Purpose One copy of the 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 LOH 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 (MSP). 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 HNSCC cell lines (52.7%) but not in the majority 18/20 (90.0%) of non-malignant lines. GALR1 methylation was also found in 38/100 (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 TSA 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 re-expression support the hypothesis that GALR1 is a tumor suppressor gene in HNSCC.

In the United States, oral cancer accounts for more deaths annually than cervical cancer, leukemias, or Hodgkin's lymphoma. Studies have shown that aberrations of chromosome 18q develop with tumor progression and are associated with significantly decreased survival in head and neck cancer patients. The G-protein-coupled receptor, galanin receptor 1 (GALR1), maps to this region of chromosome 18q. Although the role of GALR1 has been well characterized in neuronal cells, little is known regarding this receptor in nonneuronal cells. In this study, the expression, mitogenic function, and signaling mechanism of GALR1 are investigated in normal and malignant oral epithelial cells. mRNA expression was determined via reverse transcriptase-PCR. Protein quantification was done via immunoblot analysis and enzyme-linked immunosorbent assay. For functional and signaling studies, an inhibitory antibody was generated to the N-terminal ligand binding domain of GALR1. GALR1 protein and mRNA expression and GAL secretion were detected at variable levels in immortalized human oral keratinocytes and human oropharyngeal squamous cell carcinoma cell lines. Upon competitive inhibition of GALR1, proliferation was up-regulated in immortalized and malignant keratinocytes. Furthermore, studies with the inhibitory antibody and U0126, the MAPK inhibitor, show that GALR1 inhibits proliferation in immortalized and malignant keratinocytes by inactivating the MAPK pathway. GALR1s inhibitory effects on proliferation in epithelial cells raises the possibility that inactivation or disregulation of this receptor can lead to uncontrolled proliferation and neoplastic transformation.

Purpose Galanin and its three receptors (GALR1-3) are expressed in many normal tissues, but silenced in some tumors. Contradictory roles for galanin and its receptors in various tumors have been reported. To understand their function, investigations of individual GALRs are necessary. In head and neck squamous carcinoma cells (HNSCC) with silenced GALR1 and GALR2, we showed that re-expressed GALR1 suppresses tumor cell proliferation via Erk1/2-mediated effects on cdk inhibitors and cyclin D1. Others showed that GALR2 could induce apoptosis in neuroblastoma cells with wild-type p53, whereas GALR2 stimulated proliferation in small cell lung cancer. In this study, we investigated the role of GALR2 in HNSCC cells that have mutant p53 and do not express GALR1. Experimental Design and Results UM-SCC1, a human oral carcinoma cell line with a splice site mutation causing a 46-bp p53 off frame deletion, was stably transfected to express GALR2 (UM-SCC-1-GALR2). Galanin treatment of UM-SCC-1-GALR2 caused morphological changes and a marked decrease in cell number that were not observed in UM-SCC-1-mock cells. Galanin and GALR2 resulted in decreased BrdU incorporation, p27Kip1 and p57Kip2 up-regulation, and decreased cyclin D1 expression. These effects were similar to GALR1 signaling in HNSCC, however, GALR2 also induced caspase-3-dependent apoptosis, which was confirmed by annexin-V staining and DNA fragmentation analysis. These were not observed with GALR1. Conclusion This study demonstrates that GALR2 re-expression can inhibit cell proliferation and induce apoptosis in HNSCC cells with mutant p53. GALR2 may be a feasible target for HNSCC therapy.

Supplementary Table S1 from Epigenetic Inactivation of Galanin Receptor 1 in Head and Neck Cancer

<div>Abstract<p><b>Purpose:</b> One copy of the galanin receptor 1 (<i>GALR1</i>) 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 <i>GALR1</i> gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors.</p><p><b>Experimental Design:</b> Promoter methylation of <i>GALR1</i> 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.</p><p><b>Results:</b> The <i>GALR1</i> 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. <i>GALR1</i> methylation was also found in 38 of 100 (38%) primary tumor specimens. Methylation correlated with decreased <i>GALR1</i> expression. In tumors, methylation was significantly correlated with increased tumor size (<i>P</i> = 0.0036), lymph node status (<i>P</i> = 0.0414), tumor stage (<i>P</i> = 0.0037), <i>cyclin D1</i> expression (<i>P</i> = 0.0420), and <i>p16</i> methylation (<i>P</i> = 0.0494) and survival (<i>P</i> = 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 <i>GALR1</i> mRNA. Treatment with trichostatin A and 5-azacytidine restored <i>GALR1</i> expression. In UM-SCC-23 cells that have total silencing of <i>GALR1</i>, exogenous <i>GALR1</i> expression and stimulation with galanin suppressed cell proliferation.</p><p><b>Conclusions:</b> Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after reexpression support the hypothesis that <i>GALR1</i> is a tumor suppressor gene in HNSCC.</p></div>