Inactivation of RASSF1A, RARbeta2 and DAP-kinase by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma.
Epigenetic modification is one of the mechanisms leading to gene silencing in neoplastic cells. By methylation-specific PCR, we analyzed the promoter methylation of three cancer-related genes: Ras Association domain Family 1A (RASSF1A), Death Associated Protein kinase (DAP-kinase) and Retinoic Acid Receptor beta2 (RARbeta2) in two NPC xenografts (C15 and C17), 68 primary NPC tumors, and nine normal nasopharyngeal epithelia. We showed that C15 and C17 displayed a complete promoter methylation of RASSF1A, RARbeta2 and DAP-kinase genes. In primary NPC tumors, the incidence of promoter methylation was very high for all three tested genes: 91% for RASSF1A, 88% for both RARbeta2 and DAP-kinase whereas all normal nasopharyngeal epithelia were unmethylated. Interestingly, our study revealed that aberrant promoter methylation of the three genes were statistically associated with the lymph node involvement (p < 0.0001). In addition, hypermethylation of RASSF1A was correlated with age at diagnosis (p = 0.047) and T stage (p = 0.037) while the RARbeta2 hypermethylation was associated with histological type (p = 0.011). Taken together, our results demonstrate that silencing of RASSF1A and RARbeta2 expression by promoter hypermethylation is associated with highly differentiated tumors, advanced tumor stage and the presence of lymph node metastasis. To assess the functional significance of the epigenetic silencing of RARbeta2 and DAP-kinase in NPC, we analysed the expression of two downstream target genes COX-2 and p53 by reverse PCR (RT-PCR) and immunohistochemistry (IHC). We revealed a significant association between expression of COX-2 and loss of RARbeta2 through aberrant methylation (p = 0.003) in NPC biopsies. We concluded that the inactivation of RASSF1A, RARbeta2 and DAP-Kinase by hypermethylation is a key step in NPC tumorigenesis and progression.
Fendri
Ali
A
Unité de Recherche Génétique du Cancer et Production de protéines thérapeutiques, Centre de Biotechnologie de Sfax, Sfax, Tunisia.
Masmoudi
Asma
A
Khabir
Abdelmajid
A
Sellami-Boudawara
Tahia
T
Daoud
Jamel
J
Frikha
Mounir
M
Ghorbel
Abdelmonem
A
Gargouri
Ali
A
Mokdad-Gargouri
Raja
R
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D002648:Child; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D064550:Death-Associated Protein Kinases; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008207:Lymphatic Metastasis; D008297:Male; D008875:Middle Aged; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D018168:Receptors, Retinoic Acid / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D055815:Young Adult
D051017:Apoptosis Regulatory Proteins; C411762:RASSF1 protein, human; D018168:Receptors, Retinoic Acid; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C540966:retinoic acid receptor, beta2, human; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases