, or even immune response modulation. Furthermore, the crosstalk between TERT, TERC, RNA-binding proteins, and microRNAs contributes to a greater extent to cancer biology. To understand the multifaceted roles played by TERT and TERC in cancer and viral life cycles, and then to develop effective therapeutic strategies against these diseases, are fundamental for this goal. By investigating deeply, the complicated mechanisms and relationships between TERT and TERC, scientists will open the doors to new therapies. In its analysis, the review emphasizes the significance of gaining insight into the multifaceted roles that TERT and TERC play in cancer pathogenesis, as well as their involvement in the viral life cycle for designing effective anticancer therapy approaches.

Differential analysis of histopathological and genetic markers of cancer aggressiveness, and survival difference in EBV-positive and EBV-negative prostate carcinoma.

Several studies have shown an association between prostate carcinoma (PCa) and Epstein-Barr virus (EBV); however, none of the studies so far have identified the histopathological and genetic markers of cancer aggressiveness associated with EBV in PCa tissues. In this study, we used previously characterized EBV-PCR-positive (n = 39) and EBV-negative (n = 60) PCa tissues to perform an IHC-based assessment of key histopathological and molecular markers of PCa aggressiveness (EMT markers, AR expression, perineural invasion, and lymphocytic infiltration characterization). Additionally, we investigated the differential expression of key oncogenes, EMT-associated genes, and PCa-specific oncomiRs, in EBV-positive and -negative tissues, using the qPCR array. Finally, survival benefit analysis was also performed in EBV-positive and EBV-negative PCa patients. The EBV-positive PCa exhibited a higher percentage (80%) of perineural invasion (PNI) compared to EBV-negative PCa (67.3%) samples. Similarly, a higher lymphocytic infiltration was observed in EBV-LMP1-positive PCa samples. The subset characterization of T and B cell lymphocytic infiltration showed a trend of higher intratumoral and tumor stromal lymphocytic infiltration in EBV-negative tissues compared with EBV-positive tissues. The logistic regression analysis showed that EBV-positive status was associated with decreased odds (OR = 0.07; p-value < 0.019) of CD3 intratumoral lymphocytic infiltration in PCa tissues. The analysis of IHC-based expression patterns of EMT markers showed comparable expression of all EMT markers, except vimentin, which showed higher expression in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Furthermore, gene expression analysis showed a statistically significant difference (p < 0.05) in the expression of CDH1, AR, CHEK-2, CDKN-1B, and CDC-20 and oncomiRs miR-126, miR-152-3p, miR-452, miR-145-3p, miR-196a, miR-183-3p, and miR-146b in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Overall, the survival proportion was comparable in both groups. The presence of EBV in the PCa tissues results in an increased expression of certain oncogenes, oncomiRs, and EMT marker (vimentin) and a decrease in CD3 ITL, which may be associated with the aggressive forms of PCa.

Lytic and Latent Genetic Diversity of the Epstein-Barr Virus Reveals Raji-Related Variants from Southeastern Brazil Associated with Recombination Markers.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus etiologically associated with benign and malignant diseases. Since the pathogenic mechanisms of EBV are not fully understood, understanding EBV genetic diversity is an ongoing goal. Therefore, the present work describes the genetic diversity of the lytic gene BZLF1 in a sampling of 70 EBV-positive cases from southeastern Brazil. Additionally, together with the genetic regions previously characterized, the aim of the present study was to determine the impact of viral genetic factors that may influence EBV genetic diversity. Accordingly, the phylogenetic analysis of the BZLF1 indicated two main clades with high support, BZ-A and BZ-B (PP > 0.85). Thus, the BZ-A clade was the most diverse clade associated with the main polymorphisms investigated, including the haplotype Type 1 + V3 (p < 0.001). Furthermore, the multigene phylogenetic analysis (MLA) between BZLF1 and the oncogene LMP1 showed specific clusters, revealing haplotypic segregation that previous single-gene phylogenies from both genes failed to demonstrate. Surprisingly, the LMP1 Raji-related variant clusters were shown to be more diverse, associated with BZ-A/B and the Type 2/1 + V3 haplotypes. Finally, due to the high haplotypic diversity of the Raji-related variants, the number of DNA recombination-inducing motifs (DRIMs) was evaluated within the different clusters defined by the MLA. Similarly, the haplotype BZ-A + Raji was shown to harbor a greater number of DRIMs (p < 0.001). These results call attention to the high haplotype diversity of EBV in southeast Brazil and strengthen the hypothesis of the recombinant potential of South American Raji-related variants via the LMP1 oncogene.

Epstein-Barr virus-associated lymphoepithelioma-like intrahepatic cholangiocarcinoma: A report of 3 cases and literature review. / Epstein-Barrç— æ¯’ç›¸å ³æ·‹å·´ä¸Šçš®ç˜¤æ ·è‚å† èƒ†ç®¡ç™Œ3ä¾‹å¹¶æ–‡çŒ®å¤ä¹ ï¼ˆè‹±æ–‡ï¼‰.

Hepatic lymphoepithelioma-like carcinoma (LELC) is an extremely rare malignant tumor characterized by undifferentiated malignant epithelial cells and significant lymphatic infiltration. Hepatic LELC mainly includes lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) and lymphoepithelioma-like intrahepatic cholangiocarcinoma (LEL-CC). Epstein-Barr virus (EBV) infection is considered as an important factor in LELC carcinogenesis. Since 2005, Xiangya Hospital of Central South University has treated a total of 3 patients with EBV-associated LEL-CC, which all showed liver masses by CT scans. After surgical resection, the EBV encoded RNA (EBER) and CK19 expression in all 3 patients were positive, and pathological examination confirmed EBV-associated LEL-CC. Two patients had a good postoperative prognosis, while 1 patient received relevant immunotherapy and chemotherapy after surgery. Based on the analysis of existing literature, the author believes that hepatic LELC can be included in the classification of liver tumors, which will provide new ideas for the accurate diagnosis and treatment of hepatic LELC.

Clinicopathological characteristics and prognosis of Epstein-Barr virus-associated gastric cancer.

OBJECTIVE: Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct molecular subtype of gastric cancer (GC). At present, the clinical characteristics and prognostic implications of EBV infection and the potential clinical benefits of immune checkpoint blockade in GC remain to be clarified. Hence, this study was designed to analyze the clinical and pathological characteristics of GC patients with varying EBV infection states and compare their overall survival (OS). METHODS: A retrospective study was performed on 1031 consecutive GC patients who underwent gastrectomy at the Affiliated Hospital of Xuzhou Medical University from February 2018 to November 2022. EBV-encoded RNA (EBER) in situ hybridization (ISH) was used for EBV assessment, and immunohistochemical staining was used for evaluation of human epidermal growth factor receptor 2 (HER2), programmed death ligand 1 (PD-L1), and Ki67 expression. EBVaGC was defined as tumors with EBV positivity. In addition, EBV-negative GC (EBVnGC) patients were matched with EBVaGC patients based on seven clinicopathological parameters (age, gender, anatomic subsite, tumor size, Lauren classification, degree of differentiation, and tumor-node-metastasis [TNM] stage). The correlations of clinical features with HER2, PD-L1, and Ki67 expression were evaluated statistically. The survival of patients was assessed through medical records, telephone, or WeChat communication, and prognostic analysis was performed using the logrank test as well as univariable and multivariable regression analysis. RESULTS: Out of 1031 GC patients tested, 35 (3.4%) were diagnosed with EBVaGC. Notably, the EBVaGC group exhibited a distinct predominance of males and younger patients, significantly higher Ki67 and PD-L1 expression levels, and a lower prevalence of pericancerous nerve invasion than the EBVnGC group (P < 0.01). In the 35 EBVaGC cases, Ki67 expression was negatively correlated with age (P < 0.05), suggesting that a younger onset age was associated with higher Ki67 expression. In addition, PD-L1 expression was correlated with the degree of differentiation, T-stage, and clinical stage of the patient. Furthermore, PD-L1 expression was elevated in tumors with lower differentiation or at later stages (P < 0.05). Using univariate analysis, Ki67, PD-L1, and clinical stage were identified as significant factors influencing the overall survival (OS) of EBVaGC patients (P < 0.05). Moreover, multivariate survival analysis revealed that clinical stage and Ki67 expression were independent risk factors for the OS of the patients (P < 0.05), and the three-year OS rate of EBVaGC patients was 64.2%. CONCLUSION: EBV-ISH is a practical and valuable method to identify EBVaGC. Owing to its unique etiological, pathological, and clinical characteristics, patients with EBVaGC might benefit from immune checkpoint blockade therapy.

Synthetic BZLF1-targeted transcriptional activator for efficient lytic induction therapy against EBV-associated epithelial cancers.

The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.

Comparison of Whole Blood and Plasma for Monitoring Cytomegalovirus and Epstein-Barr Virus.

Monitoring Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection after transplantation is recommended to enable preemptive therapy. However, the most suitable sample type remains unclear. Patients who underwent hematopoietic stem cell or liver transplantation were included in this study. Viral loads in sequential whole-blood and plasma samples were retrospectively analyzed. EBV DNA was detected more frequently in whole blood (55%) than in plasma (18%). The detection rate of CMV DNA was similar between the two sample types. The correlation of viral loads between the two sample types were 0.515 and 0.688 for EBV and CMV, respectively. Among paired samples in which EBV DNA was detected in whole blood, the plasma EBV detection rate was significantly higher in patients who underwent hematopoietic stem cell transplantation than in those who underwent liver transplantation. The viral DNA load in whole blood and plasma showed similar trends. The EBV detection rate was higher in whole blood, and a high correlation was observed between CMV DNA loads and whole blood and plasma. These results indicate that whole blood is more sensitive for monitoring both EBV and CMV, whereas plasma is a potential alternative sample for monitoring CMV.

The nucleic acid binding protein SFPQ represses EBV lytic reactivation by promoting histone H1 expression.

Epstein-Barr virus (EBV) uses a biphasic lifecycle of latency and lytic reactivation to infect >95% of adults worldwide. Despite its central role in EBV persistence and oncogenesis, much remains unknown about how EBV latency is maintained. We used a human genome-wide CRISPR/Cas9 screen to identify that the nuclear protein SFPQ was critical for latency. SFPQ supported expression of linker histone H1, which stabilizes nucleosomes and regulates nuclear architecture, but has not been previously implicated in EBV gene regulation. H1 occupied latent EBV genomes, including the immediate early gene BZLF1 promoter. Upon reactivation, SFPQ was sequestered into sub-nuclear puncta, and EBV genomic H1 occupancy diminished. Enforced H1 expression blocked EBV reactivation upon SFPQ knockout, confirming it as necessary downstream of SFPQ. SFPQ knockout triggered reactivation of EBV in B and epithelial cells, as well as of Kaposi's sarcoma-associated herpesvirus in B cells, suggesting a conserved gamma-herpesvirus role. These findings highlight SFPQ as a major regulator of H1 expression and EBV latency.

Epstein-Barr virus-encoded BART9 and BART15 miRNAs are elevated in exosomes of cerebrospinal fluid from relapsing-remitting multiple sclerosis patients.

Epstein-Barr virus (EBV) infection is approved as the main environmental trigger of multiple sclerosis (MS). In this path, we quantified ebv-miR-BART9-3p and ebv-miR-BART15 in exosomes of cerebrospinal fluid (CSF) of untreated relapsing-remitting MS (RRMS) patients in comparison with the control group. Interestingly, patients displayed significant upregulation of ebv-miR-BART9-3p (18.4-fold) and ebv-miR-BART15 (3.1-fold) expression in CSF exosomes. Moreover, the expression levels of hsa-miR-21-5p and hsa-miR-146a-5p were found to be significantly elevated in the CSF samples obtained from the patient group compared to those obtained from the HC group. The levels of Interferon-gamma (IFN-γ), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), transforming growth factor beta (TGF-ß), and tumor necrosis factor-alpha (TNF-α) were observed to be significantly elevated in the serum and CSF exosomes of the patients. The highest increase was observed in TGF-ß (8.5-fold), followed by IL-23 (3.9-fold) in CSF exosomes. These findings are in agreement with the association between EBV infection and inflammatory cytokines induction. Furthermore, the ratios of TGF-ß: TNF-α and TGF-ß: IFN-γ attained values of 4 to 16.4 and 1.3 to 3.6, respectively, in the CSF exosomes of the patients, in comparison to those of the control group. These findings show EBV activity in RRMS patients is different from that of healthy ones. Elevation of ebv-miR-BART9-3p, ebv-miR-BART15, and inflammatory cytokines expression in CSF exosomes in RRMS patients provides a substantial link between EBV activity and the onset of the disease, as well as the transition from EBV infection to MS.

Germinal center cytokine driven epigenetic control of Epstein-Barr virus latency gene expression.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.

Cytological Features of Fibrin-associated Diffuse Large B-cell Lymphoma Arising in Retroperitoneal Pseudocyst: The First Case Report.

BACKGROUND/AIM: Fibrin-associated diffuse large B-cell lymphoma (FA-DLBCL) is frequently associated with the Epstein-Barr virus (EBV) and manifests as non-mass-forming microscopic lesions within fibrin-rich lesions. Herein, we describe the cytological features of FA-DLBCL. CASE REPORT: A 72-year-old man presented with a large retroperitoneal cystic mass that was treated by cyst aspiration and laparoscopic excision. Individually dispersed large, atypical cells in a necrotic background contained scant cytoplasm and hyperchromatic nuclei with irregular nuclear contours, frequent karyorrhectic debris, and mitotic figures. A fibrinous exudate with necrotic material attached to the inner surface of the cystic mass contained large, atypical cells that were individually scattered or arranged in small clusters. These were positive for cluster of differentiation 20 and Epstein-Barr virus-encoded RNA in situ hybridization. CONCLUSION: We cytologically characterized FA-DLBCL as large, atypical cells that were individually scattered or arranged in small clusters in a necrotic background. To the best of our knowledge, we revealed the cytological features of FA-DLBCL.

Role of plasma EBV-DNA load and EBER status on newly diagnosed peripheral T-cell lymphoma.

PURPOSE: To explore the prognostic and therapeutic role of Epstein-Barr Virus (EBV) on peripheral T-cell lymphoma (PTCL). METHODS: Totally 262 newly diagnosed PTCL patients who were hospitalized from January 2014 to December 2022 were retrospectively enrolled. Molecular analysis included 31 eligible patients. EBV-encoded RNA (EBER) presence in tumor tissue and EBV DNA levels in patients at baseline (DNA1) and after 4 cycles of chemotherapy (DNA4) were assessed. RESULTS: Our findings revealed that the EBER-positive cohort exhibited significant differences compared to counterparts in overall survival (OS, P = 0.047) and progression-free survival (PFS, P = 0.009). Both DNA1 and DNA4 were significantly associated with inferior OS. Multivariate analysis demonstrated that DNA4 independently affected PTCL prognosis for OS (hazard ratio = 5.1617; 95% confidence interval 1.1017-24.1831; P = 0.037). Treatment with the cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) plus azacytidine regimen showed a better OS compared to CHOP or CHOP plus etoposide for patients with partially positive EBER and EBER positive statuses (P = 0.192), although the improvement was not statistically significant. This study delineated the genetic paradigm of PTCL, comparing genetic differences by EBV status and found that EBER partially positive plus positive patients were more likely to have DNMT3A (P = 0.002), RHOAG17V (P = 0.023), and TET2 mutations (P = 0.032). CONCLUSION: EBER, DNA1, and DNA4 emerged as sensitive markers for prognosis. CHOP plus azacytidine might present a preferable option for PTCL patients with DNA methylation due to EBV infection.

Interferon-induced transmembrane protein-1 competitively blocks Ephrin receptor A2-mediated Epstein-Barr virus entry into epithelial cells.

Epstein-Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m6A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1's crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

Expression and prognostic significance of the PD-1/PD-L1 pathway in AIDS-related non-Hodgkin lymphoma.

OBJECTIVE: Immune tolerance and evasion play a critical role in virus-driven malignancies. However, the phenotype and clinical significance of programmed cell death 1 (PD-1) and its ligands, PD-L1 and PD-L2, in aggressive acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin lymphoma (AR-NHL) remain poorly understood, particularly in the Epstein-Barr virus (EBV)-positive subset. METHODS: We used in situ hybridization with EBV-encoded RNA (EBER) to assess the EBV status. We performed immunohistochemistry and flow cytometry analysis to evaluate components of the PD-1/PD-L1/L2 pathway in a multi-institutional cohort of 58 patients with AR-NHL and compared EBV-positive and EBV-negative cases. RESULTS: The prevalence of EBV+ in AR-NHL was 56.9% and was associated with a marked increase in the expression of PD-1/PD-L1/PD-L2 in malignant cells. Patients with AR-NHLs who tested positive for both EBER and PD-1 exhibited lower survival rates compared to those negative for these markers (47.4% vs. 93.8%, p = 0.004). Similarly, patients positive for both EBER and PD-L1 also demonstrated poorer survival (56.5% vs. 93.8%, p = 0.043). Importantly, PD-1 tissue-expression demonstrated independent prognostic significance for overall survival in multivariate analysis and was correlated to elevated levels of LDH (r = 0.313, p = 0.031), increased PD-1+ Tregs (p = 0.006), and robust expression of EBER (r = 0.541, p < 0.001) and PD-L1 (r = 0.354, p = 0.014) expression. CONCLUSIONS: These data emphasize the importance of PD-1-mediated immune evasion in the complex landscape of immune oncology in AR-NHL co-infected with EBV, and contribute to the diagnostic classification and possible definition of immunotherapeutic strategies for this unique subgroup.

OTUD1 enhances gastric cancer aggressiveness by deubiquitinating EBV-encoded protein BALF1 to stabilize the apoptosis inhibitor Bcl-2.

The Epstein-Barr virus (EBV) is implicated in several cancers, including EBV-associated gastric cancer (EBVaGC). This study focuses on EBV-encoded BALF1 (BamH1 A fragment leftward reading frame 1), a key apoptosis regulator in EBV-related cancers, whose specific impact on EBVaGC was previously unknown. Our findings indicate that BALF1 overexpression in gastric cancer cells significantly enhances their proliferation, migration, and resistance to chemotherapy-induced apoptosis, confirming BALF1's oncogenic potential. A novel discovery is that BALF1 undergoes degradation via the ubiquitin-proteasome pathway. Through analysis of 69 deubiquitinating enzymes (DUBs), ovarian tumor protease (OTU) domain-containing protein 1 (OTUD1) emerged as a vital regulator for maintaining BALF1 protein stability. Furthermore, BALF1 was found to play a role in regulating the stability of the B-cell lymphoma-2 (Bcl-2) protein, increasing its levels through deubiquitination. This mechanism reveals BALF1's multifaceted oncogenic role in gastric cancer, as it contributes both directly and indirectly to cancer progression, particularly by stabilizing Bcl-2, known for its anti-apoptotic characteristics. These insights significantly deepen our understanding of EBV's involvement in the pathogenesis of gastric cancer. The elucidation of OTUD1's role in BALF1 regulation and its influence on Bcl-2 stabilization provide new avenues for therapeutic intervention in EBVaGC, bridging the gap between viral oncogenesis and cellular protein regulation and offering a more holistic view of gastric cancer development under the influence of EBV.

Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

EBV-Associated Gastric Cancer; An In Situ Hybridization Assay on Tissue Microarray: A Multi-Region Study from Four Major Provinces of Iran.

BACKGROUND: Gastric cancer is the fourth leading cause of cancer-related deaths in the world. The identification of gastric cancer subtypes related to recognizable microbial agents may play a pivotal role in the targeted prevention and treatment of this cancer. The current study is conducted to define the frequency of Epstein-Barr virus (EBV) infection in gastric cancers of four major provinces, with different incidence rates of gastric cancers, in Iran. METHODS: Paraffin blocks of 682 cases of various types of gastric cancer from Tehran, South and North areas of Iran were collected. Twelve tissue microarray (TMA) blocks were constructed from these blocks. Localization of EBV in tumors was assessed by in situ hybridization (ISH) for EBV-encoded RNA (EBER). Chi-squared test was used to evaluate the statistical significance between EBV-associated gastric cancer (EBVaGC) and clinicopathologic tumor characteristics. RESULTS: Fourteen out of 682 cases (2.1%) of gastric adenocarcinoma were EBER-positive. EBER was positive in 8 out of 22 (36.4%) of medullary carcinomas and 6 out of 660 (0.9%) of non-medullary type, which was a statistically significant difference (P<0.001). The EBVaGCs were more frequent in younger age (P=0.009) and also showed a trend toward the lower stage of the tumor (P=0.075). CONCLUSION: EBV-associated gastric adenocarcinoma has a low prevalence in Iran. This finding can be due to epidemiologic differences in risk factors and exposures, and the low number of gastric medullary carcinomas in the population. It may also be related to gastric tumor heterogeneity not detected with the TMA technique.

The Epstein-Barr virus-miRNA-BART6-5p regulates TGF-ß/SMAD4 pathway to induce glycolysis and enhance proliferation and metastasis of gastric cancer cells.

Background: EBV-miR-BARTs exhibit significant relevance in epithelial tumors, particularly in EBV-associated gastric and nasopharyngeal cancers. However, their specific mechanisms in the initiation and progression of gastric cancer remain insufficiently explored. Material and Methods: Initially, EBV-miRNA-BART6-5p and its target gene SMAD4 expression were assessed in EBV-associated gastric cancer tissues and cell lines. Subsequent transfection induced overexpression of EBV-miRNA-BART6-5p in AGS and MKN-45, and downregulation in EBV-positive cells (SUN-719). The subsequent evaluation aimed to observe their impact on gastric cancer cell proliferation, migration, and glycolytic processes, with the TGF-ß/SMAD4 signaling pathway value clarified using a TGF-ß inhibitor. Results: EBV-miRNA-BART6-5p exhibits pronounced upregulation in EBV-associated gastric cancer tissues and EBV-positive cells, while its target gene SMAD4 demonstrates downregulated expression. Upregulation of it can promote the proliferation and migration of gastric cancer cells. Additionally, We found EBV-miRNA-BART6-5p promotes glycolysis of gastric cancer cells. Inhibition of the TGF-ß/SMAD4 signaling pathway resulted in suppressed proliferation and migration of gastric cancer cells, concomitant with a diminished glycolytic capacity. Conclusion: In this study, we found that EBV-miRNA-BART6-5p can target SMAD4, effectively increasing glycolysis in gastric cancer cells by regulating the TGF-ß/SMAD4 signaling pathway, thereby enhancing the proliferation and metastasis of gastric cancer cells. Our findings may offer new insights into the metabolic aspects of gastric cancer.

Aflatoxin B1 and Epstein-Barr virus-induced CCL22 expression stimulates B cell infection.

Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.