Abstract
PurposeAlthough promoter hypermethylation has been an accepted means of tumor suppressor gene inactivation, activation of otherwise normally repressed proto-oncogenes by promoter demethylation has been infrequently documented.Experimental DesignIn this study we performed an integrative, whole-genome analysis for discovery of epigenetically activated proto-oncogenes in head and neck cancer tumors. We used the 47K GeneChip U133 Plus 2.0 Affymetrix expression microarray platform to obtain re-expression data from 5-aza treated normal cell line and expression data from primary head and neck squamous cell carcinoma (HNSCC) tumor tissues and normal mucosa tissues. We then investigated candidate genes by screening promoter regions for CpG islands and bisulfite sequencing followed by QUMSP and RT PCR for the best candidate genes. Finally, functional studies were performed on the top candidate gene.ResultsFrom the top 178 screened candidates 96 had CpG islands in their promoter region. Seven candidate genes showed promoter region methylation in normal mucosa samples and promoter demethylation in a small cohort of primary HNSCC tissues. We then studied the demethylation of the top 3 candidate genes in an expanded cohort of 76 HNSCC tissue samples and 17 normal mucosa samples. We identified MAGEB2 as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues. We then found significantly higher expression of MAGEB2 in tumors in a separate cohort of 73 primary HNSCC tissues and 31 normal tissues. Finally, we found that MAGEB2 has growth promoting effects on minimally transformed oral keratinocyte cell lines but not a definite effect on HNSCC cell lines.ConclusionIn conclusion, we identified MAGEB2 as activated by promoter demethylation in HNSCCand demonstrates growth promoting effects in a minimally transformed oral keratinocyte cell line. More studies are needed to evaluate MAGBE2's exact role in HNSCC.
Highlights
Epigenetic modifications encompass changes such as DNA methylation, chromatin modifications, and genomic imprinting
We identified MAGEB2 as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues
We found significantly higher expression of MAGEB2 in tumors in a separate cohort of 73 primary head and neck squamous cell carcinomas (HNSCC) tissues and 31 normal tissues
Summary
Using an integrative high-throughput discovery approach, previously described [2,3] we screened for activated protooncogenes in HNSCC tissues using: 1) re expression data of 5aza treated normal minimally transformed cell line (OKF) in a GeneChip U133 Plus 2.0 Affymetrix expression microarray platform and 2) expression data from 13 primary HNSCC tumor tissues and 5 normal mucosa tissues performed on the same platform. Additional studies are needed to fully substantiate this finding and elucidate the exact role of MAGEB2 in HNSCCs. Additional studies are needed to fully substantiate this finding and elucidate the exact role of MAGEB2 in HNSCCs To summarize, in this present study we show that by using an integrative analysis approach, combining a large cohort of HNSCC and normal mucosa tissue expression arrays data and pharmacologic de-methylation re-expression arrays data from normal oral keratinocyte cell lines and a validation process on a large cohort of primary HNSCC tumor tissues and normal mucosa tissues, we were able to identify MAGEB2 as activated by promoter demethylation in HNSCC and demonstrates growth promoting effects on a minimally transformed oral keratinocye cell line Further research is needed to completely elucidate the functions and role of MAGEB2 in tumor development and whether it could be exploited as a target for therapy in HNSCC
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