title: Validation of nucleolar protein 4 as a novel methylated tumor suppressor gene in head and neck cancer.
abstract: Methylation of CpG islands in the promoter region of genes acts as a significant mechanism of epigenetic gene silencing in head and neck cancer. In the present study, we assessed the association of epigenetic alterations of a panel of 12 genes [nucleolar protein 4 (NOL4), iroquois homeobox 1 (IRX1), SLC5A8, LRRC3B, FUSSEL18, EBF3, GBX2, HMX2, SEPT9, ALX3, SOCS3 and LHX6] with head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. After the initial screening of methylated CpG islands on the promoter regions by bisulfite sequencing using salivary rinse samples, only two genes had methylated CpG dinucleotides on their promoter regions in tumor samples and absence of methylated CpGs were found in normal salivary rinse samples after bisulfite modification and bisulfite sequencing. We then performed real-time quantitative methylation-specific PCR (QMSP) on 16 salivary rinse and 14 normal mucosal samples from healthy subjects and 33 HNSCC tumor samples for the two genes selected. After validation with QMSP, one gene, NOL4, was highly methylated (91%) in tumor samples and unmethylated in normal salivary rinses and minimally methylated in normal mucosal samples demonstrating cancer-specific methylation in HNSCC tissues. Although the IRX1 gene was observed as methylated in normal mucosal and salivary rinse samples, the methylation values of these normal samples were very low (<10%). In conclusion, we identified NOL4 as a highly specific promoter methylated gene associated with HNSCC. IRX1 may have potential as a biomarker for HNSCC and should be assessed in a larger cohort.
authors: Department of Basic Oncology, Oncology Institute, Istanbul University, Capa, Istanbul 34093, Turkey.
authors: Semra
authors: S
authors: Demokan
authors: Department of Dermatology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
authors: Alice Y
authors: AY
authors: Chuang
authors: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
authors: Kavita M
authors: KM
authors: Pattani
authors: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
authors: David
authors: D
authors: Sidransky
authors: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
authors: Wayne
authors: W
authors: Koch
authors: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
authors: Joseph A
authors: JA
authors: Califano
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D014644:Genetic Variation; D006258:Head and Neck Neoplasms / Q000175:diagnosis / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D018398:Homeodomain Proteins; C501591:IRX1 protein, human; C115809:NOL4 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors