**5. DNA methylation modification as therapeutic targets in NPC**

DNA methylation plays important roles in NPC carcinogenesis, including the silencing of cellular TSGs and some EBV encoded genes. The EBV encoded oncoprotein, LMP1, has been shown to interacting with methyltransferase (DNMT) and further induce the cellular gene E-cadherin promoter methylation (Tsai et al. 2006). And DNA methylation also suppresse EBV encoded genes, including the LMP1, immediate-early lytic antigens Zta and Rta, and some EBV immunodominant antigens (EBNA2,3A, 3B, 3C) (Paulson and Speck 1999; Tierney et al. 2000; Salamon et al. 2001). Thus, DNA methylation also plays an important role in the maintenance of specific EBV latency programmers and regulating EBV lifecycle and latency in NPC cells.

DNA methylation is a reversible phenomenon. Reactivating methylated and silenced cellular tumor suppressor genes and immunodominant tumor/viral antigens by demethylating agents might restore normal cell growth control, or induce cell immunity against cancer cells. Demethylating agents would also reactivate the expression of EBV early and lytic genes in latently infected NPC cells, which will lead to further tumor cell death.

Epigenetic therapeutic agents include DNA methyltransferase inhibitors and histone deacetylase (HDAC) inhibitors. 5-Azacytidine and 5-aza-2'-deoxycytidine are the most widely studied DNMT inhibitors. Clinical trials using such agents have been carried out on a series of cancer patients. In several phase I/II/III studies, decitabine (5-aza-2' deoxycytidine) has also shown promising data in patients with MDS and AML (Kantarjian et al. 2007; Issa et al. 2004). In patients with NPC and EBV-positive AIDS-associated Burkitt lymphoma, azacitidine effectively induces demethylation of all the latent and early lytic EBV promoters and some viral antigens, indicated the potential of epigenetic therapy for NPC (Chan et al. 2004).

#### **6. References**

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

detection. The detection rate can reach 98% when combined analysis of five methylation markers (*RASSF1A, p16, WIF1, CHFR* and *RIZ1*) in a recent study (Hutajulu et al. 2011).

Cancer specific DNA methylation can be detected in tumor-derived free DNA in the bloodstream, e.g. in serum or plasma. High frequency of methylated *DAPK* gene were found not only in NPC tumors, but also could be detected in plasma and buffy coat of NPC patients (Wong et al. 2002). Methylated DNA was detectable in plasma of NPC patients before treatment including 46% for *CDH1*,42% for *CDH1*, 42% for *p16*, 20% for *DAPK* ,20% for *p15 and 5%* for *RASSF1A*. Aberrantly hypermethylated promoter DNA of at least one of the five genes was detectable in 71% of plasma of NPC patients before treatment. Hypermethylated promoter DNA of at least one of the three genes (*CDH1*, *DAPK1*, and p16) was detectable in post-treatment plasma of 38% recurrent NPC patients and none of the patients in remission. Suggesting that cell-free circulating methylated DNA might be a useful serological marker in assisting in screening of primary and potentially salvageable

**4.2 Methylation markers in circulating DNA** 

local or regional recurrent NPC (Wong et al. 2004).

for early diagnosis.

and latency in NPC cells.

**4.3 Methylation markers in other body fluids and nasopharyngeal swabs** 

**5. DNA methylation modification as therapeutic targets in NPC** 

In addition to tissue analysis, methylated DNA has been detected in the mouth and throat rinsing fluid, saliva and nasopharyngeal swabs of NPC patients. Methylated DNA found in cancer patient serum correlated reasonably well with methylation levels in tumor tissue, and it is also believed that the source of serum DNA is necrotic tumor cells. Hypermethylated *RIZ1* gene was detected in 60% of NPC primary tumors, but not in any of the normal controls. Of 30 matched body fluid samples, methylated *RIZ1* DNA was found in 37% of NP swabs, 30% of rinsing fluid, 23% of plasma, and 10% of buffy coat samples. The results in NPC tumor and NP swab samples from the same patients show good concordance. Our early study also reported that the high sensitivity (81%) and specificity (0% false positives) of detecting aberrant methylation of *CDH13* (encoded a cell adhesion molecule H-cadherin) from nasopharyngeal swabs suggested it could be utilized as a tool

DNA methylation plays important roles in NPC carcinogenesis, including the silencing of cellular TSGs and some EBV encoded genes. The EBV encoded oncoprotein, LMP1, has been shown to interacting with methyltransferase (DNMT) and further induce the cellular gene E-cadherin promoter methylation (Tsai et al. 2006). And DNA methylation also suppresse EBV encoded genes, including the LMP1, immediate-early lytic antigens Zta and Rta, and some EBV immunodominant antigens (EBNA2,3A, 3B, 3C) (Paulson and Speck 1999; Tierney et al. 2000; Salamon et al. 2001). Thus, DNA methylation also plays an important role in the maintenance of specific EBV latency programmers and regulating EBV lifecycle

DNA methylation is a reversible phenomenon. Reactivating methylated and silenced cellular tumor suppressor genes and immunodominant tumor/viral antigens by


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**2** 

 *China* 

**Pathologic Significance of EBV** 

*Center for Molecular Medicine, Xiangya Hospital, Central South University, Changsha,* 

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

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

**1. Introduction** 

of EBERs in NPC.

**Encoded RNA in NPC** 

Zhi Li, Lifang Yang and Lun-Quan Sun

