Dolly P. Huang

Focus on nasopharyngeal carcinoma.

Support from the Hong Kong Research Grant Council (CUHK 4301/99M; 4071/02M; 4067/02M; 410/03M; and HKUST 2/03C) and the Kadoorie Charitable Foundation is gratefully acknowledged.

Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus

We have established a cell line (C666‐1) from undifferentiated nasopharyngeal carcinoma (NPC). This cell line consistently carries the Epstein‐Barr virus (EBV) in long‐term cultures. C666‐1 is a subclone of its parental cell line, C666, derived from an NPC xenograft of southern Chinese origin. It grows as an adherent culture and lacks contact inhibition. In addition, it is tumorigenic in athymic nude mice. The cells consistently express EBV‐encoded RNAs and are positively stained for cytokeratin, an epithelial marker. In addition, they express EBNA1 protein, LMP1 and LMP2 transcripts and thus resemble the EBV latency II pattern. The virus genotype is EBV‐1 with the latent membrane protein 1 gene showing a 30‐bp deletion at the carboxyl terminus, both consistent with findings in southern Chinese NPC tumours. Cytogenetic analysis revealed a sub‐diploid status with a chromosomal modal number of 45. C666‐1 is unique among NPC cell lines in that it carries EBV. These cells may serve as a good investigative tool as the viral latency pattern and genotype are observed in the majority of primary NPC biopsies from Chinese patients. Int. J. Cancer 83:121–126, 1999.

Genetic and epigenetic changes in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a malignancy with remarkable racial and geographic distribution. The development of this EBV-associated cancer likely involves cumulative genetic and epigenetic changes in a background of predisposed genetic and environmental factors. Genome-wide studies have unravelled multiple chromosomal abnormalities with involvement of specific oncogenes and tumour suppressor genes. Alterations of genes such as Ras association domain family 1A (RASSF1A), p16/INK4A, p14/ARF suggest that multiple cellular pathways were dysregulated in the NPC cells. Studies on the precancerous lesions revealed early genetic changes and a critical role of EBV latent infection in the development of this cancer. Based on the existing findings, a pathogenetic model for NPC is proposed.

The significance of LMP1 expression in nasopharyngeal carcinoma

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a key effector of EBV-mediated B cell transformation. LMP1 displays potent oncogenic properties in rodent fibroblasts, and induces a wide range of effects in B cells and epithelial cells. LMP1 functions as a constitutively active tumor necrosis factor receptor (TNFR) engaging a multitude of signaling pathways that include NF-kappaB, the mitogen-activated protein kinases (MAPKs), JNK, p38, the JAK/STAT pathway and, more recently, the small Rho GTPases. The constitutive activation of these signaling cascades explains LMP1s ability to induce such a diverse array of morphological and phenotypic effects in cells and provides an insight into how LMP1 may induce cell transformation. The frequent expression of LMP1 in undifferentiated nasopharyngeal carcinoma (NPC) points to a role for this viral oncoprotein as a key effector molecule in NPC pathogenesis.

Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein-Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFbeta inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are non-tumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC.

CTL Control of EBV in Nasopharyngeal Carcinoma (NPC): EBV-Specific CTL Responses in the Blood and Tumors of NPC Patients and the Antigen-Processing Function of the Tumor Cells

Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.

Detection of recurrent chromosomal gains and losses in primary nasopharyngeal carcinoma by comparative genomic hybridisation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China but rare in Western countries. To search for genetic alterations in NPC, we examined a series of 20 primary tumours with comparative genomic hybridisation. The identified common chromosomal alterations included gain of chromosomes 1q, 8, 12, 19 and 20 as well as loss of chromosomes 1p, 3p, 9p, 9q, 11q, 13q, 14q and 16q. In concordance with our previous loss of heterozygosity studies in primary NPC, a high incidence of loss was detected on chromosomes 3p (75%), 11q (70%) and 14q (65%). Losses of 9q (60%), 13q (50%) and 16q (40%) were also identified. Novel chromosomal gains were observed on chromosome 12, with a high frequency (70%). Current analysis has revealed a comprehensive profile of the chromosomal regions showing losses and gains in primary NPC. Our findings may provide an entry point for conducting further investigations to locate the putative tumour‐suppresser genes and oncogenes that may be involved in the tumourigenesis of NPC. Int. J. Cancer 82:498–503, 1999.

RASSF1A is a target tumor suppressor from 3p21.3 in nasopharyngeal carcinoma

Deletion on the short arm of chromosome 3 is one of the most important genetic abnormalities in the tumorigenesis of nasopharyngeal carcinoma (NPC). Both physical mapping and functional studies have targeted an NPC‐related tumor suppressor gene(s) to chromosome 3p21.3. We have reported recently that RASSF1A gene, located on a 120‐kb minimal deletion region on 3p21.3, was frequently inactivated by promoter hypermethylation in NPC. We further confirmed that RASSF1A is the critical target tumor suppressor from 3p21.3, with the evidence that loss of expression and aberrant methylation of the other 8 candidate genes/transcripts (HYAL2, FUS1, RASSF1C, BLU, NPRL2, 101F6, PL6 and CACNA2D2) in this 120‐kb region were rare in NPC samples. The contribution of RASSF1A in NPC tumorigenesis was investigated by restoring its expression in a RASSF1A deficient cell line, C666‐1. Transient transfection of wild‐type RASSF1A resulted in marked growth inhibition in NPC cells. Isolated stable clones expressing wild‐type RASSF1A demonstrated retarded cell proliferation in vitro. Soft‐agar assay also showed decreased number and sizes of colony formed in these clones. In vivo nude mice assay demonstrated the dramatic reduction of tumorigenic potential in the RASSF1A‐transfected clones. Our results provide strong evidence to support RASSF1A as a target tumor suppressor gene on 3p21.3 in NPC.

Loss of heterozygosity on the short arm of chromosome 3 in nasopharyngeal carcinoma

A consistent loss of constitutional heterozygosity within a specific chromosome locus in a tumor type is suggestive of a tumor suppressor gene important in the genesis of that tumor. We studied whether such genetic alterations are involved, in the development of nasopharyngeal carcinoma (NPC). Tumor and matched blood leukocytes DNA from eleven Hong Kong Chinese patients with primary NPC stages I to IV were subjected to restriction fragment length polymorphism (RFLP) analysis using chromosome 3-specific polymorphic probes. Such probes are assigned to chromosomal region 3p25 (RAF-1), 3p24-22.1 (ERBA beta), 3p21 (DNF15S2), 3p14 (D3S3), and 3q12 (D3S1). The breakpoint varied among tumors, ranging in extent from 3p21-14. However, 100% frequency of complete loss of heterozygosity was observed at two chromosomal loci: RAF-1 locus (ten of ten cases at 3p25) and D3S3 locus (nine of nine cases at 3p14), in all evaluable NPC patients, suggesting the presence of putative tumor suppressor gene(s) within or close to these defined regions. The observed consistent deletion of alleles on the short arm of chromosome 3 in the NPC cases, which is in line with our previously reported and present cytogenetic findings, may represent a critical event in the multistep genesis of NPC. The present report also identifies defined loci for linkage studies on NPC families.

Epstein–Barr virus-encoded small RNA induces insulin-like growth factor 1 and supports growth of nasopharyngeal carcinoma-derived cell lines

To assess the role of insulin-like growth factor 1 (IGF-1) in the growth of nasopharyngeal carcinoma (NPC), three NPC-derived cell lines, C666-1, CNE1 and HONE1, were examined. C666-1 cells maintained NPC phenotype of Epstein–Barr virus (EBV) expression and were positive for IGF-1 secretion, and their growth was strikingly inhibited by treatment with an anti-IGF-1 antibody under low serum condition. On the other hand, CNE1 and HONE1 cells were EBV-negative and did not secrete IGF-1. Although they could not grow under low serum condition, addition of recombinant IGF-1 made them grow. EBV conversion of CNE1 and HONE1 cells reproduced NPC phenotype of EBV expression and accompanied IGF-1 expression. Although they could grow under low serum condition, their growth was strikingly inhibited by treatment with the anti-IGF-1 antibody. These results suggest that EBV infection induces IGF-1 in NPC cell lines, and that the secreted IGF-1 acts as an autocrine growth factor. These findings seem to be operative in vivo, as NPC biopsies consistently express IGF-1. Further studies demonstrated that increased IGF-1 expression reflected transcriptional activation, and EBV-encoded small RNA (EBER) was responsible for IGF-1 induction. EBER is invariably expressed in EBV-associated malignancies, including NPC. The present findings strongly suggest that EBER directly affects the pathogenesis of NPC.