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Head and neck squamous cell carcinoma (HNSCC) remains a morbid disease with poor prognosis and treatment that typically leaves patients with permanent damage to critical functions such as eating and talking. Currently only three targeted therapies are FDA approved for use in HNSCC, two of which are recently approved immunotherapies. In this work, we identify biological pathways involved with this disease that could potentially be targeted by current FDA approved cancer drugs and thereby expand the pool of potential therapies for use in HNSCC treatment. We analyzed 508 HNSCC patients with sequencing information from the Genomic Data Commons (GDC) database and assessed which biological pathways were significantly enriched for somatic mutations or copy number alterations. We then further classified pathways as either “light” or “dark” to the current reach of FDA-approved cancer drugs using the Cancer Targetome, a compendium of drug-target information. Light pathways are statistically enriched with somatic mutations (or copy number alterations) and contain one or more targets of current FDA-approved cancer drugs, while dark pathways are enriched with somatic mutations (or copy number alterations) but not currently targeted by FDA-approved cancer drugs. Our analyses indicated that approximately 35–38% of disease-specific pathways are in scope for repurposing of current cancer drugs. We further assess light and dark pathways for subgroups of patient tumor samples according to HPV status. The framework of light and dark pathways for HNSCC-enriched biological pathways allows us to better prioritize targeted therapies for further research in HNSCC based on the HNSCC genetic landscape and FDA-approved cancer drug information. We also highlight the importance in the identification of sub-pathways where targeting and cross targeting of other pathways may be most beneficial to predict positive or negative synergy with potential clinical significance. This framework is ideal for precision drug panel development, as well as identification of highly aberrant, untargeted candidates for future drug development. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) related tumours account for a significant proportion of head and neck squamous cell carcinomas (HNSCCs) in developed countries. They respond better to chemo- and radio-therapy, and have a better stage specific prognosis. To establish their prevalence in China, we assessed a series of histology confirmed HNSCCs collected in Zhejiang and Guangdong provinces by PCR for HPV DNA and by immunohistochemistry for p16 protein status. Among 303 HNSCCs, HPV DNA was detected in 26.4%, with HPV16 DNA in 71% of these. Of HNSCC located in the oropharynx, 38.55% (32/83) were HPV+ve. In this series, p16 status was a relatively poor predictor of HPV status as detected by PCR. The stage specific survival time of HPV+ HNSCCs was significantly longer than for HPV- HNSCC. HPV status should be assessed for oropharyngeal cancers in China to assist with appropriate management, and prophylaxis against HPV infection should be considered to reduce the incidence of this disease. [ABSTRACT FROM AUTHOR]

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease of significant mortality and with limited treatment options. Recent genomic analysis of HNSCC tumors has identified several distinct molecular classes, of which the mesenchymal subtype is associated with Epithelial to Mesenchymal Transition (EMT) and shown to correlate with poor survival and drug resistance. Here, we utilize an integrated approach to characterize the molecular function of ETS1, an oncogenic transcription factor specifically enriched in Mesenchymal tumors. To identify the global ETS1 cistrome, we have performed integrated analysis of RNA-Seq, ChIP-Seq and epigenomic datasets in SCC25, a representative ETS1high mesenchymal HNSCC cell line. Our studies implicate ETS1 as crucial regulator of broader oncogenic processes and specifically Mesenchymal phenotypes, such as EMT and cellular invasion. We found that ETS1 preferentially binds cancer specific regulator elements, in particular Super-Enhancers of key EMT genes, highlighting its role as a master regulator. Finally, we show evidence that ETS1 plays a crucial role in regulating the TGF-β pathway in Mesenchymal cell lines and in leading-edge cells in primary HNSCC tumors that are endowed with partial-EMT features. Collectively our study highlights ETS1 as a key regulator of TGF-β associated EMT and reveals new avenues for sub-type specific therapeutic intervention. [ABSTRACT FROM AUTHOR]

The overall survival for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) remains low, with little progress made over decades. Cisplatin, most frequently used for HNSCC treatment, activates mitochondria-dependent apoptosis through the BCL-2 family proteins. We have previously demonstrated that the pro-apoptotic BH3-only protein, NOXA plays a critical role in this process. NOXA binds and inactivates anti-apoptotic MCL-1, while the BCL-2 inhibitor ABT-263 is capable of inactivating anti-apoptotic BCL-2 and BCL-XL. We hypothesized that combination of NOXA and ABT-263 treatment increases cell death by simultaneously inhibiting anti-apoptotic BCL-2 family proteins in HNSCC cells. Here, we demonstrated that combination of ectopic NOXA expression and ABT-263 enhanced apoptosis in p53-inactive, p53 wild-type, and human papillomavirus (HPV)-positive HNSCC cell lines. Furthermore, a retinoid derivative and an endoplasmic reticulum stress inducer, fenretinide, induced NOXA, and combination of fenretinide and ABT-263 strongly induced apoptosis in HNSCC cells regardless of the HPV or p53 statuses. We also found that MCL-1 and BCL-XL are the primary targets of apoptosis induced by the combinations. These results will develop novel and alternative therapeutic strategies to directly modify the cell death machinery in HNSCC. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinoma (HNSCC) affects about 700.000 individuals per year worldwide with oral squamous cell carcinoma (OSCC) as a major subcategory. Despite a comprehensive treatment concept including surgery, radiation, and chemotherapy the 5-year survival rate is still only about 50 percent. Chronic inflammation is one of the hallmarks of carcinogenesis. Until now, little is known about the premalignant status of oral lichen planus (OLP) and molecular alterations in OLP are still poorly characterized. Our study aims to delineate differential DNA methylation patterns in OLP, OSCC, and normal oral mucosa. By applying a bead chip approach, we identified altered chromosomal patterns characteristic for OSCC while finding no recurrent alterations in OLP. In contrast, we identified numerous alterations in the DNA methylation pattern in OLP, as compared to normal controls, that were also present in OSCC. Our data support the hypothesis that OLP is a precursor lesion of OSCC sharing multiple epigenetic alterations with OSCC. [ABSTRACT FROM AUTHOR]

Aim: Characterizing tumor heterogeneity with textural indices extracted from 18F-fluorodeoxyglucose positron emission tomography (FDG PET/CT) is of growing interest in oncology. Several series showed promising results to predict survival in patients with head and neck squamous cell carcinoma (HNSCC), analyzing various tumor segmentation methods and textural indices. This preliminary study aimed at assessing the inter-observer and inter-segmentation method variability of textural indices in HNSCC pre-therapeutic FDG PET/CT. Materials and methods: Consecutive patients with HNSCC referred in our department for a pre-therapeutic FDG PET/CT from January to March 2016 were retrospectively included. Two nuclear medicine physicians separately segmented all tumors using 3 different segmentation methods: a relative standardized uptake value (SUV) threshold (40%SUVmax), a signal-to-noise adaptive SUV threshold (DAISNE) and an image gradient-based method (PET-EDGE). SUV and metabolic tumor volume were recorded. Thirty-one textural indices were calculated using LIFEx software (). After correlation analysis, selected indices’ inter-segmentation method and inter-observer variability were calculated. Results: Forty-three patients (mean age 63.8±9.3y) were analyzed. Due to a too small segmented tumor volume of interest, textural analysis could not be performed in 6, 11 and 15 cases with respectively DAISNE, 40%SUVmax and PET-EDGE segmentation methods. Five independent textural indices were selected (Homogeneity, Correlation, Entropy, Busyness and LZLGE). There was a high inter-contouring method variability for Homogeneity, Correlation, Entropy and LZLGE (p<0.0001 for each index). The inter-observer reproducibility analysis revealed an excellent agreement for 3 indices (Homogeneity, Correlation and Entropy) with an intraclass correlation coefficient higher than 0.90 for the 3 methods. Conclusions: This preliminary study showed a high variability of 4 out of 5 textural indices (Homogeneity, Correlation, Entropy and LZLGE) extracted from pre-therapeutic FDG PET/CT in HNSCC using 3 different contouring methods. However, for each method, there was an excellent agreement between observers for 3 of these textural indices (Homogeneity, Correlation and Entropy). [ABSTRACT FROM AUTHOR]

Background: Human papillomavirus (HPV) is the cause of a fraction of head and neck squamous cell carcinoma. Although this relation is well-known, it is still not clear the role of HPV in premalignant oral lesions such as oral lichen planus (OLP) and dysplasia. We aimed to evaluate the HPV-DNA prevalence and type distribution in a set of oral biopsies obtained from patients diagnosed with OLP and dysplasia, as well as the role of HPV in these lesions. Methods: A retrospective cohort of all premalignant oral lesions consecutively diagnosed from March 30th 1995 to May 21st 2014 at Hospital of Bellvitge and Odontological University Hospital of Bellvitge was identified and classified in four groups: OLP (groups 1 and 2) and dysplasias (groups 3 and 4) that progressed or not to invasive cancer during follow-up. A random selection targeting 25 cases was aimed to be performed for each group. All selected cases were subjected to pathological evaluation, DNA quality control and HPV-DNA detection. HPV-DNA positive samples were further subject to p16INK4a analysis. Results: A total of 83 cases yielded a valid HPV-DNA result. From those, 7 and 34 cases were OLP that progressed or not to invasive cancer during follow-up, whereas 24 and 18 cases were displasias that progressed or not to invasive cancer during follow-up, respectively. HPV-DNA was detected in 4 samples (3 dysplastic lesions and 1 OLP). Two samples were HPV16 positive (2%), 1 sample HPV18 positive (1%) and 1 sample (1%) was HPV indeterminate. Two out of four HPV-DNA positive cases had high p16INK4a expression and none of the HPV positive cases progressed to invasive cancer during long-term follow-up. Conclusions: We found a low HPV-DNA attributable fraction in premalignant lesions of the oral cavity, suggesting that HPV is unlikely to play a significant role in oral carcinogenesis in our setting. [ABSTRACT FROM AUTHOR]

Fanconi anemia is a rare genome instability disorder with extreme susceptibility to squamous cell carcinoma of the head and neck and anogenital tract. In patients with this inherited disorder, the risk of head and neck cancer is 800-fold higher than in the general population, a finding which might suggest a viral etiology. Here, we analyzed the possible contribution of human polyomaviruses to FA-associated head and neck squamous cell carcinoma (HNSCC) by a pan-polyomavirus immunohistochemistry test which detects the T antigens of all known human polyomaviruses. We observed weak reactivity in 17% of the HNSCC samples suggesting that based on classical criteria, human polyomaviruses are not causally related to squamous cell carcinomas analyzed in this study. [ABSTRACT FROM AUTHOR]

Purpose: The prognostic impact of circulating tumor cells (CTC) on disease recurrence, progression and survivals in patients with head and neck squamous cell carcinoma (HNSCC) has not been adequately described. The objective of this study was to determine the impacts of the presence of CTC on loco-regional recurrence and survival of HNSCC patients by conducting a systematic review and meta-analysis. Methods: A comprehensive search for articles published between 1990 and 2016 was conducted and data from these studies were extracted, using the MEDLINE, Cochrane Library, and EMBASE databases. The main outcomes were overall survival (OS) and recurrence-free survival (RFS) of HNSCC patients. Pooled hazard ratio (HR) and 95% confidence intervals (95%CI) were calculated using the random effect model for outcomes. The quality of the studies, heterogeneity and publication bias were assessed with the appropriate statistical methods. Results: Six eligible studies with 429 patients were identified. The presence of CTC was significantly associated shorter RFS (HR = 4.88 [95%CI: 1.93–12.35], P < 0.001). However, it could not predict patients’ OS (HR = 1.92 [95%CI: 0.93–3.96], P = 0.078). The following analyses using univariable values of each study also made the similar results (HR = 1.70 [95%CI: 0.83–3.45] for OS, HR = 3.79 [95%CI: 2.02–7.13] for RFS). Heterogeneity and publication bias were not significant, except one enrolled study. Conclusions: The presence of CTC is not a significant prognostic indicator for OS of patients with HNSCC, although it could reflect the outcomes of loco-regional disease. [ABSTRACT FROM AUTHOR]

Resistance to antitumor immunity can be promoted by the oncogenic pathways operational in human cancers, including the epidermal growth factor receptor (EGFR) pathway. Here we studied if and how EGFR downstream signaling in head and neck squamous cell carcinoma (HNSCC) can affect the attraction of immune cells. HPV-negative and HPV-positive HNSCC cell lines were analyzed in vitro for CCL2, CCL5, CXCL9, CXCL10, IL-6 and IL-1β expression and the attraction of T cells under different conditions, including cetuximab treatment and stimulation with IFNγ and TNFα using qPCR, ELISA and migration experiments. Biochemical analyses with chemical inhibitors and siRNA transfection were used to pinpoint the underlying mechanisms. Stimulation of HNSCC cells with IFNγ and TNFα triggered the production of T-cell attracting chemokines and required c-RAF activation. Blocking of the EGFR with cetuximab during this stimulation increased chemokine production in vitro, and augmented the attraction of T cells. Mechanistically, cetuximab decreased the phosphorylation of MEK1, ERK1/2, AKT, mTOR, JNK, p38 and ERK5. Chemical inhibition of EGFR signaling showed a consistent and pronounced chemokine production with MEK1/2 inhibitor PD98059 and JNK inhibitor SP600125, but not with inhibitors of p38, PI3K or mTOR. Combination treatment with cetuximab and a MEK1/2 or JNK inhibitor induced the highest chemokine expression. In conclusion, overexpression of EGFR results in the activation of multiple downstream signaling pathways that act simultaneously to suppress type 1 cytokine stimulated production of chemokines required to amplify the attraction of T cells. [ABSTRACT FROM AUTHOR]

Background: Head and neck cancers include carcinomas of the oral cavity, larynx, sinonasal tract and nasopharynx. Studies on molecular expression of prognostic tumour markers in Ghana are scarce. The purpose of this study was to determine the expression of p53, p16, EGFR, Cyclin-D1 and HER2 among patients with non-oropharyngeal head and neck squamous cell carcinoma (HNSCC). Methodology: Tissue microarrays from 154 histologically confirmed non-oropharyngeal HNSCC at the Komfo Anokye Teaching Hospital from 2006–2014 were constructed using duplicate cores of representative and viable areas from tumours. Expression of EGFR, p53, p16, Cyclin-D1 and HER2 was evaluated using immunohistochemistry. Results: For non-oropharyngeal HNSCC, majority of the cases (66.2%; 102/154) had stage IV disease. EGFR was the most expressed molecular marker (29.4%; 25/85) followed by p53 (24.0%; 29/121), p16 (18.3%; 23/126) and Cyclin-D1 (10.0%; 12/120). HER2 was not expressed in any of the cases. There was a significantly (p = 0.022) higher expression of Cyclin-D1 in tumours of the oral cavity (19.6%; 9/46) than in those of the larynx (4.7%; 2/43) and nose (3.2%; 1/31). Tumours in stages I–III were more frequently positive for p16 (28.6%; 12/42) than tumours in stage IV (13.1%; 11/84). Conclusion: Expression of p53, EGFR, p16 and Cyclin-D1 in non-oropharyngeal HNSCC in Ghana is largely similar to what has been reported in published studies from other countries. [ABSTRACT FROM AUTHOR]

Background: Limited information is available about the involvement of human papillomavirus (HPV) in head and neck squamous cell carcinomas (HNSCCs) in Romanian patients. Objective: To evaluate the HPV-attributable fraction in HNSCCs collected in Northeastern Romania. Materials and methods: In total, 189 formalin-fixed paraffin-embedded tissue samples (99 oral cavity tumors, 28 oropharynx, 48 pharynx, and 14 larynx/hypopharynx) were analyzed for HPV DNA and RNA using Luminex-based assays, and for overexpression of p16INK4a (p16) by immunohistochemistry. Results: Of the 189 cases, 23 (12.2%) were HPV DNA-positive, comprising half of the oropharyngeal cases (14/28, 50.0%) and 9/161 (5.6%) of the non-oropharyngeal cases. HPV16 was the most prevalent HPV type (20/23, 86.9%), followed by HPV18 (5/23, 21.7%) and HPV39 (1/23, 4.3%). Only two (2/189, 1.1%) HNSCC cases were HPV-driven, i.e. positive for both HPV DNA and RNA. Conclusion: A very small subset of HNSCC cases within this cohort from Northeastern Romania appeared to be HPV-driven. [ABSTRACT FROM AUTHOR]

Introduction: The transcription factor SOX2 has been identified as a lineage survival oncogene in squamous cell carcinoma and copy number gain is a common event in several human malignancies including head and neck cancer. However, the regulation and function of SOX2 during carcinogenesis as well as its prognostic value appears to be highly context dependent. As an example, high SOX2 expression in lung squamous cell carcinoma (SCC) is related to a favorable prognosis, while it is associated with poor outcome in lung adenocarcinoma. More recently, higher SOX2 levels and improved survival was also reported for head and neck SCC (HNSCC), and silencing of SOX2 expression in HNSCC cell lines revealed a mesenchymal-like phenotype with prominent vimentin expression. So far, SOX2 expression and its clinical relevance for other head and neck cancers, such as adenoid cystic carcinoma (HNACC) have not been sufficiently investigated. Material and methods: SOX2, vimentin and E-cadherin expression was assessed by immunohistochemical staining on serial sections from formalin fixed and paraffin embedded tissue samples of a patient cohort (n = 45) with primary ACC and correlated with patient and tumor characteristics as well as survival. Results: High SOX2 expression was found in 14 (31%) primary tumor specimens and was significantly correlated with a N0 lymph node status (p = 0.04), while low SOX2 expression was correlated with a solid growth pattern (p = 0.031). Of the 45 patients, 27 tumor samples resembled an EMT-like phenotype, as assessed by high vimentin and low E-cadherin levels. However, in HNACC SOX2 levels were neither correlated with vimentin nor with E-cadherin expression, further supporting a context dependent regulation and function of SOX2 in distinct tumor entities. Conclusion: The absence of SOX2 was predominantly found in solid HNACC, which are characterized by a more aggressive phenotype in ACC. However, the underlying molecular mechanisms of SOX2 regulation and function in distinct HNACC subgroups remain to be fully elucidated. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide. The pregnane X receptor (PXR) is a nuclear receptor regulating several target genes associated with cancer malignancy. We here demonstrated a significant effect of PXR on HNSCC cell growth, as evidenced in PXR knock-down experiments. PXR transcriptional activity is more importantly regulated by the presence of coactivators and corepressors than by PXR protein expression. To date, there is scarce information on the regulation of PXR in HNSCC and on its role in the pathogenesis of this disease. Coactivator and corepressor expression was screened through qRT-PCR in 8 HNSCC cell lines and correlated to PXR activity, determined by using a reporter gene assay. All cell lines considerably expressed all the cofactors assessed. PXR activity negatively correlated with nuclear receptor corepressor 2 (NCoR2) expression, indicating a major role of this corepressor in PXR modulation and suggesting its potential as a surrogate for PXR activity in HNSCC. To test the association of NCoR2 with the malignant phenotype, a subset of three cell lines was transfected with an over-expression plasmid for this corepressor. Subsequently, cell growth and chemoresistance assays were performed. To elucidate the mechanisms underlying NCoR2 effects on cell growth, caspase 3/7 activity and protein levels of cleaved caspase 3 and PARP were evaluated. In HNO97 cells, NCoR2 over-expression decreased cell growth, chemoresistance and increased cleaved caspase 3 levels, caspase activity and cleaved PARP levels. On the contrary, in HNO124 and HNO210 cells, NCoR2 over-expression increased cell growth, drug resistance and decreased cleaved caspase 3 levels, caspase activity and cleaved PARP levels. In conclusion, we demonstrated a role of PXR and NCoR2 in the modulation of cell growth in HNSCC. This may contribute to a better understanding of the highly variable HNSCC therapeutic response. [ABSTRACT FROM AUTHOR]

Introduction: The main risk factors for head and neck squamous cell carcinoma (HNSCC) are tobacco and alcohol consumption and human papillomavirus (HPV) infection. However, in a subset of patients, no risk factors can be identified. Glutathione S-transferase π (GTSP1) is a carcinogen-detoxifying enzyme that is activated by exposure to carcinogens, and it is associated with a reduction in response to toxic therapies. We studied the expression of GTSP1 in tumor and non-tumor tissue samples from patients with and without these risks to identify whether GTSP1 expression differs according to exposure to carcinogens. Materials and methods: Non-smoker/non-drinker (NSND) and smoker/drinker (SD) patients were matched according to age, gender, tumor site, TNM stage, grade and histological variants to establish 47 pairs of patients who have been previously tested for HPV. GTSP1 immunostaining was analyzed using a semi-quantitative method with scores ranging from 0 to 3 according to the area of immunostaining. Results: GTSP1 expression was detected in the tumors of both groups. GTSP1 expression was higher in the non-tumor margins of SD patients (p = 0.004). There was no association between GTSP1 expression and positivity for HPV. No differences in survival were observed according to GTSP1 staining in tumors and non-tumor margins. Conclusion: This study showed that GTSP1 was expressed in tumors of HNSCC patients regardless of smoking, drinking or HPV infection status. The difference in GTSP1 expression in non-tumor margins between the two groups may have been due to two possible reasons. First, elevated GTSP1 expression in SD patients might be the result of activation of GTSP1 in response to exposure to carcinogens. Second, alternatively, impairment in the detoxifying system of GTSP1, as observed by the reduced expression of GTSP1, might make patients susceptible to carcinogens other than tobacco and alcohol, which may be the underlying mechanism of carcinogenesis in the absence of risk factors. [ABSTRACT FROM AUTHOR]

Despite modern treatment approaches, survival of patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) remains low and it is difficult to identify patients who derive optimal benefit from treatment. We therefore analyzed which commonly available laboratory and clinical parameters may help improve the prognostication in this patient group. This retrospective monocenter analysis includes 128 patients with recurrent or metastatic SCCHN treated with cetuximab alone or in combination with polychemotherapy as first line therapy. Factors with independent prognostic power in the multivariate analysis were used to build up a score separating patient groups with different survival. Patients had a median age of 61 years and 103 patients were treated with polychemotherapy plus cetuximab. An ECOG score above 1, high CRP and leukocyte levels, less intensive treatment and a time below 12 months from primary diagnosis to relapse remained as independent negative prognostic factors in multivariate analysis. Patients with 0 to 1 risk factors had a median OS of 13.6 months compared to a median OS of less than one month for patients 4 to 5 risk factors (p<0.001). This study identifies 5 clinical and serum values that influence survival of patients with recurrent or metastatic SCCHN treated with cetuximab. By combining these factors to create a score for OS, it is possible to distinguish a group of patients with significantly improved survival and define those most likely to have no benefit from cetuximab treatment. [ABSTRACT FROM AUTHOR]

Background: Despite advances in combined modality therapy, outcomes in head and neck squamous cell cancer (HNSCC) remain dismal with five-year overall survival rates of less than 50%. Prognostic biomarkers are urgently needed to identify patients with a high risk of death after initial curative treatment. Methylation status of the paired-like homeodomain transcription factor 2 (PITX2) has recently emerged as a powerful prognostic biomarker in various cancers. In the present study, the clinical performance of PITX2 methylation was validated in a HNSCC cohort by means of an independent analytical platform (Infinium HumanMethylation450 BeadChip, Illumina, Inc.). Methods: A total of 528 HNSCC patients from The Cancer Genome Atlas (TCGA) were included in the study. Death was defined as primary endpoint. PITX2 methylation was correlated with overall survival and clinicopathological parameters. Results: PITX2 methylation was significantly associated with sex, tumor site, p16 status, and grade. In univariate Cox proportional hazards analysis, PITX2 hypermethylation analyzed as continuous and dichotomized variable was significantly associated with prolonged overall survival of HNSCC patients (continuous: hazard ratio (HR) = 0.19 [95%CI: 0.04–0.88], p = 0.034; dichotomized: HR = 0.52 [95%CI: 0.33–0.84], p = 0.007). In multivariate Cox analysis including established clinicopathological parameters, PITX2 promoter methylation was confirmed as prognostic factor (HR = 0.28 [95%CI: 0.09–0.84], p = 0.023). Conclusion: Using an independent analytical platform, PITX2 methylation was validated as a prognostic biomarker in HNSCC patients, identifying patients that potentially benefit from intensified surveillance and/or administration of adjuvant/neodjuvant treatment, i.e. immunotherapy. [ABSTRACT FROM AUTHOR]

Calcium signal plays an important role in a variety of cancer cell metabolism, but knowledge on its role in head and neck squamous cell carcinoma (HNSCC) is limited. Store-operated calcium entry (SOCE) is the principal Ca2+ entry mechanism that maintains calcium concentration and produces calcium signal in non-excitable cells. SOCE is triggered by stromal interaction molecule 1 (STIM1), which is located in endoplasmic reticulum (ER) as Ca2+ sensor. Although, many studies demonstrated that STIM1 and SOCE play important functions in the regulation of many cancer progressions, their clinical relevance in HNSCC remains unclear. In this study, STIM1 expression levels notably increased in 89% HNSCC tissues compared with those in adjacent normal tissues. Meanwhile, this overexpression was close associated with tumor size but not with neck lymph node metastasis. Thus, this study mainly focuses on STIM1 function in HNSCC tumor growth. Three HNSCC cell lines, namely, TSCCA (oral cancer cell line) and Hep2 (laryngeal cell line) with high STIM1 expression levels and Tb3.1 (oral cancer cell line) with STIM1 expression level lower than previous two cell lines, were selected for in vitro study. Downregulated STIM1 expression levels in TSCCA and Hep2 arrested cells in G0/G1 stages, promoted cell apoptosis, and inhibited cell proliferation. By contrast, upregulated STIM1 expression in Tb3.1 inhibited cell apoptosis and promoted cell proliferation. Induced by thapsigargin (TG), ER stress was amplified when STIM1 expression was downregulated but was attenuated as STIM1 expression was upregulated. Furthermore, TSCCA cell xenograft models confirmed that STIM1 could promote HNSCC tumor growth in vivo. The present study provides new insight into HNSCC molecular mechanism and potential therapeutic target through targeting SOCE-dependent process. However, whether STIM1 participates in HNSCC metastasis requires further study. [ABSTRACT FROM AUTHOR]

Background: The death-associated protein kinase (DAPK) is a tumor suppressor gene, which is a mediator of cell death of INF-γ–induced apoptosis. Aberrant methylation of DAPK promoter has been reported in patients with head and neck squamous cell carcinoma (HNSCC). However, the results of these studies are inconsistent. Hence, the present study aimed to evaluate the association between the promoter methylation of DAPK gene and HNSCC. Methods: Relevant studies were systematically searched in PubMed, Web of Science, Ovid, and Embase. The association between DAPK promoter methylation and HNSCC was assessed by odds ratio (ORs) and 95% confidence intervals (CI). To evaluate the potential sources of heterogeneity, we conducted the meta-regression analysis and subgroup analysis. Results: Eighteen studies were finally included in the meta-analysis. The frequency of DAPK promoter methylation in patients with HNSCC was 4.09-fold higher than the non-cancerous controls (OR = 3.96, 95%CI = 2.26–6.95). A significant association between DAPK promoter methylation and HNSCC was found among the Asian region and the Non-Asia region (Asian region, OR = 4.43, 95% CI = 2.29–8.58; Non-Asia region, OR = 3.39, 95% CI = 1.18–9.78). In the control source, the significant association between DAPK promoter methylation and HNSCC was seen among the autologous group and the heterogeneous group (autologous group, OR = 2.71, 95% CI = 1.49–4.93; heterogeneous group, OR = 9.50, 95% CI = 2.98–30.27). DAPK promoter methylation was significantly correlated with alcohol status (OR = 1.85, 95% CI = 1.07–3.21). Conclusion: The results of this meta-analysis suggested that aberrant methylation of DAPK promoter was associated with HNSCC. [ABSTRACT FROM AUTHOR]

Background: Geographic heterogeneity of human papillomavirus (HPV) involvement in head and neck squamous cell carcinoma (HNSCC) has been observed over the last few years. This trend has not been evaluated in the Philippines. Hence, this study aims to provide for the first time a data on the prevalence of HPV in HNSCC in the northwestern region of the Philippines. Methods: Two hundred one (201) biopsy samples (179 formalin fixed paraffin embedded and 22 fresh frozen) from 163 Filipino HNSCC cases (oral cavity = 88; larynx = 60; oropharynx = 15) diagnosed between 2003 to 2013 were initially included in this study. HPV DNA was detected by two methods: (1) BSGP5+/6+-PCR/ multiplex human papillomavirus genotyping and (2) TaqMan probes-based real-time qPCR. Presence of HPV type-specific transcripts were also analyzed by reverse transcription-PCR with subsequent hybridization to oligonucleotide probes coupled to Luminex beads. Co-amplification of the β-globin and ubiquitin C genes served as internal positive controls for DNA and RNA analyses, respectively. Results and conclusions: Of the 163, 82 (50.3%) cases had at least one tissue sample that was valid for molecular analysis. Only two of the DNA valid cases (2.4%) were HPV DNA-positive (HPV11 and HPV33). All HPV mRNA assays rendered negative results except for HPV11 transcripts. Results of this study may indicate that there is probably very low prevalence of HPV-associated HNSCC among Filipino adults living in a rural region of the Philippines. This study could serve as a benchmark for designing follow-up studies that would assess possible changes in trends of HNSCC among Filipinos in different ethnic regions of the country, especially urban areas in which the population is expected to adapt Western style sexual behavior. A prospective sampling of fresh frozen tissue is also highly recommended to ensure better molecular analyses. [ABSTRACT FROM AUTHOR]

Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”. [ABSTRACT FROM AUTHOR]

Background: Hyperactivation of STAT3 via constitutive phosphorylation of tyrosine 705 (Y705) is common in most human cancers, including head and neck squamous carcinoma (HNSCC). STAT3 is rarely mutated in cancer and the (epi)genetic alterations that lead to STAT3 activation are incompletely understood. Here we used an unbiased approach to identify genomic and epigenomic changes associated with pSTAT3(Y705) expression using data generated by The Cancer Genome Atlas (TCGA). Methods and Findings: Mutation, mRNA expression, promoter methylation, and copy number alteration data were extracted from TCGA and examined in the context of pSTAT3(Y705) protein expression. mRNA expression levels of 1279 genes were found to be associated with pSTAT3(705) expression. Association of pSTAT3(Y705) expression with caspase-8 mRNA expression was validated by immunoblot analysis in HNSCC cells. Mutation, promoter hypermethylation, and copy number alteration of any gene were not significantly associated with increased pSTAT3(Y705) protein expression. Conclusions: These cumulative results suggest that unbiased approaches may be useful in identifying the molecular underpinnings of oncogenic signaling, including STAT3 activation, in HNSCC. Larger datasets will likely be necessary to elucidate signaling consequences of infrequent alterations. [ABSTRACT FROM AUTHOR]

Objective: To determine the relationship between tumor glucose metabolism and tumor blood flow (TBF) in head and neck squamous cell carcinoma (HNSCC). Methods: We retrospectively analyzed 57 HNSCC patients. Tumor glucose metabolism was assessed by maximum and mean standardized uptake values (SUVmax and SUVmean) obtained by 18F-fluorodeoxyglucose positron-emission tomography. TBF values were obtained by arterial spin labeling with 3-tesla MRI. The correlations between both SUVs and TBF were assessed in the total series and among patients divided by T-stage (T1–T3 and T4 groups) and tumor location (pharynx/oral cavity and sinonasal cavity groups). Pearson’s correlation coefficients were calculated for significant correlations. Results: Significant correlations were detected: a negative correlation in the advanced T-stage group (TBF and SUV max: r, −0.61, SUVmean: r, −0.62), a positive correlation in the non-advanced T-stage pharynx/oral cavity group (TBF and SUVmax: r, 0.70, SUVmean: r, 0.73), a negative correlation in the advanced T-stage pharynx/oral cavity group (TBF and SUVmax: r, −0.62, SUVmean: r, −0.65), and a negative correlation in the advanced T-stage sinonasal cavity group (TBF and SUVmax: r, −0.61, SUVmean: r, −0.65). Conclusion: Significant correlations between glucose uptake and TBF in HNSCC were revealed by the division of T-stage and tumor location. [ABSTRACT FROM AUTHOR]

Various serum biomarkers have been developed for predicting head and neck squamous cell carcinoma (HNSCC) prognosis. However, none of them have been proven to be clinically significant. A recent study reported that the ratio of aspartate aminotransaminase (AST) to alanine aminotransaminase (ALT) had a prognostic effect on non-metastatic cancers. This study aimed to examine the effect of the AST/ALT ratio on the survival of patients with HNSCC. Clinical data of 356 patients with locoregionally advanced HNSCC were collected. The effect of the AST/ALT ratio on overall survival was analyzed using a Cox proportional hazard model. Moreover, recursive partitioning analysis (RPA) was used to divide the patients into groups on the basis of the clinical stage and AST/ALT ratio. The prognostic ability of this grouping was validated using an independent data set (N = 167). The AST/ALT ratio ranged from 0.42 to 4.30 (median, 1.42) and was a prognostic factor for overall survival that was independent of age, primary sites, and tumor stage (hazard ratio: 1.36, confidence interval: 1.08−1.68, P = 0.010). RPA divided patients with stage IVA into the following two subgroups: high AST/ALT (≥2.3) and low AST/ALT (<2.3) subgroups. The 5-year survival rate for patients with stage III, stage IVA with a low AST/ALT ratio, stage IVA with a high AST/ALT ratio, and stage IVB were 64.8%, 49.2%, 28.6%, and 33.3%, respectively (p < 0.001). Compared with the low AST/ALT group, the adjusted hazard ratio for death was 2.17 for high AST/ALT group (confidence interval: 1.02–.22 P = 0.045). The AST/ALT ratio was demonstrated to be a prognostic factor of HNSCC. The ratio subdivided patients with stage IVA into low- and high-risk groups. Moreover, intensified treatment for the high-risk group may be considered. [ABSTRACT FROM AUTHOR]

Background: O6-methylguanine-DNA methyl-transferase (MGMT) gene, a DNA repair gene, plays a critical role in the repair of alkylated DNA adducts that form following exposure to genotoxic agents. MGMT is generally expressed in various tumors, and its function is frequently lost because of hypermethylation in the promoter. The promoter methylation of MGMT has been extensively investigated in head and neck squamous cell carcinoma (HNSCC). However, the association between the promoter methylation of MGMT and HNSCC risk remains inconclusive and inconsistent. Therefore, we performed a meta-analysis to better clarify the association between the promoter methylation of MGMT and HNSCC risk. Methods: A systematical search was conducted in PubMed, Web of Science, EMBASE, and Ovid for studies on the association between MGMT promoter methylation and HNSCC. Odds ratio (ORs) and 95% confidence intervals (CI) were calculated to estimate association between MGMT promoter methylation and risk of HNSCC. The meta-regression and subgroup analysis were undertaken to explore the potential sources of heterogeneity. Results: Twenty studies with 1,030 cases and 775 controls were finally included in this study. The frequency of MGMT promoter methylation was 46.70% in HNSCC group and 23.23% in the control group. The frequency of MGMT promoter methylation in HNSCC group was significantly higher than the control group (OR = 2.83, 95%CI = 2.25–3.56). Conclusion: This meta-analysis indicates that aberrant methylation of MGMT promoter was significantly associated with the risk of HNSCC, and it may be a potential molecular marker for monitoring the disease and may provide new insights to the treatment of HNSCC. [ABSTRACT FROM AUTHOR]

Mitochondrial genes play important roles in cellular energy metabolism, free radical generation, and apoptosis. Dysregulation of these genes have long been suspected to contribute to the generation of reactive oxygen species (ROS), increased proliferation and progression of cancer. A family of orthologues of yeast silent information regulator 3 (SIRT3), 4 (SIRT4) and mitochondrial tumor suppressor 1 (MTUS1) are important mitochondrial tumor suppressor genes which play an important role in the progression of multiple cancers. However, their role in the development of oxidative stress, enhanced proliferation and progression of head and neck squamous cell carcinoma (HNSCC) has not yet been studied. In this study we aimed to test the association between reduced mitochondrial tumor suppressor genes’ activities and enhancement in tissue oxidative stress and cell proliferation in HNSCC cases. The expression of mitochondrial tumor suppressor genes (SIRT3, SIRT4 and MTUS1), mitochondrial DNA repair gene (OGG1-2a) and a proliferation marker (Ki-67) was studied in a study cohort of 120 HNSCC patients and controls with reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR (qPCR) in order to determine the potential prognostic significance of these genes. A statistically significant downregulation of SIRT3 (p<0.001), SIRT4 (p<0.0001), MTUS1 (p<0.002) and OGG1 (p<0.0001) was observed in HNSCC compared to control samples. Ki-67 was also overexpressed (p<0.0001) in HNSCC versus control samples. Additionally, to explore gene–gene relationship, we observed a positive spearmen correlation between SIRT3 versus SIRT4 (r = 0.523***, p<0.0001), SIRT3 versus MTUS1 (r = 0.273***, p<0.001), SIRT3 versus OGG1-2a (r = 0.213*, p<0.03), SIRT4 versus OGG1 (r = 0.338***, p<0.0001) and MTUS1 versus OGG1-2a (r = 0.215*, p<0.03) in HNSCC cases. A negative spearman correlation was observed between OGG1 versus Ki-67 (r = -0.224**, p<0.01) and OGG1-2a versus Ki-67 (r = -0.224**, p<0.01) in HNSCC cases. Here we report that the deregulation of mitochondrial tumor suppressor genes (SIRT3, SIRT4 and MTUS1) in relation to decreased expression of mitochondrial DNA repair gene OGG1-2a and increased proliferation (measured by proliferation marker Ki-67) may be considered important factors in the development of head and neck squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) is over-expressed in nearly all cases of squamous cell carcinoma of the head and neck (SCCHN), and is an important driver of disease progression. EGFR targeted therapies have demonstrated clinical benefit for SCCHN treatment. In this report, we investigated the pre-clinical efficacy of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, both alone and in combination with ionizing radiation (IR), a primary curative modality for SCCHN. One normal oral epithelial (NOE) and three SCCHN (FaDu, UT-SCC-8, UT-SCC-42a) cell lines were used to conduct cell viability, clonogenic survival, cell cycle, and immunoblotting assays in vitro, using increasing doses of Dacomitinib (10–500 nM), both with and without IR (2–4 Gy). The FaDu xenograft model was utilized for tumor growth delay assays in vivo, and immunohistochemical analyses were conducted on extracted tumors. A dose-dependent reduction in cell viability and clonogenic survival after Dacomitinib treatment was observed in all three SCCHN models. Treatment led to a significant reduction in EGFR signalling, with a subsequent decrease in phosphorylation of downstream targets such as ERK, AKT, and mTOR. In vivo, Dacomitinib treatment delayed tumor growth, while decreasing phospho-EGFR and Ki-67 immunoexpression. These effects were further enhanced when combined with IR, both in vitro and in vivo. The preclinical data support the further evaluations of Dacomitinib combined with IR for the future management of patients with SCCHN. [ABSTRACT FROM AUTHOR]

Background: Elucidating the molecular phenotype of cancers with high metastatic potential will facilitate the development of novel therapeutic approaches to the disease. Gene expression profiles link epithelial to mesenchymal transition (EMT) phenotype with high-risk HNSCC. We sought to determine the role of protein biomarkers of EMT in head and neck squamous carcinoma (HNSC) prognosis. Methods: Protein expression analysis of EGFR, β-catenin and E-cadherin was performed on a cohort of 102 patients with HNSCC recruited between 1992 and 2005 using automated quantitative protein analysis (AQUA). We evaluated associations with clinicopathological parameters and prognosis. Results: There were 67 patients with primary squamous cell carcinoma of the head and neck in this cohort who met inclusion criteria and for whom we had complete E-cadherin, beta-catenin and EGFR expression data. High E-cadherin expressers had longer 5-year progression-free survival (PFS) compared to those with low E-cadherin (59.7% versus 40.6%, p = 0.04) and overall survival (OS) (69.6% versus 44.3%, p  = 0.05). Kaplan-Meier analysis showed that patients with low beta-catenin-expressing tumors trended toward worse 5-year PFS (p = 0.057). High EGFR expressers had inferior OS compared to low EGFR expressers (27.7% vs. 54%, p = 0.029). In the multivariable analysis context, E-cadherin remained an independent predictor of improved OS (HR = 0.204, 95% CI 0.043 to 0.972, p = 0.046) while EGFR trended towards significance for OS. Conclusions: The putative markers of EMT defined within a panel of HNSCC using AQUA are associated with tumors of poor prognosis. [ABSTRACT FROM AUTHOR]

Purpose: Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors. Experimental Design: We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort. Results: After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001). Conclusion: Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression. [ABSTRACT FROM AUTHOR]

The phosphoinositol-3 kinase (PI3K) pathway is highly dysregulated in squamous cell carcinoma of the head and neck (SCCHN). While inhibitors of the PI3K/AKT pathway are being developed in cancer, their efficacy does not appear to be related to the presence of mutations or amplification in pathway genes. The PI3K pathway is a major regulator of macro-autophagy, an evolutionarily conserved catabolic process that degrades cellular materials to promote cellular homeostasis and survival under stress. Employing a panel of SCCHN cell lines, we observed a significant correlation between the activity of PI3K/AKT inhibitors and their ability to induce autophagy. More specifically, resistance to these inhibitors was associated with accumulation of p62/SQSTM1, a pleotropic protein that is consumed during autophagy, while loss of autophagy was, for the first time, found to be due to silencing of an essential autophagy gene, ATG7. Moreover, modulating ATG7 and p62/SQSTM1 could regulate sensitivity to PI3K/AKT inhibitors, underscoring a mechanistic link between autophagy and drug sensitivity. Analysis of human tissues revealed progressive accumulation of p62/SQSTM1 in a significant proportion of cancer samples compared to normal tissue, suggesting that defective autophagy has relevance to SCCHN. These findings are further validated by analysis of TCGA data confirming homozygous deletion and mRNA down-regulation of ATG7 in 10.0% of SCCHN samples. Taken together, these data indicate that p62/SQSTM1 levels modulate sensitivity to PI3K/AKT inhibitors; cancers vary in their capacity to undergo autophagy through epigenetic modification and, when deficient, accumulate p62/SQSTM1; and expression of autophagy-related proteins may serve as markers for resistance to PI3K/AKT inhibitors in SCCHN. [ABSTRACT FROM AUTHOR]

Background: Monocyte chemotactic protein-1 (MCP-1) recruits monocytes and macrophages to inflammation sites, and inflammatory infiltration correlates with the progression of head and neck squamous cell carcinoma (HNSCC). This study aims to determine whether MCP-1 expression is related to HNSCC malignancy and patient survival. We also investigated the relationship between MCP-1 expression and the phosphorylation state of the pro-survival pathway factors Akt, ERK, and STAT3. Methods: Expression of MCP-1 and related proteins in HNSCC cell lines was investigated using western blotting. HNSCC patients (34) without distant metastasis at diagnosis were recruited for tissue specimen evaluation of MCP-1 expression and clinical outcomes. The relationship between MCP-1 expression and survival was evaluated using the Cox proportional hazard model with stepwise selection. Results: High-grade HNSCC cell lines were found to have higher levels of active Akt, ERK, and/or STAT3 than did lower grade cell lines under serum-free condition. OCSL, the most malignant cell line, had the highest level of endogenous MCP-1. Administration of exogenous recombinant MCP-1 increased phosphorylation of Akt, ERK, and STAT3 in a dose- and time-dependent manner and increased cellular resistance to serum starvation. Inhibition of Akt, ERK, or STAT3 reduced cell growth and caused cell death. Long-term survival of HNSCC patients was negatively associated with the histological intensity of MCP-1, implicating MCP-1 as a potential prognostic marker for HNSCC. Conclusions: These results suggest that overexpressed MCP-1 in cancer cells may promote HNSCC progression through upregulating pro-survival signaling pathways. High cellular MCP-1 expression is related to poor overall survival rate in HNSCC patients. [ABSTRACT FROM AUTHOR]

In this study we investigated the correlation between RhoC expression and cancer stem cells (CSCs) formation in head and neck squamous cell carcinoma (HNSCC). The inhibition of RhoC function was achieved using shRNA. The expression of stem cell surface markers, ALDH and CD44 were significantly low in two RhoC depleted HNSCC cell carcinoma cell lines. Furthermore, a striking reduction in tumorsphere formation was achieved in RhoC knockdown lines. The mRNA expression of RhoC in RhoC knockdown adherent and tumorspheres are dramatically down regulated as compared with the scrambled control. The mRNA expression of stem cell transcription factors; nanog, oct3/4 (Pouf1), and sox2 were significantly depleted in RhoC knockdown clones. Further, the phosphorylation of STAT3ser727, and STAT3tyr705 were significantly down regulated in RhoC knockdown clones. The overexpression of STAT3 in RhoC knockdown did not show any change in expression patterns of either-STAT3tyr705 or stem cell transcription factors, signifying the role of RhoC in STAT3 activation and thus the expression of nanog, oct3/4 and sox2 in HNSCC. The expression of Inter leukin-6 (IL-6) in RhoC knockdown HNSCC cell lines was dramatically low as compared to the scrambled control. Further, we have shown a rescue in STAT3 phosphorylation by IL-6 stimulation in RhoC knockdown lines. This study is the first of its kind to establish the involvement of RhoC in STAT3 phosphorylation and hence in promoting the activation of core cancer stem cells (CSCs) transcription factors. These findings suggest that RhoC may be a novel target for HNSCC therapy. [ABSTRACT FROM AUTHOR]

Several epidemiological studies have investigated the relation between allergy and cancer with contradicting conclusions, and reports on immunological differences are scarce. By focusing on inflammation, the present study was designed to compare the immune response induced by allergic rhinitis (AR) and head and neck squamous cell carcinoma (HNSCC). Blood and serum was obtained from patients with symptomatic seasonal AR, and newly detected HNSCC, as well as healthy controls. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN) were isolated and cultured with or without the toll-like receptor ligands, Pam3CSK4, LPS, R837, and CpG. Cellular activation and cytokine release were assessed with ELISA, Luminex Multiplex Immunoassay, flow cytometry, and real-time RT-PCR. Sera from HNSCC patients showed elevated levels of innate immune cytokines, and exhibited a response profile consistent with an increased innate immune reaction. In contrast, sera and stimulated PBMC from AR patients displayed increased concentrations of T cell related cytokines, consistent with an adaptive immune response. The presented data demonstrate that AR and HNSCC induce two distinct immunological processes, indicating an inverse association between the immunological responses seen in patients with allergy and cancer of the upper airway. [ABSTRACT FROM AUTHOR]

Head and Neck Squamous Cell Carcinoma (HNSCC) encompasses malignancies that arise in the mucosa of the upper aerodigestive tract. Recent high throughput DNA sequencing revealed HNSCC genes mutations that contribute to several cancer cell characteristics, including dysregulation of cell proliferation and death, intracellular proinflammatory signaling, and autophagy. The PYRIN-domain containing NLR (Nucleotide-binding domain, Leucine rich Repeats – containing) proteins have recently emerged as pivotal modulators of cell death, autophagy, inflammation, and metabolism. Their close physiologic association with cancer development prompted us to determine whether mutations within the NLRP (PYRIN-containing NLR) gene family were associated with HNSCC genome instability and their clinicopathologic correlations. Catastrophic mutational events underlie cancer cell genome instability and mark a point-of-no-return in cancer cell development and generation of heterogeneity. The mutation profiles of 62 patients with primary conventional type HNSCC excluding other histologic variants were analyzed. Associations were tested using Fisher's Exact test or Mann-Whitney U test. Mutations in NLRP were associated with elevated genome instability as characterized by higher mutation rates. Clinically, NLRP mutations were more frequently found in HNSCC arising in the floor of mouth (50.0%) in comparison with HNSCC at other head and neck locations (14.8%). These mutations were clustered at the leucine rich repeats region of NLRP proteins, and affected NLRP genes were mostly localized at chromosomes 11p15.4 and 19q13.42-19q13.43. Twenty novel NLRP mutations were identified in HNSCC, and mutations in this group of genes were correlated with increased cancer cell genome mutation rates, and such features could be a potential molecular biomarker of HNSCC genome instability. [ABSTRACT FROM AUTHOR]

Tripartite motif-containing 24 (TRIM24), a member of the transcriptional intermediary factor 1 family, functions as a co-regulator that positively or negatively modulates the transcriptional activities of several nuclear receptors. The aim of this study was to investigate TRIM24 expression and its clinical significance in head and neck squamous cell carcinoma. The expression levels of TRIM24 variants were examined in head and neck squamous cell carcinoma (HNSCC) samples and cell lines by real-time PCR and WB. The expression levels of TRIM24 measured in 91 locally advanced HNSCC tumors were measured by immunohistochemistry and correlated with clinical and pathological parameters. The functional role of TRIM24 in HNSCC was further investigated by silencing its expression in HNSCC cell lines. TRIM24 variants were up-regulated in 56 HNSCC samples (P<.001) and 9 HNSCC cell lines (P<.05). TRIM24 protein was overexpressed in 6 of 8 HNSCC cell lines and in 2 of 3 HNSCC samples. Furthermore, 54.95% (50/91) of HNSCC samples exhibited remarkably elevated expression of TRIM24 by immunohistochemistry. Univariate analysis revealed that high TRIM24 expression was associated with worse overall survival (P = .020). In multivariate analysis, TRIM24 expression was identified as an independent predictor of overall survival (P = .030), after adjusting for other clinicopathological parameters. Upon TRIM24 silencing, the proliferation of HNSCC cells was notably inhibited due to the induction of apoptosis. These results suggest that aberrant TRIM24 expression may play an important role in the development of HNSCC and is a promising prognostic indicator for patients with locally advanced HNSCC. [ABSTRACT FROM AUTHOR]

The aim of this study is to understand the mechanism of EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66–84%), low frequency of gene amplification (10–32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63–77%) and CDC25A (37–64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2′-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC. [ABSTRACT FROM AUTHOR]

The genome is organized and packed into the nucleus through interactions with core histone proteins. Emerging evidence suggests that tumors are highly responsive to epigenetic alterations that induce chromatin-based events and dynamically influence tumor behavior. We examined chromatin organization in head and neck squamous cell carcinoma (HNSCC) using acetylation levels of histone 3 as a marker of chromatin compaction. Compared to control oral keratinocytes, we found that HNSCC cells are hypoacetylated and that microenvironmental cues (e.g., microvasculature endothelial cells) induce tumor acetylation. Furthermore, we found that chemical inhibition of histone deacetylases (HDAC) reduces the number of cancer stem cells (CSC) and inhibits clonogenic sphere formation. Paradoxically, inhibition of HDAC also induced epithelial-mesenchymal transition (EMT) in HNSCC cells, accumulation of BMI-1, an oncogene associated with tumor aggressiveness, and expression of the vimentin mesenchymal marker. Importantly, we observed co-expression of vimentin and acetylated histone 3 at the invasion front of human HNSCC tumor tissues. Collectively, these findings suggest that environmental cues, such as endothelial cell-secreted factors, modulate tumor plasticity by limiting the population of CSC and inducing EMT. Therefore, inhibition of HDAC may constitute a novel strategy to disrupt the population of CSC in head and neck tumors to create a homogeneous population of cancer cells with biologically defined signatures and predictable behavior. [ABSTRACT FROM AUTHOR]

The retinoic-acid-inducible gene (RIG)-like receptor (RLR) family proteins are major pathogen reorganization receptors (PRR) responsible for detection of viral RNA, which initiates antiviral response. Here, we evaluated the functional role of one RLR family member, RIG-I, in human head and neck squamous cell carcinoma (HNSCC). RIG-I is abundantly expressed both in poorly-differentiated primary cancer and lymph node metastasis, but not in normal adjacent tissues. Activation of RIG-I by transfection with low dose of 5′-triphosphate RNA (3p-RNA) induces low levels of interferon and proinflammatory cytokines and promotes NF-κB- and Akt-dependent cell proliferation, migration and invasion. In contrast, activation of RIG-I by a high dose of 3p-RNA induces robust mitochondria-derived apoptosis accompanied by decreased activation of Akt, which is independent of the interferon and TNFα receptor, but can be rescued by over-expression of constitutively active Akt. Furthermore, co-immunoprecipitation experiments indicate that the CARD domain of RIG-I is essential for inducing apoptosis by interacting with caspase-9. Together, our results reveal a dual role of RIG-I in HNSCC through regulating activation of Akt, in which RIG-I activation by low-dose viral dsRNA increases host cell surviral, whereas higher level of RIG-I activation leads to apopotosis. These findings highlight the therapeutic potential of dsRNA mediated RIG-I activation in the treatment of HNSCC. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented. [ABSTRACT FROM AUTHOR]

Purpose: Tumors with oncogenic dependencies on the HER family of receptor tyrosine kinases (RTKs) often respond well to targeted inhibition. Our previous work suggested that many cell lines derived from squamous cell carcinomas of the head and neck (SCCHNs) depend on autocrine signaling driven by HER2/3 dimerization and high-level co-expression of HRG. Additionally, results from a Phase I trial of MEHD7495A, a dual-action antibody that blocks ligand binding to EGFR and HER3, suggest that high-level HRG expression was associated with clinical response in SCCHN patients. Here we explore the hypothesis that high-level HRG expression defines a subpopulation of SCCHNs with activated HER3. Experimental Design: qRT-PCR expression profiling was performed on >750 tumors of diverse origin, including >150 therapy-naïve, primary, and recurrent SCCHNs. Activated HER3, defined by immunoprecipitation of phospho-HER3, was compared to HRG expression in SCCHN samples. Paracrine versus autocrine expression was evaluated using RNA-in situ hybridization. Results: SCCHN tumors express the highest levels of HRG compared to a diverse collection of other tumor types. We show that high HRG expression is associated with activated HER3, whereas low HRG expression is associated with low HER3 activation in SCCHN tumors. Furthermore, HRG expression is higher in recurrent SCCHN compared to patient-matched therapy naïve specimens. Conclusions: HRG expression levels define a biologically distinct subset of SCCHN patients. We propose that high-level expression of HRG is associated with constitutive activation of HER3 in SCCHN and thus defines an actionable biomarker for interventions targeting HER3. [ABSTRACT FROM AUTHOR]