Positional differences of intronic transposons in pAMT affect the pungency level in chili pepper through altered splicing efficiency.

Capsaicinoids are unique compounds that give chili pepper fruits their pungent taste. Capsaicinoid levels vary widely among pungent cultivars, which range from low pungency to extremely pungent. However, the molecular mechanisms underlying this quantitative variation have not been elucidated. Our previous study identified various loss-of-function alleles of the pAMT gene which led to low pungency. The mutations in these alleles are commonly defined by Tcc transposon insertion and its footprint. In this study, we identified two leaky pamt alleles (pamtL1 and pamtL2 ) with different levels of putative aminotransferase (pAMT) activity. Notably, both alleles had a Tcc transposon insertion in intron 3, but the locations of the insertions within the intron were different. Genetic analysis revealed that pamtL1 , pamtL2 and a loss-of-function pamt allele reduced capsaicinoid levels to about 50%, 10% and less than 1%, respectively. pamtL1 and pamtL2 encoded functional pAMT proteins, but they exhibited lower transcript levels than the functional type. RNA sequencing analysis showed that intronic transposons disrupted splicing in intron 3, which resulted in simultaneous expression of functional pAMT mRNA and non-functional splice variants containing partial sequences of Tcc. The non-functional splice variants were more dominant in pamtL2 than in pamtL1 . This suggested that the difference in position of the intronic transposons could alter splicing efficiency, leading to different pAMT activities and reducing capsaicinoid content to different levels. Our results provide a striking example of allelic variations caused by intronic transposons; these variations contribute to quantitative differences in secondary metabolite contents.

Prostanoid receptor genes confer poor prognosis in head and neck squamous cell carcinoma via epigenetic inactivation.

BACKGROUND: Chronic inflammation is a risk factor for head and neck squamous cell carcinoma (HNSCC) and other diseases. Prostanoid receptors are clearly involved in the development of many types of cancer. However, their role is not simple and is poorly understood in HNSCC. METHODS: Methylation profiles of prostanoid receptor family genes were generated for tumour samples obtained from 274 patients with HNSCC, including 69 hypopharynx, 51 larynx, 79 oral cavity, and 75 oropharynx tumour samples, by quantitative methylation-specific PCR. Promoter methylation was then evaluated with respect to various clinical characteristics and patient survival. RESULTS: The mean number of methylated genes per sample was 2.05 ± 2.59 (range 0 to 9). Promoters of PTGDR1, PTGDR2, PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, and TBXA2R were methylated in 43.8%, 18.2%, 25.5%, 17.5%, 41.2%, 8.0%, 19.3%, 20.4%, and 11.3% of the samples, respectively. Methylation indices for prostanoid receptor family genes tended to be higher as the number of TET methylation events increased. Patients with 5-9 methylated genes had a significantly lower survival rate than that of patients with 0-4 methylated genes (log-rank test, P= 0.007). In multivariate analyses, PTGDR1 methylation was most highly correlated with recurrence in patients with hypopharyngeal cancer (P = 0.014). A similar correlation was observed for PTGER4 in patients with laryngeal cancer (P = 0.046). Methylation of the PTGIR and TBXA2R promoters was positively correlated with recurrence in oropharyngeal cancer (P = 0.028 and P = 0.006, respectively). Moreover, Patients with 5-9 methylated genes were extremely lower of 5hmC levels (P = 0.035) and was correlated with increasing expression of DNMT3A and DNMT3B (P < 0.05 and P < 0.05, respectively). CONCLUSION: We characterised the relationship between the methylation status of prostanoid receptor genes and recurrence in HNSCC. These results provide new perspectives for the development of molecular targeted treatment approaches.

Aberrant Epigenetic Regulation in Head and Neck Cancer Due to Distinct EZH2 Overexpression and DNA Hypermethylation.

Enhancer of Zeste homologue 2 (EZH2) overexpression is associated with tumor proliferation, metastasis, and poor prognosis. Targeting and inhibition of EZH2 is a potentially effective therapeutic strategy for head and neck squamous cell carcinoma (HNSCC). We analyzed EZH2 mRNA expression in a well-characterized dataset of 230 (110 original and 120 validation cohorts) human head and neck cancer samples. This study aimed to investigate the effects of inhibiting EZH2, either via RNA interference or via pharmacotherapy, on HNSCC growth. EZH2 upregulation was significantly correlated with recurrence (p < 0.001) and the methylation index of tumor suppressor genes (p < 0.05). DNMT3A was significantly upregulated upon EZH2 upregulation (p = 0.043). Univariate analysis revealed that EZH2 upregulation was associated with poor disease-free survival (log-rank test, p < 0.001). In multivariate analysis, EZH2 upregulation was evaluated as a significant independent prognostic factor of disease-free survival (hazard ratio: 2.085, 95% confidence interval: 1.390⁻3.127; p < 0.001). Cells treated with RNA interference and DZNep, an EZH2 inhibitor, showed the most dramatic changes in expression, accompanied with a reduction in the growth and survival of FaDu cells. These findings suggest that EZH2 upregulation is correlated with tumor aggressiveness and adverse patient outcomes in HNSCC. Evaluation of EZH2 expression might help predict the prognosis of HNSCC patients.

Site-specific methylation patterns of the GAL and GALR1/2 genes in head and neck cancer: Potential utility as biomarkers for prognosis.

The aim of this study was to evaluate the prognostic value of the promoter methylation status of galanin (GAL) and galanin receptor 1/2 (GALR1/2) by assessing their association with disease-free survival and known prognostic factors in head and neck cancer. We generated methylation profiles of GAL and GALR1/2 in tumor samples obtained from 202 patients with head and neck squamous cell carcinoma (HNSCC); these included 43 hypopharynx, 42 larynx, 59 oral cavity, and 58 oropharynx tumor samples. CpG island hypermethylation status of the three genes was analyzed using quantitative methylation-specific PCR (Q-MSP). In order to determine the prognostic value of the methylation status of these genes, the associations between methylation index and various clinical characteristics, especially tumor site, were assessed for tumors from patients with HNSCC. The methylation index was positively correlated with female gender (P = 0.008) and disease recurrence (P = 0.01) in oral cancer and human papillomavirus (HPV)-positive (P = 0.004) status and disease recurrence (P = 0.005) in oropharyngeal cancer. Among patients with oral and oropharyngeal cancer, promoter hypermethylation of GAL, GALR1, or GALR2 was statistically correlated with a decrease in disease-free survival (log-rank test, P = 0.036 and P = 0.042, respectively). Furthermore, methylation of GAL, GALR1, or GALR2 exhibited the highest association with poor survival (log-rank test, P = 0.018) in patients with HPV-negative oropharyngeal cancers. As such, GAL and GALR1/2 methylation status may serve as an important site-specific biomarker for prediction of clinical outcome in patients with HNSCC. © 2016 Wiley Periodicals, Inc.

Evaluation of epigenetic inactivation of vascular endothelial growth factor receptors in head and neck squamous cell carcinoma.

The aim of this study was to determine the methylation status of the genes encoding the vascular endothelial growth factor receptors and to evaluate the usefulness of VEGFR methylation as a prognostic indicator in head and neck squamous cell carcinoma. VEGFR messenger RNA expression and promoter methylation were examined in a panel of cell lines via quantitative reverse transcription and methylation-specific polymerase chain reaction, respectively. Promoter methylation was compared with clinical characteristics in 128 head and neck squamous cell carcinoma samples. The normalized methylation values for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the tumour cell lines than in normal tonsil samples, whereas amounts of VEGFR1, VEGFR2 and VEGFR3 messenger RNA were significantly higher. Methylation of the VEGFR1 promoter (p = 0.003; 66/128 head and neck squamous cell carcinoma samples, 52%) and VEGFR3 promoter (p = 0.043; 53/128 head and neck squamous cell carcinoma samples, 41%) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046; 47/128 head and neck squamous cell carcinoma samples, 37%). Concurrent methylation of the VEGFR1 and VEGFR3 promoters significantly correlated with reduced disease-free survival (log-rank test, p = 0.009). In a multivariate logistic regression analysis, methylation of the VEGFR1, VEGFR3 and both the VEGFR1 and VEGFR3 promoters independently predicted recurrence (odds ratios and 95% confidence intervals: 3.19, 1.51-6.75 (p = 0.002); 2.24, 1.06-4.76 (p = 0.035); and 2.56, 1.09-6.05 (p = 0.032), respectively). Methylation of the VEGFR promoters predicts poor prognosis in head and neck squamous cell carcinoma patients.

Tumor suppressor activity and inactivation of galanin receptor type 2 by aberrant promoter methylation in head and neck cancer.

BACKGROUND: There is accumulating evidence that galanin receptors (GALRs) may be tumor suppressors in head and neck squamous cell carcinoma (HNSCC). Promoter methylation status and gene expression were assessed in a large panel of head and neck primary tumors, based on the hypothesis that cytosine-guanine dinucleotide (CpG) hypermethylation might silence the galanin receptor 2 (GALR2) gene. METHODS: GALR2 expression was examined in a panel of cell lines by using quantitative reverse transcription polymerase chain reaction (RT-PCR). The methylation status of the GALR2 promoter was studied using quantitative methylation-specific PCR (Q-MSP). UM-SCC-1 was stably transfected to express GALR2. RESULTS: GALR2 expression was suppressed in UM-SCC cell lines, whereas nonmalignant cell lines exhibited stable expression. GALR2 methylation found in 31 of 100 (31.0%) tumor specimens was significantly correlated with the methylation status of both GALR1 and Galanin. The observed GALR2 promoter hypermethylation was statistically correlated with a decrease in disease-free survival (log-rank test, P=.045). A multivariate logistic-regression analysis revealed a high odds ratio for recurring methylation of GALR2 and the gene pair GALR2 and Galanin, 8.95 (95% confidence interval, 2.29-35.03; P=.024) and 9.05 (95% confidence interval, 1.76-46.50; P=.008), respectively. In addition, exogenous expression of GALR2 suppressed cell proliferation in UM-SCC-1 cells with hypermethylated Galanin and GALR2-proficient cell lines. CONCLUSIONS: Frequent promoter hypermethylation in association with prognosis, and growth suppression after re-expression, supports the hypothesis that GALR2 may act to suppress tumor activity. GALR2 is a potentially significant therapeutic target and prognostic factor for this cancer type.

Biochemical Aspects of Putative Aminotransferase Responsible for Converting Vanillin to Vanillylamine in the Capsaicinoid Biosynthesis Pathway in Capsicum Plants.

The biosynthesis pathway of capsaicinoids includes the conversion of vanillin to vanillylamine, where putative aminotransferase (pAMT) is thought to be the enzyme responsible in Capsicum plants. The objectives of this study were to prove that pAMT is the enzyme responsible for this conversion in plants and to clarify its catalytic properties using biochemical methods. Both an extract of habanero placenta and recombinant pAMT (rpAMT) constructed by using an Escherichia coli expression system were able to convert vanillin to vanillylamine in the presence of γ-aminobutyric acid as an amino donor and pyridoxal phosphate as a coenzyme. Conversion from vanillin to vanillylamine by the placenta extract was significantly attenuated by adding an anti-pAMT antibody to the reaction system. The amino donor specificity and affinity for vanillin of rpAMT were similar to those of the placenta extract. We thus confirmed that pAMT is the enzyme responsible for the conversion of vanillin to vanillylamine in capsaicinoid synthesis in Capsicum fruits. Therefore, we propose that pAMT should henceforth be named vanillin aminotransferase (VAMT).

Novel prognostic value and potential utility of opioid receptor gene methylation in liquid biopsy for oral cavity cancer.

Opioids are a class of recreational drugs and prescription medications that bind to a group of G-protein-coupled receptors known as opioid receptors (ORs). ORs are involved in the development of many types of cancer; however, their role in head and neck squamous cell carcinoma (HNSCC) is complex and poorly understood. Here, we analyzed the methylation status of five OR genes in verification (301 HNSCC primary samples) and validation (five circulating tumor DNA [ctDNA] samples) studies using quantitative methylation-specific PCR (Q-MSP). OPRL1 and OPRM1 methylation levels were significantly higher in HNSCC tissues than in corresponding normal tissues from the same individuals (P = 0.001 and P < 0.001, respectively). In Kaplan-Meier estimate and multivariate Cox proportional hazard analyses, two genes (OPRL1 and OPRM1) were significantly associated with increased recurrence in the methylation group with oral cavity cancer. Furthermore, a validation study of ctDNA demonstrated that OPRL1 genes exhibited predictive performance as emerging biomarkers and were each capable of discriminating the plasma from tumor-free individuals. We characterized the relationship between OR gene methylation status and outcomes in oral cavity cancer. Our results highlight the potential utility of ctDNA methylation-based detection in the clinical management of oral cavity cancer.

Telomere shortening in head and neck cancer: association between DNA demethylation and survival.

A growing body of evidence indicates that telomere dysfunction is a biological marker of progression in several types of cancer. However, the association between head and neck squamous cell carcinoma (HNSCC) and telomere length (TL) remains unknown. We measured the absolute TL levels in a well-characterised dataset of 211 tumoral vs normal tissues obtained from the same patients by quantitative polymerase chain reaction assay. Normalised TL levels were significantly lower in tumour samples than in normal tissue (P < 0.001) and there was a positive correlation between tumour tissue and normal mucosal tissue (R2 = 0.176, P < 0.001). We were able to distinguish two classes, one with a tumour/normal TL ratio ≤ 0.3 (38.4%), which showed clear telomere erosion, and the other with a tumour/normal TL ratio > 0.3 (61.6%), in which the TL was slightly shorter or longer than that in normal tissue. Notably, the tumour/normal TL ratio was correlated with the likelihood of disease recurrence (P = 0.002), the 5-hydroxymethylcytosine level (P = 0.043), and expression of the ten-eleven translocation (TET) gene (P = 0.043). Our findings show that TL shortening and subsequent low levels of 5-hydroxymethylcytosine and TET expression may contribute to development of HNSCC.

Galanin receptor subtype 2 suppresses cell proliferation and induces apoptosis in p53 mutant head and neck cancer cells.

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 galanin receptors are necessary. In head and neck squamous carcinoma cells (HNSCC) with silenced GALR1 and GALR2, we showed that reexpressed 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: UM-SCC-1, 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). RESULTS: Galanin treatment of UM-SCC-1-GALR2 caused morphologic changes and a marked decrease in cell number that were not observed in UM-SCC-1-mock cells. Galanin and GALR2 resulted in decreased bromodeoxyuridine incorporation, p27(Kip1) and p57(Kip2) up-regulation, and decreased cyclin D1 expression. These effects were similar to GALR1 signaling in HNSCC, but 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 shows that GALR2 reexpression can inhibit cell proliferation and induce apoptosis in HNSCC cells with mutant p53. GALR2 may be a feasible target for HNSCC therapy.

Long interspersed nuclear element 1 hypomethylation has novel prognostic value and potential utility in liquid biopsy for oral cavity cancer.

BACKGROUND: New biomarkers are urgently needed to improve personalized treatment approaches for head and neck squamous cell carcinoma (HNSCC). Global DNA hypomethylation has wide-ranging functions in multistep carcinogenesis, and the hypomethylation of long interspersed nucleotide element-1 (LINE-1) is related to increased retrotransposon activity and induced genome instability. However, little information is available regarding LINE-1 hypomethylation and its prognostic implications in HNSCC. METHODS: In this study, we analyzed LINE-1 hypomethylation levels in a well-characterized dataset of 317 primary HNSCC tissues and 225 matched pairs of normal mucosa tissues, along with five oral cavity cancer (OCC) circulating tumor DNA (ctDNA) samples using quantitative real-time methylation and unmethylation PCR. The analysis was performed according to various clinical characteristics and prognostic implications. RESULTS: The results demonstrated that LINE-1 hypomethylation levels were significantly higher in the HNSCC tissues than in corresponding normal tissues from the same individuals (P < 0.001). Univariate analysis revealed that high levels of LINE-1 hypomethylation were correlated with poor disease-free survival (DFS; log-rank test, P = 0.038), whereas multivariate analysis demonstrated that they were significant independent prognostic factor for DFS (hazard ratio: 2.10, 95% confidence interval: 1.02-4.36; P = 0.045). Moreover, samples with high LINE-1 hypomethylation levels exhibited the greatest decrease in 5-hydroxymethylcytosine (5-hmC) levels and increase in tumor-suppressor gene methylation index (P = 0.006 and P < 0.001, respectively). Further, ctDNA studies also showed that LINE-1 hypomethylation had high predictive ability in OCC. CONCLUSIONS: LINE-1 hypomethylation is associated with a higher risk of early OCC relapse, and is hence, a potential predictive biomarker for OCC. Furthermore, 5-hmC levels also exhibited predictive potential in OCC, based on their inverse correlation with LINE-1 hypomethylation levels. LINE-1 hypomethylation analysis, therefore, has applications in determining patient prognosis and real-time surveillance of disease recurrence, and could serve as an alternative method for OCC screening. SUPPLEMENTARY INFORMATION: Supplementary information accompanies this paper at 10.1186/s40364-020-00235-y.

G Protein-Coupled Receptor Genes, PTGDR1, PTGDR2, and PTGIR, Are Candidate Epigenetic Biomarkers and Predictors for Treated Patients with HPV-Associated Oropharyngeal Cancer.

Differences in the biology of human papillomavirus (HPV)-associated oropharyngeal cancers (OPCs) and HPV-negative OPCs may have implications in patient management. Early detection is imperative to reduce HPV-associated OPC mortality. Circulating tumor DNA (ctDNA) can potentially serve as a biomarker for monitoring clinically relevant cancer-related genetic and epigenetic modifications. We analyzed the methylation status of 24 G protein-coupled receptor (GPCR) genes in verification (85 OPC primary samples) and validation (8 OPC ctDNA samples) studies using quantitative methylation-specific polymerase chain reaction (Q-MSP). The Q-MSP-based verification study with 85 OPC primary samples revealed the GPCR genes that were significantly associated with recurrence in high methylation groups (≥14 methylated genes) with OPC and HPV-associated OPC (p < 0.001). In the Kaplan-Meier estimate and multivariate Cox proportional hazard analyses, 13 GPCR genes were significantly related to increased recurrence in the methylation group. Furthermore, the validation study on ctDNA showed that three of these genes (Prostaglandin D2 receptor 1: PTGDR1, Prostaglandin D2 receptor 2: PTGDR2, and Prostaglandin I2 Receptor: PTGIR) had a prediction performance as emerging biomarkers. We characterized the relationship between the methylation status of GPCR genes and outcomes in HPV-associated OPC. Our results highlight the potential utility of ctDNA methylation-based detection for the clinical management of HPV-associated OPC.

Overexpression of Sal-like protein 4 in head and neck cancer: epigenetic effects and clinical correlations.

BACKGROUND: Sal-like protein 4 (SALL4), an embryonic stem cell factor, has been reported to play an essential role in embryogenesis and oncogenesis. As yet, however, the expression and role of this transcription factor in head and neck squamous cell carcinoma (HNSCC) has not been established. METHODS: We assessed SALL4 mRNA expression in a well-characterised dataset of 230 HNSCC samples (test cohort 110 cases and validation cohort 120 cases). We also transfected HNSCC cells (FaDu and UM-SCC-6) with SALL4 siRNA and assessed its effects on proliferation and expression of specific epigenetic factors in order to uncover the role of SALL4 in HNSCC. RESULTS: Overexpression of SALL4 was detected in tumour samples of both cohorts. HNSCC cells treated with SALL4 siRNA showed a reduction in growth and a decrease in DNA methyltransferase 3 alpha (DNMT3A) expression. In the patient cohorts, SALL4 overexpression was found to significantly correlate with disease recurrence (p < 0.001) and SALL4 methylation status (p = 0.002). We also found that DNMT3A was significantly upregulated upon SALL4 upregulation (p < 0.001). High expression levels of SALL4 correlated with decreases in disease-free survival (DFS) rates (log-rank test, p < 0.001). Multivariate analysis revealed that SALL4 expression served as an independent prognostic factor for DFS (hazard ratio: 2.566, 95% confidence interval: 1.598-4.121; p < 0.001). CONCLUSIONS: Our findings indicate that SALL4 upregulation correlates with HNSCC tumour aggressiveness and an adverse patient outcome. Our findings also indicate that DNMT3A may synergistically contribute to the regulatory effects of SALL4. Our findings provide insight into SALL4-mediated HNSCC development via epigenetic modulation.

Identification of novel methylation markers in HPV-associated oropharyngeal cancer: genome-wide discovery, tissue verification and validation testing in ctDNA.

Human papilloma virus (HPV)-associated oropharyngeal cancer (OPC) is an independent tumour type with regard to cellular, biological, and clinical features. The use of non-invasive biomarkers such as circulating tumour DNA (ctDNA) may be relevant in early diagnosis and eventually improve the outcomes of patients with head and neck squamous cell carcinoma (HNSCC). Genome-wide discovery using RNA sequencing and reduced representation bisulfite sequencing yielded 21 candidates for methylation-targeted genes. A verification study (252 HNSCC patients) using quantitative methylation-specific PCR (Q-MSP) identified 10 genes (ATP2A1, CALML5, DNAJC5G, GNMT, GPT, LY6D, LYNX1, MAL, MGC16275, and MRGPRF) that showed a significant increase recurrence in methylation groups with OPC. Further study on ctDNA using Q-MSP in HPV-associated OPC showed that three genes (CALML5, DNAJC5G, and LY6D) had a high predictive ability as emerging biomarkers for a validation set, each capable of discriminating between the plasma of the patients from healthy individuals. Among the 42 ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 31 (73.8%), 19 (45.2%), and 19 (45.2%) samples, respectively. Among pre-treatment ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 8/8 (100%), 7/8 (87.5%), and 7/8 (87.5%) samples, respectively. Methylated CALML5, DNAJC5G, and LY6D were found in 2/8 (25.0%), 0/8 (0%), and 1/8 (12.5%) of the final samples in the series, respectively. Here, we present the relationship between the methylation status of three specific genes and cancer recurrence for risk classification of HPV-associated OPC cases. In conclusion, ctDNA analysis has the potential to aid in determining patient prognosis and real-time surveillance for disease recurrences and serves as an alternative method of screening for HPV-associated OPC.

Neuropeptide receptor genes GHSR and NMUR1 are candidate epigenetic biomarkers and predictors for surgically treated patients with oropharyngeal cancer.

Pathological staging and histological grading systems are useful, but imperfect, predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Aberrant promoter methylation is the main type of epigenetic modification that plays a role in the inactivation of tumor suppressor genes. To identify new potential prognostic markers, we investigated the promoter methylation status of five neuropeptide receptor genes. The methylation status of the target genes was compared with clinical characteristics in 278 cases; 72 hypopharyngeal cancers, 54 laryngeal cancers, 75 oropharyngeal cancers, and 77 oral cavity cancers were studied. We found that the NTSR1, NTSR2, GHSR, MLNR, and NMUR1 promoters were methylated in 47.8%, 46.8%, 54.3%, 39.2%, and 43.5% of the samples, respectively. GHSR and NMUR1 promoter methylation independently predicted recurrence in HNSCC. In patients with oropharyngeal cancer (n = 75), GHSR and NMUR1 promoter methylation significantly correlates with survival in surgically treated patients. We classified our patients as having a low, intermediate, or high-risk of death based on three factors: HPV status, and GHSR and NMUR1 promoter methylation. The disease-free survival (DFS) rates were 87.1%, 42.7%, and 17.0%, respectively. Combined data analysis of the methylation status of ten-eleven translocation (TET) family genes indicated a trend toward greater methylation indices as the number of TET methylation events increased. In the current study, we presented the relationship between the methylation status of the GHSR and NMUR1 genes and recurrence in HNSCC, specifically in risk classification of oropharyngeal carcinomas cases with HPV status.

Epigenetic inactivation of galanin receptor 1 in head and neck cancer.

PURPOSE: 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. 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. 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.

Genes Located on 18q23 Are Epigenetic Markers and Have Prognostic Significance for Patients with Head and Neck Cancer.

Loss of heterozygosity (LOH) on chromosome 18q23 is associated with significantly decreased survival in head and neck cancer. In agreement with such tumor suppressive roles, the loss of function of genes located in this region can be achieved through LOH and promotor hypermethylation. In this study, the methylation status of promoters of 18q23 genes in 243 head and neck cancer patients was assessed by quantitative methylation-specific PCR. Promoter methylation was then compared to various clinical characteristics and patient survival. GALR1 and SALL3 promoter methylation correlated with reduced disease-free survival (log-rank test, p = 0.018 and p = 0.013, respectively). Furthermore, based on multivariate Cox proportional hazards analysis, these methylation events were associated with poor disease-free survival, with hazard ratios of 1.600 (95% confidence interval: CI, 1.027⁻2.493; p = 0.038) and 1.911 (95% CI, 1.155⁻3.162; p = 0.012), respectively. By comparison, GALR1 and SALL3 methylation were not prognostic for overall survival in The Cancer Genome Atlas (TCGA) cohort. Our findings suggest that the methylation status of 18q23 genes could serve as important biomarkers for the prediction of clinical outcomes in well-annotated head and neck squamous cell carcinoma cohorts. GALR1 and SALL3 methylation could thus help to facilitate risk stratification for individualized treatment.

SALL2 Is a Novel Prognostic Methylation Marker in Patients with Oral Squamous Carcinomas: Associations with SALL1 and SALL3 Methylation Status.

Staging and pathological grading systems are convenient, but imperfect predictors of recurrence of head and neck squamous cell carcinoma. Therefore, to identify potential alternative prognostic markers, we investigated the methylation status of the promoter of Sal-like protein 2 (SALL2). SALL2 mRNA expression was absent in 8/9 (88.9%) University of Michigan squamous cell carcinoma cell lines, whereas two nonmalignant cell lines had stable expression. The normalized methylation value of SALL2 in cancer cell lines was significantly higher than in normal cell lines. SALL2 methylation found in 74 of 233 (31.8%) tumor specimens was correlated with the methylation status of both SALL1 and SALL3. SALL2 methylation was not associated with any difference in disease-free survival (DFS). Therefore, the presence of SALL2 methylation was statistically correlated with a decrease in DFS in patients with oral cancer (log-rank test, p = 0.032). Furthermore, it was associated with disease recurrence in 36.2% of oral cancer cases, with an odds ratio of 2.922 (95% confidence interval = 1.198-7.130; p = 0.018) by multivariate Cox proportional hazard regression analysis. This study suggests that cytosine-phosphate- guanosine (CpG) hypermethylation is a likely mechanism of SALL2 inactivation and supports the hypothesis that SALL2 could serve as an important clinical risk assessment.

Promoter methylation of galanin receptors as epigenetic biomarkers for head and neck squamous cell carcinomas.

INTRODUCTION: While remarkable progress has been made in standard treatments for head and neck squamous cell carcinomas (HNSCCs), the long-term survival remains at an unsatisfactory 40-50%. To improve the survival rate, biomarkers for optimal treatment selection and prognostic prediction, as well as novel, low-toxicity treatment strategies, are required. Galanin receptor (GALR) 1 and GALR2 are well-studied tumor suppressors in HNSCCs. Compared with other clinicopathological factors, the epigenetic variants of GALRs have been found to be the most powerful markers to predict the prognosis of HNSCC patients. Areas covered: This review outlines the functions and signaling pathways of GALRs and explains the potential of GALR promoter methylation as a biomarker for HNSCC prognosis. We also summarize recent developments in promoter methylation studies in HNSCC and indicate future directions for GALR promoter methylation studies. Expert commentary: GALR studies have highlighted two major aspects with implications in HNSCC - that G-protein coupled receptors (GPCRs) act as tumor suppressor genes and that GALR promoter methylation is significantly related to the carcinogenesis of HNSCC. The findings of GALR studies can be applied to studies on other GPCRs and further in-depth DNA methylation studies. Deeper insights into GPCR epigenetics are expected to markedly improve HNSCC treatment.

5-Hydroxymethylcytosine and ten-eleven translocation dioxygenases in head and neck carcinoma.

Ten-eleven translocation (TET) enzymes are implicated in DNA demethylation through dioxygenase activity, which converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC). However, the specific roles of TET enzymes and 5-hmC levels in head and neck squamous cell carcinoma (HNSCC) have not yet been evaluated. In this study, we analyzed 5-hmC levels and TET mRNA expression in a well-characterized dataset of 117 matched pairs of HNSCC tissues and normal tissues. 5-hmC levels and TET mRNA expression were examined via enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. 5-hmC levels were evaluated according to various clinical characteristics and prognostic implications. Notably, we found that 5-hmC levels were significantly correlated with tumor stage (P = 0.032) and recurrence (P = 0.018). Univariate analysis revealed that low levels of 5-hmC were correlated with poor disease-free survival (DFS; log-rank test, P = 0.038). The expression of TET family genes was not associated with outcomes. In multivariate analysis, low levels of 5-hmC were evaluated as a significant independent prognostic factor of DFS (hazard ratio: 2.352, 95% confidence interval: 1.136-4.896; P = 0.021). Taken together, our findings showed that reduction of TET family gene expression and subsequent low levels of 5-hmC may affect the development of HNSCC.