**2. DNA methylation, histone modifications and chromatin structure**

DNA methylation is the only genetically programmed DNA modification in mammals. This postreplication modification is almost exclusively found on the 5′ position of the pyrimidine ring of cytosines in the context of the dinucleotide sequence CpG. 5′-methylcytosine accounts for ∼1% of all bases, varying slightly in different tissue types and the majority (75%) of CpG dinucleotides throughout mammalian genomes are methylated (Tost 2010). Sequence regions with a high density of CpG residues are termed as CpG islands. A CpG island is defined as a sequence of 200-plus base pairs with a G+C content of more than 50%, and an observed versus expected ratio for the occurrence of CpGs of more than 0.6 (Jones and Takai 2001). These CpG islands are associated with gene promoters in approximately 50% of genes and are generally maintained in an unmethylated state. DNA methylation can interfere with transcription in several ways. It can inhibit the binding of transcriptional activators with their cognate DNA recognition sequence such as Sp1 and Myc through sterical hindrance. The methylation binding proteins and the DNA methyltransferases (DNMTs) bind to methylated DNA and prevent the binding of potentially activating transcription factors. The methylation binding proteins and DNMTs also recruit additional proteins with repressive function such as histone deacetylases and chromatin remodeling complexes to the methylated DNA to establish a repressive chromatin configuration (Bird 2002).

To date, three major cellular enzymic activities associated with DNA methylation have been characterized (DNMT1, DNMT3A, and DNMT3B) (Malik and Brown 2000). They catalyze the transfer of a methyl group from SAM to the cytosine base. DNMT1 is considered as a maintenance methyltransferase, it is located at the replication fork during the S phase of the cell cycle and catalyze the methylation of the newly synthesized DNA strand using the parent strand as a template. The methyltransferases DNMT3A and DNMT3B are responsible for *De novo* methylation. These enzymes not only targeting specific sequences, they also work cooperatively to methylate the genome (Malik and Brown 2000).

Tumor-specific elevation of DNMTs is a causative step in many cancers. All three DNMTs, were observed modestly overexpressed in many types of tumor cells at the mRNA or protein level (Robertson et al. 1999). Furthermore, modest overexpression of exogenous mouse Dnmt1 in NIH 3T3 cells can promote cellular transformation (Wu et al. 1993). Additionally, genetic inactivation of Dnmt1 in mice decreases the development of gastrointestinal tumors in a mouse model of gastrointestinal cancer (Laird et al. 1995). These evidences indicate a possible role for DNMTs in tumorigenesis. However, the mechanisms that underlie such a role in cancer are still not defined.

Genomic DNA is highly folded and packaged into chromosomes or chromatin by histone and nonhistone proteins in the nuclei of all eukaryotic cells (Jenuwein and Allis 2001). The fundamental repeating unit of chromatin is the nucleosome, in which 146 DNA base pairs are wrapped left handed around a core histone protein, which consists of two of each of the four histone protein subunits: H2A, H2B, H3 and H4. Each core histone has an aminoterminal 'tail' of about 25-40 residues long, where they are frequent targets for various posttranslational modifications (Fischle, Wang, and Allis 2003). The state of chromatin is regulated largely by covalent modifications of the histone tails. The major modifications include the acetylation of specific lysine residues by histone acetyltransferases (HATs), the

DNA methylation is the only genetically programmed DNA modification in mammals. This postreplication modification is almost exclusively found on the 5′ position of the pyrimidine ring of cytosines in the context of the dinucleotide sequence CpG. 5′-methylcytosine accounts for ∼1% of all bases, varying slightly in different tissue types and the majority (75%) of CpG dinucleotides throughout mammalian genomes are methylated (Tost 2010). Sequence regions with a high density of CpG residues are termed as CpG islands. A CpG island is defined as a sequence of 200-plus base pairs with a G+C content of more than 50%, and an observed versus expected ratio for the occurrence of CpGs of more than 0.6 (Jones and Takai 2001). These CpG islands are associated with gene promoters in approximately 50% of genes and are generally maintained in an unmethylated state. DNA methylation can interfere with transcription in several ways. It can inhibit the binding of transcriptional activators with their cognate DNA recognition sequence such as Sp1 and Myc through sterical hindrance. The methylation binding proteins and the DNA methyltransferases (DNMTs) bind to methylated DNA and prevent the binding of potentially activating transcription factors. The methylation binding proteins and DNMTs also recruit additional proteins with repressive function such as histone deacetylases and chromatin remodeling complexes to the methylated DNA to establish a repressive chromatin configuration (Bird

To date, three major cellular enzymic activities associated with DNA methylation have been characterized (DNMT1, DNMT3A, and DNMT3B) (Malik and Brown 2000). They catalyze the transfer of a methyl group from SAM to the cytosine base. DNMT1 is considered as a maintenance methyltransferase, it is located at the replication fork during the S phase of the cell cycle and catalyze the methylation of the newly synthesized DNA strand using the parent strand as a template. The methyltransferases DNMT3A and DNMT3B are responsible for *De novo* methylation. These enzymes not only targeting specific sequences, they also

Tumor-specific elevation of DNMTs is a causative step in many cancers. All three DNMTs, were observed modestly overexpressed in many types of tumor cells at the mRNA or protein level (Robertson et al. 1999). Furthermore, modest overexpression of exogenous mouse Dnmt1 in NIH 3T3 cells can promote cellular transformation (Wu et al. 1993). Additionally, genetic inactivation of Dnmt1 in mice decreases the development of gastrointestinal tumors in a mouse model of gastrointestinal cancer (Laird et al. 1995). These evidences indicate a possible role for DNMTs in tumorigenesis. However, the mechanisms

Genomic DNA is highly folded and packaged into chromosomes or chromatin by histone and nonhistone proteins in the nuclei of all eukaryotic cells (Jenuwein and Allis 2001). The fundamental repeating unit of chromatin is the nucleosome, in which 146 DNA base pairs are wrapped left handed around a core histone protein, which consists of two of each of the four histone protein subunits: H2A, H2B, H3 and H4. Each core histone has an aminoterminal 'tail' of about 25-40 residues long, where they are frequent targets for various posttranslational modifications (Fischle, Wang, and Allis 2003). The state of chromatin is regulated largely by covalent modifications of the histone tails. The major modifications include the acetylation of specific lysine residues by histone acetyltransferases (HATs), the

work cooperatively to methylate the genome (Malik and Brown 2000).

that underlie such a role in cancer are still not defined.

**2. DNA methylation, histone modifications and chromatin structure** 

2002).

methylation of lysine and arginine residues by histone methyltransferases (HMTs), and the phosphorylation of specific serine groups by histone kinases (HKs). Other histone modifications include attachment of ubiquitination, and sulmolation. Enzymes responsible for the cleavage of some histone modifications, such as histone deacetylases (HDACs), histone phosphatases (PPs), ubiquitin hydrolases (Ubps) and poly (ADPribose)glycohydrolases (PARGs), have already been identified (Biel, Wascholowski, and Giannis 2005).

Posttranslational modifications are closely related to fundamental cellular events like the activation and repression of transcription. In the case of histone H3, in general, acetylation of H3 at lysine 14 (H3-K14), phosphorylation of serine 10 (H3-S10), and methylation of H3-K4 leads to transcriptional activation. In contrast, the repression of certain genes is linked to deacetylation of H3-K14 and methylation of H3-K9. The specific combination of these modifications has been termed the histone code, that determines histone–DNA and histone–histone contacts, which may in turn regulate the on or off state of genes or unfolding/folding state of the chromatin structure (Jenuwein and Allis 2001; Esteller 2007).

Histone modifications and other epigenetic mechanisms such as DNA methylation appear to work together in a coordinated and orderly fashion, to establishing and maintaining gene activity states, thus regulating gene transcription (Fischle, Wang, and Allis 2003; Biel, Wascholowski, and Giannis 2005). In the past decade, more and more attention has been paid on histone modifications, which led to the discovery and characterization of a large number of histone-modifying molecules and protein complexes. Alterations of histonemodifying complexes are believed to disrupt the pattern and levels of histone marks and consequently dysregulate the normal control of chromatin-based cellular processes, ultimately leading to oncogenic transformation and the development of cancer (Esteller 2007).

## **3. NPC as an epigenetic disease**

#### **3.1 Hypermethylation of cellular tumor suppressor genes and the dysregulation of the corresponding cellular pathways**

NPC distinguish itself from other malignancies by the number of genes targeted for silencing by promoter methylation. Several classic tumor suppressor genes, such as p53 and Rb, are found to be mutated in more than 50% of all the tumors, but were rarely found to be mutated in NPC (Burgos 2003; Chang et al. 2002; Tao and Chan 2007). On the contrary, hypermethylation of known or candidate tumor suppressor genes involved in various fundamental pathways has been reported in NPC, such as apoptosis, DNA damage repair, tumor invasion and metastasis. The full list of genes which have been found to be aberrantly methylated in NPC was summarized in table 1.

#### **Ras signalling**

Activated Ras proteins has been shown to play a key role in the development of human cancers (Bos 1989). Ras proteins serve as a node in the transduction of information from a variety of cell surface receptors to an array of intracellular signaling pathways. Mutated variants of Ras (mutations at residues 12, 13 or 61) are found in 30% of all human cancers

Epigenetics of Nasopharyngeal Carcinoma 5

Ras-mediated signalling pathways (Seng et al. 2007). Recently, a novel isoform of the *DLC1* gene was identified, which suppresses tumor growth and frequently silenced in multiple common tumors including NPC. This novel isoform encodes an 1125-aa (amino acid) protein with distinct N-terminus as compared with other known *DLC1* isoforms. Similar to other isoforms, *DLC1-i4* is expressed ubiquitously in normal tissues, and epigenetically inactivated by promoter hypermethylation in NPC. The differential expression of various *DLC1* isoforms suggests interplay in modulating the complex activities of *DLC1* during

Altered p53 pathway is common detected in NPC, even though NPC rarely presents abnormality in the p53 gene itself, p53 function may be inactivated by either overexpression of ΔN-p63 or loss of p14/ARF. ΔN-p63 is a p53 homolog. It can block p53's function as transcription factor. P14 functions as a stabilizer of p53 as it can interact with, and sequester, MDM1, a protein responsible for the degradation of p53 (Ozenne et al. 2010). *p14* is methylated in 20% on NPC, the epigenetic inactivation of *p14/ARF* may facilitate p53 degradation in NPC cells (Kwong et al. 2002). Loss of p53 function may affect cell cycle arrest at the G1 or G2/M phase and p53-mediated apoptosis in response to DNA damage

Recently, Qian Tao et al. found that *UCHL1* was frequently silenced by promoter CpG methylation in nasopharyngeal carcinoma; and acts as a functional tumor suppressor gene for NPC through stabilizing p53 through deubiquitinating p53 and p14ARF and ubiquitinating MDM2, which is mediated by its hydrolase and ligase activities, further

The Wnt signalling pathway is important for normal development and is frequently aberrantly activated in a variety of cancers. Although the role of the Wnt pathway in NPC has not been fully explored, there is abundant evidence that aberrant Wnt signalling plays a role in NPC development. In a recent study by gene expression profiling, the aberrant expression of the Wnt signalling pathway components, such as wingless-type MMTV integration site family, member 5A, Frizzled homolog 7, casein kinase II beta, β-catenin, CREB-binding protein, and dishevelled-associated activator of morphogenesis 2 was identified and further validated on NPC tissue microarrays (Zeng et al. 2007). Furthermore, most NPC tumors exhibit Wnt pathway protein dysregulation: 93% have increased Wnt protein expression and 75% have decreased expression of Wnt inhibitory factor (WIF), an endogenous Wnt antagonist (Shi et al. 2006; Zeng et al. 2007). These results indicate that

The Wnt inhibitory factor 1 (*WIF1*) gene acts as a Wnt antagonist factor by direct binding to Wnt ligands. In NPC, methylation was frequently observed in 85% of NPC primary tumors, with *WIF1* expressed and unmethylated in normal cell lines and normal tissues. Ectopic expression of WIF1 in NPC cells resulted in significant inhibition of tumor cell colony formation, and significant downregulation of β-catenin protein level in NPC cells. Indicates that epigenetic inactivation of *WIF1* contributes to the aberrant activation of Wnt pathway

carcinogenesis (Low et al. 2011).

(Kwong et al. 2002; Crook et al. 2000).

resulting in the induction of tumor cell apoptosis (Li et al. 2010).

aberrant Wnt signalling is a critical component of NPC.

and is involved in the pathogenesis of NPC (Chan et al. 2007).

**P53 signalling** 

**Wnt signalling** 

(Bos 1989). Mutations at residues 12, 13 or 61 might lock Ras protein in the active state, which mediate a variety of biological effects associated with enhanced growth and transformation. Ras activity is regulated by cycling between inactive GDP-bound and active GTP-bound forms. When GTP-bound, Ras binds to and activates a plethora of effector molecules. GTPase-activating proteins (GAPs), such as p120GAP and NF1, trigger the hydrolysis of GTP back to the inactive GDP-bound form (Boguski and McCormick 1993). Because Ras GAPs switch off Ras signalling, they have always been considered as potential tumor suppressor genes. Recent study reveal that the Ras GTPase-activating-like protein (RASAL), a Ca2+-regulated Ras GAP that decodes the frequency of Ca2+ oscillations, is silenced through CpG methylation in multiple tumors including NPC (Jin, Wang, Ying, Wong, Cui, et al. 2007). In addition, ectopic expression of catalytically active RASAL leads to growth inhibition of NPC cells by Ras inactivation, thus, epigenetically silencing of RASAL is an alternative mechanism of aberrant Ras activation in NPC (Jin, Wang, Ying, Wong, Cui, et al. 2007).

Although it is widely accepted that Ras functions as an oncoprotein, more and more evidence show that Ras proteins may also induces growth arrest properties of cells, such as senescence, apoptosis, terminal differentiation (Spandidos et al. 2002). The growth inhibitory effects of Ras were induced by a group of proteins with Ras binding domain. These proteins were identified as negative effectors of Ras and designated as Ras association domain family (RASSF). Within this super family, the *RASSF1A* and *RASSF2A* gene are frequently inactivated by promoter hypermethylation (Lo et al. 2001; Zhang et al. 2007), functional studies also support their role as putative tumor suppressors in NPC.

The induction of invasiveness and metastasis by Ras were mediated by downstream effectors which are involved in the regulation of cell adhesion, cell-matrix interaction and cell motility, such as RhoGTPases, RalGEF and components of PI3K pathways (Giehl 2005). Recent studies have further indicated that the Ras/PI3K/AKT pathway is associated in several human cancers. Activation of the Ras/PI3K/AKT pathway can occur by many mechanisms, which include activation of Ras, mutation or amplification of *PI3K*, amplification of *AKT*, and mutation/decreased expression of the tumor-suppressor genes *PTEN* and *HIN-1*. The *HIN-1* gene has various biological functions, including inhibiting cell cycle reentry, suppressing migration and invasion, and inducing apoptosis; these effects are mediated by inhibiting AKT signalling pathway (Krop et al. 2005). *HIN-1* gene is hypermethylated in human NPC. Methylated *HIN-1* promoter was found in 77% of primary NPC tumors and not found in the normal nasopharyngeal biopsies. Moreover, methylated *HIN-1* promoter can be detected in 46% of nasopharyngeal swabs, 19% of throat-rinsing fluids, 18% of plasmas, and 46% of buffy coats of peripheral blood of the NPC patients but was not detectable in all normal controls (Wong, Kwong, et al. 2003).

The Ras family shares at least 30% sequence identity with several other small monomeric G protein families, such as the Rho/Rac/CDC42, Rab/Ypt, Ran, Arf, and Rad families (Adjei 2001). The major 8p22 tumor suppressor Deleted in Liver Cancer 1 (*DLC1*) gene is a homologue of rat p122RhoGAP. It was identified as a major downregulated gene in NPC by expression subtraction. By expression subtraction, Qian Tao's group identified that *DLC1* is an 8p22 TSG as a major downregulated gene in NPC. Their study also demonstrated *DLC1* is hypermethylated not only in NPC, but also in esophageal and cervical carcinomas. Downregulation of *DLC1* contributes to NPC oncogenesis by disrupting Ras-mediated signalling pathways (Seng et al. 2007). Recently, a novel isoform of the *DLC1* gene was identified, which suppresses tumor growth and frequently silenced in multiple common tumors including NPC. This novel isoform encodes an 1125-aa (amino acid) protein with distinct N-terminus as compared with other known *DLC1* isoforms. Similar to other isoforms, *DLC1-i4* is expressed ubiquitously in normal tissues, and epigenetically inactivated by promoter hypermethylation in NPC. The differential expression of various *DLC1* isoforms suggests interplay in modulating the complex activities of *DLC1* during carcinogenesis (Low et al. 2011).

#### **P53 signalling**

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

(Bos 1989). Mutations at residues 12, 13 or 61 might lock Ras protein in the active state, which mediate a variety of biological effects associated with enhanced growth and transformation. Ras activity is regulated by cycling between inactive GDP-bound and active GTP-bound forms. When GTP-bound, Ras binds to and activates a plethora of effector molecules. GTPase-activating proteins (GAPs), such as p120GAP and NF1, trigger the hydrolysis of GTP back to the inactive GDP-bound form (Boguski and McCormick 1993). Because Ras GAPs switch off Ras signalling, they have always been considered as potential tumor suppressor genes. Recent study reveal that the Ras GTPase-activating-like protein (RASAL), a Ca2+-regulated Ras GAP that decodes the frequency of Ca2+ oscillations, is silenced through CpG methylation in multiple tumors including NPC (Jin, Wang, Ying, Wong, Cui, et al. 2007). In addition, ectopic expression of catalytically active RASAL leads to growth inhibition of NPC cells by Ras inactivation, thus, epigenetically silencing of RASAL is an alternative mechanism of aberrant Ras activation in NPC (Jin, Wang, Ying, Wong, Cui,

Although it is widely accepted that Ras functions as an oncoprotein, more and more evidence show that Ras proteins may also induces growth arrest properties of cells, such as senescence, apoptosis, terminal differentiation (Spandidos et al. 2002). The growth inhibitory effects of Ras were induced by a group of proteins with Ras binding domain. These proteins were identified as negative effectors of Ras and designated as Ras association domain family (RASSF). Within this super family, the *RASSF1A* and *RASSF2A* gene are frequently inactivated by promoter hypermethylation (Lo et al. 2001; Zhang et al. 2007),

The induction of invasiveness and metastasis by Ras were mediated by downstream effectors which are involved in the regulation of cell adhesion, cell-matrix interaction and cell motility, such as RhoGTPases, RalGEF and components of PI3K pathways (Giehl 2005). Recent studies have further indicated that the Ras/PI3K/AKT pathway is associated in several human cancers. Activation of the Ras/PI3K/AKT pathway can occur by many mechanisms, which include activation of Ras, mutation or amplification of *PI3K*, amplification of *AKT*, and mutation/decreased expression of the tumor-suppressor genes *PTEN* and *HIN-1*. The *HIN-1* gene has various biological functions, including inhibiting cell cycle reentry, suppressing migration and invasion, and inducing apoptosis; these effects are mediated by inhibiting AKT signalling pathway (Krop et al. 2005). *HIN-1* gene is hypermethylated in human NPC. Methylated *HIN-1* promoter was found in 77% of primary NPC tumors and not found in the normal nasopharyngeal biopsies. Moreover, methylated *HIN-1* promoter can be detected in 46% of nasopharyngeal swabs, 19% of throat-rinsing fluids, 18% of plasmas, and 46% of buffy coats of peripheral blood of the NPC patients but

The Ras family shares at least 30% sequence identity with several other small monomeric G protein families, such as the Rho/Rac/CDC42, Rab/Ypt, Ran, Arf, and Rad families (Adjei 2001). The major 8p22 tumor suppressor Deleted in Liver Cancer 1 (*DLC1*) gene is a homologue of rat p122RhoGAP. It was identified as a major downregulated gene in NPC by expression subtraction. By expression subtraction, Qian Tao's group identified that *DLC1* is an 8p22 TSG as a major downregulated gene in NPC. Their study also demonstrated *DLC1* is hypermethylated not only in NPC, but also in esophageal and cervical carcinomas. Downregulation of *DLC1* contributes to NPC oncogenesis by disrupting

functional studies also support their role as putative tumor suppressors in NPC.

was not detectable in all normal controls (Wong, Kwong, et al. 2003).

et al. 2007).

Altered p53 pathway is common detected in NPC, even though NPC rarely presents abnormality in the p53 gene itself, p53 function may be inactivated by either overexpression of ΔN-p63 or loss of p14/ARF. ΔN-p63 is a p53 homolog. It can block p53's function as transcription factor. P14 functions as a stabilizer of p53 as it can interact with, and sequester, MDM1, a protein responsible for the degradation of p53 (Ozenne et al. 2010). *p14* is methylated in 20% on NPC, the epigenetic inactivation of *p14/ARF* may facilitate p53 degradation in NPC cells (Kwong et al. 2002). Loss of p53 function may affect cell cycle arrest at the G1 or G2/M phase and p53-mediated apoptosis in response to DNA damage (Kwong et al. 2002; Crook et al. 2000).

Recently, Qian Tao et al. found that *UCHL1* was frequently silenced by promoter CpG methylation in nasopharyngeal carcinoma; and acts as a functional tumor suppressor gene for NPC through stabilizing p53 through deubiquitinating p53 and p14ARF and ubiquitinating MDM2, which is mediated by its hydrolase and ligase activities, further resulting in the induction of tumor cell apoptosis (Li et al. 2010).

#### **Wnt signalling**

The Wnt signalling pathway is important for normal development and is frequently aberrantly activated in a variety of cancers. Although the role of the Wnt pathway in NPC has not been fully explored, there is abundant evidence that aberrant Wnt signalling plays a role in NPC development. In a recent study by gene expression profiling, the aberrant expression of the Wnt signalling pathway components, such as wingless-type MMTV integration site family, member 5A, Frizzled homolog 7, casein kinase II beta, β-catenin, CREB-binding protein, and dishevelled-associated activator of morphogenesis 2 was identified and further validated on NPC tissue microarrays (Zeng et al. 2007). Furthermore, most NPC tumors exhibit Wnt pathway protein dysregulation: 93% have increased Wnt protein expression and 75% have decreased expression of Wnt inhibitory factor (WIF), an endogenous Wnt antagonist (Shi et al. 2006; Zeng et al. 2007). These results indicate that aberrant Wnt signalling is a critical component of NPC.

The Wnt inhibitory factor 1 (*WIF1*) gene acts as a Wnt antagonist factor by direct binding to Wnt ligands. In NPC, methylation was frequently observed in 85% of NPC primary tumors, with *WIF1* expressed and unmethylated in normal cell lines and normal tissues. Ectopic expression of WIF1 in NPC cells resulted in significant inhibition of tumor cell colony formation, and significant downregulation of β-catenin protein level in NPC cells. Indicates that epigenetic inactivation of *WIF1* contributes to the aberrant activation of Wnt pathway and is involved in the pathogenesis of NPC (Chan et al. 2007).

Epigenetics of Nasopharyngeal Carcinoma 7

cadherins, connexins, and other components of cell adhesion are dysregulated (Du et al. 2011; Sun et al. 2007; Ying et al. 2006; Huang et al. 2001; Lou, Chen, Lin, et al. 1999; Xiang et

Cadherins have strong implications in tumorigenesis through cadherin-mediated cell–cell adhesion, which maintains tissue integrity and homeostasis. Disruption of this organized adhesion by genetic and epigenetic mechanisms during carcinogenesis might result in changes in signal transduction, loss of contact inhibition, and altered cell migration and stromal interactions. Some of the cadherins, such as E-cadherin and H-cadherin, were characterized as TGSs, which inhibit tumor invasion and metastasis (Berx and van Roy 2009; Jeanes, Gottardi, and Yap 2008). Disruption of cadherin expression and inappropriate switching among cadherin family members by genetic or epigenetic mechanisms are key events in the acquisition of the invasive phenotype for many tumors. The E-cadherin gene is silenced by promoter hypermethylation in human NPC because of aberrant expression of DNMT induced by the Epstein-Barr virus-encoded oncoprotein latent membrane 1 (Tsai et al. 2002). Moreover, loss of E-cadherin expression is significantly associated with histological grade, intracranial invasion and lymph node and distant metastasis (Lou, Chen, Sheen, et al. 1999). Three other members of the cadherin family: *CDH13*, *CDH4* and *PCDH10*, are involved in NPC owing to promoter methylation (Sun et al. 2007; Ying et al. 2006; Du et al. 2011). This evidence indicates a deep involvement of epigenetic regulation of

Intercellular communication through gap junction (GJIC) have a significant role in maintaining tissue homeostasis and has long been proposed as a mechanism to regulate growth control, development and differentiation. Reduced GJIC activity has long been implicated in carcinogenesis. Loss of GJIC leads to aberrant proliferation and an enhanced neoplastic phenotype. Reduced expression of the connexin (Cx) genes dysregulation of GJIC activity were observed in a series of human cancers. Thus, some Cx genes have been suggested as tumor suppressor genes (Pointis et al. 2007). Down-regulation of connexin 43 (*Cx43*) expression and dysfunctional GJIC were demonstrated in NPC tissues and cells, suggesting that dysfunctional GJIC plays a key role in nasopharyngeal carcinogenesis (Shen et al. 2002; Xiang et al. 2002). Further study revealed that inactivation of *Cx43* gene was mediated by epigenetic mechanism of promoter hypermethylation in NPC. Treatment of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine could induce restoration of GJIC

MMPs are type IV collagenases whose overexpression has been implicated in a number of cancers. MMPs can not only degrade basement membranes and extracellular matrices to allow for tumor invasion, they are also involved in activation of growth factors to promote cell growth and angiogenesis, and also protect tumor cells from apoptotic signals (Gialeli, Theocharis, and Karamanos 2011). In NPC, MMP1, MMP3 and MMP9 were shown to be upregulated by LMP1 (Stevenson, Charalambous, and Wilson 2005; Kondo et al. 2005; Lee et al. 2007). While MMP19 appears to be down-regulated in 69.7% of primary NPC specimens (Chan et al. 2010). Allelic deletion and promoter hypermethylation contribute to MMP19 down-regulation. The catalytic activity of MMP19 plays an important role in anti-tumor and

al. 2002).

the cadherin family in the carcinogenesis of NPC.

anti-angiogenesis activities (Chan et al. 2010).

and an inhibition of tumor phenotype of CNE-1 cells (Yi et al. 2007).

#### **Cell cycle and DNA repair**

Aberrant apoptosis, as in all malignancies, is also required for NPC development. Inhibition of apoptosis seems to be critical to NPC tumorigenesis. Death-associated protein kinase (*DAPK*) is a Ca/calmodulin-regulated serine/threonine kinase and a positive mediator of apoptosis. Loss of *DAPK* expression was shown to be associated with promoter region methylation in NPC. Methylation of the promoter was found in 76% of NPC, as well as plasma of patients with NPC (Chang et al. 2003). A demethylating agent, 5-aza-2' deoxycytidine, might slow the growth of NPC cells in vitro and in vivo by reactivating the *DAPK* gene silenced by de novo methylation (Kong et al. 2006).

Like all cancers, development of NPC requires the derangement of the normal cell cycle. Several classical CDK inhibitors in G1 -S checkpoint, such as p16/INK4A, p15/INK4A, and p14/ARF, were demonstrated to be hypermethylated in NPC and act as tumor suppressors during NPC development (Li, Shu, et al. 2011).

Dysregulation of the DNA repair system by DNA methylation is also an essential event in NPC development (Lo, To, and Huang 2004; Tao and Chan 2007). MGMT is a DNA repair protein that removes mutagenic and cytotoxic adducts from O6-guanine in DNA. Frequent methylation of *MGMT* associated with gene silencing occurs in human cancers. However, only a small portion (28%) of primary NPC were *MGMT* hypermethylated (Wong, Tang, et al. 2003). A rather high frequency (40%) of hypermethylation of the DNA mismatch repair gene *hMLH1* was observed in NPC primary tumors (Wong, Tang, et al. 2003). But methylation of *hMLH1* cannot be detected in the plasma of NPC patients (Wong et al. 2004).

Chromosomal instability (CIN) is a cytogenetic hallmark of human cancers (Cheung et al. 2005; Lengauer, Kinzler, and Vogelstein 1998). Increasing evidence suggests that impairment of mitotic checkpoint is causally associated with CIN. Several chromosomal aberrations have been identified in NPC. Some sites correspond to proteins key to NPC development, including p16, RASSF1A, and CKIs, while a number of sites do not correspond to any known tumor suppressors or oncogenes (Li, Shu, et al. 2011). CHFR is one of the mitotic checkpoint regulators and it delays chromosome condensation in response to mitotic stress. *CHFR* mRNA was significantly decreased or undetectable in NPC cell lines as well as human NPC xenografts, hypermethylation of *CHFR* promoter was strongly correlated with decreased CHFR expression in NPC cell lines and xenografts (Cheung et al. 2005). And hypermethylation of *CHFR* promoter region was detected in 61.1% (22 out of 36) of primary NPC tumors while it was absent in non-malignant tissues (Cheung et al. 2005).

#### **Cell adhesion**

Multiple cell adhesion molecules involve in intercellular and cell-extracellular matrix interactions of cancer. Cancer progression is a multi-step process in which some adhesion molecules play a pivotal role in the development of recurrent, invasion, and metastasis. Alterations in the adhesion properties of cancer cells play an essential role in the development and progression of cancer. Loss of intercellular adhesion allows malignant cells to escape from their site of origin, degrade the extracellular matrix, acquire a more motile and invasion phenotype, and finally, invade and metastasize. In NPC, epigenetic mechanism was involved in the abnormal cell adhesion, a diverse of molecules such as

Aberrant apoptosis, as in all malignancies, is also required for NPC development. Inhibition of apoptosis seems to be critical to NPC tumorigenesis. Death-associated protein kinase (*DAPK*) is a Ca/calmodulin-regulated serine/threonine kinase and a positive mediator of apoptosis. Loss of *DAPK* expression was shown to be associated with promoter region methylation in NPC. Methylation of the promoter was found in 76% of NPC, as well as plasma of patients with NPC (Chang et al. 2003). A demethylating agent, 5-aza-2' deoxycytidine, might slow the growth of NPC cells in vitro and in vivo by reactivating the

Like all cancers, development of NPC requires the derangement of the normal cell cycle. Several classical CDK inhibitors in G1 -S checkpoint, such as p16/INK4A, p15/INK4A, and p14/ARF, were demonstrated to be hypermethylated in NPC and act as tumor

Dysregulation of the DNA repair system by DNA methylation is also an essential event in NPC development (Lo, To, and Huang 2004; Tao and Chan 2007). MGMT is a DNA repair protein that removes mutagenic and cytotoxic adducts from O6-guanine in DNA. Frequent methylation of *MGMT* associated with gene silencing occurs in human cancers. However, only a small portion (28%) of primary NPC were *MGMT* hypermethylated (Wong, Tang, et al. 2003). A rather high frequency (40%) of hypermethylation of the DNA mismatch repair gene *hMLH1* was observed in NPC primary tumors (Wong, Tang, et al. 2003). But methylation of *hMLH1* cannot be detected in the plasma of NPC patients (Wong et al. 2004). Chromosomal instability (CIN) is a cytogenetic hallmark of human cancers (Cheung et al. 2005; Lengauer, Kinzler, and Vogelstein 1998). Increasing evidence suggests that impairment of mitotic checkpoint is causally associated with CIN. Several chromosomal aberrations have been identified in NPC. Some sites correspond to proteins key to NPC development, including p16, RASSF1A, and CKIs, while a number of sites do not correspond to any known tumor suppressors or oncogenes (Li, Shu, et al. 2011). CHFR is one of the mitotic checkpoint regulators and it delays chromosome condensation in response to mitotic stress. *CHFR* mRNA was significantly decreased or undetectable in NPC cell lines as well as human NPC xenografts, hypermethylation of *CHFR* promoter was strongly correlated with decreased CHFR expression in NPC cell lines and xenografts (Cheung et al. 2005). And hypermethylation of *CHFR* promoter region was detected in 61.1% (22 out of 36) of primary NPC tumors while it was absent in non-malignant tissues

Multiple cell adhesion molecules involve in intercellular and cell-extracellular matrix interactions of cancer. Cancer progression is a multi-step process in which some adhesion molecules play a pivotal role in the development of recurrent, invasion, and metastasis. Alterations in the adhesion properties of cancer cells play an essential role in the development and progression of cancer. Loss of intercellular adhesion allows malignant cells to escape from their site of origin, degrade the extracellular matrix, acquire a more motile and invasion phenotype, and finally, invade and metastasize. In NPC, epigenetic mechanism was involved in the abnormal cell adhesion, a diverse of molecules such as

*DAPK* gene silenced by de novo methylation (Kong et al. 2006).

suppressors during NPC development (Li, Shu, et al. 2011).

**Cell cycle and DNA repair** 

(Cheung et al. 2005).

**Cell adhesion** 

cadherins, connexins, and other components of cell adhesion are dysregulated (Du et al. 2011; Sun et al. 2007; Ying et al. 2006; Huang et al. 2001; Lou, Chen, Lin, et al. 1999; Xiang et al. 2002).

Cadherins have strong implications in tumorigenesis through cadherin-mediated cell–cell adhesion, which maintains tissue integrity and homeostasis. Disruption of this organized adhesion by genetic and epigenetic mechanisms during carcinogenesis might result in changes in signal transduction, loss of contact inhibition, and altered cell migration and stromal interactions. Some of the cadherins, such as E-cadherin and H-cadherin, were characterized as TGSs, which inhibit tumor invasion and metastasis (Berx and van Roy 2009; Jeanes, Gottardi, and Yap 2008). Disruption of cadherin expression and inappropriate switching among cadherin family members by genetic or epigenetic mechanisms are key events in the acquisition of the invasive phenotype for many tumors. The E-cadherin gene is silenced by promoter hypermethylation in human NPC because of aberrant expression of DNMT induced by the Epstein-Barr virus-encoded oncoprotein latent membrane 1 (Tsai et al. 2002). Moreover, loss of E-cadherin expression is significantly associated with histological grade, intracranial invasion and lymph node and distant metastasis (Lou, Chen, Sheen, et al. 1999). Three other members of the cadherin family: *CDH13*, *CDH4* and *PCDH10*, are involved in NPC owing to promoter methylation (Sun et al. 2007; Ying et al. 2006; Du et al. 2011). This evidence indicates a deep involvement of epigenetic regulation of the cadherin family in the carcinogenesis of NPC.

Intercellular communication through gap junction (GJIC) have a significant role in maintaining tissue homeostasis and has long been proposed as a mechanism to regulate growth control, development and differentiation. Reduced GJIC activity has long been implicated in carcinogenesis. Loss of GJIC leads to aberrant proliferation and an enhanced neoplastic phenotype. Reduced expression of the connexin (Cx) genes dysregulation of GJIC activity were observed in a series of human cancers. Thus, some Cx genes have been suggested as tumor suppressor genes (Pointis et al. 2007). Down-regulation of connexin 43 (*Cx43*) expression and dysfunctional GJIC were demonstrated in NPC tissues and cells, suggesting that dysfunctional GJIC plays a key role in nasopharyngeal carcinogenesis (Shen et al. 2002; Xiang et al. 2002). Further study revealed that inactivation of *Cx43* gene was mediated by epigenetic mechanism of promoter hypermethylation in NPC. Treatment of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine could induce restoration of GJIC and an inhibition of tumor phenotype of CNE-1 cells (Yi et al. 2007).

MMPs are type IV collagenases whose overexpression has been implicated in a number of cancers. MMPs can not only degrade basement membranes and extracellular matrices to allow for tumor invasion, they are also involved in activation of growth factors to promote cell growth and angiogenesis, and also protect tumor cells from apoptotic signals (Gialeli, Theocharis, and Karamanos 2011). In NPC, MMP1, MMP3 and MMP9 were shown to be upregulated by LMP1 (Stevenson, Charalambous, and Wilson 2005; Kondo et al. 2005; Lee et al. 2007). While MMP19 appears to be down-regulated in 69.7% of primary NPC specimens (Chan et al. 2010). Allelic deletion and promoter hypermethylation contribute to MMP19 down-regulation. The catalytic activity of MMP19 plays an important role in anti-tumor and anti-angiogenesis activities (Chan et al. 2010).

Epigenetics of Nasopharyngeal Carcinoma 9

*LTF* Lactoferrin 3p21.3 Cell cycle regulation (Yi et al. 2006;

through G2

9p34.1 Positive mediator of

apoptosis

11q13 Apoptosis, metobolism, energy pathways

16q21 Induces apoptosis with caspase-3 activation

14q11.2 Induces apoptosis with

with TNF-α.

19q13.12 Key regulator of cell proliferation, differentiation, and apoptosis, repress NF-kB and AP-1 signaling

in bones

PP2A

11q12.3 Proapoptotic function

8q24 Induced apoptosis,inhibits tumor growth specifically

through the inhibition of

activation of caspase 3, 8 and 9, synergistic effects

D1

3p14-21 Cell cycle regulator via inhibition of pRB phosphorylation through down-regulation of cyclin

> response, apoptosis, transcription factor

gamma-interferon induced

2q33-q34 Apoptosis (Li, Shu, et al.

**Function Refs** 

An important regulator of the cell cycle required for S phase and passage

Zhang et al. 2011)

 (Yanatatsaneejit et al. 2008)

 (Wong, Tang, et al. 2003)

 (Wong et al. 2004; Chang et al. 2003; Kwong et al. 2002; Li, Shu, et al. 2011)

2011; Wong, Tang, et al. 2003)

 (Kwong et al. 2002; Li, Shu, et al. 2011)

 (Wang et al. 2009)

(Shao et al. 2007)

(Lu et al. 2009)

 (Yanatatsaneejit et al. 2008)

(Cheng et al. 2010)

 (Cheung et al. 2008)

**somal location**

q13

*TP73* Tumor protein p73 1p36.3 Cell cycle, DNA damage

**Gene Full name Chromo-**

*CCNA1* Cyclin A1 13q12.3-

tyrosine-protein phosphatase gamma

protein kinase

Caspase 8, apoptosis-related cysteine peptidase

Glutathione Stransferase pi 1

MARVEL transmembrane domain-containing

member 3

MARVEL transmembrane domain-containing

member 5

Tumor necrosis factor receptor superfamily, member 11b

Phospholipase A2, group XVI

*ZNF382* Zinc finger protein 382

*PTPRG* Receptor-type

**Apoptosis** *DAPK* Death-associated

*CMTM3* CKLF like

*CMTM5* CKLF like

*TNFRSF11B /OPG* 

*PLA2G16/H RASLS3* 

*CASP8/CAP 4/MACH/M CH5/ FLICE*

*GSTP1/ DFN7/ GST3*

**Cancerrelated process** 

*OPCML* (opioid binding protein/cell adhesion molecule-like gene), also known as *OBCAM* (opioid binding cell adhesion molecule), belonging to the IgLON family of glycosylphosphatidylinositol (GPI)-anchored cell adhesion molecules involved in cell adhesion and cell-cell recognition. Located at 11q25, *OPCML* was the first IgLON member linked to tumorigenesis. In NPC, the *OPCML-v1* were observed to be epigenetically inactivated, what's more, the methylation was detected in a remarkable frequency: 98% of NPC tumor tissues. The high incidence of epigenetic inactivation of *OPCML* in NPC indicates that *OPCML* methylation could be an epigenetic biomarker for the molecular diagnosis of NPC (Cui et al. 2008).


*OPCML* (opioid binding protein/cell adhesion molecule-like gene), also known as *OBCAM* (opioid binding cell adhesion molecule), belonging to the IgLON family of glycosylphosphatidylinositol (GPI)-anchored cell adhesion molecules involved in cell adhesion and cell-cell recognition. Located at 11q25, *OPCML* was the first IgLON member linked to tumorigenesis. In NPC, the *OPCML-v1* were observed to be epigenetically inactivated, what's more, the methylation was detected in a remarkable frequency: 98% of NPC tumor tissues. The high incidence of epigenetic inactivation of *OPCML* in NPC indicates that *OPCML* methylation could be an epigenetic biomarker for the molecular

> **somal location**

**Function Refs** 

9p21 Cell cycle regulation (Wong et al.

 (Wong, Tang, et al. 2003; Chang et al. 2003; Wong et al. 2004; Li, Shu, et al. 2011)

2004; Chang et al. 2003; Wong, Tang, et al. 2003; Lo et al. 1996; Li, Shu, et al. 2011)

 (Cheung et al. 2005; Li, Shu, et al. 2011)

(Liu et al. 2008)

(Loyo et al. 2011)

(Ying et al. 2005)

2008)

 (Cheung et al. 2009)

et al. 2003)

9p21 Cyclin-dependent kinase inhibitor for CDK4 and CDK6, a cell growth regulator of cell cycle G1 progression

12q24.33 Mitotic checkpoint regulator early in G2-M transition

16q12 Transcriptional regulation, inhibits G1-S transition

3P14.2 Cell-cycle regulation, G1-S phase checkpoint, DNA-damage response, nucleotide and nucleic acid

metabolism

14q13.1 Negative regulator of G1 progression

9q22 Inhibits G1-S and G2-M transition, apoptosis

G1 cell cycle arrest (Ayadi et al.

3P21.3 Cell cycle (Liu et al. 2003)

1p36.21 G2-M cell cycle arrest (Chang

diagnosis of NPC (Cui et al. 2008).

*P15/MTS2/T P15/ INK4B*

*CDKN2A/ P16/INK4A/ MTS1/ CDK4I/ CDKN2*

*CHFR/ RNF116/ RNF196*

*FHIT/ FRA3B/ 3P3Aase*

*ZMYND10/ BLU* 

*PRDM2/ PRDM2*

**Gene Full name Chromo-**

Cyclin-dependent kinase inhibitor 2B

Cyclin-dependent kinase inhibitor 2A

Checkpoint with forkhead and ring finger domains

containing 7

Fragile histidine triad gene

DNA-damageinducible, gamma

and esophageal cancer1

Zinc finger, MYND-type containing 10

polydactyly1

PR domain containing 2, with ZNF domain 3p22- 21.3

*BRD7* Bromodomain

*GADD45G* Growth arrest and

*DLEC1* Deleted in lung

*MIPOL1* Mirror-image

**Cancerrelated process** 

**Cell cycle** *CDKN2B/* 



Epigenetics of Nasopharyngeal Carcinoma 11

**Function Refs** 

12q14 Antagonist of Wnt signaling (Lin et al. 2006;

GTPase-activating protein specific for RhoA and

5p13 Adaptor molecule involved in multiple receptormediated signaling

> Ras GTPase-activating protein, negatively regulates RAS signaling

negative regulator of G2-M phase checkpoint

3p14.1 Anti-angiogenesis (Lung, Lo, Xie, et

4p14 Stabilize p53 and activate the p14ARF-p53 signaling

pathway

13q22 Negative regulator of ET/ETAR pathway

via concomitant

downregulation of vascular endothelial growth factor and matrix metalloproteinase 2

 (Li, Shu, et al. 2011; Wong, Tang, et al. 2003)

 (Chow et al. 2004; Zhou et al.

 (Zhang et al. 2007)

Chan et al. 2007)

(Peng et al. 2006)

(Tong et al. 2010)

 (Jin, Wang, Ying, Wong, Cui, et al.

(Li et al. 2010)

(Yi et al. 2009)

 (Lo et al. 2002; Zhou et al. 2007)

(Law et al. 2011)

al. 2008)

2007)

2005)

9p21 Stabilizes p53, interacts with MDM2

3p21.3 Regulate Ras signaling pathway

20p12.1 Regulate Ras signaling pathway

Cdc42

pathways

**somal location**

8p21.3- 22

12q23 q24

*SFN/14-3-3 σ* Stratifin 1p36.11 Downstream target of p53,

*FBLN2* Fibulin 2 3p25.1 Angiogenesis suppression

**Gene Full name Chromo-**

reading frame

(RalGDS/AF-6) domain family member 1A

(RalGDS/AF-6) domain family member 2A

*ARF/P14* Alternate open

*RASSF1A* Ras association

*RASFF2A* Ras association

*WIF-1* Wnt inhibitory factor-1

*DLC-1* Deleted in liver cancer-1

*DAB2* Disabled homolog 2, mitogenresponsive phosphoprotein (Drosophila)

> activator like 1 (GAP1 like)

carboxyl-terminal esterase L1

receptor type B

metallopeptidase

thrombospondin type 1 motif 9

*RASAL1* RAS protein

*UCHL1* Ubiquitin

*EDNRB* Endothelin

*ADAMTS9* A disintegrin-like and

with

**Cancerrelated process** 

**Signal transduction** 

**Angiogenesis** 


Epigenetics of Nasopharyngeal Carcinoma 11

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

16q22.1 Calcium-dependent

16q23.3 Calcium-dependent

20q13.3 Calcium-dependent

11q25 Cell adhesion, cell-cell recognition

**Function Refs** 

 (Wong, Tang, et al. 2003; Wong et al. 2004)

(Sun et al. 2007)

(Ying et al. 2006)

(Du et al. 2011)

(Cui et al. 2008)

 (Wong, Tang, et al. 2003)

(Yi et al. 2007)

 (Hui et al. 2003; Lung et al. 2004)

 (Jin, Wang, Ying, Wong, Li, et al. 2007; Wei et al.

(Lung et al. 2005)

et al. 2002)

 (Wong, Tang, et al. 2003; Wong et al. 2004)

2010)

2010)

adhesion and cell migration

adhesion and cell migration

adhesion and cell migration

adhesion and cell migration

7q22 Serine protease inhibitor (Wang et al.

that mediates cell-to-cell and cell-to-matrix interactions

intercellular communication

mediate cell-cell interaction

organization, focal adhesion

10q26 Repair alkylated guanine (Kwong

11q23 Cell adhesion molecules,

16q23.1 Cell adhesion modulator, inhibits growth factorindependent cell proliferation

11q23.3 Regulates cytoskeletal

3p21.3 DNA mismatch repair protein, cell cycle G2-M arrest

and migration by modulating the activity of p190 RhoGAP and Rho GTPase

12q14 Extra cellular matrix (Chan et al. 2010)

**somal location**

Protocadherin 10 4q28.3 Calcium-dependent

*THBS1* Thrombospondin 1 15q15 An adhesive glycoprotein

*Cx43* Connexin 43 20q11 Gap junction and

**Gene Full name Chromo-**

cadherin (heart)

*CDH1* Cadherin 1, type 1, E-cadherin (epithelial)

*CDH4* Cadherin 4, type 1, R-cadherin (retinal)

*OPCML* Opioid binding

*TFPI-2* Tissue factor

2

19

*MMP19* Matrix

*ADAMTS18* ADAM

*MGMT* O-6-

*MLH1 /hMLH1 /HNPCC /FCC2* 

with

*THY1/CD90* Thy-1 cell surface antigen

DNA

2 (E. coli)

*TSLC1 /CADM1* 

**DNA repair**  protein/cell adhesion molecule

pathway inhibitor-

metalloproteinase-

Tumor suppressor in lung cancer 1

metallopeptidase

thrombospondin type 1 motif, 18

methyoguanine-

methyltransferase

MutL homolog 1, colon cancer, nonpolyposis type

*CDH13* Cadherin 13, H-

*PCDH10 /OL-PCDH /KIAA1400* 

**Cancerrelated process** 

**Invasion and metastasis** 



Epigenetics of Nasopharyngeal Carcinoma 13

EBV is a prototype of gamma herpes virus which was discovered more than 40 years ago from Burkitt's lymphoma, a childhood tumor that is common in sub-Saharan Africa. Further studies reveal that EBV was widespread in all human populations, which infects more than 90% of the world's adult population. Human are the only natural host for EBV. Once infected with EBV, the individual remains a lifelong asymptomatic carrier of the virus

EBV was implicated in a variety of human malignancies, such as post-transplant lymphoma, AIDS-associated lymphomas, Burkitt lymohoma, Hodgkin's disease, T-cell lymphoma, NPC, parotid gland carcinoma and gastric carcinoma (Young and Rickinson 2004; Pattle and Farrell 2006). The association between EBV infection and NPC was well documented by the fact that EBV genome presents in virtually all the NPC cells (Lo and Huang 2002; Lo, To, and Huang 2004). Tumorigenesis of NPC is proposed to be a multistep process. EBV may play an important role in the etiology of the NPC, involving activation of oncogenes and/or the inactivation of tumor suppressor genes. Early genetic changes may predispose the epithelial cells to EBV infection or persistent maintenance of latent cycle. Expression of latent genes in the EBV-infected cells may enhance its transformation capacities, and subsequently, clonal expansion may result in the rapid progression to invasive carcinoma. There are two alternative states of EBV infection: lytic and latent (Young and Rickinson 2004; Fernandez et al. 2009). In EBV-infected cells, virus replication with production of infectious virus is a rare event. Typically, EBV establishes a latent infection. This is characterized by the expression of a limited set of viral products, including six EBV-encoded nuclear antigens (EBNA1, 2, 3A, 3B, 3C, -LP), three latent membrane proteins (LMP1, 2A, 2B) and two EBV-encoded nuclear RNAs (EBER1, EBER2). Expression of different panels of latent gene transcripts is controlled by usage of three distinct EBV nuclear antigen (EBNA) promoters (Wp, Cp, and Qp). In established lymphoblastoid cell lines (LCLs), the EBNA transcripts are initiated at the C promoter, Cp, located to the BamHI C fragment of the viral genome. In EBV genome, W promoter (Wp) is the first promoter to be activated immediately after EBV infection of human B cells, but it undergoes progressively methylation and switches off in LCLs. In parallel, an unmethylated promoter, Cp, is switched on. In other EBV-carrying cell types, Cp is switched off. These include memory B cells, Burkitt's lymphomas (BLs), EBV-associated carcinomas (NPC, gastric carcinoma) and Hodgkin's lymphomas; these cells typically use the Q promoter (Qp) for expression of EBNA1 transcripts, but not the transcripts coding for the other five EBNAs, and may differ from each other regarding the expression of LMPs, BARTs (BARF0 and BARF1) and EBV-encoded microRNAs (Li and Minarovits 2003). LMP1 is the major EBV oncoprotein in NPC (Tao and Chan 2007; Lo, To, and Huang 2004). By activating several important cellular signalling pathways like NF-B, JNK, JAK/STAT and PI-3K pathway, LMP1 could upregulate antiapoptotic gene products, such as BCL2, A20, AP-1, CD40, CD54 and also cytokines IL-6 and IL-8; thereby exhibit its oncogenic characteristics (Eliopoulos and Young 2001). LMP1 expressing NPCs show different growth pattern and prognosis from those without LMP1 expression (Hu et al. 1995). Although EBV genome presents in virtually all the NPC cells, expression of LMP1 is variable in NPC: LMP1 is expressed in only approximately 65% of NPC biopsies (Fahraeus et al. 1988; Young et al. 1988). This variability can be related to the

**3.2 Epstein-Barr virus and DNA methylation** 

(Young and Rickinson 2004).

Table 1. List of methylated tumor suppressor genes involved in nasopharyngeal carcinoma (NPC)

3q24 Binds retinoic acid to

**Function Refs** 

 (Kwong, Lo, Chow, To, et al. 2005; Kwong et al. 2002; Seo, Kim, and Jang 2008)

et al. 2008; Kwong, Lo, Chow, Chan, et al. 2005; Kwok et al. 2009)

 (Kwong, Lo, Chow, To, et al.

Chow, To, et al.

(Chen et al. 2011)

 (Wong, Kwong, et al. 2003)

 (Li, Li, et al. 2011)

(Zhou et al. 2009)

 (Lung, Lo, Wong, et al. 2008)

2005)

2005)

mediates cellular signaling during embryonic

morphogenesis, cell growth

stream into cells, solubilizes retinol and retinal, protects cells from membranolytic

1p36.22 Similar to CRBP1 (Kwong, Lo,

involved in smooth muscle cell differentiation

differentiation, cell-cycle reentry regulator, suppresses tumor cell migration and invasion, induces apoptosis

OSCP/ATP5O protein,a stress-responsive gene

intron RNA splicing and protein synthesis within the mitochondria,indirectly required for mitochondrial genome maintenance

in maintaining genomic

1p34.3 Interaction partner of the mitochondrial ATP synthase subunit

3p21.3 Essential roles in group I

integrity

3q25 Retinoic acid target gene (Yanatatsaneejit

and differentiation

3q23 Draws retinol from blood

retinoid action

**somal location**

**Gene Full name Chromo-**

receptor beta 2

Retinoic acid receptor responder (tazarotene induced) 1

Cellular retinol binding protein 1

binding protein 4

domain-contrining

protein 1

synthetase 2, mitochondrial

*LARS2* Leucyl-tRNA

*Myocd* Myocardin 17p11.2 Transcription factor,

*CRYAB* Crystallin,alpha B 11q23.1 An important nuclear role

Table 1. List of methylated tumor suppressor genes involved in nasopharyngeal carcinoma

High-in-normal-1 5q35 Involved in epithelial cell

Cellular retinol

*RARβ2* Retinoic acid

*RARRES1 /TIG1* 

*CRBP*Ⅰ

*/RBP1* 

*CRBP*Ⅳ

*HIN1 /SCGB3A1* 

**Others** *NOR1* Oxidored-nitro

**Cancerrelated process** 

**Vitamin response** 

**Tissue development and differentiation**

(NPC)

#### **3.2 Epstein-Barr virus and DNA methylation**

EBV is a prototype of gamma herpes virus which was discovered more than 40 years ago from Burkitt's lymphoma, a childhood tumor that is common in sub-Saharan Africa. Further studies reveal that EBV was widespread in all human populations, which infects more than 90% of the world's adult population. Human are the only natural host for EBV. Once infected with EBV, the individual remains a lifelong asymptomatic carrier of the virus (Young and Rickinson 2004).

EBV was implicated in a variety of human malignancies, such as post-transplant lymphoma, AIDS-associated lymphomas, Burkitt lymohoma, Hodgkin's disease, T-cell lymphoma, NPC, parotid gland carcinoma and gastric carcinoma (Young and Rickinson 2004; Pattle and Farrell 2006). The association between EBV infection and NPC was well documented by the fact that EBV genome presents in virtually all the NPC cells (Lo and Huang 2002; Lo, To, and Huang 2004). Tumorigenesis of NPC is proposed to be a multistep process. EBV may play an important role in the etiology of the NPC, involving activation of oncogenes and/or the inactivation of tumor suppressor genes. Early genetic changes may predispose the epithelial cells to EBV infection or persistent maintenance of latent cycle. Expression of latent genes in the EBV-infected cells may enhance its transformation capacities, and subsequently, clonal expansion may result in the rapid progression to invasive carcinoma.

There are two alternative states of EBV infection: lytic and latent (Young and Rickinson 2004; Fernandez et al. 2009). In EBV-infected cells, virus replication with production of infectious virus is a rare event. Typically, EBV establishes a latent infection. This is characterized by the expression of a limited set of viral products, including six EBV-encoded nuclear antigens (EBNA1, 2, 3A, 3B, 3C, -LP), three latent membrane proteins (LMP1, 2A, 2B) and two EBV-encoded nuclear RNAs (EBER1, EBER2). Expression of different panels of latent gene transcripts is controlled by usage of three distinct EBV nuclear antigen (EBNA) promoters (Wp, Cp, and Qp). In established lymphoblastoid cell lines (LCLs), the EBNA transcripts are initiated at the C promoter, Cp, located to the BamHI C fragment of the viral genome. In EBV genome, W promoter (Wp) is the first promoter to be activated immediately after EBV infection of human B cells, but it undergoes progressively methylation and switches off in LCLs. In parallel, an unmethylated promoter, Cp, is switched on. In other EBV-carrying cell types, Cp is switched off. These include memory B cells, Burkitt's lymphomas (BLs), EBV-associated carcinomas (NPC, gastric carcinoma) and Hodgkin's lymphomas; these cells typically use the Q promoter (Qp) for expression of EBNA1 transcripts, but not the transcripts coding for the other five EBNAs, and may differ from each other regarding the expression of LMPs, BARTs (BARF0 and BARF1) and EBV-encoded microRNAs (Li and Minarovits 2003). LMP1 is the major EBV oncoprotein in NPC (Tao and Chan 2007; Lo, To, and Huang 2004). By activating several important cellular signalling pathways like NF-B, JNK, JAK/STAT and PI-3K pathway, LMP1 could upregulate antiapoptotic gene products, such as BCL2, A20, AP-1, CD40, CD54 and also cytokines IL-6 and IL-8; thereby exhibit its oncogenic characteristics (Eliopoulos and Young 2001). LMP1 expressing NPCs show different growth pattern and prognosis from those without LMP1 expression (Hu et al. 1995). Although EBV genome presents in virtually all the NPC cells, expression of LMP1 is variable in NPC: LMP1 is expressed in only approximately 65% of NPC biopsies (Fahraeus et al. 1988; Young et al. 1988). This variability can be related to the

Epigenetics of Nasopharyngeal Carcinoma 15

pathogenesis. In the same study, they also identified two novel and highly abundant EBV miRNA genes, namely, miR-BART21 and miR-BART22 (Zhu et al. 2009). A parallel study demonstrated that LMP2A is the putative target of miR-BART22 in NPC. LMP2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells, down-modulation of LMP2A expression by miR-BART22 may permit escape of EBV-infected cells from host immune surveillance (Lung et al. 2009). Similar regulations were also addressed on LMP1: EBV-encoded BART miRNAs target the 3′ UTR of the LMP1 gene and negatively regulate LMP1 protein expression. These miRNAs also modulate LMP1-induced NF-κB signalling

and alleviate the cisplatin sensitivity of LMP1-expressing NPC cells (Lo et al. 2007).

**roles as biomarkers** 

(Balch et al. 2009; Laird 2003).

**4.1 DNA methylation, results from tumor tissues** 

(Schulz 2005).

**4. Epigenetic alternations in relation to clinical parameters of NPC, and their** 

Frequent aberrantly methylated TSGs in tumors have been used as molecular markers for the detection of malignant cells from various clinical materials. It provides possibilities of both cancer early detection and dynamic monitoring of cancer patients after treatment

DNA methylation biomarkers hold a number of advantages over other biomarker types, such as proteins, gene expression and DNA mutations (Balch et al. 2009; Laird 2003). Methylated DNA sequences are more chemically and biologically stable, and more easier to be amplified, thus greatly enhancing detection sensitivity. DNA methylation are often cancer specific, and restriction to limited regions of DNA in the CpG islands. Compared to genetic alternations such as gene mutation or amplification, aberrant methylation on TSG promoters is rather prevalent and tumor-specific among NPCs. As mentioned above, NPC tumor progression is well characterized by a number of combinatorial epigenetic aberrations distinct to other malignancy, including DNA methylation of more than 30 genes. Consequently, these methylated DNA sequences represent potential biomarkers for diagnosis, staging, prognosis and monitoring of response to therapy or tumor recurrence

It has been shown that some genes are high frequently methylated in tumor tissue DNA obtained from NPC primary tumors, but not in normal tissues (Pan et al. 2005; Sun et al. 2007; Zhang et al. 2007; Li, Shu, et al. 2011). These genes are ideal candidate to serve as biomarkers for detection of NPC. Some of these TSGs are not only methylated in NPC, but also commonly methylated in other cancers. So methylation assessment of single genes lacks sufficient specificity for NPC diagnosis. It is believed that panels of multiple methylation biomarkers may achieve higher accuracy required for discriminate NPC from other cancers (Kwong et al. 2002; Hutajulu et al. 2011). This notion was supported by a study of Esteller et al, which showed that a panel of three to four markers could define an abnormality in 70– 90% of each cancer type through detecting their aberrant methylation (Esteller et al. 2001). Some studies have been conducted using different combination of gene panels, though there is overlap among them. Combination of methylation markers not only improved the discrimination between NPC and non-NPC diseases, but also the sensitivity of cancer

methylation status of the regulatory sequences (LRS, LMP1 regulatory sequence) located 5' from LMP1p, as LMP1 is expressed in NPCs with unmethylated LRS but is absent from NPCs with highly methylated LRS. A good correlation exists between LRS methylation and silencing of LMP1p in EBV-carrying lymphoid cell lines and tumors as well (Li and Minarovits 2003)).

On the other hand, EBV regulates the expression of critical cellular genes using cellular DNA methylation machinery. LMP1 has been shown to interacting with methyltransferase and further induce the cellular gene E-cadherin (*CDH1*) promoter methylation. Increased methylation may occur through the activity of DNA methyltransferases 1, 3a, and 3b that in turn are induced through JNK/AP1 signalling by LMP1. Transfection of LMP1 into cancer cells suppressed E-cadherin expression, thereby facilitating a more invasive growth of NPC cells (Tsai et al. 2006). It will be interesting to discover novel target genes regulated by epigenetic mechanism of EBV.
