**1. Introduction**

26 Carcinogenesis, Diagnosis, and Molecular Targeted Treatment for Nasopharyngeal Carcinoma

Zhou, W., X. Feng, H. Li, L. Wang, B. Zhu, W. Liu, M. Zhao, K. Yao, and C. Ren. 2009.

Zhu, J. Y., T. Pfuhl, N. Motsch, S. Barth, J. Nicholls, F. Grasser, and G. Meister. 2009.

*Neoplasia* 7 (9):809-15.

*Biophys Sin (Shanghai)* 41 (1):54-62.

carcinomas. *J Virol* 83 (7):3333-41.

Virus DNA in nasopharyngeal carcinoma and matched tumor-adjacent tissues.

Inactivation of LARS2, located at the commonly deleted region 3p21.3, by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma. *Acta Biochim* 

Identification of novel Epstein-Barr virus microRNA genes from nasopharyngeal

The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 107 copies per cell) during latent infection by EBV in a variety of cells. Owing to its expression abundance and universal existence in all of the 3 forms of latent infection, EBERs have been under intensive studies since they were discovered by Lernar (Lerner et al., 1981) for the first time. Looking back over the past 30 years, great efforts have been made to unveil the accurate role of EBERs in the latency and transformation process, the definite secondary structure and the signaling pathways they participate in. Despite significant achievements were achieved in these fields, most pioneer work was conducted in lymphoma cells. Bearing this in mind, we explore the similarities between lymphoma and carcinoma to fill the gaps in our knowledge of EBERs' roles in nasopharyngeal carcinoma (NPC). However, it remains to be clarified whether the same scenario accurately applies to the pathological significance of EBERs in NPC.

Epstein-Barr virus (EBV) is consistently detected in NPC from regions of both high and low incidence. In EBV infected cells, there exist some polyribosomal virus-specific RNAs which are the most abundant RNAs (Rymo, 1979). Initial transcription mapping studies by Kieff and colleagues indicated that polyribosomal virus-specific RNA was encoded primarily by the internal repeat region of EBV DNA and, to a lesser extent, by certain other regions of the genome (Orellana & Kieff, 1977; Powell et al., 1979). Making use of cloned restriction endonuclease fragments of EBV, Arrand discovered that the major cytoplasmic RNA in these cells was specified by part of the EcoRI J fragment, which was consistent with Rymo's observation (Arrand & Rymo, 1982). Meanwhile, there were reports that revealed SLE antibodies anti-La, but not the other sera tested, identified two new small RNAs, which corresponded to the most actively transcribed portion of EBV DNA in Rymo's investigation and they were termed EBERs for the first time. In the following 1980's, emphasis were put on the structure, transcription regulation and the function of EBER-La complex. After these preliminary explorations, intensive research was focused on the role of EBERs in the oncogenesis of lymphoma, the involvement of EBERs in the process of lymphoblastoid cell line (LCL) transformation and the potential anti-apoptosis response triggered by EBERs. With these inspiring achievements, some scholars were intrigued by the autocrine growth of several tumor cells and successfully discovered the link between cytokine induction and EBERs in B and T lymphocyte, gastric carcinoma and nasopharyngeal carcinoma in the

Pathologic Significance of EBV Encoded RNA in NPC 29

which is undergoing the germinal center (GC) reaction. This single translocated and surviving cell is the founder cell of an endemic BL, which accounts for the oncogenic role of EBV in lymphoma (Niller et al., 2003). What's more, Ferenc Banati found that in vitro methylation blocked binding of the cellular proteins c-Myc and ATF to the 50-region of the EBER-1 gene, which indicated a complicated transcription regulation of EBERs (Banati et al., 2008). With the special transcription elements of EBERs, Choy had devised shRNA plasmid to silence gene expression, which achieves better effect in some cases (Choy et al., 2008).

Fig. 1. Potential secondary structures of EBERs. The arrows indicate alternate 3' termini.

EBER in situ hybridization is considered the gold standard for detecting and localizing latent EBV in tissue samples (Ambinder & Mann, 1994). After all, EBER transcripts are consistently expressed in virtually all the EBV positive tumors, and they are likewise expressed in lymphoid tissues taken from patients with infectious mononucleosis, and in the rare infected cell representing normal flora in healthy virus carriers. The only EBV-related lesion that lacks EBERs is oral hairy leukoplakia, a purely lytic infection of oral epithelial

(A) EBER 1; (B) EBER 2. Adapted from Rosa *et al*. (1981)

following decade. More recently, our knowledge has been deepened by unveiling the TLR3 and RIG-I signaling pathways induced by EBERs, which are responsible for the autocrine growth of lymphomas and some EBV associated pathogenesis (Iwakiri et al., 2005; Samanta et al., 2008). However, the accurate role of EBERs in the pathogenesis of NPC is still obscure. There have been some contradictory reports with respect to the contribution of EBERs to the oncogenesis of NPC and the relationship between EBERs and anti-apoptosis response. What makes these dilemmas more complicated is the existence of EBERs in various stages of NPC. Interestingly, expression of the EBERs seems to be down-regulated during differentiation. Thus examples of NPC that have differing degrees of differentiation lack EBER expression in differentiated areas (Pathmanathan et al., 1995). The EBERs are also not detected in the permissive EBV infection, hairy leukoplakia, and are downregulated during viral replication (Gilligan et al., 1990). Collecting the previous data together, despite that the EBERs have been studied over 3 decades and some observations indicate they may play important roles in the transformation of lymphoma (Yajima et al., 2005) and NPC (Yoshizaki et al., 2007), the exact function of EBERs in NPC are still controversial.

## **2. Structure, transcription and clinical significance of EBERs**

EBERs, the most abundant cytoplasmic RNA species identified in five lymphoid cell lines and a Burkitt lymphoma biopsy, are encoded by the right-hand 1,000 base pairs of the EcoRI J fragment of EBV DNA (Rosa et al., 1981). EBER1 is 166 (167) nucleotides long and EBER2 is 172 ± 1 nucleotides long with the heterogeneity resides at the 3' termini (Fig. 1). Striking similarities are apparent both between the EBERs and the two adenovirus-associated RNAs, VAI and VAII, and between the regions of the two viral genomes that specify these small RNAs (Arrand et al., 1989). The EBER genes are separated by 161 base pairs and are transcribed from the same DNA strand. Both EBER genes carry intragenic transcription control regions A and B boxes which can be transcribed by RNA polymerase III (pol III). However, both EBER1 and 2 contain upstream elements and TATA-like sequences typical of polⅡ promoters including Sp1 and ATF binding sites (Howe & Shu, 1989). Within 1 kilo base EBER region, 10 single base changes which group the strains into two families (1 and 2) have been identified. The EBER1 sequences are completely conserved, two base changes are within EBER2-coding sequence and eight are outside the coding regions (Arrand et al., 1989). EBV has been shown to induce the cellular transcription factors TFIIIB and TFIIIC (leading to induction of general pol III-mediated transcription) and the typical pol II transcription factor ATF-2, that enhance expression of EBER1 and EBER2 (Felton-Edkins et al., 2006), which may account for the low expression of transfected EBERs plasmids in EBVnegative cells (Komano et al., 1999). To elucidate transcription regulation of EBERs more exactly, Thomas J Owen discovered that transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 through transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc (Owen et al., 2010). To shed more light on the transcription of EBERs, Hans Helmut Niller analyzed protein binding at the EBER locus of EBV by genomic footprinting electrophoretic mobility shift, reporter gene assay, and chromatin immunoprecipitation in a panel of six B-cell lines. With these methods, 130 base pairs upstream of the EBER1 gene, contains two E-boxes providing a consensus sequence for binding of the transcription factor and oncoprotein c-Myc to the EBV genome. Translocated and deregulated c-myc directly activates and maintains the antiapoptotic functions of the EBER locus in a single EBV-infected B cell

following decade. More recently, our knowledge has been deepened by unveiling the TLR3 and RIG-I signaling pathways induced by EBERs, which are responsible for the autocrine growth of lymphomas and some EBV associated pathogenesis (Iwakiri et al., 2005; Samanta et al., 2008). However, the accurate role of EBERs in the pathogenesis of NPC is still obscure. There have been some contradictory reports with respect to the contribution of EBERs to the oncogenesis of NPC and the relationship between EBERs and anti-apoptosis response. What makes these dilemmas more complicated is the existence of EBERs in various stages of NPC. Interestingly, expression of the EBERs seems to be down-regulated during differentiation. Thus examples of NPC that have differing degrees of differentiation lack EBER expression in differentiated areas (Pathmanathan et al., 1995). The EBERs are also not detected in the permissive EBV infection, hairy leukoplakia, and are downregulated during viral replication (Gilligan et al., 1990). Collecting the previous data together, despite that the EBERs have been studied over 3 decades and some observations indicate they may play important roles in the transformation of lymphoma (Yajima et al., 2005) and NPC (Yoshizaki et al., 2007), the

EBERs, the most abundant cytoplasmic RNA species identified in five lymphoid cell lines and a Burkitt lymphoma biopsy, are encoded by the right-hand 1,000 base pairs of the EcoRI J fragment of EBV DNA (Rosa et al., 1981). EBER1 is 166 (167) nucleotides long and EBER2 is 172 ± 1 nucleotides long with the heterogeneity resides at the 3' termini (Fig. 1). Striking similarities are apparent both between the EBERs and the two adenovirus-associated RNAs, VAI and VAII, and between the regions of the two viral genomes that specify these small RNAs (Arrand et al., 1989). The EBER genes are separated by 161 base pairs and are transcribed from the same DNA strand. Both EBER genes carry intragenic transcription control regions A and B boxes which can be transcribed by RNA polymerase III (pol III). However, both EBER1 and 2 contain upstream elements and TATA-like sequences typical of polⅡ promoters including Sp1 and ATF binding sites (Howe & Shu, 1989). Within 1 kilo base EBER region, 10 single base changes which group the strains into two families (1 and 2) have been identified. The EBER1 sequences are completely conserved, two base changes are within EBER2-coding sequence and eight are outside the coding regions (Arrand et al., 1989). EBV has been shown to induce the cellular transcription factors TFIIIB and TFIIIC (leading to induction of general pol III-mediated transcription) and the typical pol II transcription factor ATF-2, that enhance expression of EBER1 and EBER2 (Felton-Edkins et al., 2006), which may account for the low expression of transfected EBERs plasmids in EBVnegative cells (Komano et al., 1999). To elucidate transcription regulation of EBERs more exactly, Thomas J Owen discovered that transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 through transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc (Owen et al., 2010). To shed more light on the transcription of EBERs, Hans Helmut Niller analyzed protein binding at the EBER locus of EBV by genomic footprinting electrophoretic mobility shift, reporter gene assay, and chromatin immunoprecipitation in a panel of six B-cell lines. With these methods, 130 base pairs upstream of the EBER1 gene, contains two E-boxes providing a consensus sequence for binding of the transcription factor and oncoprotein c-Myc to the EBV genome. Translocated and deregulated c-myc directly activates and maintains the antiapoptotic functions of the EBER locus in a single EBV-infected B cell

exact function of EBERs in NPC are still controversial.

**2. Structure, transcription and clinical significance of EBERs** 

which is undergoing the germinal center (GC) reaction. This single translocated and surviving cell is the founder cell of an endemic BL, which accounts for the oncogenic role of EBV in lymphoma (Niller et al., 2003). What's more, Ferenc Banati found that in vitro methylation blocked binding of the cellular proteins c-Myc and ATF to the 50-region of the EBER-1 gene, which indicated a complicated transcription regulation of EBERs (Banati et al., 2008). With the special transcription elements of EBERs, Choy had devised shRNA plasmid to silence gene expression, which achieves better effect in some cases (Choy et al., 2008).

Fig. 1. Potential secondary structures of EBERs. The arrows indicate alternate 3' termini. (A) EBER 1; (B) EBER 2. Adapted from Rosa *et al*. (1981)

EBER in situ hybridization is considered the gold standard for detecting and localizing latent EBV in tissue samples (Ambinder & Mann, 1994). After all, EBER transcripts are consistently expressed in virtually all the EBV positive tumors, and they are likewise expressed in lymphoid tissues taken from patients with infectious mononucleosis, and in the rare infected cell representing normal flora in healthy virus carriers. The only EBV-related lesion that lacks EBERs is oral hairy leukoplakia, a purely lytic infection of oral epithelial

Pathologic Significance of EBV Encoded RNA in NPC 31

Hovanessian, 1989). Considering the resemblance, Bhat and Thimmappaya successfully proved that EBERs can functionally substitute for the VA RNAs in the lytic growth of Ad5 (Bhat & Thimmappaya, 1983, 1985). What's more, EBERs could directly bind PKR and inhibit its activity, then block phosphorylation of eIF2a, thus resulting in the blockage of inhibition of protein synthesis by eIF2a (Clarke et al., 1991; Sharp et al., 1993). When added to reticulocyte lysates at high concentrations, EBER-1 could prevent inhibition of translation by double-stranded RNA (Clarke et al., 1990). However, EBER-1 enhanced overall protein synthesis in the absence of PKR expression (Laing et al., 2002; Laing et al., 1995). In support of EBERs function regardless of PKR, EBER-deleted recombinant EBV transformed primary B lymphocytes into LCLs, which were indistinguishable from LCLs transformed by wildtype EBV in their proliferation, in latency-associated EBV gene expression, and in their permissiveness for EBV replication cycle gene expression (Swaminathan et al., 1991). Especially, another publication indicated EBERs could support replication of the defective adenovirus in vivo but PKR phosphorylation status wasn't influenced (Wang et al., 2005). This difference is likely a result of distinct subcellular compartmentalization of these two molecules, with the EBERs being exclusively nuclear, while PKR is predominately found in

Furthermore, it was speculated EBERs could partly restores resistance to both spontaneous and interferon-induced apoptosis (Komano et al., 1999) and PKR probably act as the mediator of the EBER protective effect against apoptosis despite controversial observation provided by Ruf et al. (Ruf et al., 2000). According to Komano et al., Transfection of the EBER genes into EBV-negative Akata clones restored the capacity for growth in soft agar, tumorigenicity in SCID mice, resistance to apoptotic inducers, and upregulated expression of bcl-2 oncoprotein that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. To support this hypothesis, researchers have made it clear that when EBV-negative Akata cells transfected with EBERs were analysed, PKR autophosphorylation *in vitro* was inhibited (Nanbo et al., 2002). However, Ruf reported that EBERs did posses a modest ability to protect the cell against interferon-induced apoptosis, but this process was independent of PKR-eIF-2α activation (Ruf et al., 2005). Thus Swaminathan suggested that EBERs might inhibit apoptosis while it was unlikely that

inhibition of PKR was the primary mechanism for this effect (Swaminathan, 2010).

EBER-1 also interacts with the ribosomal protein L22, a componenet of the 60S eukaryotic ribosomal subunit unique to eukaryotes (Dobbelstein & Shenk, 1995; Toczyski et al., 1994; Toczyski & Steitz, 1991, 1993). In EBV-infected BL cells, roughly 50% of the cellular pool of L22 is found in association with EBER-1 ribonucleoprotein (RNP) particles, and a substantial fraction of L22 is physically relocalized from nucleoli to the nucleoplasm. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed (Gregorovic et al., 2011). Binding to 28S rRNA likely serves to target L22 to nucleoli, while binding to EBER-1 RNA likely results in sequestration or retention of L22 in the nucleoplasm. In truth, BL cells expressing mutated EBER-1 RNAs incapable of binding to and relocalizing L22 have significantly reduced capacity to enhance cell growth potential relative to BL cells expressing wild-type EBERs (Houmani et al., 2009), which indicated that the EBER1-L22 complex may be beneficial for

the cytoplasm.

lymphoma growth.

cells. (Gilligan et al., 1990). Recently, researchers have discovered that EBERs could be used as a sensitive marker to monitor NPC cells at various metastatic sites by techniques of in situ hybridization. In cases of metastatic cancer of unknown origin, it is thus reasonable to consider NPC if EBV is present in the tumor cells (Chao et al., 1996). Kimura has established a novel flow cytometric in situ hybridization assay to detect EBV+ suspension cells using a peptide nucleic acid probe specific for EBERs. With this method, they can not only decide the EBV load but also locate EBV-infected cells, which will be beneficial for diagnosis of Epstein-Barr virus (EBV)–associated diseases and exploration of the pathogenesis of EBV infection (Kimura et al., 2009).
