Head And Neck Squamous Cell Carcinoma Carcinogenesis Research Articles

Transforming growth factor-β signaling in head and neck squamous cell carcinoma: Insights into cellular responses.

Head and neck squamous cell carcinoma (HNSCC) arises in the oral cavity, salivary glands, larynx, pharynx, nasal cavity and paranasal sinuses, and is characterized by high morbidity and metastasis rates. Transforming growth factor-β (TGF-β) is a homodimeric protein known to be a multifunctional regulator in target cells and to serve a pivotal role in numerous types of cancer, including HNSCC. The role of TGF-β signaling in carcinogenesis can change from tumor-suppressing to tumor-promoting. In addition, TGF-β induces epithelial-mesenchymal transition and restrains immune surveillance on malignant cells. In the present review, the effects of TGF-β signaling at a cellular level were discussed, which includes the regulation of tumor cells, immune cells and other stromal cells, as well as the possible mechanisms underlying the conversion from a tumor suppressor to a tumor promoter in HNSCC. Further research is required to improve the understanding on how this network is involved in carcinogenesis, progression and metastases in HNSCC.

FAT1 somatic mutations in head and neck carcinoma are associated with tumor progression and survival.

In recent years, the incidence and mortality rates of head and neck squamous cell carcinoma (HNSCC) have increased worldwide. Therefore, understanding genomic alterations in HNSCC carcinogenesis is crucial for appropriate diagnosis and therapy. Protocadherin FAT1, which encodes 4588 amino acid residues, regulates complex mechanisms to promote oncogenesis or suppression of malignancies. Multiplex PCR-based next-generation sequencing (NGS) revealed FAT1 somatic mutations. The clinicopathologic implications of FAT1 in HNSCC were investigated using expression assays, and the functional role of FAT1 in HNSCC pathogenesis was determined using ectopic expression and knockdown experiments. Approximately 29% patients with HNSCC harbored damaging FAT1 mutations. InVEx algorithm identified FAT1 as a significant functional mutation burden. Each type of mutation (missense, nonsense and frameshift) accounted for nearly one-third of deleterious mutations. FAT1 mutations correlated with lower FAT1 expression in tumors. The knockdown of the endogenous expression of FAT1 and exogenous expression of crucial FAT1 domains unequivocally indicated that FAT1 suppressed the migration and invasion capability of HNSCC cells. Functional analysis suggested that nonsense mutations in FAT1 result in the loss of the suppression of tumor progression. FAT1 mutations and downregulation defined nodal involvement, lymphovascular permeation and tumor recurrence. In addition, FAT1 mutations and downregulation are independent predictors of poor disease-free survival in patients with HNSCC. This study is the first to perform multiplex PCR-based NGS to indicate marked non-synonymous FAT1 mutations in HNSCC, which are prognostic indicators. The gene analysis strategy proposed for detecting FAT1 mutations may be a valid method for mutation screening.

Viral DNA integration and methylation of human papillomavirus type 16 in high-grade oral epithelial dysplasia and head and neck squamous cell carcinoma.

This study evaluated the integration and methlyation of human papillomavirus type 16 (HPV16) in head and neck squamous cell carcinoma (HNSCC) and its oral precursor, high-grade oral epithelial dysplasia (hgOED). Archival samples of HPV16-positive hgOED (N = 19) and HNSCC (N = 15) were evaluated, along with three HNSCC (UMSCC-1, -47 and -104) and two cervical cancer (SiHa and CaSki) cell lines. HgOED cases were stratified into three groups with increasing degrees of cytologic changes (mitosis, karyorrhexis and apoptosis). The viral load was higher and the E2/E6 ratio lower (indicating a greater tendency toward viral integration) in group 3 than in groups 1 or 2 (p = 0.002, 0.03). Methylation was not observed in hgOED cases and occurred variably in only three HNSCC cases (26.67%, 60.0% and 93.3%). In HNSCC cell lines, lower E7 expression correlated with higher levels of methylation. HgOED with increased cytologic change, now termed HPV-associated oral epithelial dysplasia (HPV-OED), exhibited an increased viral load and a tendency toward DNA integration, suggesting a potentially increased risk for malignant transformation. More detailed characterization and clinical follow-up of HPV-OED patients is needed to determine whether HPV-OED is a true precursor to HPV-associated HNSCC and to clarify the involvement of HPV in HNSCC carcinogenesis.

Detection of Human Papilloma Virus and Risk Factors among Patients with Head and Neck Squamous Cell Carcinoma Attending a Tertiary Referral Centre in South India

Background:Head and Neck Squamous Cell Carcinomas (HNSCC) is the sixth most common cancer globally. In India, on an average 25-30% of all cancer cases affect the head and neck. The etiological factors associated with HNSCC are tobacco, alcohol and environmental carcinogens. However there are few cases, where there are no obvious risk factors involved. In western counties, there are many reports of human papilloma virus (HPV) association with HNSCC. Hence, we conducted a study to determine the role of HPV infection and risk factors among patients with HNSCC.Materials and Methods:A prospective, cross-sectional study was conducted in a tertiary referral centre from January 2014 to March 2016. 88 patients were enrolled in the study. Socio- demographic, behavioural data, site and subsite involvement, histopathology, staging and treatment were documented. Polymerase chain reaction (PCR) was performed to detect the presence of HPV DNA using consensus primers MY 09/11 and GP5+/GP6+ and further the samples were subjected to PCR for detecting HPV type 16 and 18.Results:The study included 88 participants with HNSCC. 57 had oral and oropharyngeal squamous cell carcinoma, 11 with laryngeal malignancy and 20 involving hypopharynx. Among the participants buccal mucosa (n=22) was the most common subsite involved, majority (50%) had moderately differentiated squamous cell carcinoma and 53.4% presented in stage IV. 2 (2.6%) cases were positive for HPV consensus and both were positive for HPV 16, one case each in larynx and hypopharynx. There was statistical significance in the association between betel nut chewing, cigarette smoking and alcohol intake as risk factors in the carcinogenesis of HNSCC.Conclusion:In our setting in South India, HPV does not play a major role in the carcinogenesis of HNSCC but betel nut chewing, tobacco exposure and alcohol consumption remain major risk factors for HNSCC.

The Association and Clinical Significance of CDKN2A Promoter Methylation in Head and Neck Squamous Cell Carcinoma: a Meta-Analysis

Background/Aims: The association between cyclin-dependent kinase inhibitor 2A (CDKN2A) hypermethylation and head and neck squamous cell carcinoma (HNSCC) risk has been investigated by a number of studies. However, these studies have not demonstrated consistent results. Moreover, the role of CDKN2A methylation in HNSCC carcinogenesis and its clinical significance remain unclear. Methods: We performed a systematic meta-analysis based on 72 articles (including 3399 HNSCCs, 668 premalignant lesions, and 2393 normal controls) from the PubMed, Google Scholar, Web of Science, Embase, China National Knowledge Infrastructure and Wanfang databases. Results: Our study showed a significant increase in the frequency of CDKN2A methylation during HNSCC carcinogenesis (HNSCC vs. normal controls, odds ratio (OR) = 6.72, P < 0.01; HNSCC vs. precancerous lesions, OR = 1.89, P < 0.05; precancerous lesions vs. normal controls, OR = 14.70, P < 0.01). Moreover, CDKN2A methylation was significantly associated with gender (OR = 1.34; P < 0.05) and lymph node metastasis (OR = 2.32; P < 0.01). The area under summary receiver operating characteristic curve (AUC) for diagnosis of HNSCC based on all samples and saliva sample subgroup were 0.77 and 0.96, respectively. Additionally, CDKN2A hypermethylation was significantly associated with shorter overall survival (OS) (hazard ratio (HR) = 1.01, P < 0.05) and recurrence-free survival (RFS) (HR = 1.77, P < 0.05). Conclusion: Our findings indicate CDKN2A methylation is involved in the carcinogenesis, progression, and metastasis of HNSCC. Furthermore, methylated CDKN2A could be a potential diagnostic and prognostic biomarker for HNSCC.

Evaluation of epidermal growth factor receptor expression by a new scoring system in head-and-neck squamous cell carcinoma and its association with various pathological prognostic factors.

Context:Squamous cell carcinoma is an aggressive epithelial malignancy of the upper aerodigestive tract comprising 90% of all head-and-neck squamous cell carcinoma (HNSCC). It is the sixth leading cancer worldwide, with approximately 600,000 cases reported annually. It is one of the most common cancers in India.Aims and Objective:Epidermal growth factor receptor (EGFR) being the most promising marker has potentially offered new methods to prognosticate and plays an essential role in early diagnosis and treatment apart from tumor, node and metastasis staging which has been used till now. Therefore, this study has been undertaken to evaluate the expression of EGFR in HNSCC cases, according to the new scoring system and find its association with various pathological prognostic factors.Materials and Methods:Forty-eight resected specimens of oral squamous cell carcinoma were received. Cases were appropriately staged, and paraffin-embedded tumor sections, stained with hematoxylin and eosin, were graded. EGFR expression was evaluated as extent score, intensity score and total score (TS).Statistical Analysis:Data obtained were transferred on to an excel sheet. Chi-square test with and without Yate's correction was employed to compare various parameters. P ≤ 0.05 was taken as critical level of significance.Results:A significant association was observed between TS of EGFR expression and tumor grade but not with tumor stage or lymph node metastasis.Conclusion:A significant association of EGFR expression exists with tumor grade as per the new scoring system adopted. High EGFR expression suggests uncontrolled growth which depicts that EGFR upregulation may be an early event during HNSCC carcinogenesis.

Expression profile analysis identifies a two-gene signature for prediction of head and neck squamous cell carcinoma patient survival.

The aim of this study is to identify a gene prognostic signature for the head-and-neck squamous cell carcinoma (HNSCC). HNSCC is one of the most common malignancies worldwide; however, the molecular mechanisms underlying the malignancy are unclear. We analyzed the gene expression profiles of GSE2379, GSE53819, and GSE59102 derived from the gene expression omnibus, and the cancer genome atlas (TCGA) HNSC databases. The R software was used to identify the differentially expressed genes (DEGs) between HNSCC tissues and normal controls. Gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway, protein-protein interactions network, and survival analyses of common DEGs were also performed. A total of 52 upregulated and 31 downregulated DEGs were identified. Functional analyses demonstrated that these DEGs were mainly enriched in extracellular matrix-receptor interaction, focal adhesion, tyrosine metabolism, and cytokine-cytokine receptor interaction. According to the survival analyses, PLAU and SERPINE1 could predict the overall survival of HNSCC patients from the TCGA cohort. Multivariable Cox regression analyses showed that the PLAU and SERPINE1 were independent prognostic factors for HNSCC patients. The prediction power of this two-gene signature was evaluated through receiver operating characteristic curve analysis and achieved a better prognostic value than PLAU (area under curve 0.613 [95% confidence interval 0.569-0.656] vs. 0.577 [0.533-0.621]; P = 0.008) or SERPINE1 (0.613 [0.569-0.656] vs. 0.586 [0.541-0.629]; P = 0.043) when considered alone. The study has identified a set of novel genes and pathways that play significant roles in the carcinogenesis and progression of HNSCC. This two-gene signature may prove to be a useful therapeutic target for HNSCC.

Abstract 46: Validation of potential predictive DNA methylation biomarkers for head and neck squamous cell carcinoma anatomic subsite

Abstract Introduction: We previously identified by a genome-wide approach, differentially methylated genes in head and neck squamous cell carcinoma (HNSCC), including genes uniquely methylated in each HNSCC anatomic subsite. To define their potential prognostic value the validation of uniquely methylated genes in a cohort of HNSCC patients, according to the anatomic subsite, is warranted. Thus, here we present validation data of three potential DNA methylation biomarkers in a cohort comprising primary tumors of 54 HNSCC patients. Methods: Tumor DNA was extracted from HNSCC patients, stratified by the primary tumor anatomic subsite, which included 21 laryngeal cancers, 21 oral cavity cancer, and 12 pharyngeal cancers. The DNA methylation status of three candidate biomarkers, CDH1 for laryngeal cancer, PITX2 for oral cavity cancer, and SFRP1 for pharyngeal cancer were assessed. DNA methylation analysis was performed using qMSP MethyLight assay and quantitative methylation value for each gene was expressed as the Percentage of Methylation Reference (PMR). A PMR value cut-off was established to classify samples as positively or negatively methylated. PMR values &amp;lt;10 were classified as negatively methylated and PMR values of ≥10 were classified as positively methylated for each gene. Results: Positive methylation (PMR ≥10) was detected in 71% of laryngeal samples for CDH1 and, in pharyngeal cancer, 66% showed positive methylation for SFRP1. 90% of oral cavity samples showed positive methylation for PITX2. No association with clinicopathologic characteristics (stage, differentiation, recurrence) was found. However, high PMR values (PMR ≥20) were observed in early stage tumors (I and II) as for late stage tumors (III and IV) for all candidate genes tested. Conclusion: HNSCC subsites (larynx, oral cavity and pharynx) show differential DNA methylation patterns for selected genes. Aberrant methylation of CDH1, PITX2 and SFRP1 was established in a high percentage of tumors, suggesting these candidate genes might be useful as molecular classifiers for HNSCC subsite. In addition, aberrant methylation of CDH1, PITX2, and SFRP1 was detected in early stage tumors; which might be indicators of hypermethylation as an early event in HNSCC carcinogenesis, and in association to the anatomic subsite. Analysis of these unique DNA methylation biomarkers for HNSCC may be valuable for an accurate disease prognosis and for design of novel treatments. Citation Format: Bianca L. Rivera, Jaime Aponte, Sol Nadal, Sebastian Oliva, Sebastian Bravo, Rafael Guerrero, Juan Trinidad, Adriana Baez. Validation of potential predictive DNA methylation biomarkers for head and neck squamous cell carcinoma anatomic subsite [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 46.

Abstract 68: Functional interactions of HPV and PARP-1 in head and neck cancer

Abstract Though the overall incidence of head and neck cancer has been declining over the last several decades, there has been a concomitant and worrisome rise in human-papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). HPV infection is a necessary but not sufficient condition for cancer development. Unlike in cervical cancer, where several co-factors including smoking and co-infection with other agents are well established, how HPV drives malignant transformation in head and neck cancer and which cellular factors are required are not well characterized. Recent evidence from our group has shown that poly (ADP-ribose) polymerase-1 (PARP-1) is overexpressed and overactive in HPV-positive (HPV(+)), but not HPV-negative (HPV(-)), oropharyngeal squamous cell carcinoma (OPSCC). The significance of PARP-1 overexpression and hyperactivation in cancer is only beginning to emerge. High levels of PARP-1 are found in a fraction of hepatocellular and breast cancers and correlate with poor prognosis. Recently, PARP-1 was implicated in prostate cancer progression. This study explores the role of PARP-1 in HPV infection and HNSCC carcinogenesis. Biochemical methods were used to determine PARP activity in vitro, in head and neck tumors, and during infection with HPV16 pseudovirus (PsV) or overexpression of HPV16 proteins in epithelial cells. The response to PARP inhibition was evaluated with clonogenic survival assays. HPV L1 mRNA in HNSCC was detected using quantitative real-time PCR and HPV L1 protein levels were determined by immunohistochemistry. Here, we show that the HPV16 major capsid protein L1 directly binds PARP-1 and activates poly(ADP-ribosyl)ation in the absence of DNA damage in vitro and in HPV(-) cells. Using PARP-1 knockout cells, we found that PARP-1 expression and enzymatic activity supports initial HPV infection. Furthermore, HPV(-) cells were sensitized to PARP inhibition both after L1 overexpression and after infection with HPV16 PsV particles. In conclusion, we report functional interactions between the viral capsid and cellular protein, signifying a vital role played by PARP-1 in HPV infection and carcinogenesis. Importantly, our data provide a mechanistic explanation for pharmacological PARP inhibition as an intriguing therapeutic opportunity for HPV(+) head and neck cancers. Citation Format: Cyril S. Gary, Michael Hajek, Asel Biktasova, Andrew Sewell, Yarbrough G. Wendell, Natalia Issaeva. Functional interactions of HPV and PARP-1 in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 68.

TRAF3/CYLD mutations identify a distinct subset of human papillomavirus-associated head and neck squamous cell carcinoma.

BACKGROUNDThe incidence of human papillomavirus (HPV)‐associated (HPV‐positive) head and neck squamous cell carcinoma (HNSCC) of the oropharynx has dramatically increased over the last decade and continues to rise. Newly diagnosed HPV‐positive HNSCCs in the United States currently outnumber any other HPV‐associated cancers, including cervical cancer. Despite introduction of the HPV vaccine, the epidemic of HPV‐positive HNSCC is expected to continue for approximately 60 years. Compared with patients who have tobacco‐associated HNSCC, those who have HPV‐positive HNSCC have better overall survival and response to treatment. Current treatment, including chemotherapy and radiation therapy, is associated with lifelong morbidity, and there are limited treatments and no curative options for patients who develop recurrent metastatic disease. Therapeutic de‐escalation (decreased radiation dose) is being tested through clinical trials; however, those studies select patients based solely on tumor and patient smoking characteristics. Mechanisms of HPV‐driven carcinogenesis in HNSCC are not well understood, which limits new therapeutic strategies and hinders the appropriate selection of patients for de‐escalation therapy.METHODSThe authors analyzed HNSCC data from The Cancer Genome Atlas to identify molecular characteristics that correlate with outcomes and integration status of the HPV genome.RESULTSThe current investigations identified a subset of HPV‐positive HNSCCs with mutations in the genes TRAF3 (tumor necrosis factor receptor‐associated factor 3) and CYLD (cylindromatosis lysine 63 deubiquitinase). Defects in TRAF3 and CYLD correlated with the activation of transcriptional factor nuclear factor κB, episomal HPV status of tumors, and improved patient survival.CONCLUSIONSDefects in TRAF3/CYLD were accompanied with the activation of nuclear factor κB signaling and maintenance of episomal HPV in tumors, suggesting that these mutations may support an alternative mechanism of HPV tumorigenesis in head and neck tumors. Cancer 2017;123:1778–1790. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

TREATMENT OF HEAD AND NECK SQUAMOUS CELL CARCINOMA ACCORDING ON THE SPECIFIC MOLECULAR FEATURES OF THE TUMOR (A LITERATURE REVIEW)

Despite the achieved progress in radiotherapy, chemotherapy, and surgery, head and neck squamous cell carcinoma (HNSCC) still remains the sixth most common cause of death from cancer worldwide. The division of HNSCC into 2 large groups with different survival rates is a significant achievement made during the last decades in cancer research and treatment of head and neck cancer. In 45 % – 90 % of cases, oropharyngeal squamous cell carcinoma is presumably associated with human papillomavirus (HPV). A recent whole-exome sequencing study on HNSCC helped to develop new principles of treatment that will allow to increase the effectiveness of conventional therapy. The study demonstrated that inactivating mutations in the p53 gene trigger carcinogenesis. The majority of tumors have such mutations that inactivate the p53 tumor suppressor gene. According to the results of sequencing, HPV-positive and HPV-negative tumors have completely different mutation profiles. Intratumoral heterogeneity should be taken into account when implementing new treatment approaches. We present an overview of studies published between 1989 and 2014. Current review briefly describes molecular mechanisms of carcinogenesis in HNSCC in the light of genetic and biochemical features of the tumor, paying particular attention to the most significant scientific achievements in this field. Moreover, we outline the advancements of wholeexome sequencing in HNSCC and give an overview of recent studies devoted to new therapeutic approaches. The process of carcinogenesis in HNSCC is often initiated by tumor suppressors. In this case, the development of target-based drugs is problematic. Target therapy focused on the ways of tumor growth suppression is a much more serious challenge than inhibition of oncogenic signals, because it requires reactivation of tumor suppressors and restoration of their functions, which is more difficult than conventional chemical and biological blockage. Poor survival of patients with HNSCC, which is usually associated with a small size of recurrent tumors, their latent growth, and localization in various anatomical areas, shows that there is an urgent need for developing new therapeutic approaches for the disease. The study was aimed to analyze specific molecular features of head and neck tumors and to explore the opportunities of providing personalized care for these patients.

STAT3 as a Chemoprevention Target in Carcinogen-Induced Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal disease due, in large part, to a high rate of second primary tumor (SPT) formation. The 4-nitroquinoline 1-oxide (4-NQO) mouse model of oral carcinogenesis provides a robust system in which to study chemopreventive agents in the context of chemically induced HNSCC tumors. STAT3 is a potent oncogene that is hyperactivated by tyrosine phosphorylation early in HNSCC carcinogenesis and is a rational therapeutic target. We recently reported that loss-of-function of the STAT3 phosphatase PTPRT promotes STAT3 activation in HNSCC tumors and preclinical models and may serve as a predictive biomarker of response to STAT3 inhibitors, including the small-molecule Stattic. We therefore investigated the hypothesis that Ptprt-knockout (KO) mice would be more susceptible to 4-NQO-induced oral carcinogenesis and more sensitive to Stattic-mediated chemoprevention compared with wild-type (WT) mice. Herein, we demonstrate that Ptprt WT and KO mice develop similar spectra of HNSCC disease severity upon 12 weeks of 4-NQO administration, with no apparent effect of Ptprt genotype on carcinogenesis or treatment outcome. Targeting of STAT3 with Stattic resulted in a chemopreventive effect against 4-NQO-induced oral cancer (P = 0.0402). While these results do not support a central role for PTPRT in 4-NQO-induced HNSCC carcinogenesis, further investigation of STAT3 as a chemoprevention target in this cancer is warranted. Cancer Prev Res; 9(8); 657-63. ©2016 AACR.

Abstract 5243: STAT3 inhibition as chemoprevention in a chemically induced mouse model of HNSCC

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is frequently fatal due to carcinogen-induced field cancerization, leading to second primary tumor formation in ∼4% of patients per year. Effective chemoprevention could improve survival. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor and rational therapeutic target in HNSCC. We recently reported that loss of function of PTPRT (protein tyrosine phosphatase receptor type T), a direct STAT3 phosphatase, by frequent somatic mutation or promoter methylation leads to hyper-phosphorylation of STAT3 and may predict sensitivity to STAT3 inhibition using Stattic. In the present study, we sought to evaluate the contribution of PTPRT to chemical carcinogenesis and sensitivity to Stattic-mediated HNSCC chemoprevention in the 4-NQO (4-nitroquinoline 1-oxide) model in vivo using PTPRT wild-type (WT) or knockout (KO) mice. Methods: PTPRT WT or KO mice received 4-NQO-containing water (100 μg/mL) for 12 weeks. At experiment initiation, a subset of mice received vehicle (PBS) or 50 mg/kg Stattic by oral gavage Q5D. After 12 weeks of 4-NQO administration, mice were randomized to receive vehicle/Stattic for 12 additional weeks. Mice were then sacrificed followed by tongue excision for H&amp;E and IHC analysis. Histologic assessment of dysplasia was scored from 0 to 6 (normal to invasive SCC). Results: By week 3, we observed unexpected combined toxicity of 4-NQO and Stattic, with 3 mouse deaths. Combined treatment was suspended until the end of the 12-week 4-NQO administration period, and no further toxicity was noted. At the end of the 24-week experiment, we observed a diverse range of neoplasias in the control group, with 7/13 (53.8%) exhibiting SCC of which 4 (30.8%) were invasive. Using a rank transformation statistic, we detected no significant effect of PTPRT status (P = 0.9985) or interaction between PTPRT status and treatment (P = 1.0) with respect to pathology scores. Further analysis revealed a trend toward effective Stattic-mediated chemoprevention (P = 0.0831). Interestingly, the only two mice with normal-appearing oral epithelia were both PTPRT KO and treated with Stattic. Conclusions: PTPRT status does not appear to predict susceptibility to 4-NQO-mediated carcinogenesis, nor sensitivity to Stattic-mediated chemoprevention in this mouse model of HNSCC initiation. Nevertheless, we observed a trend toward effective chemoprevention in Stattic-treated mice supporting a role for STAT3 in HNSCC carcinogenesis. Citation Format: Noah D. Peyser, Lin Wang, Marie Acquafondata, Maria Freilino, Hua Li, Yan Zeng, Malabika Sen, William E. Gooding, Daniel E. Johnson, Jennifer R. Grandis. STAT3 inhibition as chemoprevention in a chemically induced mouse model of HNSCC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5243.

The molecular mechanism of human papillomavirus-induced carcinogenesis in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Recently, the incidence of oropharyngeal cancer (OPC) has increased markedly in comparison to that of HNSCC, which is associated with the use of tobacco or alcohol or both. This increase has resulted mainly from the global rise in the number of human papillomavirus (HPV)-related oropharyngeal cancers (HPV-OPCs). HPV-OPC has several unique characteristics, including presentation in younger patients, better response rates to treatment, and better prognosis compared to alcohol- and smoking-related HNSCC. HPV infection status is now an independent prognostic factor for survival in patients with OPC. In general, HPV oncoproteins E6 and E7 are the primary viral factors responsible for the initiation and progression of HPV-related cancers via the inactivation of p53 and pRb. However, alterations in additional factors, including genomic instability, HPV DNA integration, and epigenetic alterations, could be equally important for neoplastic transformation and tumor progression. The impact of genomic instability and external environmental factors on the initiation of cervical cancer development through high-risk HPV infection has been well characterized, although less is known about the mechanism underlying HPV-induced carcinogenesis in HNSCC. This review provides an overview of the biology and molecular mechanisms of HPV-related cancers, including a particular focus on several recent studies on the comprehensive characterization of genomic alterations in HPV-associated HNSCC.

Human papilloma virus prevalence in HIV patients with head and neck squamous cell carcinoma.

The implication of human papilloma virus (HPV) in head and neck squamous cell carcinoma (HNSCC) is well established, especially in oropharyngeal SCC. HIV patients have a higher risk of persistent HPV infection. We investigated the role of HPV in HNSCC carcinogenesis in HIV population. Retrospective monocentric study. We studied HIV patients who presented with HNSCC between 1994 and 2014. For each patient, tumor characteristics, HIV disease, and survival information were collected. Tumor HPV testing was performed using p16 immunohistochemistry (IHC), in-situ hybridization and PCR. We assessed the percentage of HPV in this population of HIV patients with HNSCC and compared HIV disease characteristics based on HPV status. Forty-seven patients were included: 11 women/36 men, the median age was 50 years. Tumor HPV testing was performed in 40 patients. Tumors were located in oropharynx (32%), oral cavity (32%), larynx (21%), and hypopharynx (11%). At the time of diagnosis, median CD4 level was 385 cells/μl, 31% of the patients were stage (Centers for Disease Control, stage C). The percentage of HPV linked to HNSCC for all locations in HIV patients was 30% (n = 12). HPV16 accounted for 50% of all HPV genotypes. HPV positive status was associated with a CD4 nadir of less than 200 (P = 0.026), but not with CD4 level at time of diagnosis (P = 0.414). HPV-negative tumors tend to be associated with poorer 5-year overall survival (hazard ratio = 2.9, P = 0.0711). HPV plays a critical role in HNSCC development in HIV population. HIV immunodeficiency may increase HPV persistence and progression of HNSCC.

Emerging role of sphingosine-1-phosphate signaling in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer type, with an annual incidence of approximately half a million people worldwide. It has a high recurrence rate and an extremely low survival rate. This is due to limited availability of effective therapies to reduce the rate of recurrence, resulting in high morbidity and mortality of patients with advanced stages of the disease. HNSCC often develops resistance to chemotherapy and targeted drug therapy. Thus, to overcome the problem of drug resistance, there is a need to explore novel drug targets. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in inflammation, tumor progression, and angiogenesis. S1P is synthesized intracellularly by two sphingosine kinases (SphKs). It can be exported to the extracellular space, where it can activate a family of G-protein-coupled receptors. Alternatively, S1P can act as an intracellular second messenger. SphK1 regulates tumor progression, invasion, metastasis, and chemoresistance in HNSCC. SphK1 expression is highly elevated in advanced stage HNSCC tumors and correlates with poor survival. In this article, we review current knowledge regarding the role of S1P receptors and enzymes of S1P metabolism in HNSCC carcinogenesis. Furthermore, we summarize the current perspectives on therapeutic approaches for targeting S1P pathway for treating HNSCC.

Abstract A34: The discovery of novel noncoding circular RNAs generated by high-risk human papillomavirus type 16

Abstract Recently, two independent research teams discovered thousands of conserved non-coding human circular RNAs (circRNAs), however, there are no studies suggesting the existence of circRNAs produced by human viruses. The objective of this study is to explore our hypothesis that human papillomavirus (HPV)-16 creates viral circRNAs that may alter host activity in order to promote viral replication and induction of carcinogenesis. CircRNAs are non-coding RNAs generated by the alternative splicing of a 5' acceptor and 2' or 3' donor sites, creating a “head” to “tail” covalent junction of exonic and/or intronic regions. Although the role of circRNAs is largely unknown, studies have demonstrated several functions for circRNAs including sequestering microRNAs (miRNAs) and regulation of transcription. Recently, circRNAs have also been associated with several cancers such as colorectal, breast, and gastric cancers. Infections with high-risk HPVs, such as types 16 and 18, are responsible for the majority of cervical cancers and a subset of head and neck squamous cell carcinomas (HNSCC). HPV is a small double-stranded circular DNA encoding six early proteins (E-1, -2, -4, -5, -6, and -7) and two late proteins (L-1, -2). HPV DNA is tightly regulated and undergoes extensive and complex splicing to achieve the expression of these proteins. Our preliminary data suggest that HPV-16 extensive splicing generates at least two viral circRNAs (hpv-circRNAs) that could potentially regulate host cellular processes promoting tumorigenesis. We demonstrated the existence of the “head” to “tail” junctions of hpv-circRNAs in HPV-16 viral RNA with reverse transcription polymerase chain reaction (RT-PCR) amplification using divergent primers in RNA extracted from cervical tumor cell lines. Quantitative RT-PCR (qRT-PCR) analysis showed the retention of the hpv-circRNAs in RNase R treated samples suggesting the absence of free RNA ends in these hpv-circRNAs. Moreover, these viral circRNAs persisted even after the cervical tumor cells were exposed to Actinomycin D for 48 hrs (to inhibit RNA synthesis), confirming the stability of these hpv-circRNAs in comparison to the short-lived lariat structures that also can occur during splicing. Together, our data suggest the existence of viral circRNAs produced in HPV-16. To the best of our knowledge, these are the first described human viral generated circRNAs. Currently, we are characterizing these hpv-circRNAs by conducting knockdown and rescue experiments followed by behavior assays (e.g. invasion, angiogenesis, and proliferation). We believe this project will be significant in that it will identify and show the importance of viral circRNAs in HPV-related cancers as well as give us new information about a novel mechanism by which high-risk HPV infection contributes to cervical and HNSCC carcinogenesis. Citation Format: Karen E. Hayes, Jamie A. Barr, Jeremy E. Wilusz, Ivan Martinez. The discovery of novel noncoding circular RNAs generated by high-risk human papillomavirus type 16. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A34.

Abstract A26: Importance of long noncoding RNAs in human papillomavirus-related cancers

Abstract The objective of this study is to identify the importance of long non-coding RNAs (lncRNAs) in the induction of carcinogenesis by high-risk human papillomavirus (HPV)-16 oncoprotein E6 in HPV-related cancers. Dysregulation of long non-coding RNAs (lncRNAs) occurs in most human cancers, however, there are only a few studies showing dysregulation of specific lncRNAs in human papillomavirus (HPV)-related cancers. High-risk HPV infection (e.g. HPV-16 and HPV-18) is one the most common causes of cervical cancer, as well as a subset of head and neck squamous cell carcinoma (HNSCC). It is well known that one of the main factors contributing to HPV-related carcinogenesis is the expression of high-risk HPV E6 viral oncoprotein and its interaction with several human proteins, such as the tumor suppressor p53 and the anti-apoptotic protein Bak. It is hypothesized that HPV-16 E6 changes the expression of host lncRNAs to regulate downstream processes important in the induction of carcinogenesis. To test this hypothesis, we stably express high-risk HPV-16 E6 in primary human keratinocytes and conducted high-throughput RNA sequencing analysis to identify HPV-16 E6-mediated changes in lncRNAs expression. Our preliminary data shows over 500 lncRNAs up- or down- regulated by greater than 2-fold with the expression of HPV-16 E6 compared to control. We validated the expression changes of many individual lncRNAs after the expression of HPV-16E6, via q-RT-PCR and Northern blots. In addition we observe similar expression patterns when comparing normal cells to HPV+ cervical cancer cell lines. Currently, we are characterizing specific lncRNAs of interest that are altered with expression of HPV-16 E6 by using 5' and 3' rapid amplification of cDNA ends (RACE) experiments as well as determining certain lncRNA's mechanism of action contributing to cancer by conducting knockdown and rescue experiments followed by behavior assays (e.g. invasion, angiogenesis, and proliferation). To aid in the determination of the function of the lncRNAs, we plan to use Fluorescence in situ Hybridization (FISH) assays to identify their localization. We believe this project will be significant in that it will identify and show the importance of lncRNAs in HPV-related cancers as well as give us new information about a novel mechanism by which high-risk HPV infection contributes to cervical and HNSCC carcinogenesis. Citation Format: Jamie A. Barr, Karen E. Hayes, Ivan Martinez. Importance of long noncoding RNAs in human papillomavirus-related cancers. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A26.

Dynamic cellular and molecular modulations of diabetes mediated head and neck carcinogenesis

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent neoplasms worldwide. While numerous potent dietary insults were considered as oncogenic players for HNSCC development, the impact of metabolic imbalance was less emphasized during HNSCC carcinogenesis. Previous preclinical and epidemiological investigations showed that DM could possibly be correlated with greater incidence and poorer prognosis in HNSCC patients; however, the outcomes from different groups are contradictive and underlying mechanisms remains elusive. In the present study, the changes of cellular malignancy in response to prolonged glucose incubation in HNSCC cells were examined. The results demonstrated that hyperglycemia enhanced HNSCC cell malignancy over time through suppression of cell differentiation, promotion of cell motility, increased resistance to cisplatin, and up-regulation of the nutrient-sensing Akt/AMPK-mTORC1 pathway. Further analysis showed that a more aggressive tongue neoplastic progression was found under DM conditions compared to non-DM state whereas DM pathology led to a higher percentage of cervical lymph node metastasis and poorer prognosis in HNSCC patients. Taken together, the present study confirms that hyperglycemia and DM could enhance HNSCC malignancy and the outcomes are of great benefit in providing better anti-cancer treatment strategy for DM patients with HNSCC.

Dynamic cellular and molecular modulations of diabetes mediated head and neck carcinogenesis.

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent neoplasms worldwide. While numerous potent dietary insults were considered as oncogenic players for HNSCC development, the impact of metabolic imbalance was less emphasized during HNSCC carcinogenesis. Previous preclinical and epidemiological investigations showed that DM could possibly be correlated with greater incidence and poorer prognosis in HNSCC patients; however, the outcomes from different groups are contradictive and underlying mechanisms remains elusive. In the present study, the changes of cellular malignancy in response to prolonged glucose incubation in HNSCC cells were examined. The results demonstrated that hyperglycemia enhanced HNSCC cell malignancy over time through suppression of cell differentiation, promotion of cell motility, increased resistance to cisplatin, and up-regulation of the nutrient-sensing Akt/AMPK-mTORC1 pathway. Further analysis showed that a more aggressive tongue neoplastic progression was found under DM conditions compared to non-DM state whereas DM pathology led to a higher percentage of cervical lymph node metastasis and poorer prognosis in HNSCC patients. Taken together, the present study confirms that hyperglycemia and DM could enhance HNSCC malignancy and the outcomes are of great benefit in providing better anti-cancer treatment strategy for DM patients with HNSCC.