Gene promoter methylation signature predicts survival of head and neck squamous cell carcinoma patients

Infection with high-risk types of human papilloma virus (HPV) is currently the best-established prognostic marker for head and neck squamous cell carcinoma (HNSCC), one of the most common and lethal human malignancies worldwide. Clinical trials have been launched to address the concept of treatment de-escalation for HPV-positive HNSCC with the final aim to reduce treatment related toxicity and debilitating long-term impacts on the quality of life. However, HPV-related tumors are mainly restricted to oropharyngeal SCC (OPSCC) and there is an urgent need to establish reliable biomarkers for all patients at high risk for treatment failure. A patient cohort (n = 295) with mainly non-OPSCC (72.9%) and a low prevalence of HPV16-related tumors (8.8%) was analyzed by MassARRAY to determine a previously established prognostic methylation score (MS). Kaplan-Meier revealed a highly significant correlation between a high MS and a favorable survival for OPSCC (P = 0.0004) and for non-OPSCC (P<0.0001), which was confirmed for all HNSCC by multivariate Cox regression models (HR: 9.67, 95% CI [4.61–20.30], P<0.0001). Next, we established a minimal methylation signature score (MMSS), which consists of ten most informative of the originally 62 CpG units used for the MS. The prognostic value of the MMSS was confirmed by Kaplan-Meier analysis for all HNSCC (P<0.0001) and non-OPSCC (P = 0.0002), and was supported by multivariate Cox regression models for all HNSCC (HR: 2.15, 95% CI [1.36–3.41], P = 0.001). In summary, the MS and the MMSS exhibit an excellent performance as prognosticators for survival, which is not limited by the anatomical site, and both could be implemented in future clinical trials.     

DNA methylation biomarkers for head and neck squamous cell carcinoma

DNA methylation plays an important role in the etiology and pathogenesis of head and neck squamous cell carcinoma (HNSCC). The current study aimed to identify aberrantly methylated-differentially expressed genes (DEGs) by a comprehensive bioinformatics analysis. In addition, we screened for DEGs affected by DNA methylation modification and further investigated their prognostic values for HNSCC. We included microarray data of DNA methylation (GSE25093 and GSE33202) and gene expression (GSE23036 and GSE58911) from Gene Expression Omnibus. Aberrantly methylated-DEGs were analyzed with R software. The Cancer Genome Atlas (TCGA) RNA sequencing and DNA methylation (Illumina HumanMethylation450) databases were utilized for validation. In total, 27 aberrantly methylated genes accompanied by altered expression were identified. After confirmation by The Cancer Genome Atlas (TCGA) database, 2 hypermethylated-low-expression genes (FAM135B and ZNF610) and 2 hypomethylated-high-expression genes (HOXA9 and DCC) were identified. A receiver operating characteristic (ROC) curve confirmed the diagnostic value of these four methylated genes for HNSCC. Multivariate Cox proportional hazards analysis showed that FAM135B methylation was a favorable independent prognostic biomarker for overall survival of HNSCC patients.     

Identification of 4-lncRNA prognostic signature in head and neck squamous cell carcinoma

Deregulated long noncoding RNAs (lncRNA) have been critically implicated in tumorigenesis and serve as novel diagnostic and prognostic biomarkers. Here we sought to develop a prognostic lncRNA signature in patients with head and neck squamous cell carcinoma (HNSCC). Original RNA-seq data of 499 HNSCC samples were retrieved from The Cancer Genome Atlas database, which was randomly divided into training and testing set. Univariate Cox regression survival analysis, robust likelihood-based survival model and random sampling iterations were applied to identify prognostic lncRNA candidates in the training cohort. A prognostic risk score was developed based on the Cox coefficient of four individual lncRNA imputed as follows: (0.14546 × expression level of RP11-366H4.1) + (0.27106 × expression level of LINC01123) + (0.54316 × expression level of RP11-110I1.14) + (−0.48794 × expression level of CTD-2506J14.1). Kaplan-Meier analysis revealed that patients with high-risk score had significantly reduced overall survival as compared with those with low-risk score when patients in training, testing, and validation cohorts were stratified into high- or low-risk subgroups. Multivariate survival analysis further revealed that this 4-lncRNA signature was a novel and important prognostic factor independent of multiple clinicopathological parameters. Importantly, ROC analyses indicated that predictive accuracy and sensitivity of this 4-lncRNA signature outperformed those previously well-established prognostic factors. Noticeably, prognostic score based on quantification of these 4-lncRNA via qRT-PCR in another independent HNSCC cohort robustly stratified patients into subgroups with high or low survival. Taken together, we developed a robust 4-lncRNA prognostic signature for HNSCC that might provide a novel powerful prognostic biomarker for precision oncology.     

SUZ12 is a novel putative oncogene promoting tumorigenesis in head and neck squamous cell carcinoma

The suppressor of zest 12 (SUZ12), one of the core polycomb repressive complex 2 (PRC2) components, has increasingly appreciated as a key mediator during human tumorigenesis. However, its expression pattern and oncogenic roles in head and neck squamous cell carcinoma (HNSCC) remain largely unexplored yet. Here, we sought to determine its expression pattern, clinicopathological significance and biological roles in HNSCC. Through data mining and interrogation from multiple publicly available databases, our bioinformatics analyses revealed that SUZ12 mRNA was significantly overexpressed in multiple HNSCC patient cohorts. Moreover, SUZ12 protein was markedly up‐regulated in primary HNSCC samples from our patient cohort as assessed by immunohistochemical staining and its overexpression significantly associated with cervical node metastasis and reduced overall and disease‐free survival. In the 4‐nitroquinoline 1‐oxide (4NQO)‐induced HNSCC mouse model, increased SUZ12 immunostaining was observed along with disease progression from epithelial hyperplasia to squamous cell carcinoma in tongue. Furthermore, shRNA‐mediated SUZ12 knock‐down significantly inhibited cell proliferation, migration and invasion in HNSCC cells, and resulted in compromised tumour growth in vivo. Collectively, our data reveal that SUZ12 might serve as a putative oncogene by promoting cell proliferation, migration and invasion, and also a novel biomarker with diagnostic and prognostic significance for HNSCC.     

Hypoxia-related LncRNAs signature predicts prognosis and is associated with immune infiltration and progress of head and neck squamous cell carcinoma

BackgroundDisclosing prognostic information is necessary to enable good treatment selection and improve patient outcomes. Previous studies suggest that hypoxia is associated with an adverse prognosis in patients with HNSCC and that long non-coding RNAs (lncRNAs) show functions in hypoxia-associated cancer biology. Nevertheless, the understanding of lncRNAs in hypoxia related HNSCC progression remains confusing.MethodsData were downloaded from TCGA and GEO database. Bioinformatic tools including R packages GEOquery, limma, pheatmap, ggplot2, clusterProfiler, survivalROC and survcomp and LASSO cox analysis were utilized. Si-RNA transfection, CCK8 and real-time quantified PCR were used in functional study.ResultsGEO data (GSE182734) revealed that lncRNA regulation may be important in hypoxia related response of HNSCC cell lines. Further analysis in TCGA data identified 314 HRLs via coexpression analysis between differentially expressed lncRNAs and hypoxia-related mRNAs. 23 HRLs were selected to build the prognosis predicting model using lasso Cox regression analyses. Our model showed excellent performance in predicting survival outcomes among patients with HNSCC in both the training and validation sets. We also found that the risk scores were related to tumor stage and to tumor immune infiltration. Moreover, LINC01116 were selected as a functional study target. The knockdown of LINC01116 significantly inhibited the proliferation of HNSCC cells and effected the hypoxia induced immune and the NF-κB/AKT signaling.ConclusionsData analysis of large cohorts and functional experimental validation in our study suggest that hypoxia related lncRNAs play an important role in the progression of HNSCC, and its expression model can be used for prognostic prediction.     

A robust six-miRNA prognostic signature for head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) remains a major health problem worldwide. We aimed to identify a robust microRNA (miRNA)-based signature for predicting HNSCC prognosis. The miRNA expression profiles of HNSCC were obtained from The Cancer Genome Atlas (TCGA) database. The TCGA HNSCC cohort was randomly divided into the discovery and validation cohort. A miRNA-based prognostic signature was built up based on TGCA discovery cohort, and then further validated. The downstream targets of prognostic miRNAs were subjected to functional enrichment analyses. The role of miR-1229-3p, a prognosis-related miRNA, in tumorigenesis of HNSCC was further evaluated. A total of 305 significantly differentially expressed miRNAs were found between HNSCC samples and normal tissues. A six-miRNA prognostic signature was constructed, which exhibited a strong association with overall survival (OS) in the TCGA discovery cohort. In addition, these findings were successfully confirmed in TCGA validation cohort and our own independent cohort. The miRNA-based signature was demonstrated as an independent prognostic indicator for HNSCC. A risk signature-based nomogram model was constructed and showed good performance for predicting the OS for HNSCC. The functional analyses revealed that the downstream targets of these prognostic miRNAs were closely linked to cancer progression. Mechanistically, in vitro analysis revealed that miR-1229-3p played a tumor promoting role in HNSCC. In conclusion, our study has developed a robust miRNA-based signature for predicting the prognosis of HNSCC with high accuracy, which will contribute to improve the therapeutic outcome.     

Downregulation of the circadian Period family genes is positively correlated with poor head and neck squamous cell carcinoma prognosis

ABSTRACT

We investigated the relationship between head and neck squamous cell carcinoma (HNSCC) and the mRNA and protein expression levels of the circadian genes of the Period (Per) family, Per1, Per2 and Per3. Tissue sections of HNSCC and normal head and neck tissues from two patient cohorts from two different hospitals were collected to assess the mRNA and protein expressions of the three Per family genes using real-time quantitative PCR (RT-PCR) and immunohistochemistry (IHC). The clinicopathological features and disease prognosis for the latter cohort were analyzed through IHC and statistical methods. Protein positive expression levels of the three Per family genes in HNSCC tissues was found to be approximately two times lower than that in normal tissues (p < .01). Moreover, patients with locally advanced HNSCC showed significantly greater downregulation of Per1, Per2 and Per3 mRNA expression levels as compared to patients with early-stage cancer (p < .05). Immunohistochemical examination of HNSCC patient tissues revealed a positive correlation between the Per family protein expression and the clinical tumor staging (p < .05). In addition, the Per protein-positive expression group showed higher 3-year survival rates [overall survival (OS) and progression-free survival (PFS)] as assessed by Kaplan-Meier plots and statistical analysis (p < .05). Our findings confirm the positive correlation between Per family gene expression and survival outcomes and support their role as prognostic markers for HNSCC.     

CD147 promotes progression of head and neck squamous cell carcinoma via NF‐kappa B signaling

CD147/basigin (BSG) is highly upregulated in many types of cancer, our previous study has found that CD147/BSG is highly expressed in head and neck squamous cell carcinoma (HNSCC) stem cells, but its role in HNSCC and the underlying mechanism is still unknown. In this study, we investigated the role of CD147 in the progression of HNSCC. Real‐time PCR, western blot and immunohistochemistry were used to detect the expression of CD147 in total 189 HNSCC tissues in compared with normal tissues. In addition, we used proliferation, colony formation, cell cycle and apoptosis, migration and invasion as well as wound‐healing assay to determine the biological roles of CD147 in HNSCC. Then, a xenograft model was performed to evaluate tumor‐promoting and metastasis‐promoting role of CD147 in HNSCC. The results showed that upregulated CD147 expression was associated with aggressive clinicopathologic features in HNSCC. In addition, CD147 promoted proliferation, migration and reduced the apoptosis phenotype of HNSCC cells in vitro as well as tumor initiation and progression in vivo. Furthermore, we demonstrated that CD147 promoted HNSCC progression through nuclear factor kappa B signaling. Therefore, we concluded that CD147 promoted tumor progression in HNSCC and might be a potential prognostic and treatment biomarker for HNSCC.     

Wnt3a promotes radioresistance via autophagy in squamous cell carcinoma of the head and neck

The canonical Wnt/β‐catenin signalling pathway and autophagy play critical roles in cancer progression. However, the role of Wnt‐mediated autophagy in cancer radioresistance remains unclear. In this study, we found that irradiation activated the Wnt/β‐catenin and autophagic signalling pathways in squamous cell carcinoma of the head and neck (SCCHN). Wnt3a is a classical ligand that activated the Wnt/β‐catenin signalling pathway, induced autophagy and decreased the sensitivity of SCCHN to irradiation both in vitro and in vivo. Further mechanistic analysis revealed that Wnt3a promoted SCCHN radioresistance via protective autophagy. Finally, expression of the Wnt3a protein was elevated in both SCCHN tissues and patients' serum. Patients showing high expression of Wnt3a displayed a worse prognosis. Taken together, our study indicates that both the canonical Wnt and autophagic signalling pathways are valuable targets for sensitizing SCCHN to irradiation.     

Prediction of head and neck squamous cell carcinoma survival based on the expression of 15 lncRNAs

Recent evidence suggests that long noncoding RNAs (lncRNAs) are essential regulators of many cancer-related processes, including cancer cell proliferation, invasion, and migration. There is thus a reason to believe that the detection of lncRNAs may be useful as a diagnostic and prognostic strategy for cancer detection, however, at present no effective genome-wide tests are available for clinical use, constraining the use of such a strategy. In this study, we performed a comprehensive assessment of lncRNAs expressed in samples in the head and neck squamous cell carcinoma (HNSCC) cohort available in The Cancer Genome Atlas database. A risk score (RS) model was constructed based on the expression data of these 15 lncRNAs in the validation data set of HNSCC patients and was subsequently validated in validation data set and the entire data set. We were able to stratify patients into high- and low-risk categories, using our lncRNA expression panel to determine an RS, with significant differences in overall survival (OS) between these two groups in our test set (median survival, 1.863 vs. 5.484 years; log-rank test, p < 0.001). We were able to confirm the predictive value of our 15-lncRNA signature using both a validation data set and a full data set, finding our signature to be reproducible and effective as a means of predicting HNSCC patient OS. Through the multivariate Cox regression and stratified analyses, we were further able to confirm that the predictive value of this RS was independent of other predictive factors such as clinicopathological parameters. The Gene set enrichment analysis revealed potential functional roles for these 15 lncRNAs in tumor progression. Our findings indicate that an RS established based on a panel of lncRNA expression signatures can effectively predict OS and facilitate patient stratification in HNSCC.     

Epiberberine suppresses the metastasis of head and neck squamous cell carcinoma cells by regulating the MMP-13 and JNK pathway

Head and neck squamous cell carcinoma (HNSCC) is one of the most common histological types of head and neck cancer. Epiberberine is a potent antitumour agent for several types of cancer. This study is aimed at investigating the regulatory and molecular mechanism of epiberberine on HNSSC cell metastasis. The results showed that epiberberine inhibited the motility of Ca9-22 and FaDu cell lines at nontoxicity doses. Moreover, the epithelial-mesenchymal transition (EMT)-related proteins, vimentin, snail and slug, were found suppressing after epiberberine treatments. In addition, the JNK signalling cascade and the metalloproteinase 13 (MMP-13) expression were also found downregulated by epiberberine. In conclusion, the present study demonstrates that epiberberine suppresses cell migration and invasion by regulating the JNK pathway and MMP-13. These results suggest that epiberberine could be a potential antimetastatic agent in HNSCC cells.     

Human head and neck squamous cell carcinoma cells are both targets and effectors for the angiogenic cytokine, VEGF

Former vascular endothelial growth factor (VEGF)-head and neck squamous cell carcinoma (HNSCC) studies have focused on VEGF's contributions toward tumor-associated angiogenesis. Previously, we have shown that HNSCC cells produce high levels of VEGF. We therefore hypothesized that VEGF serves a biphasic role, that is, pro-angiogenic and pro-tumorigenic in HNSCC pathogenesis. Western blots confirmed the presence of VEGF's primary mitogenic receptors, VEGFR-2/KDR and VEGFR-1/Flt-1 in cultured HNSCC cells. Subsequent studies evaluated VEGF's effects on HNSCC intracellular signaling, mitogenesis, invasive capacities, and matrix metalloproteinases (MMPs) activities. Introduction of hrVEGF(165) initiated ROS-mediated intracellular signaling, resulting in kinase activation and phosphorylation of KDR and Erk1/2. As high endogenous VEGF production rendered HNSCC cells refractory to exogenous VEGF's mitogenic effects, siRNA was employed, inhibiting endogenous VEGF production for up to 96 h. Relative to transfection vector matched controls, siRNA treated HNSCC cells showed a significant decrease in proliferation at both 30 and 50 nM siRNA doses. Addition of exogenous hrVEGF(165) (30 and 50 ng/ml) to siRNA-silenced HNSCC cells resulted in dose-dependent increases in cell proliferation. Cell invasion assays showed VEGF is a potent HNSCC chemoattractant and demonstrated that VEGF pre-treatment enhanced invasiveness of HNSCC cells. Conditioned media from VEGF challenged HNSCC cells showed a moderate increase in gelatinase activity. Our results demonstrate, for the first time, that HNSCC cells are both targets and effectors for VEGF. These data introduce the prospect that VEGF targeted therapy has the potential to fulfill both anti-angiogenic and anti-tumorigenic functions.     

LRP1B mutation is associated with tumor HPV status and promotes poor disease outcomes with a higher mutation count in HPV-related cervical carcinoma and head & neck squamous cell carcinoma

Human papillomavirus (HPV) infection and gene mutations were reputed as key factors in cervical carcinoma (CC) and head and neck squamous cell carcinoma (HNSCC). However, the associations of HPV status and gene mutations remain to be determined. This study aims to identify molecular patterns of LRP1B mutation and HPV status via rewiring tumor samples of HNSCC (n=1478) and CC (n=178) from the TCGA dataset. Here, we found that LRP1B mutation was associated with HPV status in CC (P=0.040) and HNSCC (P=0.044), especially in HPV 16 integrated CC (P=0.036). Cancer survival analysis demonstrated that samples with LRP1B mutation showed poor disease outcomes in CC (P=0.013) and HNSCC (P=0.0124). In addition, the expression status of LPR1B was more favorable for prediction than TP53 or RB1 in CC and HNSCC. Mutation clustering analysis showed that samples with LRP1B mutation showed higher mutation count in CC (P=1.76e-67) and HNSCC (P<10e-10). Further analysis identified 289 co-occurrence genes in these two cancer types, which were enriched in PI3K signaling, cell division process, and chromosome segregation process, et al. The 289-co-occurrence gene signature identified a cluster of patients with a higher portion of copy number variation (CNV) lost in the genome, different tumor HPV status (P<10e-10), higher mutation count (P<10e-10), higher fraction genome altered value (P=2.078e-4), higher aneuploidy score (P=3.362e-4), and earlier started the smoking year (P=2.572e-4), which were associated with shorter overall survival (P=0.0103) in CC and HNSCC samples. Overall, LRP1B mutation was associated with tumor HPV status and was an unfavorable prognostic biomarker for CC and HNSCC.     

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC.     

An 11-lncRNA expression could be potential prognostic biomarkers in head and neck squamous cell carcinoma

The aim of our study is to construct the competing endogenous RNA (ceRNA) network of head and neck squamous cell carcinoma (HNSCC) and identify key long noncoding RNAs (lncRNAs) to predict prognosis. The genes whose expression were differentially in HNSCC and normal tissues were explored by the Cancer Genome Atlas database. The ceRNA network was constructed by the Cytoscape software. The lncRNAs which could estimate the overall survival were explored from Cox proportional hazards regression. There are 1997, 589, and 82 mRNAs, lncRNAs, and miRNAs whose expression were statistically significant different, respectively. Then, the network between miRNA and mRNA or miRNA and lncRNA was constructed by miRcode, miRDB, TargetScan, and miRanda. Five mRNAs, 10 lncRNAs, and 3 miRNAs were associated with overall survival. Then, 11-lncRNAs were found to be prognostic factors. Therefore, our research analyzed the potential signature of novel 11-lncRNA as candidate prognostic biomarker from the ceRNA network for patients with HNSCC.