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Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. To enhance molecular knowledge of HNSCC and define a non-coding RNA (ncRNA) landscape of the disease, we profiled the transcriptome-wide dysregulation of long non-coding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) using RNA-sequencing data from 422 HNSCC patients in The Cancer Genome Atlas (TCGA). 307 non-coding transcripts differentially expressed in HNSCC were significantly correlated with patient survival, and associated with mutations in TP53, CDKN2A, CASP8, PRDM9, and FBXW7 and copy number variations in chromosomes 3, 5, 7, and 18. We also observed widespread ncRNA correlation to concurrent TP53 and chromosome 3p loss, a compelling predictor of poor prognosis in HNSCCs. Three selected ncRNAs were additionally associated with tumor stage, HPV status, and other clinical characteristics, and modulation of their expression in vitro reveals differential regulation of genes involved in epithelial-mesenchymal transition and apoptotic response. This comprehensive characterization of the HNSCC non-coding transcriptome introduces new layers of understanding for the disease, and nominates a novel panel of transcripts with potential utility as prognostic markers or therapeutic targets.

Head and neck squamous cell carcinoma persists as one of the most common and deadly malignancies, with early detection and effective treatment still posing formidable challenges. To expand our currently sparse knowledge of the noncoding alterations involved in the disease and identify potential biomarkers and therapeutic targets, we globally profiled the dysregulation of small nucleolar and long noncoding RNAs in head and neck tumors. Using next-generation RNA-sequencing data from 40 pairs of tumor and matched normal tissues, we found 2808 long noncoding RNA (lncRNA) transcripts significantly differentially expressed by a fold change magnitude ≥2. Meanwhile, RNA-sequencing analysis of 31 tumor-normal pairs yielded 33 significantly dysregulated small nucleolar RNAs (snoRNA). In particular, we identified two dramatically down-regulated lncRNAs and one down-regulated snoRNA whose expression levels correlated significantly with overall patient survival, suggesting their functional significance and clinical relevance in head and neck cancer pathogenesis. We confirmed the dysregulation of these noncoding RNAs in head and neck cancer cell lines derived from different anatomic sites, and determined that ectopic expression of the two lncRNAs inhibited key EMT and stem cell genes and reduced cellular proliferation and migration. As a whole, noncoding RNAs are pervasively dysregulated in head and squamous cell carcinoma. The precise molecular roles of the three transcripts identified warrants further characterization, but our data suggest that they are likely to play substantial roles in head and neck cancer pathogenesis and are significantly associated with patient survival.

Alcohol consumption, tobacco use, and human papillomavirus infection have been well-established as the primary risk factors for head and neck squamous cell carcinoma (HNSCC). However, the surge in popularity of e-cigarettes (e-cigs) has prompted speculation of e-cig use potentially emerging as a new risk factor. With previous research on the safety of e-cigs still collectively inconclusive, a comprehensive study of the carcinogenicity of these devices remains urgently necessary. We therefore investigated the potential genotoxic, cytotoxic, and invasive and migratory effects of e-cig vapor exposure on human epithelial cells. A panel of normal epithelial and head and neck cancer cell lines was treated with 0.5%, 1.0%, and 2.0% by volume e-cig vapor from two popular e-cig brands, over periods ranging from 24 hours to 4 weeks. Neutral comet assay and immunostaining for γ-H2AX foci revealed significant induction of DNA double-stranded breaks (up to 3-fold increase, p < 0.05) in cell lines incubated with both nicotinized and nicotine-free e-cig vapor. To evaluate the cytotoxicity of e-cigarettes, trypan blue exclusion and clonogenic assays were performed following short-term e-vapor exposure. Our results indicate that in epithelial cells, short-term treatment induces up to a 5-fold increase in cell death without nicotine, and up to a 10-fold increase with nicotine as compared to untreated controls (p < 0.001). We subsequently assessed the effects of e-cigarettes on HNSCC progression and metastatic potential through exposure of established HNSCC cell lines to e-cigarette vapor. Wound healing assays revealed increased migration of HNSCC cells following e-cigarette treatment, with significant upregulation of key EMT-promoting genes observed via qRT-PCR. We will next examine the effects of long-term (6-9 months) e-cig vapor exposure in vivo using mouse models. Mouse oral epithelium will be harvested for histologic analysis and sequenced to determine the mutagenic potential of long-term e-cig vapor exposure. Nevertheless, our present findings based on short-term e-vapor exposure alone already pose alarming implications for the carcinogenic effects of e-cigarette use. Citation Format: Avinaash Korrapati, Vicky Yu, Maarouf A. Saad, Mehran Rahimy, Yinan Xuan, Angela Zou, Aswini Krishnan, Kevin Brumund, Weg M. Ongkeko. The carcinogenic effects of electronic cigarettes in oral cancer. [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 4069.

Head and neck squamous cell carcinoma (HNSCC) persists as one of the most common and deadly malignancies, with early detection and effective treatment still posing formidable challenges. To expand our currently sparse knowledge of the epigenetic alterations involved in this disease and identify potential biomarkers and therapeutic targets, we globally profiled the dysregulation of small nucleolar (snoRNA) and long non-coding RNAs (lncRNA) in head and neck tumors. Using next-generation RNA-sequencing data from 40 pairs of tumor and matched normal tissues, we profiled lncRNA expression by mapping each RNA-seq library to a reference annotation containing 32,108 lncRNAs. Using edgeR, we found 19,681 lncRNA transcripts significantly differentially expressed by a fold change magnitude ≥ 4 with a false discovery rate Note: This abstract was not presented at the meeting. Citation Format: Thomas K. Honda, Angela Zou, Vicky Yu, Hao Zheng, Selena Kuo, Maarouf Saad, Yinan Xuan, Pranav Singh, Jessica Wang-Rodriguez, Weg M. Ongkeko. Transcriptome-wide expression profiling of long noncoding and small nucleolar RNAs in head and neck squamous cell carcinomas identifies novel transcripts associated with survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 151. doi:10.1158/1538-7445.AM2015-151