Epigenetic inactivation of galanin eceptor 1 in head and neck cancer

Kiyoshi Misawa, Yo Ueda, Takeharu Kanazawa, Yuki Misawa, Ilwhan Jang, John Chadwick Brenner, Tetsuya Ogawa, [No Value] SatoruTakebayashi, Reidar A. Grenman, James G. Herman, Hiroyuki Mineta, Thomas E. Carey

Research output: Contribution to journalArticle

Abstract

Purpose: One copy of the galanin receptor1 (GALR1) locus on 18q is often deleted and expression is absentin 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. Experimental Design: 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. Treatmentwith 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.

Original languageEnglish (US)
Pages (from-to)7604-7613
Number of pages10
JournalClinical Cancer Research
Volume14
Issue number23
DOIs
StatePublished - Dec 1 2008

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Galanin
Head and Neck Neoplasms
Epigenomics
Methylation
Neoplasms
Cell Line
trichostatin A
Azacitidine
Loss of Heterozygosity
Transcription Initiation Site
Cyclin D1
Gene Silencing
Tumor Cell Line
Tumor Suppressor Genes
Real-Time Polymerase Chain Reaction

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Misawa, K., Ueda, Y., Kanazawa, T., Misawa, Y., Jang, I., Brenner, J. C., ... Carey, T. E. (2008). Epigenetic inactivation of galanin eceptor 1 in head and neck cancer. Clinical Cancer Research, 14(23), 7604-7613. https://doi.org/10.1158/1078-0432.CCR-07-4673

Epigenetic inactivation of galanin eceptor 1 in head and neck cancer. / Misawa, Kiyoshi; Ueda, Yo; Kanazawa, Takeharu; Misawa, Yuki; Jang, Ilwhan; Brenner, John Chadwick; Ogawa, Tetsuya; SatoruTakebayashi, [No Value]; Grenman, Reidar A.; Herman, James G.; Mineta, Hiroyuki; Carey, Thomas E.

In: Clinical Cancer Research, Vol. 14, No. 23, 01.12.2008, p. 7604-7613.

Research output: Contribution to journalArticle

Misawa, K, Ueda, Y, Kanazawa, T, Misawa, Y, Jang, I, Brenner, JC, Ogawa, T, SatoruTakebayashi, NV, Grenman, RA, Herman, JG, Mineta, H & Carey, TE 2008, 'Epigenetic inactivation of galanin eceptor 1 in head and neck cancer', Clinical Cancer Research, vol. 14, no. 23, pp. 7604-7613. https://doi.org/10.1158/1078-0432.CCR-07-4673
Misawa K, Ueda Y, Kanazawa T, Misawa Y, Jang I, Brenner JC et al. Epigenetic inactivation of galanin eceptor 1 in head and neck cancer. Clinical Cancer Research. 2008 Dec 1;14(23):7604-7613. https://doi.org/10.1158/1078-0432.CCR-07-4673
Misawa, Kiyoshi ; Ueda, Yo ; Kanazawa, Takeharu ; Misawa, Yuki ; Jang, Ilwhan ; Brenner, John Chadwick ; Ogawa, Tetsuya ; SatoruTakebayashi, [No Value] ; Grenman, Reidar A. ; Herman, James G. ; Mineta, Hiroyuki ; Carey, Thomas E. / Epigenetic inactivation of galanin eceptor 1 in head and neck cancer. In: Clinical Cancer Research. 2008 ; Vol. 14, No. 23. pp. 7604-7613.
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abstract = "Purpose: One copy of the galanin receptor1 (GALR1) locus on 18q is often deleted and expression is absentin 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. Experimental Design: 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. Treatmentwith 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.",
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AU - Misawa, Kiyoshi

AU - Ueda, Yo

AU - Kanazawa, Takeharu

AU - Misawa, Yuki

AU - Jang, Ilwhan

AU - Brenner, John Chadwick

AU - Ogawa, Tetsuya

AU - SatoruTakebayashi, [No Value]

AU - Grenman, Reidar A.

AU - Herman, James G.

AU - Mineta, Hiroyuki

AU - Carey, Thomas E.

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N2 - Purpose: One copy of the galanin receptor1 (GALR1) locus on 18q is often deleted and expression is absentin 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. Experimental Design: 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. Treatmentwith 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.

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