DNA Methylation as a Biomarker in Nasopharyngeal Carcinoma … · Histology of NPC Arising in the epithelial lining of the nasopharynx, NPC comprises the vast majority of nasopharyngeal

DNA Methylation as a Biomarker in Nasopharyngeal Carcinoma

Feryel Ksiaa and Mounir Trimeche*Department of Pathology, University Hospital Farhat Hached, Sousse, Tunisia

Abstract

Nasopharyngeal carcinoma (NPC) is a malignancy with remarkable ethnic and geographic distribution.Initially, point mutations and chromosomal deletions were considered to be the major events involved inthe inactivation of tumor-suppressor genes in NPC. The discovery that many tumor-suppressor genes canalso be inactivated by hypermethylation of the CpG islands in their promoter region clearly indicates thatepigenetic events also play an important role as alternative mechanisms in NPC carcinogenesis.

In this chapter, we update current information on methylated genes associated with the developmentand progression of NPC. Promoter hypermethylation of critical genes could be potential biomarkers andtherapeutic targets for NPC.

Several genes have been investigated for methylation in the promoter region in NPC. These methylatedgenes are involved in critical pathways, such as DNA repair, cell cycle regulation, and invasion/metastasis.

The role of hypermethylated genes in the deregulation of critical pathways in NPC is now well known.Besides their role on the pathogenesis of NPC, results from many investigations have provided additionalinformation on the potential role of hypermethylated genes as predictive biomarkers in the developmentand progression of NPC.

List of Abbreviations

CDH1 Epithelial E-CadherinCGH Comparative Genomic HybridizationDAB2 Human Disabled-2DAP-kinase Death-Associated Protein KinaseDLC-1 Deleted in Liver Cancer-1DLEC1 Deleted in Lung and Esophageal Cancer 1DNMT DNA MethyltransferaseEBV Epstein–Barr VirusGSTP1 Glutathione S-Transferase P1HIN-1 High in Normal-1HLA Histocompatibility Leukocyte AntigensIARC International Agency for Research on CancerLARS2 Leucyl-tRNA Synthetase 2, MitochondrialLOH Loss of HeterozygosityMGMT O6-Methylguanine-DNA MethyltransferaseMSP Methylation-Specific PCRNPC Nasopharyngeal Carcinoma

*Email: [email protected]

Biomarkers in CancerDOI 10.1007/978-94-007-7744-6_43-1# Springer Science+Business Media Dordrecht 2014

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PCR Polymerase Chain ReactionRARb2 Retinoic Acid Receptor b2RASSF1A Ras Association Domain Family 1ATFPI-2 Tissue Factor Pathway Inhibitor-2TIMP-3 Tissue Inhibitor of Metalloproteinase-3TSG Tumor-Suppressor GenesTSLC1 Tumor Suppressor in Lung Cancer 1WHO World Health Organization

Key Facts of Methylation

The following are the key facts of DNA methylation including the location and the mechanism of thismain epigenetic change, its role in the control of gene expression during embryogenesis, its implication incancer development, and its importance as a potential biomarker in human cancers:

• DNA methylation is a biological process consisting in the addition of a methyl (CH3) group at thecarbon 5 position of cytosines (C) located 50 of guanidines (G).

• This process often occurs in cytosine–guanine-rich regions of DNA (CpG islands), which are commonin the promoter regions of genes.

• DNA methylation involves an enzymatic reaction catalyzed by a group of enzymes known as DNAmethyltransferases (DNMT1, DNM3a, and DNMT3b).

• DNA methylation is one of the main epigenetic modifications implicated in the control of geneexpression during the embryonic development of organisms.

• The presence of aberrant methylation in the promoter region of genes is associated with the suppressionof gene expression in several types of cancer and in developmental disorders.

• DNA methylation is a reversible mechanism, making it an interesting therapeutic target.• Several methods have been developed to investigate DNA methylation in tumors as well as in body

fluids.• The detection of DNAmethylation has emerged as a potential biomarker for several types of cancer and

a promising therapeutic target.

Definitions of Words and Terms

Undifferentiated Carcinoma A term used to describe poorly differentiated malignant epithelial tumorthat does not have specialized structures. Undifferentiated cancer cells often grow and spread quickly.

Gene Polymorphism Difference in DNA sequence among individuals. Genetic variations occurring inmore than 1 % of a population would be considered useful polymorphisms for genetic analysis.

Familial Predisposition Individuals with a family history of cancer in a first-degree or second-degreerelative may have a genetic predisposition to developing the disease. Familial predisposition is aninherited risk of developing a cancer. Having a familial predisposition for a disease does not mean thatyou will get that disease, but your risk may be higher than that of the general population.


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Risk Factor A risk factor is a condition that is known to increase susceptibility to a disease or outcome.Risk factors are usually determined through epidemiological studies, which track diseases or health eventsthrough a given population. Examples of risk factors are tobacco smoking, which can increase your risk ofgetting nasopharyngeal carcinoma.

Endemic Population A disease that is prevalent in a certain population or people or region. Diseases inendemic population are not always present at high levels. They can be relatively rare, but the definingfeature of a regional endemic disease is that it can always be found in the population that lives there.

Epigenetic Epigenetic changes modify the physical structure of DNA and may indirectly influence theexpression of the genome. One example of an epigenetic change is DNA methylation – the addition of amethyl group – of the DNA molecule, which prevents certain genes from being expressed. For instance,an epigenetic change that silences genes could lead to uncontrolled cellular growth or to an increase inDNA damage, which, in turn, increases cancer risk.

Cancer Biomarker A distinctive biological indicator used for cancer. A handful of cancer biomarkersare currently used routinely for population screening, disease diagnosis, prognosis, monitoring of therapy,and prediction of therapeutic response. Although it is highly desirable to have biomarkers suitable forpopulation screening and early diagnosis, none of the current biomarkers has adequate sensitivity,specificity, and predictive value for population screening.

Apoptosis It refers to the death of a cell resulting from a normal series of genetically programmed events,when a cell is no longer needed. Apoptosis is a natural process that removes cells that have becomeunhealthy. Apoptosis is important in understanding cancer, because cancer cells have developed ways toavoid apoptosis. Newer treatments are being studied that interfere with the cancer cell’s ability to avoidapoptosis.

Tumor-Suppressor Genes Genes that regulate the growth of cells. When these genes are functioningproperly, they can prevent and inhibit the growth of tumors. When tumor-suppressor genes are altered orinactivated (by genetic or epigenetic alterations), they lose the ability to make a protein that controls cellgrowth. Cells can then grow uncontrolled and develop into a cancer.

Noninvasive Screening Tests Procedures that does not require insertion of an instrument or devicethrough the skin or a body orifice for diagnosis or treatment.

Tumor Aggressiveness It is the ability of cancer cells to spread in the body.

Introduction

Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor with remarkableracial and geographic distribution. In 2002, according to the data of the International Agency for Researchon Cancer (IARC), 80,000 cases of NPC were newly diagnosed worldwide (Parkin et al. 2005).

NPC is very different from other head and neck cancers not only because of its geographic distributionbut also because of its specific multifactorial etiology. Epstein–Barr virus (EBV) infection, intake ofsalted-preserved foods, tobacco and alcohol consumption, and inherited susceptibilities such as genepolymorphisms or familial predisposition are major factors associated with the development of NPC.


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Approximately 80 % of patients with NPC are diagnosed at an advanced tumor stage. The delay in thedisease discovery is mainly due to the deep-cited location and nonspecific symptoms of the tumor. Withthe introduction of new treatment modalities, survival has steadily improved over recent decades.However, cancer relapse is still the leading cause of cancer-related deaths in patients with NPC.

The early detection, prognosis, and monitoring of therapy are especially important parameters for NPCbecause of the high propensity for spreading to more sites. Knowledge about new biomarkers in NPC isimportant in improving optimal patient treatment. In this context, the epigenetic alterations, especiallymethylation of DNA, emerge as a promising track.

Regional Variation in the Incidence of NPC

There are remarkable regional variations in the incidence of NPC worldwide. In southern China andSoutheast Asia, the NPC is the most common malignant tumor with an incidence rate ranging from 20 to50/100,000 people per year. Hong Kong detains the highest NPC incidence. NPC is the major cause ofcancer morbidity and mortality in these endemic regions. Moderately higher incidence of NPC is reportedin some other population groups also such as Eskimos in the Arctic, Arabs in North Africa, and few otherpopulation groups of Southeast Asia. Conversely, the estimated incidence of NPC is lower than 1/100,000people per year in most part of the world (Chang and Adami 2006).

In areas of low tomoderate risk for NPC, a bimodal age distribution is always depicted. The first peak inincidence is observed in adolescents of each sex (10–19 years) and the second peak, higher than the firstone, in the age group 50–59 years. This relatively high frequency of NPC among young population seemsto be a characteristic feature of areas of moderate incidence of NPC.

Another important characteristic of NPC epidemiology is sex distribution. In virtually all of thepopulations studied, the rates are higher among males than females. In most populations, the male/femaleratio is 2–3:1.

Histology of NPC

Arising in the epithelial lining of the nasopharynx, NPC comprises the vast majority of nasopharyngealcancers in both high- and low-incidence populations. The World Health Organization (WHO) histopath-ological classification of NPC (Chan et al. 2005) has been adopted by several investigators. It comprisesthree histological types, (1) keratinizing squamous-cell carcinoma (type 1) and nonkeratinizing carci-noma. The nonkeratinizing group can further be separated into differentiated carcinoma (type 2) andundifferentiated carcinoma (type 3) group (Fig. 1).

The proportions of the histological types of NPC appear to vary with geographical location and race.However, undifferentiated carcinoma or type 3 is the major histopathological type of NPC. In the low-riskareas of Japan, type 1 NPC accounts for 12 % of cases, whereas undifferentiated carcinoma or type3 predominates in high-risk areas and takes up 92 % of all cases.

Types 2 and 3 NPC are Epstein–Barr virus (EBV) associated and have better prognoses than type 1;EBV infection is generally absent in type1 (Wei et al. 2011).


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Etiological Factors in NPC

It is believed that a number of etiological environmental factors along with genetic/host factors might beresponsible for the causation of NPC. At least three etiological factors are strongly suspected in NPCcarcinogenesis: the ubiquitous EBV infection, genetically determined susceptibility, and associatedenvironmental factors. Figure 2 summarizes the main factors related to the development of nasopharyn-geal carcinoma.

EBV is a member of the large herpesvirus family and distributed ubiquitously. It is found throughout allhuman populations, with a prevalence of over 90 % in adult. Initially, the association between NPC and

Fig. 1 Histology of nasopharyngeal carcinoma. (a, b) Undifferentiated carcinoma featuring cohesive sheet of tumor cells(asterisk) with plentiful intermixed lymphocytes (double asterisks) (hematoxylin and eosin staining, original magnification:a �200 and b �400). (c, d) Keratinizing squamous-cell carcinoma showing cohesive tumor cells with well-defined cellborders (hematoxylin and eosin, original magnification: c �200, d �400)

NPCEBVinfection

Genetic alterations

Tobacco

Family history

Genepolymorphisms

Epigeneticalterations

Diets

Premalignants lesions

Fig. 2 Proposed causal factors of nasopharyngeal carcinoma. The interaction between host factors, such as inheritedsusceptibilities, and environmental factors, including Epstein–Barr virus (EBV), diet, and tobacco, initiates the progressionfrom normal to premalignant disease stages. These factors combined with genetic and epigenetic alterations lead to thedevelopment of nasopharyngeal carcinoma (NPC)


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EBV was revealed by results of seroepidemiological studies from different parts of the world. It wasconfirmed subsequently by the demonstration of the persistence of EBV DNA and/or antigens in NPCtumor cells (Frappier 2012; Fig. 3). EBV is intimately related to NPC and elicits the formation ofantibodies that are useful for diagnosis and follow-up study. Numerous studies have reported thatantibodies to EBV antigens can be used in the early detection of NPC. Serological assays are beingutilized in mass screening of high-risk populations for NPC. The serology value of EBV was alsoobserved for the early detection of NPC relapse (Han et al. 2012; Chan et al. 2013).

From the viewpoint of genetic susceptibilities, a growing number of reports have confirmed anassociation between the histocompatibility leukocyte antigens (HLA) gene polymorphism and increasedrisk for NPC. Simons et al. (1978) were the first to describe the association of HLA antigens with NPC insouthern Chinese and demonstrated that HLA antigens play a role in determining both susceptibility forNPC and survival after diagnosis. Later, several studies have supported the strong genetic etiology of NPC(Li et al. 2009). Studies are in progress in different laboratories to elucidate the exact location and functionof these genes.

Although most NPCs are sporadic cases, the familial clustering of NPC has been also demonstratedworldwide. Such clustering can result from shared genetic susceptibility and/or shared environmental riskfactors. The excess risk was generally four- to tenfold among individuals with a first-degree relative withNPC, compared with those without a family history (Bei et al. 2012).

Some dietary habits are reported to be associated with the increased risk for NPC. Salted fish andsmoking have been suspected as possible etiological factors in the development of NPC. In Asiancountries, intake of salted fish and other preserved foods is particularly high in families of lowersocioeconomic status (Jia et al. 2010). Furthermore, several studies have shown that childhood exposure

Fig. 3 Detection of Epstein–Barr virus by chromogenic in situ hybridization (CISH) for Epstein–Barr virus-encodedRNA (EBER) in nasopharyngeal carcinoma. Dark blue/black positive staining for EBER in the nuclei of almost all tumorcells, whereas the normal epithelial cells (asterisk) and stromal lymphocytes (double asterisk) remain negative (originalmagnification, a �100, b �400)


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and increasing duration and frequency of consumption of salt-preserved foods are independently associ-ated with elevated risk for NPC. In contrast, consumption of fresh fruits and/or vegetables, especiallyduring childhood, is associated with a lower risk for NPC. The noticeable protective effect of fruits andvegetables may be attributed to antioxidant effects, prevention of nitrosamine formation, and otheranticarcinogenic properties (Polesel et al. 2013). It has been also reported that persons who have smokedcigarettes for many years have a higher risk for NPC, particularly for the well-differentiated subtype(Xu et al. 2012).

Genetic Alterations in NPC

The viral causative, genetic, and environmental factors, either acting alone or in combination, would leadto multiple genetic and epigenetic alterations. The development of NPC involves accumulation of geneticchanges leading to the evolution of clonal cell population that possesses growth advantages over othercells.

NPC exhibits numerous genetic abnormalities, namely, chromosomal deletions, gene amplifications,and mutations. By comparative genomic hybridization (CGH) analyses, a large number of primary NPChave been examined for a gain and loss of genetic material in the genome, including gain at chromosomes12p, 1q, 3q, 8q, 5p, and 7q as well as loss at chromosomes 3p, 11q, 13q, 14q, 16q, 16p, 1p, 9q, and 22q.

Among these allelic loss, 3p and 9q detain the highest frequencies and were detected in almost95–100 % of primary NPC biopsies and in 75 % of precancerous lesions (Li et al. 2006). It is wellknown that the 3p chromosomal arm contains some important tumor-suppressor genes (TSGs). Thus, lossof heterozygosity (LOH) on 3p in premalignant lesions indicates that this alteration could be an early andcritical molecular event in the carcinogenesis of NPC. Despite the many efforts provided, the sequence inwhich these genetic alterations participate in the evolution of NPC has not been established as clearly as inother cancers such as colon cancer (Chen et al. 2012).

Besides genetic deletion and mutation, it has been identified a second mechanism, termed epigeneticalterations, potentially responsible for NPC carcinogenesis.

Role of DNA Methylation in NPC

Epigenetic and DNA MethylationThe term epigenetic designs an inheritable change in the pattern of gene expression without a change inDNA sequence. The methylation of DNA is an important epigenetic modification that has physiologiceffects on embryogenesis, cellular development, differentiation, senescence, and aging. Three welldefinite DNA methyltransferases (DNMTs), DNMT1, DNMT3a, and DNMT3b, are responsible forDNA methylation. These enzymes act by adding a methyl group to the carbon 5 position of a cytosinealready present in the DNA strand and therefore transform the cytosine residues to 5-methylcytosineforms (Robertson 2005). DNA methyltransferases use S-adenosylmethionine as the methyl donor.Approximately 3–5 % of the cytosine residues in genomic DNA are of the 5-methylcytosine forms andare principally scattered in selected regions, called CpG islands. CpG islands of many tissue-specificgenes are methylated, except in the tissue in which they are expressed, while unmethylated CpG islandsare associated with housekeeping genes.

Nevertheless, aberrant and/or excessive DNA methylation can lead to neoplastic transformation.Indeed, several reports have demonstrated that hypermethylation in particular genes, namely, in TSGs,can be one of the main driving forces in carcinogenesis. The presence of 5-methylcytosines into the


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promoter region of genes prevents transcription factors from binding, by steric hindrance, and conse-quently reduces and even inhibits gene transcription (Figure 4).

Several studies investigating methylation profile in various tumors have shown that thehypermethylation of a specific gene depends on tumor type (Esteller 2002). For example,hypermethylation of p14 (ARF) and APC is most prevalent in gastric and colon cancers, whereas thatof GSTP1 is characteristic of steroid-related tumors such as the breast, liver, and prostate. Other genes,such as p16 (INK4A) and O6-methylguanine-DNA methyltransferase (MGMT), are less exclusive and arehypermethylated across many tumor types.

Hypermethylated Genes in NPCIn primary NPC tissues, many TSGs exhibit high frequencies of methylation. Moreover, NPC has its ownunique and specific methylation profile, slightly different to that described for other cancers. Indeed,results from numerous investigations in NPC have shown aberrant methylation at CpG islands in agrowing list of genes, including the human Ras association domain family 1A (RASSF1A); retinoic acidreceptor b2 (RARb2); cell cycle regulator p16 (INK4A), p15 (INK4b), and p14 (ARF) genes; the DNArepairMGMT; the apoptosis-related and potential metastasis inhibitor (DAP-kinase); and the detoxifying(GSTP1) genes and others. These genes belong to different pathways in cells. Epigenetic inactivation ofthem may affect all of the molecular pathways involved in cell immortalization and transformation.

Methylation-specific PCR (MSP) was the main methodology applied to access data about DNAmethylation in the promoter genes. MSP is a powerful and sensitive technique used to detecthypermethylation based on primer annealing during PCR. However, such a qualitative technique isonly able to detect methylation present in more than one CpG in the primer set and is susceptible tofalse-positive results (Cottrell 2004).

The Human Ras Association Domain Family (RASSF) GenesRASSF1, a critical tumor-suppressor gene, has been initially isolated from the lung tumor-suppressorlocus 3p21.3. RASSF1 gene encodes more than seven isoforms, including RASSF1A, RASSF1B, andRASSF1C, which are derived from alternative mRNA splicing and promoter usage (Van der Weyden andAdams 2007). RASSF1A protein is involved in the Ras signaling pathway mediating the apoptotic effectsof oncogenic Ras. RASSF1A protein structure also suggests that it may participate in the DNA damageresponse (Donninger et al. 2007).

RASSF1A is commonly inactivated through its promoter methylation (Dammann et al. 2005; Chowet al. 2004). Indeed, high frequency of promoter hypermethylation of RASSF1A has been reported inprimary NPCs and varied from 67 % to 91 % (Fendri et al. 2009; Lo et al. 2001; Challouf et al. 2012).These data suggest that promoter hypermethylation of RASSF1A has a critical role in NPC carcinogenesis.Interestingly, aberrant methylation of RASSF1A correlates with lymph node metastasis in NPC (Fendriet al. 2009).

Because of its consistently frequent promoter hypermethylation in NPC and its correlation withclinicopathological parameters of tumor aggressiveness, RASSF1 may be a useful cancer biomarker forthe identification of the aggressive NPC that may benefit from different therapeutic modalities.

Similarly, RASSF2A, an isoform of RASSF2, has been reported to be inactivated by its promotermethylation in 61 % of NPC and correlates with lymph node metastasis (Zhang et al. 2007).

The Retinoic Acid Receptor b2 (RARb2) GeneRetinoids are known to possess antiproliferative, differentiative, immunomodulatory, and apoptosis-inducing properties. The regulation of cell growth and differentiation of normal, premalignant, andmalignant cells by retinoids are thought to result from the direct and indirect effects of retinoids on


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gene expression. These effects are mediated by the nuclear receptors, including retinoic acid receptor b2(RARb2) located at 3p24 (Mark et al. 2006).

It has been suggested that methylation of RARb2 may block or interfere with the retinoid signalingpathways in NPC. Studies performed in primary NPC have reported high frequency of promoterhypermethylation for RARb2, varying from 81 % to 88 % (Kwong et al. 2002; Fendri et al. 2009).Hypermethylation of the RARb2 promoter has been correlated with lymph node metastasis in NPC(Fendri et al. 2009; Challouf et al. 2012).

The p16INK4a GeneOne of the most important pathways affected in NPC is the INK4a/ARF pathway. The INK4a/ARF locusencodes two cell cycle regulatory proteins, p16INK4a and p14ARF, which share an exon using differentreading frames. p16INK4a and p14ARF act through the Rb-CDK4 and p53 pathways.

p16INK4a is a cyclin-dependant protein kinase 4 (CDK4), which inhibits the ability of CDK4 to interactwith cyclin D1. Loss of p16INK4a function would be expected to lead to uncontrolled cell growth. In NPC,loss of p16INK4a is mainly caused by aberrant promoter methylation, whereas deletion andmutation of thisgene locus are infrequently seen (Lo et al. 1995). CpG island promoter methylation of p16INK4a has beenreported in 22–65 % of primary NPC but not in biopsies of normal nasopharynx (Lo et al. 1996; Ayadiet al. 2008; Challouf et al. 2012). These data suggest that p16INK4a methylation may be a potentialbiomarker for NPC diagnosis. Furthermore, Chang et al. (2003) have observed moderate methylation ratefor p16INK4a (20 %) in body fluids from NPC patients. They suggested that p16INK4a methylationdetection in body fluids may have a potential application in noninvasive screening of NPC.

The p14ARF GeneThe main known function of p14ARF is to prevent the p53 degradation through its binding to MDM-2which induces cell cycle arrest. Compared to p16INK4a methylation, p14ARF promoter methylation hasbeen observed less frequently in NPC (18 %) (Kwong et al. 2002). It has been speculated that epigeneticinactivation of the p14ARF gene may interfere with the p53 network in a subset of NPC tumors. Due to itslow frequency of methylation in tumors, p14ARF needs to be further tested for a potential biomarkerfor NPC.

CpG island Gene

CpG island Gene

CpG island Gene

M M M M

Promoter region

Tumor-suppressor gene canbe expressed

Tumor-suppressor genes isinactivated by mutation ordeletion

Tumor-suppressor gene isinactivated by DNA methylation

Unmethylated CpG Methylated CpG Mutation

Gene expression Gene silencing

M

X

Fig. 4 A mechanism for gene silencing. Different mechanisms by which tumor-suppressor genes could be inactivated in thetumor


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The p15 INK4B GeneThe p15 INK4B gene is also an inhibitor of cyclin-dependent kinase 4, which is an important mediator ofcell cycle control, especially in a pathway stimulated by the transforming growth factor b (TGF b)(Hannon and Beach 1994). The p15 INK4B gene has been reported to be inactivated by promoterhypermethylation in 21 % of primary NPCs (Kwong et al. 2002), which implied that p15INK4B genepromoter methylation may play a role in NPC tumorigenesis.

The Death-Associated Protein Kinase (DAP-Kinase) GeneDeath-associated protein, also known as DAP-kinase, is a Ca2+-/calmodulin-dependent cytoskeletal-associated protein kinase, with an apoptosis-inducing function (Cohen et al. 1997). DAP-kinase expres-sion may affect apoptosis and contributes to immortalization. It is an essential mediator involved in theIFN-g-induced programmed cell death in HeLa cells. DAP-kinase is also involved in tumor necrosisfactor-a (TNF-a)- and Fas-induced apoptosis (Cohen et al. 1999).

Hypermethylation of DAP-kinase promoter has been initially demonstrated in human neoplasm-derived cell lines (Kissil et al. 1997). Subsequently, several types of cancers including NPC have alsoreported a repression in DAP-kinase expression (Kong et al. 2006; Fendri et al. 2009; Kwong et al. 2002;Challouf et al. 2012). Accordingly, high methylation frequencies have been shown in NPC for DAP-kinase ranging from 47% to 88% (Fendri et al. 2009; Challouf et al. 2012).Wong et al. (2002) have foundthat hypermethylation of DAP-kinase promoter occurs in early-stage NPC carcinogenesis. Moreover,Tong et al. (2002) have observed a significant frequency of DAP-kinase promoter methylation (50 %) inbrushing samples from NPC patients. Taken together, these data suggest that DAP-kinase gene methyl-ation may be an ideal biomarker candidate for the early detection of NPC. Similarly, Wong et al. (2002)have found a high frequency of hypermethylatedDAP-kinase promoter in the plasma of NPC patients andsuggested that DAP-kinase may be one of the potentially useful genes in the clinical monitoring ofresidual or recurrent disease after treatment in NPC.

The O6-Methylguanine-DNA Methyltransferase (MGMT ) GeneO6-methylguanine-DNA methyltransferase (MGMT) is an important DNA repair with the highest activityin the liver. MGMT protects cells from DNA damage caused bymutagenic and cytotoxic agents leading toalkylation at O6-guanine (Pegg 1990). Loss or reducedMGMT expression due to CpG island methylationoccurs in several kinds of human cancers (Esteller et al. 1999). In NPC, aberrant promoter methylation forMGMT has been reported with frequencies ranging from 5.5 % to 15 % in primary tumor samples(Challouf et al. 2012; Kwong et al. 2002). Interestingly, Kwong et al. (2002) have reported that MGMTmethylation may be correlated with the aggressiveness of tumor such as the development of metastasis(Kwong et al. 2002). However, in the study performed by Challouf et al. (2012), the authors did not findany significant relations between theMGMTmethylation status and clinical characteristics of NPC. Theseobservations need, however, to be confirmed by a larger study with more NPC patients.

The Glutathione S-Transferase P1 (GSTP1) GeneThe detoxifying glutathione S-transferase P1 (GSTP1) gene protects cells from cytotoxic and carcino-genic agents by conjugation with glutathione (Daniel 1993). Many tumor types including prostate cancer,breast cancers, and cholangiocarcinomas showed GSTP1 promoter hypermethylation (Estelleret al. 1998). In NPC, methylation of GSTP1 gene occurred less frequently in only 19 % of cases(Challouf et al. 2012). Kwong et al. (2002) have not, however, detected methylation ofGSTP1 in primarytumor samples. Based on its low frequency of methylation in tumor tissues, the value of GSTP1 as apotential biomarker for NPC carcinogenesis seems to be limited.


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The High-in-Normal-1 (HIN-1) GeneHigh in normal-1 (HIN-1) is a putative cytokine gene located at 5q35-tel. HIN-1 plays a role in theregulation of epithelial cell proliferation, differentiation, or morphogenesis. The expression of HIN-1 issignificantly downregulated in human breast cancer and its preinvasive lesions. Loss ofHIN-1 expressionis caused by DNA methylation of the promoter region (Krop et al. 2001). Methylation of HIN-1 has beenalso reported in NPC. Wong et al. (2003b) found HIN-1 hypermethylation in all NPC cell lines (100 %)and in 77 % of primary NPC. Accordingly, it seems that transcriptional silencing ofHIN-1 pathway mightbe involved in NPC tumorigenesis.Wong et al. reported also high frequency of methylation in early stages(I and II) of NPC and suggested that methylation of HIN-1 might be an early event in NPC development(Wong et al. 2003). Moreover, they also detected methylated HIN-1 promoter in 46 % (12 of 26) ofnasopharyngeal swabs, 19 % (5 of 26) of throat-rinsing fluids, 18 % (2 of 11) of plasmas, and 46 % (5 of11) of buffy coats of peripheral blood of the NPC patients, but it was not detectable in all normal controls.The authors have suggested that detection of methylated promoter DNA in nasopharyngeal swab, throat-rinsing fluid, and peripheral blood might be potentially useful as tumor marker for early detection and forscreening of NPC.

Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) GeneThe TIMP family has four protein members: TIMP-1, TIMP-2, TIMP-3, and TIMP-4. TIMP-3 is anon-soluble protein that combines with the extracellular matrix. The function of TIMP-3 is the inhibitionof tumor necrosis factor-a (TNF-a)-converting enzymes and the induction of programmed cell deaththrough the stable cell surface TNF-a receptor (Mannello and Gazzanelli 2001). Reduced expression ofTIMP-3 by promoter hypermethylation has been reported in several types of tumors. Furthermore, manyauthors suggest that hypermethylation of TIMP-3 occurs in the early stages and tends to accumulate withthe multistep tumor progression. In NPC, TIMP-3 is less frequently methylated. Indeed, Challoufet al. (2012) found that in 19 % of NPC cases, the TIMP-3 gene 50 CpG islands exhibited aberrantmethylation. Contrary to other cancers, in NPC, promoter hypermethylation of TIMP-3 is only detectablein tumor tissue and not in adjacent non-tumor tissue. As a diagnostic biomarker, the value of TIMP-3maybe limited due to its low frequency of methylation in NPC.

Leucyl-tRNA Synthetase 2, Mitochondrial (LARS2) GeneLeucyl-tRNA synthetase 2, mitochondrial (LARS2) gene is located at 3p21.3. It encodes the precursor ofmitochondrial leucyl-tRNA synthetase which catalyzes the charging of tRNALeu(UUR) with leucine, anessential step in protein synthesis. LARS2 performs essential roles in protein synthesis within themitochondria and is indirectly required for mitochondrial genome maintenance. Promoterhypermethylation of LARS2 has been found in 64 % of NPC samples and in only 12.5 % of non-tumorsamples (chronic nasopharyngitis) (Zhou et al. 2009). This data indicates that DNA methylation in thepromoter region of LARS2 appears to occur at early stages of carcinogenesis and may serve as targets forthe development of a novel screening test for cancer (Zhou et al. 2009).

The Deleted in Liver Cancer-1 (DLC-1) GeneThe deleted in liver cancer-1 (DLC-1) was first isolated from human hepatocellular carcinoma. TheDLC-1gene, located at the human chromosome region 8p22, behaves like a tumor-suppressor gene.DLC-1 shareshigh sequence similarity with Rho family proteins which play essential roles in regulating diversebiological functions, including cytoskeletal organization, cell adhesion, and cell cycle progression(Etienne-Manneville and Hall 2002). The DLC-1 gene has been frequently methylated in diverse tumorsand cell lines (Wong et al. 2003; Kim et al. 2003). Methylation of theDLC-1 gene is also a common eventin NPC (Peng et al. 2006; Seng et al. 2007; Feng et al. 2013). Indeed, 80–89 % of primary NPCs have


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showed methylation in DLC-1 promoter (Peng et al. 2006; Seng et al. 2007), which strongly suggestedthat hypermethylation in the DLC-1 promoter might perform an important role in the transcriptionalsilencing of the gene and NPC carcinogenesis. Moreover, based on its high frequency of methylation intumors, DLC-1 may be a good biomarker candidate for NPC diagnosis.

Tissue Factor Pathway Inhibitor-2 (TFPI-2) GeneTissue factor pathway inhibitor-2 (TFPI-2) is located on chromosome 7q22 and functions as a proteaseinhibitor. TFPI-2 acts in the maintenance of the stability of the tumor environment and inhibits the growthof neoplasms. TFPI-2 is a candidate TSG with important roles in carcinogenesis and metastasis in humancancers. Transcriptional silencing by promoter hypermethylation of TFPI-2 has been observed in somehuman cancers including NPC (Wong et al. 2007; Wang et al. 2010). Using methylation-specific PCR andbisulfate genomic sequencing, Wang et al. (2010) found that TFPI-2was aberrantly methylated in 88.6 %(62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. The authors suggested thatepigenetic inactivation of TFPI-2 by promoter hypermethylation is common and can be a specific markerfor the diagnosis of NPC.

The Tumor Suppressor in Lung Cancer 1 (TSLC1) GeneThe tumor suppressor in lung cancer 1 (TSLC1) is reported to be a putative TSG on 11q23. The TSLC1gene encodes an immunoglobulin superfamily cell adhesion molecule (IgCAM), which is a membraneprotein involved in cell–cell interactions (Masuda et al. 2002). Promoter methylation and loss ofexpression of the TSLC1 gene have been reported in 20–60 % of cancers from the esophagus, stomach,pancreas, nasopharynx, breast, lung, liver, and uterine cervix, as well as meningiomas (Murakami 2005).Moreover, clinicopathological analyses have revealed that the inactivation of TSLC1 is likely to beinvolved in the biological aggressiveness of tumors, including invasion or metastasis (Murakami2005). Loss of TSLC1 gene expression by promoter hypermethylation has been also found in34.2–68 % of primary NPC (Hui et al. 2003; Zhou et al. 2005). No aberrant promoter methylation was,however, found in any of the investigated normal nasopharyngeal epithelia (Hui et al. 2003; Zhouet al. 2005). Lung et al. (2004) reported that the TSLC1 promoter region is hypermethylated in all NPCcell lines and re-expression of the gene occurs in HONE1 cells after 5-aza-20-deoxycytidine treatment.Results from fine deletion mapping in 11q22–23 regions and the high inactivation rate of TSLC1 gavesufficient evidence to consider TSLC1 as a potential biomarker strongly implicated in NPC development(Lung et al. 2004; Hui et al. 2003).

The Human Disabled-2 (DAB2) GeneHuman disabled-2 (DAB2) plays an important regulatory role in cellular differentiation. DAB2 functionsalso as a negative regulator of canonical Wnt signaling by stabilized beta-catenin degradation complex(Hocevar et al. 2003). Downregulation ofDAB2 has been reported in several cancer types. Although somestudies have suggested that DNA methylation of DAB2 is infrequent, Tong et al. (2010) found promotermethylation of DAB2 in 65.2 % (30/46) of primary NPC. Being high methylated, Tong et al. (2010)hypothesized that DAB2 might be a tumor-suppressor gene strongly implicated in NPC carcinogenesis.

The Deleted in Lung and Esophageal Cancer 1 (DLEC1) GeneDeleted in lung and esophageal cancer 1 (DLEC1) is located at 3p22.2.DLEC1 is identified as a candidatetumor-suppressor gene in lung, esophageal, and renal cancers. Downregulation by promoter methylationof DLEC1 has been reported in various types of tumors. In NPC, hypermethylation of the promoter ofDLEC1 has been observed in 71–86 % of cases (Kwong et al. 2007; Ayadi et al. 2008). Methylation ofDLEC1 promoter has been also detected in NPC cell lines and xenografts but not in normal


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nasopharyngeal epithelial cells. Moreover, it has been demonstrated that treatment of NPC cell lines withdemethylating agent or histone deacetylase inhibitor reversed the methylation and restored DLEC1expression. Re-expression of DLEC1 suppressed the growth and reduced the invasiveness of NPCcells. Taken together, these data strongly suggest that silencing of DLEC1 expression by promoterhypermethylation can be a biomarker for NPC diagnosis or prevention.

The BLU GeneThe BLU gene is a candidate tumor-suppressor gene, located at the commonly deleted region 3p21.3. BLUgene is an E2F-regulated, stress-responsive gene (Qiu et al. 2004). BLU gene is inactivated by bothepigenetic and genetic mechanisms in carcinomas (Qiu et al. 2004; Agathanggelou et al. 2003). In NPC,methylation-specific PCR analysis revealed that the BLU promoter was highly methylated in 34–74 % ofprimary NPC (Ayadi et al. 2008; Liu et al. 2003) and in 80 % of nasopharyngeal tumor cell lines(Agathanggelou et al. 2003) but not detected in normal nasopharyngeal tissues. The high incidence ofBLU alterations suggests that it may be one of the critical tumor-suppressor genes on chromosome 3p21.3involved in the development of NPC.

The Epithelial (E)-Cadherin (CDH1) GeneCadherins are a family of transmembrane glycoproteins, which mediates Ca2+-dependent intercellularadhesion. Epithelial (E)-cadherin (CDH1) is an important member of this family, which is expressedpredominantly on the surface of epithelial cells. The CDH1 gene acts as an invasion-/metastasis-suppressor gene (Van Roy and Berx 2008). CDH1 has also a role in organogenesis and morphogenesis(Takeichi 1991). In humans, the CDH1 gene is considered a tumor suppressor and is located onchromosome 16q22.1. CDH1 is frequently inactivated by genetic alterations, such as loss of heterozy-gosity (LOH) and mutations, and also by epigenetic changes (Strathdee 2002). In NPC, hypermethylationof CDH1 was found in 11–79 % of patient samples (Challouf et al. 2012; Ayadi et al. 2008). It isnoteworthy that hypermethylation of CDH1 was more frequently detected in advanced stages comparedto those in early stages of NPC (Niemhom et al. 2008) and was tightly associated with tumor invasionand lymph node metastasis (Li et al. 2004; Ayadi et al. 2008). These data suggested that CDH1methylation might be a useful biomarker to assess progression and for the identification of the aggressiveNPC. Interestingly, several studies have reported a relation between downregulation of CDH1 expressionand the presence of EBV, the principal etiological factor, in NPC (Krishna et al. 2005; Niemhomet al. 2008). Ran et al. (2011) found high methylation frequency of CDH1 in NPC clinical specimens(65 %) and in peripheral blood (45 %) of NPC patients and suggest the potential clinical application ofCDH1 as an early diagnostic or predictive marker.

The Stratifin (SFN/14-3-3 Sigma) GeneThe p53-regulated gene 14-3-3 sigma is a putative tumor-suppressor gene involved in cell cycleregulation and apoptosis following DNA damage. 14-3-3 sigma undergoes frequent epigenetic silencingin several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of14-3-3 sigma expression may be causally involved in tumor progression (Lodygin and Hermeking 2005).Similarly, 14-3-3 sigma promoter methylation occurred at a higher frequency in NPC, 84 % compared toadjacent noncancerous nasopharyngeal epithelial tissue. In addition, Yi et al. (2009) showed a significantcorrelation between DNA methylation of the 14-3-3 sigma gene and advanced clinical stage, lymph nodeinvolvement, and distant metastasis in NPC.


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Summary and Future Perspectives

Aberrant methylation of tumor-related genes is a common event in NPC. Methylation of genes occurs notonly in advanced tumor stages, but it is a frequent and early event. Moreover, the frequency of aberrantpromoter methylation increases during the progression from precancerous lesion to NPC. Promotermethylation of different kinds of tumor-suppressor genes including RASSF1A, RARbb-2, p16, or DAP-kinase has been demonstrated in body fluids from patients with NPC. Therefore, epigenetic changes inpreneoplastic or early neoplastic stages may serve as indicator or “biomarker” for screening of individualswith an increased risk for NPC.

Potential Application to Prognosis and Other Diseases or Conditions

It was demonstrated that re-expression of tumor-suppressor genes that are epigenetically silenced ispossible by using demethylating and histone-modifying agents. In the next years, this might be a possibletherapeutic approach, but the used therapeutic agents that influence DNA hypermethylation are toxic andlead to genome-wide alteration of the methylation pattern with the possibility of activating oncogenes orimprinted genes. Another possible aspect of chemotherapy might be to modulate the epigeneticallyinvolved pathways by using small molecules that are more specific. But further investigations in clinicaltrials are needed to prove and integrate epigenetic pathway-modulating agents.

Summary Points

• Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor with remark-able geographic distribution.

• The undifferentiated carcinoma is the major histopathological type of NPC.• A number of etiological environmental factors, especially the Epstein–Barr virus, along with genetic/

host factors might be responsible for the causation of NPC.• NPC exhibits numerous genetic abnormalities, namely, chromosomal deletions, gene amplifications,

and mutations.• The methylation of DNA is an important epigenetic modification in NPC mediated by three well

definite DNA methyltransferases (DNMT), DNMT1, DNMT3a, and DMNT3b.• NPCs have shown aberrant methylation at CpG islands in a growing list of genes, those belonging to

different pathways in cells.• Promoter methylation of different kinds of tumor-suppressor genes including RASSF1A, p16, and

DAP-kinase has been demonstrated in body fluids from patients with NPC and then may serve asbiomarker for screening of individuals with an increased risk for NPC.

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Documents

DNA Methylation as a Biomarker in Nasopharyngeal Carcinoma … · Histology of NPC Arising in the epithelial lining of the nasopharynx, NPC comprises the vast majority of nasopharyngeal