Data from Epigenetic Inactivation of Galanin Receptor 1 in Head and Neck Cancer

Abstract

<div>Abstract<p><b>Purpose:</b> One copy of the galanin receptor 1 (<i>GALR1</i>) locus on 18q is often deleted and expression is absent in some head and neck squamous cell carcinoma (HNSCC) cell lines. To determine if loss of heterozygosity and hypermethylation might silence the <i>GALR1</i> gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors.</p><p><b>Experimental Design:</b> Promoter methylation of <i>GALR1</i> in 72 cell lines and 100 primary tumor samples was analyzed using methylation-specific PCR. GALR1 expression and methylation status were analyzed further by real-time PCR and bisulfite sequencing analysis.</p><p><b>Results:</b> The <i>GALR1</i> promoter was fully or partially methylated in 38 of 72 (52.7%) HNSCC cell lines but not in the majority 18 of 20 (90.0%) of nonmalignant lines. <i>GALR1</i> methylation was also found in 38 of 100 (38%) primary tumor specimens. Methylation correlated with decreased <i>GALR1</i> expression. In tumors, methylation was significantly correlated with increased tumor size (<i>P</i> = 0.0036), lymph node status (<i>P</i> = 0.0414), tumor stage (<i>P</i> = 0.0037), <i>cyclin D1</i> expression (<i>P</i> = 0.0420), and <i>p16</i> methylation (<i>P</i> = 0.0494) and survival (<i>P</i> = 0.045). Bisulfite sequencing of 36 CpG sites upstream of the transcription start site revealed that CpG methylation within transcription factor binding sites correlated with complete suppression of <i>GALR1</i> mRNA. Treatment with trichostatin A and 5-azacytidine restored <i>GALR1</i> expression. In UM-SCC-23 cells that have total silencing of <i>GALR1</i>, exogenous <i>GALR1</i> expression and stimulation with galanin suppressed cell proliferation.</p><p><b>Conclusions:</b> Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after reexpression support the hypothesis that <i>GALR1</i> is a tumor suppressor gene in HNSCC.</p></div>