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Epigenetic Screen of Human DNA Repair Genes Identifies Aberrant Oncogene (2012) 31, 5108 --5116 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Epigenetic screen of human DNA repair genes identifies aberrant promoter methylation of NEIL1 in head and neck squamous cell carcinoma J Chaisaingmongkol1, O Popanda1,RWarta2,3, G Dyckhoff2, E Herpel4, L Geiselhart1, R Claus1, F Lasitschka5, B Campos3, CC Oakes1, JL Bermejo6, C Herold-Mende2,3, C Plass1 and P Schmezer1 Aberrant promoter methylation of different DNA repair genes has a critical role in the development and progression of various cancer types, including head and neck squamous cell carcinomas (HNSCCs). A systematic analysis of known human repair genes for promoter methylation is however missing. We generated quantitative promoter methylation profiles in single CpG units of 160 human DNA repair genes in a set of DNAs isolated from fresh frozen HNSCC and normal tissues using MassARRAY technology. Ninety-eight percent of these genes contained CpG islands (CGIs) in their promoter region; thus, DNA methylation is a potential regulatory mechanism. Methylation data were obtained for 145 genes, from which 15 genes exhibited more than a 20% difference in methylation levels between tumor and normal tissues, manifested either as hypermethylation or as hypomethylation. Analyses of promoter methylation with mRNA expression identified the DNA glycosylase NEIL1 (nei endonuclease VIII-like 1) as the most prominent candidate gene. NEIL1 promoter hypermethylation was confirmed in additional fresh frozen HNSCC samples, normal mucosa, HNSCC cell lines and primary human skin keratinocytes. The investigation of laser-microdissected tissues further substantiated increased methylation levels in tumor versus matched non-tumor cells. Immunohistological analysis revealed significantly less NEIL1 protein expression in tumor tissues. 5-Aza-20-deoxycytidine treatment and DNMT1 knockdown resulted in the re-expression of NEIL1 in HNSCC cell lines, which initially carried hypermethylated promoter regions. In conclusion, our results suggest that DNA methylation contributes to the downregulation of NEIL1 expression and might thus have a role in modulating the response to therapies of HNSCC. Oncogene (2012) 31, 5108--5116; doi:10.1038/onc.2011.660; published online 30 January 2012 Keywords: HNSCC; DNA methylation; NEIL1 expression; DNA glycosylase INTRODUCTION Aberrant DNA methylation of DNA repair genes MGMT, MLH1 or DNA repair has an important role in genetic instability, is involved MSH2 has been shown to have a critical role in the development 12 --14 in tumor initiation and progression and can modulate cancer and progression of HNSCC. A systematic screen of repair therapy outcome.1 Changes in DNA repair function were found to genes for promoter methylation is however missing. We therefore be induced by mutations and epigenetic mechanisms. It is now performed quantitative DNA methylation analyses in the promoter 15,16 recognized that epigenetic changes such as aberrant DNA region of all known DNA repair and DNA repair-related genes. methylation could be included as one of the ‘hits’ in Knudson’s We identified novel genes showing aberrant methylation in two-hit hypothesis. Epigenetic modifications, especially DNA HNSCC. The DNA glycosylase NEIL1 (nei endonuclease VIII-like 1) methylation, leading to gene silencing have been shown to be a was among those genes showing significant hypermethylation relevant factor in the disease progression of many cancers in tumor tissues. Our results suggest that DNA methylation including head and neck squamous cell carcinoma (HNSCC). contributes to the regulation of NEIL1 expression and might have HNSCC of the oral cavity, oropharynx, hypopharynx or larynx is a role in modulating the response to therapies of HNSCC. the sixth most common cancer by incidence worldwide, with B600 000 expected cases in 2011.2 Despite the treatment options of surgery, radiation and chemoradiation therapy, o50% of all RESULTS patients survive for 5 years, and this situation has not much Screening of promoter methylation in HNSCC revealed aberrantly improved within the last 20 years.3 The role of genetic alterations methylated target genes in the development of HNSCC has been extensively studied; Using evidence from the UCSC CpG Islands Track (NCBI build however, the role of many affected genes is still unknown.4,5 36/hg18 human assembly) we found that 158 out of 160 Several epigenetically silenced genes have been described in investigated genes contained CpG island (CGI) in the 50 regulatory HNSCC including CDKN2A, LHX6, TCF21, CEBPA, SFRP genes, and region offering the potential for epigenetic gene regulation members of the Fanconi anemia/BRCA pathway.6--11 as a regulatory mechanism for DNA repair genes. Two genes 1Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Department of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, Heidelberg, Germany; 3Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany; 4NCT Tissue Bank, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; 5Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany and 6Institute of Medical Biometry and Informatics, University Hospital Heidelberg, Heidelberg, Germany. Correspondence: Dr P Schmezer, Division of Epigenomics and Cancer Risk Factors (C010), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: [email protected] Received 18 September 2011; revised 9 December 2011; accepted 21 December 2011; published online 30 January 2012 The DNA repair methylome in head and neck cancer J Chaisaingmongkol et al 5109 (POLN and RPA4) contained no CGI within 10 kb upstream or differences lower than 10% between normal and tumor tissues. downstream of the transcription start site. The screen of promoter Thirty-six genes showed methylation differences 410% DNA methylation of 158 genes was performed using a set of 20 (Figure 1a). Out of these, 15 genes showed a higher than 20% HNSCC tissues from patients and 5 normal head and neck mucosa difference in their DNA methylation level and were classified as samples from non-cancer patients. For 13 genes, methylation data aberrantly methylated. Ten of them were located on autosomes could not be obtained due to problematic sequence composition and five genes on the X chromosome. of the region of interest. At the end, quantitative promoter For validation, these 15 genes were analyzed for promoter methylation data in single CpG resolution were obtained for 145 methylation in 47 tumor and 31 normal tissue samples (Table 1). DNA repair genes (5353 CpGs in total). Because of the low number Four genes showed an increased methylation (SALL3, NEIL1, FANCB of samples used in the screen, any statistical analysis would not be and MGMT) and three genes showed a decreased methylation in conclusive. Therefore, we decided to use a non-statistical tumor samples versus controls (APEX2, TREX2, MSH4; probability approach based on the range of methylation values in each values from Wilcoxon rank-sum tests under 0.05). The methylation group. A gene was defined as aberrantly hypermethylated when differences of X-linked DNA repair genes in males and females the maximum methylation value of tumors was 420% higher were separately calculated and displayed (FANCB, APEX2 and than the maximum of normal tissues, and as aberrantly TREX2). There was no indication that methylation levels of hypomethylated when the minimum value of tumors was autosomal genes were affected by gender. Methylation levels of 420% lower than the minimum value of normal tissues. The long interspersed nucleotide elements (LINE-1) were analyzed to majority of screened genes (109 genes) showed methylation obtain an estimate for global DNA methylation changes.17,18 Figure 1. (a) High-throughput methylation analysis of DNA repair genes. Graphical display of differentially methylated genes of 20 HNSCC samples. Each row of the heat map represents a tumor sample and each column represents a methylation difference between tumor and normal tissue. Methylation differences were estimated using the range of methylation values of each group (tumor versus normal), as described in the text. Red color indicates hypermethylation. Green color indicates hypomethylation in tumor compared with normal tissues. Gray indicates unavailable data. (b) mRNA expression of aberrantly methylated genes in HNSCC. Box-and-whisker plot of mRNA expression in a subset of HNSCC (T; n ¼ 27) compared with normal mucosa (N; n ¼ 18) as measured by qRT-PCR. Normalized expression levels were determined using HPRT1, GAPDH and ACTB as reference genes. The differences of mRNA expression between tumor and normal in NEIL1, APEX2 and MSH4 were statistically significant (Wilcoxon rank-sum test; *Po0.01; **Po0.001). Whisker indicates 10--90% percentile. Correlation between methylation and mRNA expression was estimated by Spearman’s rank correlation (DPo0.01). & 2012 Macmillan Publishers Limited Oncogene (2012) 5108 --5116 The DNA repair methylome in head and neck cancer J Chaisaingmongkol et al 5110 Table 1. DNA repair and repair-related genes with aberrant promoter methylation in HNSCC Gene Chromosomal Accession Methylation Tissue Number Methylation P-valuea Frequency of sample name location number status in type of samples level in percent with aberrant cancer tissue (mean±s.d.) methylationb SALL3 18q23 NM_171999 Hypermeth. Tumor n ¼ 43 26.3±21.1 o0.0001 35/43 (81%) Normal n ¼ 24 3.1±1.7 NEIL1 15q24.2 NM_024608 Hypermeth. Tumor n ¼ 45 30.9±22.1 0.0027 28/45 (62%) Normal n ¼ 23 11.4±8.9 FANCB Xp22.31 NM_152633 Hypermeth. Tumor (#) n ¼ 37 8.2±8.2 0.0009 17/37 (46%) Normal (#) n ¼ 12 3.8±2.1 Tumor (~) n ¼ 5 46.8±20.8 0.4185 --- Normal (~) n ¼ 11 42.7±14.5 MGMT 10q26.3 NM_002412 Hypermeth. Tumor n ¼ 44 8.0±18.4 0.0313 11/44 (25%) Normal n ¼ 23 0.04±0.21 APEX2 Xp11.21 NM_014481 Hypometh. Tumor (#) n ¼ 41 3.4±4.2 0.3226 --- Normal (#) n ¼ 15 5.0±8.1 Tumor (~) n ¼ 5 20.5±4.1 0.0012 5/5 (100%) Normal (~) n ¼ 15 33.7±7.0 TREX2 Xq28 NM_007205 Hypometh.
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