The Way to Malignant Transformation: Can Epigenetic Alterations Be Used to Diagnose Early-Stage Head and Neck Cancer?

Identifying and treating tumors early is the key to secondary prevention in cancer control. At present, prevention of oral cancer is still challenging because the molecular drivers responsible for malignant transformation of the 11 clinically defined oral potentially malignant disorders are still unknown. In this review, we focused on studies that elucidate the epigenetic alterations demarcating malignant and nonmalignant epigenomes and prioritized findings from clinical samples. Head and neck included, the genomes of many cancer types are largely hypomethylated and accompanied by focal hypermethylation on certain specific regions. We revisited prior studies that demonstrated that sufficient uptake of folate, the primary dietary methyl donor, is associated with oral cancer reduction. As epigenetically driven phenotypic plasticity, a newly recognized hallmark of cancer, has been linked to tumor initiation, cell fate determination, and drug resistance, we discussed prior findings that might be associated with this hallmark, including gene clusters (11q13.3, 19q13.43, 20q11.2, 22q11-13) with great potential for oral cancer biomarkers, and successful examples in screening early-stage nasopharyngeal carcinoma. Although one-size-fits-all approaches have been shown to be ineffective in most cancer therapies, the rapid development of epigenome sequencing methods raises the possibility that this nonmutagenic approach may be an exception. Only time will tell.

Identification of Prognostic Biomarkers Originating From the Tumor Stroma of Betel Quid-Associated Oral Cancer Tissues.

BACKGROUND: Partial epithelial-mesenchymal transition (p-EMT) is a distinct clinicopathological feature prevalent in oral cavity tumors of The Cancer Genome Atlas. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion. The pathological roles of otherwise nonmalignant cancer-associated fibroblasts (CAFs) in cancer progression are emerging. METHODS: Gene set enrichment analysis was used to reveal differentially enriched genes and molecular pathways in OC3 and TW2.6 xenograft tissues, representing mesenchymal and p-EMT tumors, respectively. R packages of genomic data science were executed for statistical evaluations and data visualization. Immunohistochemistry and Alcian blue staining were conducted to validate the bioinformatic results. Univariate and multivariate Cox proportional hazards models were performed to identify covariates significantly associated with overall survival in clinical datasets. Kaplan-Meier curves of estimated overall survival were compared for statistical difference using the log-rank test. RESULTS: Compared to mesenchymal OC3 cells, tumor stroma derived from p-EMT TW2.6 cells was significantly enriched in microvessel density, tumor-excluded macrophages, inflammatory CAFs, and extracellular hyaluronan deposition. By translating these results to clinical transcriptomic datasets of oral cancer specimens, including the Puram single-cell RNA-seq cohort comprising ~6000 cells, we identified the expression of stromal TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types and HYAL1 was a good indicator for four tumor cohorts, suggesting common stromal effects existing in different cancer types. CONCLUSIONS: As the tumor stroma coevolves with cancer progression, the cellular origins of molecular markers identified from conventional whole tissue mRNA-based analyses should be cautiously interpreted. By incorporating disease-matched xenograft tissue and single-cell RNA-seq results, we suggested that TGFBI and HYAL1, primarily expressed by stromal CAFs and endothelial cells, respectively, could serve as robust prognostic biomarkers for oral cancer control.

c-MYC-directed NRF2 drives malignant progression of head and neck cancer via glucose-6-phosphate dehydrogenase and transketolase activation.

Rationale: NRF2, a redox sensitive transcription factor, is up-regulated in head and neck squamous cell carcinoma (HNSCC), however, the associated impact and regulatory mechanisms remain unclear. Methods: The protein expression of NRF2 in HNSCC specimens was examined by IHC. The regulatory effect of c-MYC on NRF2 was validated by ChIP-qPCR, RT-qPCR and western blot. The impacts of NRF2 on malignant progression of HNSCC were determined through genetic manipulation and pharmacological inhibition in vitro and in vivo. The gene-set enrichment analysis (GSEA) on expression data of cDNA microarray combined with ChIP-qPCR, RT-qPCR, western blot, transwell migration/ invasion, cell proliferation and soft agar colony formation assays were used to investigate the regulatory mechanisms of NRF2. Results: NRF2 expression is positively correlated with malignant features of HNSCC. In addition, carcinogens, such as nicotine and arecoline, trigger c-MYC-directed NRF2 activation in HNSCC cells. NRF2 reprograms a wide range of cancer metabolic pathways and the most notable is the pentose phosphate pathway (PPP). Furthermore, glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) are critical downstream effectors of NRF2 that drive malignant progression of HNSCC; the coherently expressed signature NRF2/G6PD/TKT gene set is a potential prognostic biomarker for prediction of patient overall survival. Notably, G6PD- and TKT-regulated nucleotide biosynthesis is more important than redox regulation in determining malignant progression of HNSCC. Conclusions: Carcinogens trigger c-MYC-directed NRF2 activation. Over-activation of NRF2 promotes malignant progression of HNSCC through reprogramming G6PD- and TKT-mediated nucleotide biosynthesis. Targeting NRF2-directed cellular metabolism is an effective strategy for development of novel treatments for head and neck cancer.

Index of Cancer-Associated Fibroblasts Is Superior to the Epithelial-Mesenchymal Transition Score in Prognosis Prediction.

In many solid tumors, tissue of the mesenchymal subtype is frequently associated with epithelial-mesenchymal transition (EMT), strong stromal infiltration, and poor prognosis. Emerging evidence from tumor ecosystem studies has revealed that the two main components of tumor stroma, namely, infiltrated immune cells and cancer-associated fibroblasts (CAFs), also express certain typical EMT genes and are not distinguishable from intrinsic tumor EMT, where bulk tissue is concerned. Transcriptomic analysis of xenograft tissues provides a unique advantage in dissecting genes of tumor (human) or stroma (murine) origins. By transcriptomic analysis of xenograft tissues, we found that oral squamous cell carcinoma (OSCC) tumor cells with a high EMT score, the computed mesenchymal likelihood based on the expression signature of canonical EMT markers, are associated with elevated stromal contents featured with fibronectin 1 (Fn1) and transforming growth factor-ß (Tgfß) axis gene expression. In conjugation with meta-analysis of these genes in clinical OSCC datasets, we further extracted a four-gene index, comprising FN1, TGFB2, TGFBR2, and TGFBI, as an indicator of CAF abundance. The CAF index is more powerful than the EMT score in predicting survival outcomes, not only for oral cancer but also for the cancer genome atlas (TCGA) pan-cancer cohort comprising 9356 patients from 32 cancer subtypes. Collectively, our results suggest that a further distinction and integration of the EMT score with the CAF index will enhance prognosis prediction, thus paving the way for curative medicine in clinical oncology.

Discoidin Domain Receptor-1 (DDR1) is Involved in Angiolymphatic Invasion in Oral Cancer.

The discoidin domain receptor-1 (DDR1) is a non-integrin collagen receptor recently implicated in the collective cell migration of other cancer types. Previously, we identified an elevated expression of DDR1 in oral squamous cell carcinoma (OSCC) cells. Through the data mining of a microarray dataset composed of matched tumor-normal tissues from forty OSCC patients, we distilled overexpressed genes statistically associated with angiolymphatic invasion, including DDR1, COL4A5, COL4A6 and PDPN. Dual immunohistochemical staining further confirmed the spatial locations of DDR1 and PDPN in OSCC tissues indicative of collective cancer cell invasion. An elevated DDR1 expression at both the transcription and protein level was observed by treating keratinocytes with collagen of fibrillar or basement membrane types. In addition, inhibition of DDR1 kinase activity in OSCC TW2.6 cells disrupted cell cohesiveness in a 2D culture, reduced spheroid invasion in a collagen gel matrix, and suppressed angiolymphatic invasion in xenograft tissues. Taken together, these results suggest that collagen deposition in the affected tissues followed by DDR1 overexpression could be central to OSCC tumor growth and angiolymphatic invasion. Thus, DDR1 inhibitors are potential therapeutic compounds in restraining oral cancer, which has not been previously explored.

Emodin Inhibits EBV Reactivation and Represses NPC Tumorigenesis.

Nasopharyngeal carcinoma (NPC) is a unique malignancy derived from the epithelium of the nasopharynx. Despite great advances in the development of radiotherapy and chemotherapy, relapse and metastasis in NPC patients remain major causes of mortality. Evidence accumulated over recent years indicates that Epstein-Barr virus (EBV) lytic replication plays an important role in the pathogenesis of NPC and inhibition of EBV reactivation is now being considered as a goal for the therapy of EBV-associated cancers. With this in mind, a panel of dietary compounds was screened and emodin was found to have potential anti-EBV activity. Through Western blotting, immunofluorescence, and flow cytometric analysis, we show that emodin inhibits the expression of EBV lytic proteins and blocks virion production in EBV- positive epithelial cell lines. In investigating the underlying mechanism, reporter assays indicated that emodin represses Zta promoter (Zp) and Rta promoter (Rp) activities, triggered by various inducers. Mapping of the Zp construct reveals that the SP1 binding region is important for emodin-triggered repression and emodin is shown to be able to inhibit SP1 expression, suggesting that it likely inhibits EBV reactivation by suppression of SP1 expression. Moreover, we also show that emodin inhibits the tumorigenic properties induced by repeated EBV reactivation, including micronucleus formation, cell proliferation, migration, and matrigel invasiveness. Emodin administration also represses the tumor growth in mice which is induced by EBV activation. Taken together, our results provide a potential chemopreventive agent in restricting EBV reactivation and NPC recurrence.

MicroRNA-486-3p functions as a tumor suppressor in oral cancer by targeting DDR1.

BACKGROUND: Discoidin domain receptor-1 (DDR1) tyrosine kinase is highly expressed in a variety of human cancers and involved in various steps of tumorigenesis. However, the precise mechanisms underlying the abnormal expression of DDR1 in oral squamous cell carcinoma (OSCC) has not been well investigated. METHODS: The expression of DDR1 on OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Specific targeting by miRNAs was determined by software prediction, luciferase reporter assay, and correlation with target protein expression. The functions of miR-486-3p and DDR1 were accessed by MTT and Annexin V analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) and methylation specific PCR (MSP) were performed to explore the molecular mechanisms by arecoline treatment. RESULTS: Here, we reported that DDR1 was significantly upregulated in OSCC tissues and its levels were inversely correlated with miR-486-3p expression. The experimental results in vitro confirmed that miR-486-3p decreased DDR1 expression by targeting the 3'-UTR of DDR1 mRNA. Overexpression of miR-486-3p led to growth inhibition and apoptosis induction with a similar function by knockdown of DDR1. Aberrant methylation of ANK1 promoter was a highly prevalent in OSCC and contributes to oral carcinogenesis by epigenetic silencing of ANK1 and miR-486-3p. We found that miR-486-3p can be transcriptionally co-regulated with its host gene ANK1 through epigenetic repression. DNA methylation inhibitor treatment re-expressed ANK1 and miR-486-3p. Importantly, arecoline, a major betel nut alkaloid, recruited DNMT3B binding to ANK1 promoter for DNA methylation and then attenuated the expression of miR-486-3p in OSCC. CONCLUSION: This study was the first to demonstrate that betel nut alkaloid may recruit DNMT3B to regulate miR-486-3p/DDR1 axis in oral cancer andmiR-486-3p and DDR1 may serve as potential therapeutic targets of oral cancer.

Epstein-Barr virus BRLF1 induces genomic instability and progressive malignancy in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a serious health problem in China and Southeast Asia. Relapse is the major cause of mortality, but mechanisms of relapse are mysterious. Epstein-Barr virus (EBV) reactivation and host genomic instability (GI) have correlated with NPC development. Previously, we reported that lytic early genes DNase and BALF3 induce genetic alterations and progressive malignancy in NPC cells, implying lytic proteins may be required for NPC relapse. In this study, we show that immediate early gene BRLF1 induces chromosome mis-segregation and genomic instability in the NPC cells. Similar phenomenon was also demonstrated in 293 and zebrafish embryonic cells. BRLF1 nuclear localization signal (NLS) mutant still induced genomic instability and inhibitor experiments revealed that BRLF1 interferes with chromosome segregation and induces genomic instability by activating Erk signaling. Furthermore, the chromosome aberrations and tumorigenic features of NPC cells were significantly increased with the rounds of BRLF1 expression, and these cells developed into larger tumor nodules in mice. Therefore, BRLF1 may be the important factor contributing to NPC relapse and targeting BRLF1 may benefit patients.

Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes.

Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.

The SWI/SNF Chromatin Regulator BRG1 Modulates the Transcriptional Regulatory Activity of the Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1.

During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1.IMPORTANCE The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.

The Ubiquitin Ligase Itch and Ubiquitination Regulate BFRF1-Mediated Nuclear Envelope Modification for Epstein-Barr Virus Maturation.

UNLABELLED: The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE: The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.

Luteolin inhibits Epstein-Barr virus lytic reactivation by repressing the promoter activities of immediate-early genes.

The lytic reactivation of Epstein-Barr virus (EBV) has been reported to be strongly associated with several human diseases, including nasopharyngeal carcinoma (NPC). Inhibition of the EBV lytic cycle has been shown to be of great benefit in the treatment of EBV-associated diseases. The administration of dietary compounds is safer and more convenient than other approaches to preventing EBV reactivation. We screened several dietary compounds for their ability to inhibit EBV reactivation in NPC cells. Among them, the flavonoid luteolin showed significant inhibition of EBV reactivation. Luteolin inhibited protein expression from EBV lytic genes in EBV-positive epithelial and B cell lines. It also reduced the numbers of EBV-reactivating cells detected by immunofluorescence analysis and reduced the production of virion. Furthermore, luteolin reduced the activities of the promoters of the immediate-early genes Zta (Zp) and Rta (Rp) and also inhibited Sp1-luc activity, suggesting that disruption of Sp1 binding is involved in the inhibitory mechanism. CHIP analysis revealed that luteolin suppressed the activities of Zp and Rp by deregulating Sp1 binding. Taken together, luteolin inhibits EBV reactivation by repressing the promoter activities of Zp and Rp, suggesting luteolin is a potential dietary compound for prevention of virus infection.

EBV reactivation as a target of luteolin to repress NPC tumorigenesis.

Nasopharyngeal carcinoma (NPC) is a malignancy derived from the epithelial cells of the nasopharynx. Although a combination of radiotherapy with chemotherapy is effective for therapy, relapse and metastasis after remission remain major causes of mortality. Epstein-Barr virus (EBV) is believed to be one of causes of NPC development. We demonstrated previously that EBV reactivation is important for the carcinogenesis of NPC. We sought, therefore, to determine whether EBV reactivation can be a target for retardation of relapse of NPC. After screening, we found luteolin is able to inhibit EBV reactivation. It inhibited EBV lytic protein expression and repressed the promoter activities of two major immediate-early genes, Zta and Rta. Furthermore, luteolin was shown to reduce genomic instability induced by recurrent EBV reactivation in NPC cells. EBV reactivation-induced NPC cell proliferation and migration, as well as matrigel invasiveness, were also repressed by luteolin treatment. Tumorigenicity in mice, induced by EBV reactivation, was decreased profoundly following luteolin administration. Together, these results suggest that inhibition of EBV reactivation is a novel approach to prevent the relapse of NPC.

Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A)-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

Epstein-Barr virus BALF3 has nuclease activity and mediates mature virion production during the lytic cycle.

UNLABELLED: Epstein-Barr virus (EBV) lytic replication involves complex processes, including DNA synthesis, DNA cleavage and packaging, and virion egress. These processes require many different lytic gene products, but the mechanisms of their actions remain unclear, especially for DNA cleavage and packaging. According to sequence homology analysis, EBV BALF3, encoded by the third leftward open reading frame of the BamHI-A fragment in the viral genome, is a homologue of herpes simplex virus type 1 UL28. This gene product is believed to possess the properties of a terminase, such as nucleolytic activity on newly synthesized viral DNA and translocation of unit length viral genomes into procapsids. In order to characterize EBV BALF3, the protein was produced by and purified from recombinant baculoviruses and examined in an enzymatic reaction in vitro, which determined that EBV BALF3 acts as an endonuclease and its activity is modulated by Mg(2+), Mn(2+), and ATP. Moreover, in EBV-positive epithelial cells, BALF3 was expressed and transported from the cytoplasm into the nucleus following induction of the lytic cycle, and gene silencing of BALF3 caused a reduction of DNA packaging and virion release. Interestingly, suppression of BALF3 expression also decreased the efficiency of DNA synthesis. On the basis of these results, we suggest that EBV BALF3 is involved simultaneously in DNA synthesis and packaging and is required for the production of mature virions. IMPORTANCE: Virus lytic replication is essential to produce infectious virions, which is responsible for virus survival and spread. This work shows that an uncharacterized gene product of the human herpesvirus Epstein-Barr virus (EBV), BALF3, is expressed during the lytic cycle. In addition, BALF3 mediates an endonucleolytic reaction and is involved in viral DNA synthesis and packaging, leading to influence on the production of mature virions. According to sequence homology and physical properties, the lytic gene product BALF3 is considered a terminase in EBV. These findings identify a novel viral gene with an important role in contributing to a better understanding of the EBV life cycle.

Reactive oxygen species mediate Epstein-Barr virus reactivation by N-methyl-N'-nitro-N-nitrosoguanidine.

N-nitroso compounds (NOCs) and Epstein-Barr virus (EBV) reactivation have been suggested to play a role in the development of nasopharyngeal carcinoma (NPC). Although chemicals have been shown to be a risk factor contributing to the carcinogenesis of NPC, the underlying mechanism is not fully understood. We demonstrated recently that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhances the genomic instability and tumorigenicity of NPC cells via induction of EBV reactivation. However, the mechanisms that trigger EBV reactivation from latency remain unclear. Here, we address the role of ROS in induction of EBV reactivation under MNNG treatment. EBV reactivation was induced in over 70% of EBV-positive NA cells and the promoter of Rta (Rp) was activated after MNNG treatment. Inhibitor experiments revealed ATM, p38 MAPK and JNK were activated by ROS and involved in MNNG-induced EBV reactivation. Significantly, ROS scavengers N-acetyl-L-cysteine (NAC), catalase and reduced glutathione inhibited EBV reactivation under MNNG and H2O2 treatment, suggesting ROS mediate EBV reactivation. The p53 was essential for EBV reactivation and the Rp activation by MNNG. Moreover, the p53 was phosphorylated, translocated into nucleus, and bound to Rp following ROS stimulation. The results suggest ROS play an important role in initiation of EBV reactivation by MNNG through a p53-dependent mechanism. Our findings demonstrate novel signaling mechanisms used by NOCs to induce EBV reactivation and provide a novel insight into NOCs link the EBV reactivation in the contribution to the development of NPC. Notably, this study indicates that antioxidants might be effective for inhibiting N-nitroso compound-induced EBV reactivation and therefore could be promising preventive and therapeutic agents for EBV reactivation-associated malignancies.

Identification of targetable FGFR gene fusions in diverse cancers.

Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2, including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Because of the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts, which incorporate transcriptome analysis for gene fusions, are poised to identify rare, targetable FGFR fusions across diverse cancer types.

Inhibition of Epstein-Barr virus reactivation in nasopharyngeal carcinoma cells by dietary sulforaphane.

Epstein-Barr virus (EBV) has been associated with several human malignancies including nasopharyngeal carcinoma (NPC). Reactivation of latent EBV has been considered to contribute to the carcinogenesis of NPC. Blocking the EBV lytic cycle has been shown effective in the treatment of EBV-associated diseases. We have searched for natural dietary compounds inhibiting EBV reactivation in NPC cells. Among them, sulforaphane (SFN) was found to be effective in the inhibition of EBV reactivation in latent EBV-positive NPC cells, NA and HA. SFN is a histone deacetylase (HDAC) inhibitor and has been recognized as an antioxidant and antitumor compound for chemoprevention. However, its antiviral effect is less well elucidated. In this study, after determination of the cytotoxicity of SFN on various epithelial cells, we showed that SFN treatment inhibits EBV reactivation, rather than induction, by detection of EBV lytic gene expression in EBV-positive NPC cells. We also determined that the number of cells supporting the EBV lytic cycle is decreased using immunofluorescence and flow cytometric analysis. Moreover, we have found that this inhibitory effect decreases virus production. To elucidate the inhibitory mechanism of SFN on the EBV lytic cycle, luciferase reporter assays were carried out on the Zta and Rta promoters. The results show that SFN inhibits transactivation activity of the EBV immediate-early gene Rta but not Zta. Together, our results suggest that SFN has the capability to inhibit EBV lytic cycle and the potential to be taken as a dietary compound for prevention of EBV reactivation.

Epstein-Barr virus BGLF4 kinase retards cellular S-phase progression and induces chromosomal abnormality.

Epstein-Barr virus (EBV) induces an uncoordinated S-phase-like cellular environment coupled with multiple prophase-like events in cells replicating the virus. The EBV encoded Ser/Thr kinase BGLF4 has been shown to induce premature chromosome condensation through activation of condensin and topoisomerase II and reorganization of the nuclear lamina to facilitate the nuclear egress of nucleocapsids in a pathway mimicking Cdk1. However, the observation that RB is hyperphosphorylated in the presence of BGLF4 raised the possibility that BGLF4 may have a Cdk2-like activity to promote S-phase progression. Here, we investigated the regulatory effects of BGLF4 on cell cycle progression and found that S-phase progression and DNA synthesis were interrupted by BGLF4 in mammalian cells. Expression of BGLF4 did not compensate Cdk1 defects for DNA replication in S. cerevisiae. Using time-lapse microscopy, we found the fate of individual HeLa cells was determined by the expression level of BGLF4. In addition to slight cell growth retardation, BGLF4 elicits abnormal chromosomal structure and micronucleus formation in 293 and NCP-TW01 cells. In Saos-2 cells, BGLF4 induced the hyperphosphorylation of co-transfected RB, while E2F1 was not released from RB-E2F1 complexes. The E2F1 regulated activities of the cyclin D1 and ZBRK1 promoters were suppressed by BGLF4 in a dose dependent manner. Detection with phosphoamino acid specific antibodies revealed that, in addition to Ser780, phosphorylation of the DNA damage-responsive Ser612 on RB was enhanced by BGLF4. Taken together, our study indicates that BGLF4 may directly or indirectly induce a DNA damage signal that eventually interferes with host DNA synthesis and delays S-phase progression.

Ser-634 and Ser-636 of Kaposi's Sarcoma-Associated Herpesvirus RTA are Involved in Transactivation and are Potential Cdk9 Phosphorylation Sites.

The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal ((527)KKRK(530)) and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ∼30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that (634)SPSP(637) motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ∼25% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full transcriptional potency of K-RTA.