Epigenetic inactivation of galanin receptor 1 in head and neck cancer.
OBJECTIVE
One copy of the galanin receptor 1 (GALR1) 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 GALR1 gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors.


METHODS
Promoter methylation of GALR1 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.


RESULTS
The GALR1 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. GALR1 methylation was also found in 38 of 100 (38%) primary tumor specimens. Methylation correlated with decreased GALR1 expression. In tumors, methylation was significantly correlated with increased tumor size (P = 0.0036), lymph node status (P = 0.0414), tumor stage (P = 0.0037), cyclin D1 expression (P = 0.0420), and p16 methylation (P = 0.0494) and survival (P = 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 GALR1 mRNA. Treatment with trichostatin A and 5-azacytidine restored GALR1 expression. In UM-SCC-23 cells that have total silencing of GALR1, exogenous GALR1 expression and stimulation with galanin suppressed cell proliferation.


CONCLUSIONS
Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after reexpression support the hypothesis that GALR1 is a tumor suppressor gene in HNSCC.
Misawa
Kiyoshi
K
Laboratory of Head and Neck Cancer Biology, Comprehensive Cancer Center, Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor, Michigan 48109-5616, USA.
Ueda
Yo
Y
Kanazawa
Takeharu
T
Misawa
Yuki
Y
Jang
Ilwhan
I
Brenner
John Chadwick
JC
Ogawa
Tetsuya
T
Takebayashi
Satoru
S
Grenman
Reidar A
RA
Herman
James G
JG
Mineta
Hiroyuki
H
Carey
Thomas E
TE
D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics*; D015153:Blotting, Western; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015870:Gene Expression; D016147:Genes, Tumor Suppressor; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D019656:Loss of Heterozygosity; D008297:Male; D011401:Promoter Regions, Genetic; D044091:Receptor, Galanin, Type 1 / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D014408:Biomarkers, Tumor; D044091:Receptor, Galanin, Type 1