Abstract 4019: Galanin has a tumor suppressor activity and is frequently inactivated by aberrant promoter methylation in head and neck cancer

Abstract

Abstract Purpose: Chromosome 11q13 alterations have repeatedly been reported to be associated with bad prognosis in head and neck squamous cell carcinoma (HNSCC). Our recent findings support a growth regulatory function for GALR1 since antibody blockade of this receptor enhances proliferation of on HNSCC cells and Galanin and GALR1 induce a marked and prolonged ERK1/2 activation, up-regulation of p27Kip1 and p57Kip2, down-regulation of cyclin D1, and consequent inhibition of cell proliferation. Furthermore, expression-negative squamous cell cancers and cell lines exhibit hypermethylation of CpG islands in the GALR1 promoter region. Based on the hypothesis that CpG hypermethylation might silence the Galanin gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and primary tumors. Experimental Design: Promoter methylation of Galanin in 62 UM-SCC and 10 UT-SCC cell lines and 100 primary tumor samples using methylation-specific PCR. Galanin expression and methylation status of 12 UM-SCC cell lines were analyzed further by Quantitative real-time PCR and bisulfite sequencing analysis. To establish that methylation was responsible for silencing Galanin gene expression, we treated three cell lines (UM-SCC-2, -10B and -54). These cell lines that showed absence of Galanin expression and hypermethylation were cultured with 5-Azacytidine alone, TSA alone, or 5-Azacytidine plus TSA. UM-SCC-54 cell line was used in a relative colony-forming assay. Wild-type preprogalanin in the pCMV-SPORT6 construct or empty pCMV-SPORT6 construct were transfected into UM-SCC-54 cell line exhibiting Galanin hypermethylation and loss of Galanin expression. Results: MSP analysis revealed that the Galanin promoter was fully or partially methylated in 24 of 72 cell lines (33.3%) but was unmethylated in the majority 19/20 (95.0%) of non-malignant lines. Methylation correlated with decreased Galanin expression. Galanin methylation found in 24/100 HNSCC tumor specimens significantly correlated with GALR1 methylation status (P<0.001). The presence of Galanin promoter hypermethylation statistically correlated with a decrease in disease-free survival (DFS) (log-rank test, P <0.01).). In multivariate logistic-regression analysis, methylation of Galanin and methylation of the gene pair Galanin and GALR1 were associated with an odds ratio for recurrence of 8.95 (95% CI, 2.29 to 35.03; P=0.002) and 23.84 (95% CI, 2.74 to 207.17; P=0.004), respectively. In UM-SCC-54 cells that have hypermethylated Galanin and GALR1-proficient cell lines, exogenous expression of Galanin and stimulation suppressed cell proliferation. Conclusions: Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after reexpression support the hypothesis that Galanin may act as a tumor suppressor activity in HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4019. doi:1538-7445.AM2012-4019