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.     

Type II diabetes and metabolic syndrome in relation to head and neck squamous cell carcinoma risk: A SEER-Medicare database study

BackgroundDiabetes and metabolic syndrome have been found to increase the risk of various cancers. Previous studies indicated that diabetes may increase the risk of head and neck squamous cell carcinoma (HNSCC). Metabolic syndrome has not been investigated as a risk factor. We tested whether type II diabetes or metabolic syndrome were associated with HNSCC using a very large database of medical administrative records, providing the ability to investigate relatively weak effects and stratify by subgroups.MethodsWe identified persons diagnosed with HNSCC between 1994 and 2007 in the Surveillance, Epidemiology, and End Results (SEER)-Medicare database. We selected controls from a 5% sample of Medicare beneficiaries and frequency matched to cases on sex and duration of enrollment. We estimated odds ratios (OR) and 95% confidence intervals (CI) for the association between type II diabetes/metabolic syndrome and HNSCC, adjusted for potential confounders, among 14,022 cases and 42,066 controls.ResultsWe observed a very weak inverse association between type II diabetes and HNSCC (OR = 0.92; 95% CI, 0.88–0.96) and a moderate inverse association for metabolic syndrome (OR = 0.81; 95% CI, 0.78–0.85). Associations were modified by tobacco use, with null results for type II diabetes among never users (OR = 1.00; 95% CI, 0.95–1.06) and inverse associations among ever users (OR = 0.80; 95% CI, 0.75–0.86).ConclusionsWe observed moderate inverse associations between metabolic syndrome and HNSCC and weak inverse associations between type II diabetes and HNSCC, which was contrary to the evidence to date. Inadequate control for confounding factors, such as overweight/obesity, may have influenced results.     

Chromatin dysregulation associated with NSD1 mutation in head and neck squamous cell carcinoma

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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.     

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.     

Proliferation of hematopoietic cell lines induced by a soluble factor derived from human squamous cell carcinomas of the head and neck

The supernatant of a cell line of squamous cell carcinoma of the head and neck (SCCHN), PCI-50, was previously shown to induce activation, promote proliferation and increase antitumor cytotoxicity of freshly purified human natural killer (NK) cells and CD4⁺ T lymphocytes [Arch Otolaryngol Head Neck Surg (1994) in press]. This supernatant was found also to promote the growth of a variety of hematopoietic cell lines, including Jurkat, THP-1, K562, NK-92 or Epstein-Barr-virus-transformed B cell lines. The Jurkat cell line was selected as a reporter cell in an 18-h proliferation assay established to measure the growth-promoting activity of PCI-50 supernatant. The presence of soluble tumor-derived factors able to induce proliferation of Jurkat cells was demonstrated in the supernatant produced by several other SCCHN cell lines but not in that produced by a gastric cancer cell line (HR) or renal cell carcinoma line (5117G8). The growth-promoting PCI-50 supernatant was shown to contain 28±0.5 pg/ml interleukin-6, (IL-6) in vitro but was negative for interferon , IL-1, IL-2, IL-4, tumor necrosis factor , granulocyte/macrophage-colony-stimulating factor and IL-12. The addition of any of these recombinant cytokines to Jurkat cell cultures did not significantly promote growth, while PCI-50 supernatant was consistently growth-stimulatory. This supernatant neither enhanced intracellular Ca²⁺ concentration in Jurkat cells nor induced up-regulation of activation antigens on the cell surface, although it supported growth of Jurkat cells in the absence of IL-2. The growth-promoting activity in the PCI-50 supernatant was acid-labile at pH 2 for 4 h, heat-resistant at 96 °C for 1 h and sensitive to treatments with trypsin and pepsin. Preincubation of the PCI-50 producer cells with tunicamycin or cyclohexamide reduced the level of growth-promoting activity in the supernatant. A partial purification of this activity was achieved using Amicon filtration, chromatography on concanavalin-A-Sepharose and then a hydroxyapatite column and high-pressure liquid chromatography gel filtration. The partially purified glycoprotein had a molecular mass of 50–70 kDa, as detemined by gel filtration.This work was supported in part by the Pathology Education and Research Foundation and American Cancer Society grant IM-696 to TLW     

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.     

Potential for targeting head and neck squamous cell carcinoma with monoclonal antibody K984

Summary In previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts [28, 30]. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratios, as based on the biodistribution data, were at least ten times higher in case of the specific mAb K984 when compared to another non-specific, isotypematched control antibody. mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targeting.     

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.     

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.     

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.     

Circ_0000052/miR-382-3p axis induces PD-L1 expression and regulates cell proliferation and immune evasion in head and neck squamous cell carcinoma

A better understanding of the mechanisms underlying PD-L1 aberrant expression in head and neck squamous cell carcinoma (HNSCC) will help reveal predictive biomarkers and overcome resistance to treatment. In this study, the prognostic significance of PD-L1 in forty-five HNSCC archival samples was determined by qRT-PCR. The biological function associated with malignant behaviour was assessed by PD-L1 depletion, miR-382-3p re-expression and regulation of circ_0000052. The interactions of PD-L1-miRNA and miRNA-circRNA were determined by qRT-PCR, Western blot analysis, dual-luciferase reporter assays and RNA immunoprecipitation assays. PD-L1 was highly expressed in patient samples and cancer cell lines. Higher levels of PD-L1 were associated with patient recurrences and play a pivotal role in regulating cell proliferation, migration, invasion, clonogenicity and apoptosis. In addition to demonstrating that the IFN-γ/JAK2/STAT1 signalling pathway can induce PD-L1 overexpression in HNSCC, a novel mechanism by which upregulated circ_0000052 mediates PD-L1 overexpression was also demonstrated. To do this, circ_0000052 competitively binds to miR-382-3p and alleviates its repression of PD-L1. This leads to overexpression of PD-L1, causing the aggressiveness of the cells. Our data demonstrate that circ_0000052 is oncogenic, and the circ_0000052/miR-382-3p/PD-L1 axis is critical in HNSCC progression. The manipulation of circRNAs/miRNAs in combination with anti-PD-L1 therapy may improve personalized disease management.     

Correlation of the expression of double-stranded RNA-dependent protein kinase (p68) with differentiation in head and neck squamous cell carcinoma

p68 is an inducible protein kinase which is believed to be an important factor in the regulation of both viral and cellular protein synthesis. We have produced a monoclonal antibody (TJ4C4) which specifically detects p68, and which can be used to detect this antigen in formalin-fixed, paraffin-embedded tissues. Because p68 plays an important role in cellular protein synthesis, we hypothesized that it may correlate with normal and neoplastic cellular differentiation. One hundred and seventy-seven head and neck squamous cell carcinoma specimens, representing 82 patients, were studied. The relative amount, frequency, and distribution of p68 expression were determined by microscopic evaluation of ABC immunoperoxidase-stained specimens. A spectrum of immunoreactivity was detected in 156 of 177 tumors, as well as within the normal squamous epithelium. Normal, actively proliferating cells, such as the basal layer of squamous epithelium, expressed comparatively little p68. Increased p68 expression was noted to parallel the morphologic features of cellular differentiation. In neoplastic tissue, p68 expression also increased with the degree of cellular differentiation. These data demonstrate that the expression of p68 parallels the degree of cellular differentiation in squamous cell carcinoma of the head and neck region, as well as within normal squamous mucosa. Therefore, p68 may provide an objective biologic measure of cellular differentiation which does not depend on morphologic features.     

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.     

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.     

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.