<em>MAGEB2</em> is Activated by Promoter Demethylation in Head and Neck Squamous Cell Carcinoma

<div><h3>Purpose</h3><p>Although 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.</p> <h3>Experimental Design</h3><p>In 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.</p> <h3>Results</h3><p>From 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 <em>MAGEB2</em> as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues. We then found significantly higher expression of <em>MAGEB2</em> in tumors in a separate cohort of 73 primary HNSCC tissues and 31 normal tissues. Finally, we found that <em>MAGEB2</em> has growth promoting effects on minimally transformed oral keratinocyte cell lines but not a definite effect on HNSCC cell lines.</p> <h3>Conclusion</h3><p>In conclusion, we identified <em>MAGEB2</em> 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 <em>MAGBE2's</em> exact role in HNSCC.</p> </div

Detailed list of candidate gene promoters that were found to be consistently methylated (>75% of CpGs methylated) in <i>normal</i> tissue samples.

<p>Detailed list of candidate gene promoters that were found to be consistently methylated (>75% of CpGs methylated) in <i>normal</i> tissue samples.</p

RTPCR <i>MAGEB2</i> expression in HNSCC and normal mucosa tissues.

<p>RTPCR <i>MAGEB2</i> expression in HNSCC and normal mucosa tissues.</p

Anchorage dependent growth assays of <i>MAGEB2</i> in NOKSI cell line.

<p>NOKSI AD Growth at 72 hours post transient transfection with <i>MAGEB2</i>.</p

Promoter methylation status in preliminary cohort of 10 normal mucosa and 20 primary HNSCCS tissue samples.

<p>A sample was considered methylated when >75% of the sequenced CpGs were methylated. Spearmen rank correlation was used to analyze the association between percentage of methylated CpGs and expression. Gray = methylated, White = unmethylated, ND = undetermined.</p

Scatter plot of QUMSP of MAGEB2.

<p>Scatter plot of QUMSP of MAGEB2.</p