Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. Aberrant hypermethylation in the promoter region of some known or putative tumor suppressor genes occurs frequently during the development of various types of cancer including head and neck squamous cell carcinoma (HNSCC). In this study we used an expanded mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes in HNSCC. Two HNSCC cell lines were treated with 5-aza-2'-deoxycytidine followed by microarray analysis to identify epigenetically silenced genes in HNSCC. We found 1,960, 614 and 427 genes were upregulated in the HNSCC cell lines JHU-012, JHU-011 and the combination of both cell lines, respectively. HNSCC tumor and normal mucosal samples were used for gene profiling by a 47K mRNA gene expression array and we found 7,140 genes were downregulated in HNSCC tumors compared to normal mucosa, as determined by microarray analysis, and were integrated with cell line data. Integrative analysis defined 126 candidate genes, of which only seven genes showed differential methylation in tumors and no methylation in normal mucosa after bisulfite sequencing. Following validation by QMSP, one gene, guanine nucleotide-binding protein γ-7 (GNG7), was confirmed to be highly methylated in tumors and unmethylated in normal mucosal and salivary rinse samples demonstrating cancer-specific methylation in HNSCC tissues. TXNIP and TUSC2 were partially methylated in tumors and normal salivary rinses but unmethylated in normal mucosa. We concluded that GNG7 is a highly specific promoter methylated gene associated with HNSCC. In addition, TXNIP and TUSC2 are also potential biomarkers for HNSCC.

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.

Gamma-glutamyltransferase,glutathione,molecular subgroups of breast cancer

Oral squamous cell carcinoma (OSCC) remains a major global health burden, often diagnosed at advanced stages, with limited survival improvement. Epigenetic dysregulation, particularly promoter hypermethylation, plays a significant role in OSCC pathogenesis. Previous study by Demokan et al. identified Gamma-Aminobutyric Acid Receptor Beta-2 (GABRB2) as a candidate gene subject to methylation-dependent transcriptional silencing. This study aimed to evaluate the methylation and expression profiles of GABRB2 in OSCC and to explore its potential as a diagnostic biomarker. Tissue and serum samples from 48 OSCC patients and 15 healthy controls were analyzed for GABRB2 promoter methylation via quantitative methylation-specific PCR and for gene expression levels via quantitative real-time PCR. Relationships between methylation, expression levels, clinicopathological parameters, and patient outcomes were statistically assessed. GABRB2 promoter hypermethylation was detected in 14.6% of tumor samples, predominantly in floor of the mouth and buccal tumors. Decreased GABRB2 expression was observed in 52.1% of tumor tissues compared to matched-normal tissues, while 22.9% of tumors exhibited increased expression. Methylation-dependent expression loss was confirmed in 71.4% of methylated tumors. Notably, decreased expression and hypermethylation of GABRB2 were correlated with poor prognosis parameters (p < 0.05). ROC analysis showed moderate discrimination for GABRB2 expression in distinguishing tumor from normal tissues (AUC = 0.635, p = 0.022), while serum-based analysis demonstrated poor diagnostic performance. Survival analysis revealed no statistically significant prognostic impact of GABRB2 expression levels (p > 0.05). However, alcohol consumption (p = 0.006) and recurrence developed (p = 0.030) as independent predictors of poor prognosis in multivariate analysis. Our study suggests that there was association between methylation-based expression loss of GABRB2 with OSCC’ subgroups. To our knowledge, this is the first study to investigate GABRB2 gene methylation and expression profiling in both invasive/non-invasive samples from OSCC patients. Hypermethylation in the newly identified candidate GABRB2 gene may play a role in the development of tumors originating from the mouth floor and buccal anatomical regions of the oral cavity. However, since the loss of expression seen in the majority of our patients, we thought that only methylation may not the inhibition mechanism for GABRB2 gene decreased expression. The new identified candidate gene may be specific for the OSCC’s subgroups.