**1. Introduction**

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

Yang, L., Aozasa, K., Oshimi, K. & Takada, K. (2004). Epstein-Barr virus (EBV)-encoded

Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira,

Yoshizaki, T., Endo, K., Ren, Q., Wakisaka, N., Murono, S., Kondo, S., Sato, H. & Furukawa,

nasopharyngeal carcinoma. *Auris Nasus Larynx*, Vol. 34, No. 1, pp. 73-78

*Cancer Res*, Vol. 64, No. 15, pp. 5332-5337

7, pp. 730-737

RNA promotes growth of EBV-infected T cells through interleukin-9 induction.

K., Akira, S. & Fujita, T. (2004). The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. *Nat Immunol*, Vol. 5, No.

M. (2007). Oncogenic role of Epstein-Barr virus-encoded small RNAs (EBERs) in

Approximately 15% of all cancers worldwide appear to be associated with viral infections, and several human DNA viruses are now accepted as causative factors of specific malignancies. Human papillomaviruses (HPVs) cause cervical and anogenital cancers (zur Hausen 1999) and is now associated with oral cancers (Gillison & Shah 2001), but, the natural history of oncogenic HPV infections in the oral cavity is poorly understood. Epstein-Barr virus (EBV) causes infectious mononucleosis and is closely associated with Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), and Hodgkin's disease (Raab-Traub 1996).

NPC is a malignant tumour that originates within the post nasal space (Pathmanathan et al. 1995). The etiologic factors of endemic NPC include environmental risk factors, genetic susceptibility and viral infection (Yu 1991). Evidence of EBV DNA in almost all NPC cells that were studied supports the association of NPC with EBV, while, HPV has been detected in a variety of head and neck tumours including NPC. Current data suggest that approximately 15–20% of head and neck squamous cell carcinomas (HNSCC) are linked to HPV infection. To date, different degrees of associations between HPV and NPC have been described, yet no conclusive data have been obtained. Given the particular characteristics of NPC in the Moroccan population in terms of incidence, age distribution and the predominance of specific EBV strains, and HPV genotype we describe in this chapter the role of the Epstein-Barr virus ZEBRA protein and HPV in the carcinogenesis of NPC.

#### **1.1 Nasopharyngeal carcinoma**

NPC is a malignancy of the head and neck region that arises in the epithelium surface of the posterior nasopharynx, and shows a peculiar geographic and ethnic distribution. The

Role of the Epstein-Barr Virus ZEBRA Protein

cell cycle progression and immunomodulation.

**2.2 ZEBRA structure** 

(Busch & Sassone-Corsi 1990).

**2.1 ZEBRA on the scene** 

and HPV in the Carcinogenesis of Nasopharyngeal Carcinoma 45

genes, BZLF1 and BRLF1, whose protein products (Zta and Rta) function as transcriptional transactivators and induce the lytic cascade of viral gene expression (Flemington & Speck 1990). Interestingly, a variety of important proteins encoded by EBV show the homology of sequences and functions to diverse human cellular proteins. Furthermore, the EBV proteins

In cancer cells, EBV is also present in a latent state. During latency, EBV is effectively hidden from the immune system but if viral replication is initiated and lytic replication ensues, the cells express EBV genes that are more readily recognized by the immune system. The lytic DNA replication of EBV requires many viral proteins, including ZEBRA, polymerase, polymerase processivity factor, single-stranded DNA binding protein, primase, helicase and primase-associated factor. Among them ZEBRA (also called BZLF1, EB1 and Zta) is a lytic switch transactivator for expression of many early lytic genes and plays a critical role in both viral gene transcription and viral replication (Fixman, Hayward & Hayward 1995). Of all the viral transactivators, ZEBRA is unique in initiation of the ordered cascade of EBV gene expression, resulting in the expression of an estimated over 100 viral replication associated genes including those encoding early antigens, viral capsid antigens and membrane antigens (Baer et al. 1984). Many target genes of ZEBRA, such as BZLF1, BRLF1 and BMLF1 encoding the transactivators, BHRF1 and BHLF1 encoding the viral homologues of Bcl-2 (Marshall et al. 1999), and BMRF1 encoding EBV DNA polymerase accessory protein (Zhang et al. 1997), have been identified in the EBV genome. Through binding to cisacting AP-1 or ZEBRA responsive elements (ZREs) in lytic cycle promoters ZEBRA activates the transcription of the target genes (Lieberman et al. 1990). Recently, some cellular genes modulated by ZEBRA have also been revealed. The products of these cellular genes are fundamentally linked to the viral life cycle, virus-host interactions, hosT-cell environment,

ZEBRA or Zta is a member of the family of bZIP transcription factors (Sinclair 2003); it contains adjacent DNA contact (approximately amino acids 175 to 195) and multimerization domains (approximately amino acids 196 to 245) (figure 1) (Sinclair 2006) and can interact directly with specific DNA sequence elements, i.e., ZREs as a multimer . By analogy with other members of the bZIP family, the multimerization interface of ZEBRA has been predicted to fold through a coiled-coil structure (Sinclair & Farrell 1992). Biophysical

The DNA binding region and dimerization region partly conform to the well-characterized bZIP (basic/leucine zipper) domain that is found in a family of cellular transcription factors such as fos/jun, C/EBPa and GCN4. Interestingly, ZEBRA recognizes a wider range of DNA binding sites than other bZIP members. bZIP proteins are homo- or heterodimers that contain highly basic DNA binding regions adjacent to regions of a-helix that fold together as coiled coils (Sinclair 2006); the interaction with DNA is dependent on dimer formation

evidence that this prediction holds true was recently provided (Hicks et al. 2001).

can modulate the expressions of a large number of cellular proteins.

highest incidence rates of NPC are found among the southern Chinese population and in isolated northern populations such as Eskimos and Greenlanders (30 to 80 cases per 100,000 per year) (Parkin & Muir 1992). Intermediate incidence (8 to 12 cases per 100,000 per year) was reported in the Mediterranean basin, especially among the Arabic populations of North Africa (7-10% of all cancers among men), where NPC is also the commonest tumour of the ear, nose and throat region (Benider et al. 1995). The aetiology of NPC seems to be multifactorial with evidence that genetic susceptibility, environmental factors and viral infection with EBV reactivation and HPV infection are involved together or separately, simultaneously or consecutively (Hildesheim & Levine 1993).

The increased risk of NPC in North African population was associated with the consumption of rancid butter and rancid sheep fat. In fact, higher level of N-nitrosamines in rancid fat has not been demonstrated, which suggests some other disease causing chemicals in this population. A possible compound is butyric acid, which is also named n-Butanoic Acid. The glyceride form of butyric acid makes up 3 to 4% of butter, and is released into free butyric acid by hydrolysis when it becomes rancid (Feng et al. 2007). Butyric acid is known to be able to activate EBV in the B-lymphoid cells into lyric cycle (Takimoto et al. 1984), and therefore, could be related to NPC. In addition, Marijuana smoking was associated significantly to high NPC risk independently of cigarette smoking which suggests dissimilar carcinogenic mechanisms between cannabis and tobacco.

Genetic traits play a significant role in the development of NPC. Specific human leukocyte antigen (HLA) haplotypes have been reported to be associated with high risk for NPC, namely HLA-B13 in Tunisians, HLA-A3, B5 and B15 in Algerians and HLA-B18 allele in Moroccans population. In contrast, HLA-Aw33, -B14 and A9 were associated to low risk of NPC in Tunisians, Algerians and Moroccans, respectively.

Retrospectives and prospectives epidemiologic studies have indicated that assocaition between EBV, an ubiquitous human herpesvirus, and the development of different malignancies, such as Burkitt's lymphoma, 40%–50% of Hodgkin's disease, B-cell lymphoma in immunocompromised individuals, and NPC (Rickinson 2002). Undifferentiated NPC is one of the most striking examples of human malignancies that have been found strongly associated with the EBV, and interest in HPV as a cofactor in NPC occurrences has emerged over the last few years (Punwaney et al. 1999).
