Aberrant Methylation Inactivates Somatostatin and Somatostatin Receptor Type 1 in Head and Neck Squamous Cell Carcinoma

<div><p>Purpose</p><p>The aim of this study was to define somatostatin (<i>SST</i>) and somatostatin receptor type 1 (<i>SSTR1</i>) methylation profiles for head and neck squamous cell carcinoma (HNSCC) tumors at diagnosis and follow up and to evaluate their prognostic significance and value as a biomarker.</p><p>Methods</p><p>Gene expression was measured by quantitative RT-PCR. Promoter methylation status was determined by quantitative methylation-specific PCR (Q-MSP) in HNSCC.</p><p>Results</p><p>Methylation was associated with transcription inhibition. <i>SST</i> methylation in 81% of HNSCC tumor specimens significantly correlated with tumor size (<i>P</i> = 0.043), stage (<i>P</i> = 0.008), galanin receptor type 2 (<i>GALR2</i>) methylation (<i>P</i> = 0.041), and tachykinin-1 (<i>TAC1</i>) (<i>P</i> = 0.040). <i>SSTR1</i> hypermethylation in 64% of cases was correlated with tumor size (<i>P</i> = 0.037), stage (<i>P</i> = 0.037), <i>SST</i> methylation (<i>P</i> < 0.001), and expression of <i>galanin</i> (<i>P</i> = 0.03), <i>GALR2</i> (<i>P</i> = 0.014), <i>TAC1</i> (<i>P</i> = 0.023), and tachykinin receptor type 1 (<i>TACR1</i>) (<i>P</i> = 0.003). <i>SST</i> and <i>SSTR1</i> promoter hypermethylation showed highly discriminating receiver operator characteristic curve profiles, which clearly distinguished HNSCC from adjacent normal mucosal tissues. Concurrent hypermethylation of <i>galanin</i> and <i>SSTR1</i> promoters correlated with reduced disease-free survival (log-rank test, <i>P</i> = 0.0001). Among patients with oral cavity and oropharynx cancer, methylation of both <i>SST</i> and <i>SSTR1</i> promoters correlated with reduced disease-free survival (log-rank test, P = 0.028). In multivariate logistic-regression analysis, concomitant methylation of <i>galanin</i> and <i>SSTR1</i> was associated with an odds ratio for recurrence of 12.53 (95% CI, 2.62 to 59.8; <i>P</i> = 0.002).</p><p>Conclusions</p><p>CpG hypermethylation is a likely mechanism of <i>SST</i> and <i>SSTR1</i> gene inactivation, supporting the hypothesis that <i>SST</i> and <i>SSTR1</i> play a role in the tumorigenesis of HNSCC and that this hypermethylation may serve as an important biomarker.</p></div

Additional file 5: of The neuropeptide genes SST, TAC1, HCRT, NPY, and GAL are powerful epigenetic biomarkers in head and neck cancer: a site-specific analysis

Figure S3. Kaplan-Meier survival curves for the 58 patients with hypopharyngeal cancer, according to the methylation status of the five target genes. Disease-free survival for (a) SST, (b) TAC1, (c) HCRT, (d) NPY, and (e) GAL in the case of methylated (red lines) and unmethylated (blue lines) genes. (f) Joint analysis of the 5 genes. Blue line: patients with 0–2 methylated genes; red line: patients with 3–5 methylated genes. A probability of < 0.05 (*P < 0.05) was considered to represent a statistically significant difference. (EPS 1460 kb

Additional file 2: of The neuropeptide genes SST, TAC1, HCRT, NPY, and GAL are powerful epigenetic biomarkers in head and neck cancer: a site-specific analysis

Figure S1. Schematic representation of methylation analysis of 0 on 5 genes by qMSP. Schematic representation of (a) SST, (b) TAC1, (c) HCRT, (d) NPY, and (e) GAL genes. Exon structures and CpG sites within expanded views of the promoter region relative to the transcription start site (TSS). Vertical lines, individual CpG sites; straight arrows, relative location of the primers used for qMSP; bent arrow, TSS; arrowhead, translation start site (ATG). (EPS 1793 kb

Hypermethylation patterns in matched pairs of head and neck tumors and adjacent normal mucosal tissues.

<p><b>(A)</b><i>SST</i> NMVs of head and neck tumors were significantly higher than those of paired adjacent normal mucosal tissues (<i>P</i> < 0.001). <b>(B)</b> A higher frequency and quantity of <i>SSTR1</i> methylation was noted in head and neck tumors than in matched normal mucosal tissues (<i>P</i> < 0.01). <b>(C)</b> The area under the ROC curve (AUROC) value for the <i>SST</i> gene was 0.9375. At the cutoff value of 0.046, sensitivity was 80.6% and specificity was 94.4%. <b>(D)</b> The AUROC value for the <i>SSTR1</i> gene was 0.9522. At the cutoff value of 0.012, sensitivity was 61.1% and specificity was 100%.</p

Additional file 3: of The neuropeptide genes SST, TAC1, HCRT, NPY, and GAL are powerful epigenetic biomarkers in head and neck cancer: a site-specific analysis

Figure S2. ROC curves for the methylation markers in head and neck carcinomas versus adjacent normal mucosal tissue. On the basis of the ROC curve analysis, the sensitivity, specificity, and cutoff level were determined to be 80.6%, 94.4%, and 0.046 for SST (a); 72.2%, 97.2%, and 0.08 for TAC1 (b); 67.6%, 97.2%, and 0.099 for HCRT (c); 50.0%, 97.2%, and 0.041 for NPY (d); and 25.0%, 86.1%, and 0.100 for GAL (e). (EPS 1057 kb

Additional file 4: of The neuropeptide genes SST, TAC1, HCRT, NPY, and GAL are powerful epigenetic biomarkers in head and neck cancer: a site-specific analysis

Table S2. Results of log-rank tests for the effect of number of methylated genes on disease-free survival in 230 HNSCC cases. (DOCX 14 kb

Odds ratios for recurrence based on multivariate logistic-regression adjusted for stage (I, II, III vs. IV), age (65 and older vs. <65), sex, alcohol exposure, and smoking status.

<p>Methylation of <i>SSTR1</i> and <i>galanin</i> in the primary tumor was associated with the most significant odds ratios of recurrence.</p

Diagrammatic representation of <i>SST</i> methylation analysis by quantitative-MSP, expression analysis by quantitative-RT-PCR, and bisulfite sequencing analysis in UM-SCC cell lines.

<p><b>(A)</b> Relative mRNA expression of <i>SST</i> revealed lower expression in cancer cell lines than in normal fibroblasts (<i>P</i> < 0.01). The housekeeping gene <i>GAPDH</i> was run as a control for RNA integrity. <b>(B)</b> Relative mRNA expression of <i>TACR1</i> revealed lower expression in cancer cell lines than in normal fibroblasts (<i>P</i> < 0.01). <b>(C)</b> Mean <i>SST</i> NMV was significantly higher in cancer cell lines (<i>P</i> < 0.001). <b>(D)</b> Representative examples of quantitative-MSP of <i>TACR1</i> in cancer and normal fibroblasts and keratinocytes; there was a significant difference in NMV (<i>P</i> < 0.05).</p

Additional file 10: of The neuropeptide genes SST, TAC1, HCRT, NPY, and GAL are powerful epigenetic biomarkers in head and neck cancer: a site-specific analysis

Figure S8. Methylation and expression status of the five neuropeptide genes in HNSCCs in the TCGA database. Scatter plot analysis for (A) SST, (B) TAC1, (C) HCRT, (D) NPY, and (E) GAL. Spearman rank correlation coefficient (R2) and P values are shown. (EPS 2568 kb

SST and SSTR1 Genes Methylation Status in HNSCC Primary Samples.

<p>†Fisher’s exact probability test</p><p>SST and SSTR1 Genes Methylation Status in HNSCC Primary Samples.</p