Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer

Abstract

PURPOSE: There is accumulating evidence that galanin receptors (GALRs) may be tumor suppressors in head and neck squamous cell carcinoma (HNSCC). Promoter methylation status and gene expression were assessed in a large panel of primary tumors, based on the hypothesis that CpG hypermethylation might silence the galanin gene. EXPERIMENTAL DESIGN: Galanin expression was examined using reverse transcription-polymerase chain reaction (PCR). The methylation status of the galanin promoter was studied using bisulfate sequencing and methylation-specific PCR. UM-SCC-54 was stably transfected to express galanin. RESULTS: Galanin expression was absent in 3/12 (25.0%) UM-SCC cell lines, whereas three nonmalignant cell lines had stable expression. Galanin methylation was found in 24/100 (24.0%) cases. HNSCC tumor specimens was significantly correlated with the GALR1 methylation status (P = 1.88E-06). The presence of galanin promoter hypermethylation was statistically correlated with a decrease in disease-free survival (log-rank test, P = 6.02E-05). A multivariate logistic regression analysis showed that methylation of galanin and methylation of the gene pair galanin and GALR1 had an odds ratio for recurrence of 8.95 [95% confidence interval (CI), 2.29–35.03] and 23.84 (95% CI, 2.74–207.17), respectively. UM-SCC-54 cells that are GALR1-proficient but have hypermethylated galanin exhibited suppressed cell proliferation following exogenous expression of galanin. CONCLUSIONS: Association of frequent promoter hypermethylation and gene silencing with poor survival, combined with growth suppression of HNSCC cells after forced gene expression, supports the hypothesis that galanin acts as a tumor suppressor. These data suggest that galanin and GALR1 are potential therapeutic targets and prognostic factors.