title: Galanin has tumor suppressor activity and is frequently inactivated by aberrant promoter methylation in head and neck cancer.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Misawa
authors.forename: Takeharu
authors.initials: T
authors.lastname: Kanazawa
authors.forename: Yuki
authors.initials: Y
authors.lastname: Misawa
authors.forename: Takayuki
authors.initials: T
authors.lastname: Uehara
authors.forename: Atsushi
authors.initials: A
authors.lastname: Imai
authors.forename: Goro
authors.initials: G
authors.lastname: Takahashi
authors.forename: Satoru
authors.initials: S
authors.lastname: Takebayashi
authors.forename: Andrew
authors.initials: A
authors.lastname: Cole
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Carey
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mineta