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Epigenetic changes have been implicated in the pathogenesis of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Prior efforts have primarily examined regional promoter hypermethylation as a silencer of tumor suppressor gene expression. To analyze the global state of methylation in the HNSCC genome, we utilize pyrosequencing of repetitive elements (LINEs) to compare the state of global methylation in HNSCC to normal aerodigestive mucosa. 137 samples (119 HNSCC tumors and 18 normal mucosal tissues) were digested to extract DNA and subjected to bisulfite treatment. Treated DNA was amplified using PCR primers for the repetitive LINEs sequence and produced a heterogeneous sample of products, from many genomic loci. These products were pyrosequenced to quantitatively evaluate their global genomic methylation status. HNSCC specimens showed global hypomethylation, with a mean level of genomic methylation of 46.8% methylated with a standard deviation of 9.0. Conversely, the normal upper airway mucosa had a global methylation level of 54.0 and a standard deviation of 4.6 (Mann–Whitney p value < 0.001). The tumor specimens also showed an increasing degree of hypomethylation associated with advanced tumor stage (ANOVA p-value of 0.003). About 67% of HNSCC's are globally hypomethylated when evaluated against the minimum level of methylation in the normal mucosal specimens. Degree of global hypomethylation was associated with smoking history, alcohol use and stage in univariate analysis (p-value 0.02), however, only HNSCC diagnosis remained significant on multivariate analysis. Despite the presence of regional promoter hypermethylation, HNSCC demonstrates global genomic hypomethylation. The effects of stage, alcohol use and smoking on global hypomethylation were not independently significant. © 2007 Wiley-Liss, Inc.
Head and neck squamous cell carcinomas (HNSCCs) account for 3% of all cancers in the United States and 40,000 new cases each year.1 Although significant progress has been made in the areas of early detection, diagnosis and treatment, the 5-year survival rate for patients with HNSCC has shown only modest improvement in the past 40 years.2 Comprehensive analysis of clinical and treatment factors has shown tumor-site specific improvements in 5-year survival for cancers of the nasopharynx, oropharynx and hypopharynx, and late-stage laryngeal cancer.3
The molecular mechanisms of HNSCC carcinogenesis are undergoing intensive investigation. Despite significant genetic/epigenetic alterations found in these cancers, few known alterations correlate with the clinical outcomes of the disease. Epigenetic changes have been characterized in the pathogenesis of HNSCC. By far, the most studied epigenetic mechanism has been promoter hypermethylation of tumor suppressor genes including: Cyclin A1, MGMT, DCC and p16.4 Methylation of cytosine-guanine dinucleotides by the enzyme class of DNA methyltransferases transfers a methyl group from S-adenosyl-methionine and is associated with gene silencing. Promoter hypermethylation has shown marked promise for identification of biomarkers and functional tumor suppressor genes implicated in the carcinogenesis of HNSCC.
Reports have noted a paradox between regional DNA hypermethylation in the promoters of tumor suppressor genes and a state of global genomic hypomethylation in solid tumors over the last few years.5, 6, 7 In fact, there are several reasons to believe that hypomethylation may be a significant factor driving oncogenesis. Mice with disruption of DNA methyltransferase 1 (DNMT1) function demonstrate significant genomic hypomethylation in all tissues and develop aggressive T-cell lymphomas with chromosomal instability.8 Murine embryonic cells with homozygous deletion of DNMT1 exhibit significantly elevated rates of genetic deletions.9 DNMT mutation can lead to chromosomal instability in humans as well. Mutation of DNMT3b leads to numerous chromosome aberrations.10 Meta-analysis of DNA global hypomethylation across various cancer types suggest a correlation between global hypomethylation and tumor stage.7 Additionally, extensive demethylation of centromeric sequences is common in human tumors and may play a role in aneuploidy.11
To date, the global methylation status of head and neck squamous cell cancer has not been adequately studied in patients. In a limited cohort of only 6 tumor samples, Chalitchagorn et al.12 showed a promising trend toward hypomethylation in HNSCC versus normal mucosal tissues using combined bisulfite restriction analysis. In contrast, Piyathilake et al. found a higher percentage of cells positive for 5-methyl-cytosine immunostaining in 39 HNSCC samples versus normal oral epithelial cells. This evaluation was looking at the percentage of cells positive for staining and staining score, and does not necessarily correlate to the degree of DNA methylation per cell. To quantitate this finding globally, our group utilized the technique of pyrosequencing of LINE sequences to examine the degree of global genomic hypomethylation in head and neck cancer tumor specimens compared to normal control mucosal tissue in an effort to determine if global DNA hypomethylation exists in head and neck cancer.
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- Material and methods
DNA hypermethylation studies continue to elucidate epigenetic silencing of novel candidate tumor suppressor genes. However, evidence exists showing a decrease in the global genomic methylation state in solid tumors. Using LINE elements, we examined the global level of DNA methylation in primary HNSCC and compared it with normal aerodigestive mucosa. LINE elements have been used as a surrogate for global methylation level because of their marked presence in the genome, and this reproducible assay has been shown to correlate with methylation-specific restriction digests.14 There were differences in patient ages reflecting the fact that HNSCC has a median presentation in the late 50s versus the normal samples from a population that presented for benign disease. About 67% of head and neck squamous tumors in our study showed a degree of global hypomethylation that exceeded the measure of any normal mucosal specimen. Although there is a prominent degree of hypomethylation in the tumor samples, a few samples are actually hypermethylated. Our results suggest that, despite promoter hypermethylation of individual tumor suppressor genes, head and neck cancers are globally hypomethylated. These findings are actually consistent with previous reports of other solid tumors that have shown global DNA hypomethylation, and regional areas of promoter hypermethylation.7 These findings suggest an opposite conclusion to the report by Piyathilake et al.15 showing a higher percentage of cells with antibody staining to a5-methyl-CpG antibody in cancers versus normal mucosa. Although these results were not accounted for by increased DNA content of the cancer, it would require further efforts to see if this was a result of reduced chromosomal compaction, as well as the known presence of regional hypermethylation in cancers, or variation from qualitative assessment. Also, a small subset of our tumors did indeed have hypermethylation, indicating what may be an overall dysregulation of methylation in the genome of cancer cells.
To further elucidate the potential role of global hypomethylation in carcinogenesis, we assessed the relationship between increasing tumor stage and the degree of hypomethylation. Global methylation mean levels were reduced in the Stage IV lesions 45.7 compared to the Stage I/II/III (48.4/47.0/48.8), suggesting this epigenetic change worsens as tumorigenesis progresses. Although many molecular alterations continue to increase in frequency as stage increases, this does not necessarily imply causation, but remains an association.
The association between smoking status and global state of methylation in tumors suggest tobacco exposure may be causing genome-wide damage apparent in this epigenetic assay. In our study, patients who had quit smoking (p = 0.07), and current smokers (p = 0.02), with HNSCC, had lower levels of global methylation. This association was present on univariate analysis. Smoking has previously been linked to promoter methylation of genes such as SFRP in HNSCC16 and TSLC1/IGSF4, P16, MGMT in nonsmall cell lung cancer.17, 18, 19 Smoking has not been previously shown to cause global genomic hypomethylation. It has been reported that smoking is associated with reduced levels of vitamins including B12, which is required for synthesis of S-adenyl-methionine.20 This might be one factor causing the association of hypomethylation and smoking, but will require further study. Subset analysis within the tumor group also showed an association of alcohol use with hypomethylation, although it is difficult to separate tobacco and alcohol effects as they tend to be confounding characteristics in this patient population. Alcohol ingestion can also affect the B12 pathway. Dietary differences were not collected here, which can also affect methylation, especially when considered globally. Subsequent studies are planned to look at the causality of the 2 main head and neck cancer risk factors: tobacco use and ethanol in contributing to global hypomethylation.
To analyze the possible functional role of DNA hypomethylation, several theories exist supporting its contribution to molecular pathogenesis of cancer.21 Prominent theories include the direct reactivation of transposable repetitive elements, such as LINEs and Alus which have been shown to be activated by hypomethylation,22 and which commonly have deleterious effects such as functional repression through insertion into transcribed sequences. Hypomethylation may also represent a loss of imprinting in the genome that causes activation of a wide spectrum of genes that convey various growth advantages or even possible oncogenes.21 Alternatively, global hypomethylation may be an epiphenomenon associated with genomic alterations common to most solid tumors. Subsequent work will be necessary to determine the causation of global hypomethylation, and factors that influence it (e.g., tobacco exposure).