1999 Media Guide

1999 Men\u27s Track and Field Media Guide, George Fox College

<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

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

Expression Microarray Analysis Reveals Alternative Splicing of <i>LAMA3</i> and <i>DST</i> Genes in Head and Neck Squamous Cell Carcinoma

<div><p>Purpose</p><p>Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.</p><p>Experimental Design</p><p>We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.</p><p>Results</p><p>After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes <i>LAMA3, DST, VEGFC, SDHA, RASIP1</i>, and <i>TP63</i> were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed <i>TP63</i> as having dominant expression of the short <i>DeltaNp63</i> isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (<i>LAMA3</i> and <i>DST</i>) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001).</p><p>Conclusion</p><p>Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.</p></div

Validation tumor clinical characteristics.

<p>Validation tumor clinical characteristics.</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

Scatter plot of QUMSP of MAGEB2.

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

Demographic characteristics of validation tumor and UPPP normal tissue samples.

<p>Demographic characteristics of validation tumor and UPPP normal tissue samples.</p

Figure 1 demonstrates the experimental flow from tissue procurement to validation of tumor-specific alternative splicing events in our study.

<p>Figure 1 demonstrates the experimental flow from tissue procurement to validation of tumor-specific alternative splicing events in our study.</p