RESUMO
Nasopharyngeal carcinoma (NPC), a cancer that is etiologically associated with the Epstein-Barr virus (EBV), is endemic in Southern China and Southeast Asia. The scarcity of representative NPC cell lines owing to the frequent loss of EBV episomes following prolonged propagation and compromised authenticity of previous models underscores the critical need for new EBV-positive NPC models. Herein, we describe the establishment of a new EBV-positive NPC cell line, designated NPC268 from a primary non-keratinizing, differentiated NPC tissue. NPC268 can undergo productive lytic reactivation of EBV and is highly tumorigenic in immunodeficient mice. Whole-genome sequencing revealed close similarities with the tissue of origin, including large chromosomal rearrangements, while whole-genome bisulfite sequencing and RNA sequencing demonstrated a hypomethylated genome and enrichment in immune-related pathways, respectively. Drug screening of NPC268 together with six other NPC cell lines using 339 compounds, representing the largest high-throughput drug testing in NPC, revealed biomarkers associated with specific drug classes. NPC268 represents the first and only available EBV-positive non-keratinizing differentiated NPC model, and extensive genomic, methylomic, transcriptomic, and drug response data should facilitate research in EBV and NPC, where current models are limited. SIGNIFICANCE: NPC268 is the first and only EBV-positive cell line derived from a primary non-keratinizing, differentiated nasopharyngeal carcinoma, an understudied but important subtype in Southeast Asian countries. This model adds to the limited number of authentic EBV-positive lines globally that will facilitate mechanistic studies and drug development for NPC.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Animais , Camundongos , Carcinoma Nasofaríngeo/genética , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/genética , Infecções por Vírus Epstein-Barr/complicações , Linhagem Celular TumoralRESUMO
A micro-array analysis using biopsies from patients with EBV-positive undifferentiated nasopharyngeal carcinoma (NPC) and from cancer-free controls revealed down-regulation of tumour suppressor genes (TSG) not previously associated with this disease; one such gene was the ataxia telangiectasia mutated (ATM) gene. Q-PCR confirmed down-regulation of ATM mRNA and ATM protein expression in tumour cells was weak or absent in almost all cases. In NPC cell lines, however, ATM was down-regulated only in the EBV-positive line, C666.1, and in none of five EBV-negative lines. In vitro infection of EBV-negative NPC cell lines with a recombinant EBV was followed by the down-regulation of ATM mRNA and protein, and only EBV-positive cells showed a defective DNA damage response following gamma-irradiation. Our data suggest that loss of ATM function could be an important step in the pathogenesis of NPC, and may have implications for the treatment of this disease.
Assuntos
Adenocarcinoma/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Neoplasias Nasofaríngeas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/virologia , Proteínas Mutadas de Ataxia Telangiectasia , Estudos de Casos e Controles , Proteínas de Ciclo Celular/análise , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/análise , Perfilação da Expressão Gênica/métodos , Herpesvirus Humano 4 , Humanos , Imuno-Histoquímica , Neoplasias Nasofaríngeas/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/análise , Infecções Tumorais por Vírus/complicações , Infecções Tumorais por Vírus/genéticaRESUMO
BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr Virus (EBV)-associated cancer that is the fifth most common cancer in Malaysia. Early and accurate diagnoses are critical for patient prognosis. Unfortunately, early detection of NPC is still a challenge and the cost of more accurate imaging protocols is prohibitive in developing countries like Malaysia. OBJECTIVES: To evaluate the clinical values of pre-treatment plasma EBV DNA levels in Malaysian NPC patients. STUDY DESIGN: Plasma EBV DNA levels were measured by quantitative PCR (Q-PCR) in a large and multi-ethnic cohort of Malaysian patients with NPC (n=459) and 72 control subjects. RESULTS: We show for the first time that, compared to controls, NPC patients with stage I disease had significantly higher levels of EBV DNA (p<0.001). Further, the median level of plasma EBV DNA in stage IV patients with distant metastasis was >9-fold higher than those without systemic spread (p=0.001), suggesting plasma EBV DNA measurement could aid in the diagnosis of metastatic disease in advanced cases. Further, using a cut-off value of 8000 copies/mL, we demonstrate that EBV DNA level is a strong predictor for overall survival of NPC patients. CONCLUSIONS: Our data show that pre-treatment plasma EBV DNA is a potential biomarker for early stage and metastatic NPC. We conclude that the quantification of plasma EBV DNA is a useful tool in developing countries to stratify patients for MRI or PET/CT scans where such imaging protocol is not routinely applied.