INTERNATIONAL JOURNAL OF ONCOLOGY 42: 1427-1436, 2013 Identification of guanine nucleotide-binding proteinγ -7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling SEMRA DEMOKAN1,5, ALICE Y. CHUANG2, XIAOFEI CHANG1, TANBIR KHAN1, IAN M. SMITH1, KAVITA M. PATTANI1, SANTANU DASGUPTA1,6, SHAHNAZ BEGUM3, ZUBAIR KHAN1, NANETTE J. LIEGEOIS2, WILLIAM H. WESTRA3, DAVID SIDRANSKY1, WAYNE KOCH1 and JOSEPH A. CALIFANO1,4 Departments of 1Otolaryngology-Head and Neck Surgery, 2Dermatology and 3Pathology, Johns Hopkins Medical Institutions, Baltimore, MD; 4Milton J. Dance Head and Neck Center, Greater Baltimore Medical Center, Baltimore, MD, USA; 5Department of Basic Oncology, Oncology Institute, Istanbul University, Capa, Istanbul, Turkey; 6Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA Received November 1, 2012; Accepted December 10, 2012 DOI: 10.3892/ijo.2013.1808 Abstract. Silencing of tumor suppressor genes plays a vital specific methylation in HNSCC tissues. TXNIP and TUSC2 role in head and neck carcinogenesis. Aberrant hypermethyl- were partially methylated in tumors and normal salivary rinses ation in the promoter region of some known or putative tumor but unmethylated in normal mucosa. We concluded that GNG7 suppressor genes occurs frequently during the development is a highly specific promoter methylated gene associated with of various types of cancer including head and neck squamous HNSCC. In addition, TXNIP and TUSC2 are also potential cell carcinoma (HNSCC). In this study we used an expanded biomarkers for HNSCC. mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes Introduction in HNSCC. Two HNSCC cell lines were treated with 5-aza- 2'-deoxycytidine followed by microarray analysis to identify Among human malignancies, head and neck cancer is the sixth epigenetically silenced genes in HNSCC. We found 1,960, most common cancer in the world (1). Head and neck cancer is 614 and 427 genes were upregulated in the HNSCC cell lines an aggressive and life-threatening disease with poor morbidity JHU-012, JHU-011 and the combination of both cell lines, and high mortality in advanced disease. More than 40,000 new respectively. HNSCC tumor and normal mucosal samples cases of head and neck squamous cell carcinoma (HNSCC) were used for gene profiling by a 47K mRNA gene expres- are diagnosed in the United States each year, with 12,000 US sion array and we found 7,140 genes were downregulated in deaths annually. Survival rates have not improved significantly HNSCC tumors compared to normal mucosa, as determined for patients with HNSCC in the past thirty years despite active by microarray analysis, and were integrated with cell line clinical and basic research addressing this issue. Treatment data. Integrative analysis defined 126 candidate genes, of for HNSCC includes surgical resection, chemotherapy and which only seven genes showed differential methylation in radiation therapy; however, approximately 50% of all patients tumors and no methylation in normal mucosa after bisulfite have advanced disease at the time of diagnosis often requiring sequencing. Following validation by QMSP, one gene, guanine use of all three treatment modalities. Therefore, it is important nucleotide-binding protein γ-7 (GNG7), was confirmed to to discover new biomarkers in a cancer-specific manner and be highly methylated in tumors and unmethylated in normal to develop new methods that provide sensitive and reliable mucosal and salivary rinse samples demonstrating cancer- biomarkers of HNSCC for detection, treatment response and prognosis. Genetic alterations are a hallmark of human cancer, with the activation of proto-oncogenes and inactivation of tumor Correspondence to: Dr Semra Demokan, Department of Basic suppressor genes, either through deletion or inactivating point Oncology, Oncology Institute, Istanbul University, Capa, 34093 mutations, being well defined (2). In addition to these genetic Istanbul, Turkey alterations, changes in DNA methylation, an epigenetic process E-mail: [email protected] present in mammalian cells, are also a hallmark of human cancer (3). Silencing of tumor suppressor genes by means of Key words: guanine nucleotide-binding protein γ-7, gene expression, promoter hypermethylation plays an important role in head silencing, head and neck squamous cell carcinoma, epigenetics and neck carcinogenesis (4). Methylation of the CpG islands in the promoter regions of tumor suppressor genes is frequently observed with resultant reduced gene expression (5,6). 1428 DEMOKAN et al: GUANINE NUCLEOTIDE-BINDING PROTEIN γ-7 IN HNSCC To discover the new cancer-specific hypermethylated genes, a control, to investigate the normal promoter methylation gene expression profiling via oligonucleotide microarray-based status of seven newly identified candidate genes, MAP2K3 approach is a reliable technology for whole genome epigenetic (mitogen-activated protein kinase kinase 3) (n=18), MAP3K3 research (7). Measuring promoter hypermethylation by using (mitogen-activated protein kinase kinase kinase 3) (n=18), real-time quantitative methylation-specific PCR (QMSP) GNG7 (guanine nucleotide-binding protein, γ-7) (n=17), allows an objective, robust, and rapid assessment of promoter GALNT10 (UDP-N-acetyl-α-D-galactosamine:polypeptide methylation status (8-11). N-acetylgalactosaminyltransferase 10) (n=18), PPFIBP2 Previously, we employed a pharmacologic unmasking (PTPRF interacting protein, binding protein 2) (n=17), TUSC2 expression array technique using a 12K gene expression (tumor suppressor candidate 2) (n=16) and TXNIP (thioredoxin array to identify epigenetically inactivated genes in HNSCC interacting protein) (n=16). The methylation status of these (12). In this study, we expanded this approach using a whole genes was analyzed in 33 fresh HNSCC tumor samples. genome 47K array platform and then performed bisulfite DNA sequencing for 126 selected genes and QMSP for seven RNA isolation, cDNA synthesis and probe hybridization. selected genes after evaluating the bisulfite sequencing results, We prepared total RNA from 13 HNSCC tumors, 5 normal to validate HNSCC-specific methylation in novel genes. mucosa samples and cell lines using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). Total RNA quality was checked Materials and methods via the Nanodrop Spectrophotometer and Agilent Bioanalyzer total RNA series II kit. Total RNA (1 µg) was combined with Cell lines. We used 2 human head and neck cancer cell lines, 2 µl T7 oligo(dT) primer and 2 µl Poly-A controls and brought JHU-011 and JHU-012, which were developed from a laryn- to a volume of 12 µl. The samples were incubated at 70˚C for geal primary and a neck node metastasis of different HNSCC 10 min. A master mix of 4 µl first strand buffer, 2 µl DTT patients, at the Department of Otolaryngology-Head and Neck and 1 µl 10 mM dNTPs was added to the samples, followed Surgery, Johns Hopkins University. Cell lines were cultured by a 2-min incubation at 42˚C. Superscript II (Invitrogen, in RPMI-1640 medium supplemented with 10% fetal bovine Carlsbad, CA, USA) (1 µl) was added to each sample and the serum and 1% penicillin-streptomycin. All media components samples were incubated at 42˚C for 1 h for first strand cDNA were obtained from Life Technologies Invitrogen Corp. All synthesis. A master mix of 91 µl water, 30 µl 5X second strand cell lines tested negative for any mycoplasma contamination. buffer, 3 µl 10 mM dNTPs, 4 µl DNA polymerase I, 1 µl E. coli DNA ligase and 1 µl RNase H was added to each sample and 5-aza-2'-deoxycytidine treatment. Cell lines were treated with the samples were incubated for 2 h at 16˚C for second strand 5-aza-2'-deoxycytidine (5-aza-dC, a demethylating agent) and cDNA synthesis. T4 DNA polymerase (2 µl) was added trichostatin A (TSA, a histone deacetylase inhibitor) as previ- followed by a 5-min incubation at 16˚C. EDTA (10 µl, 0.5 M) ously described (7,12). Briefly, we seeded all cell lines (1x106) was added to stop the reaction. cDNA cleanup was performed in their respective culture medium and maintained them with the Affymetrix GeneChip Sample Cleanup Module, for 24 h before treating them with 5 µM 5-aza-dC (Sigma, according to the manufacturer's instructions. The cDNA St. Louis, MO, USA) for 5 days and 300 nM for the final 24 h. was eluted in 14 µl elution buffer. The final elution volume We renewed medium containing 5-aza-dC every 24 h during (~12 µl) was combined with 28 µl of IVT mix master mix the treatment and handled control cells similarly, without (4 µl 10X IVT labeling buffer, 12 µl labeling NTP mix, 4 µl adding 5-aza-dC. Stock solutions of 5-aza-dC (Sigma) and labeling enzyme mix, 8 µl RNAse-free water) for the in vitro TSA (Sigma) were dissolved in DMSO (Sigma) and ethanol transcription cRNA synthesis reaction. The samples were (100%), respectively. incubated overnight for 16 h at 37˚C followed by a hold at 4˚C. Cleanup was performed as per the manufacturer's protocol Tissue samples. After obtaining institutional review board using the Affymetrix GeneChip Sample Cleanup Module. The approval and appropriate informed consent, the HNSCC final elution volume was ~19 µl. Concentration was checked patients and control population (healthy subjects enrolled in via a nanodrop spectrophotometer. Fragmentation buffer (5X) a community screening study) were recruited at the Johns (6 µl) was then combined with 15 µg cRNA and incubated at Hopkins School of Medicine, Department of Otolaryngology- 95˚C for 35 min to fragment the cRNA. The samples were ice Head and Neck Surgery. Five normal mucosa samples from quenched and combined with the hybridization cocktail. After healthy individuals by uvulopalatopharyngoplasty (UPPP) 10 min of pre-hybridizing Human U133 Plus 2.0 Genome technique, 13 HNSCC tumors for mRNA expression array array at 45˚C, 60 rpm, 200 µl of cocktail was loaded onto each experiments, 22 salivary rinses and 14 mucosal samples from array and the arrays were hybridized for 16 h at 45˚C, 60 rpm.