**4. Epigenetic changes in nasopharyngeal carcinoma**

Nasopharyngeal carcinoma (NPC) is rare in most part of the world but prevalent in southern China, including Guangdong and Hong Kong, and Southeast Asia, with an incidence rate of 20 to 30 per 100 000 people/year (Li et al, 2011). The unique ethnic and geographic distribution of NPC indicates its unusual etiology. Three major etiologic factors, genetic, environmental, and viral factors – Epstein-Barr virus (EBV), have been identified to lead to multiple genetic and epigenetic alterations during NPC pathogenesis by either acting alone or in synergy.

EBV is a prototype of gamma herpes virus which infects >90% of the world adult population (Liebowitz, 1994). Humans are the only natural host for EBV. Primary infection with EBV normally occurs in early childhood and is usually asymptomatic in most underdeveloped countries. But when the exposure to EBV is delayed until adolescence it occasionally presents as mononucleosis. Long-term EBV coexists with most human hosts without overt serious consequences. However, in some individuals, the virus is implicated in the development of malignancy. EBV has a strong tropism for human lymphocytes and for epithelium of the upper respiratory tract (Young & Rickinson, 2004). EBV was the first human virus identified to be associated with human lymphomas as well as epithelial tumors, such as post-transplant lymphoma, AIDS-associated lymphomas, Burkitt lymphoma, Hodgkin's disease, T-cell lymphoma, nasopharyngeal carcinoma, parotid gland carcinoma, and gastric carcinoma. (Murray & Young, 2002)

Tumorigenesis of nasopharyngeal carcinoma is a multistep process. EBV may play an important role in the progression of NPC, involving activation of oncogenes and/or the inactivation of tumor suppressor genes (TSGs). Early genetic changes may predispose the epithelial cells to EBV infection or persistent maintenance of latent cycle.

Nasopharyngeal carcinoma also distinguishes itself from other tumors by the number of genes targeted for silencing by promoter methylation. The key tumor suppressor genes like p53 or Rb which are found to be mutated in 50% of all the tumors were rarely found to be mutated in nasopharyngeal carcinoma (Wensing & Farrel, 2000). On the contrary, hypermethylation of known or candidate tumor suppressor genes involved in various

Epigenetics in Head and Neck Squamous Cell Carcinoma 185

pool, and to differential expression of the enzymes involved in chondroitin/dermatan

The purpose of Kalathas and colab. (2010) was to examine the expression of the various chondroitin/dermatan sulfate synthesizing and modifying enzymes in laryngeal cartilage in healthy, macroscopically normal and cancerous specimens by RT-PCR analysis and western blotting. Furthermore, methylation specific PCR (MSP) was used to find out if DNA methylation is a regulative mechanism of their expression in laryngeal cancer. C4ST1 gene expression was very low in healthy specimens (20 times lower compared to the GAPDH gene), and increased in patients' specimens as indicated by both RT-PCR and western blotting. Its expression seemed to be controlled via methylation of a CpG island, since hypomethylation of the gene was observed in the pathologic samples compared to the macroscopically normal samples. D4ST1 gene was about equally expressed with the GAPDH gene and possessed its highest expression in the healthy tissues. In cancer, its expression was decreased 4 to 5 times and it was about equal between normal and pathologic samples. The CpG island near the promoter region was fully unmethylated therefore it did not affect the enzyme expression. DSE expression was not detected in the macroscopically normal samples, and the highest levels of it were observed in the pathologic samples, as indicated by RT-PCR, being about 10-times more compared to healthy. DSE expression seemed to be controlled by methylation of the promoter region in certain samples; the pathologic samples were hypomethylated compared to the macroscopically normal. The differential modification of the various glycosaminoglycans during cancer reflected differential expression of the enzymes involved in their biosynthesis. In their study, the clearest observations in laryngeal cancer were the significant decrease of CHSY3, CHST3 and D4ST1, and the significant increase of DSE. DSE is responsible for the epimerization of glucuronic acid in dermatan sulfate chains, which in addition require D4ST1 for their sulfation. The differential expression of only these two enzymes, which are highly responsible for the biosynthesis of dermatan sulfate, a glycosaminoglycan with tumor-inhibitory activity, indicates that a simple imbalance in enzymes' expression may

Different environmental factors are able to modulate the epigenetic information. It is widely accepted that the aberrant DNA methylation changes can be induced by both cigarette smoke and alcohol. Alcohol consumption and smoking induced the hypermethylation of p15 in the upper respiratory tract cells (Chang et al, 2004), while the hypermethylation of MGMT, p16 and DAPK was associated with tobacco-chewing induced oral cancers (Vuillemont et al, 2004). A correlation was also found between cigarette smoking and the hypermethylation of CDH1, RARbeta and FHIT (Van Engeland et al, 2003). Alcohol consumption was associated with the methylation of APC, p14, p16, MGMT, RASSF1A and hMLH1 (Van Engeland et al, 2003). Since alcohol and tobacco are the major risk factors for the development of laryngeal squamous cell carcinomas (LSCC), one can predict the

Since Slaughter's proposal of a genetic field defect concept for the explanation of the local relapse occurrence, much evidence has accumulated for its confirmation. The last ten years brought evidence that the genetic changes in the field are frequently accompanied by epigenetic aberrations. The epigenetic field defect was observed for oesophageal (Oka et al, 2009), lung (Guo et al, 2004) or stomach (Ushijima, 2007) cancers. Similarly, epigenetic

frequent occurrence of the methylome aberrations in LSCC patients.

sulfate biosynthesis.

affect tumor progression.

fundamental pathways has been reported in NPC, such as apoptosis, DNA damage repair, tumor invasion and metastasis. Many TSGs were aberrantly methylated in their 5'CpG islands: 84% of the RASSF1A, 80% of the RARβ2, 76% of the DAP-kinase, 46% of the p16, 89,7% of the CDH13, 65% of the CHFR, 50,9% of the RASSF2A (Li et al, 2011). DNA methylation may play an important role in the maintenance of specific EBV latency programmes in the nasopharyngeal carcinoma cells. Methylation of both viral and cellular genes may be involved in the transformation of nasopharyngeal epithelial cells. Induction of epigenetic alterations in certain cellular genes was proposed as one of the mechanisms for enhancing the transformation of nasopharyngeal epithelial cells by EBV infection.

Loss of heterozygosity at 3p21 is common in various cancers including nasopharyngeal carcinoma (NPC). BLU is one of the candidate tumor suppressor genes (TSGs) in this region. Ectopic expression of BLU results in the inhibition of colony formation of cancer cells, suggesting that BLU is a tumor suppressor. Qiu and colab. (2004) identified a functional BLU promoter and found that it can be activated by environmental stresses such as heat shock, and is regulated by E2F. The promoter and first exon are located within a CpG island. BLU is highly expressed in testis and normal upper respiratory tract tissues including nasopharynx. However, in all seven NPC cell lines examined, BLU expression was downregulated and inversely correlated with promoter hypermethylation. Biallelic epigenetic inactivation of BLU was also observed in three cell lines. Hypermethylation was further detected in 19/29 (66%) of primary NPC tumors, but not in normal nasopharyngeal tissues. Treatment of NPC cell lines with 5-aza-20-deoxycytidine activated BLU expression along with promoter demethylation. Although hypermethylation of RASSF1A, another TSG located immediately downstream of BLU, was detected in 20/27 (74%) of NPC tumors, no correlation between the hypermethylation of these two TSGs was observed (Qiu et al, 2004).

Aberrant "epigenetic code" of cell signaling facilitates the subsequent selection of genetic mutations of certain signaling pathways in the initiation and progression of NPC. As more epigenetic alterations of cell signaling genes are found, we will obtain systematic understanding of the molecular features of NPC. Study of epigenetically silenced cell signaling regulators in NPC will lead to the further development of clinical strategies of NPC prevention and therapy. Moreover, promoter methylation of cell signaling regulators could serve as diagnostic biomarkers for NPC risk assessment, early detection, and prognosis (Li et al, 2011).
