DNA methylation regulated microRNAs in HPV-16-induced head and neck squamous cell carcinoma (HNSCC).

INTRODUCTION: Epigenetic modifications have been reported to play an important role in regulating gene expression and these modifications become critical when they have a role in controlling another important layer of epigenetic regulation namely microRNAs. In the present study, we have identified the microRNAs that may be regulated by promoter DNA methylation and histone acetylation in Human papilloma virus-positive head and neck squamous cell carcinoma. METHODOLOGY: HPV-negative cell line (UPCI:SCC-116) and HPV-16 +ve cell line (UPCI:SCC-090) were treated with methylation inhibitor (5-aza-2'-deoxycytidine, AZA) and acetylation inhibitor (Trichostatin-A, TSA), followed by micro-array analysis. The differentially expressed miRNAs were validated in control (n = 10), HPV-16 +ve (n = 30), and HPV -ve (n = 30) HNC, TCGA (n = 529) tissue samples, and two HPV -ve (SCC116 and Hacat) and two HPV +ve (SCC090 and SiHa) cell lines. Methylation-specific PCR (MSP) and chromatin immunoprecipitation assay (CHIP) were performed to validate their regulation. In silico and in vitro analyses of identified miRNAs were done to study putative pathways they target and their possible role in carcinogenesis. RESULTS: Among 10 miRNAs specifically up-regulated in microarray analysis of AZA-treated SCC090 cells, we observed significantly decreased expression of hsa-miR-181c-5p, hsa-miR-132-5p, hsa-miR-658 in HPV +ve HNC cohort, TCGA tissue samples, and cell lines as compared to their HPV -ve counterpart, and their promoter region also possesses CpG islands. MSP and analysis of TCGA data (MethHC) revealed increased frequency of methylation at the promoter of hsa-miR-132-5p that is negatively correlated with its expression. In TSA-treated SCC090 cells, out of 7 miRNAs, two namely Hsa-miR-129-2-3p and Hsa-miR-449a were found to be up-regulated as compared to HPV -ve cells. However, the levels of enrichment by anti-acetyl-H3 and anti-acetyl-H4 were significantly low in cell lines compared to respective controls and both were up-regulated in HPV +ve compared to HPV -ve TCGA tissue samples. In silico analysis revealed hsa-miR-132-5p targeted canonical ß-catenin/wnt pathway and modulation of down-stream genes of the pathway was observed on over-expression/inhibition of hsa-miR-132-5p. CONCLUSION: This study suggests the role of epigenetic modifications in regulating expression of miRNAs in HPV +ve HNSCC.

Role of non-coding RNAs in head and neck squamous cell carcinoma: A narrative review.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with high recurrence, metastasis, and poor treatment outcome. Recent studies have reported that non-coding RNA (ncRNA) might play critical role in regulating different types of cancer. MicroRNAs (miRs) are short ncRNAs (20-25 nucleotides) responsible for post-transcriptional regulation of gene expression and may have a role in oncogenesis by acting as oncomiRs or tumor suppressor miRs. Long non-coding RNAs (lncRNAs) are heterogenous group of ncRNAs more than 200 nucleotides long, can act in cis and/or in trans, and have been also implicated in carcinogenesis. These molecules have been suggested to be promising candidates as diagnostic and prognostic biomarkers and for development of novel therapeutic approaches. In this review, we have summarized recent findings on role of these ncRNAs in HPV-negative (HPV-ve) and HPV-positive (HPV+ve) HNSCC. The available literature supports differential expression of both microRNAs and long non-coding RNAs, which include oncogenic ncRNAs (miR-21, miR-31, miR-155, miR-211, HOTAIR, and MALAT1) and tumor suppressor ncRNAs (let7d, miR-17, miR-375, miR-139, and MEG3) in HPV+ve HNSCC tumors as compared to HPV-ve tumors and they have distinct role in the pathophysiology of these two types of HNSCCs.

Detection of active human papilloma virus-16 in head and neck cancers of Asian North Indian patients.

BACKGROUND: Head and neck cancers (HNC) are one of the most common cancers in India. Human papillomavirus (HPV) has been identified as an emerging risk factor for HNC. METHODS: The present study was carried out to determine the active form of HPV-16 using a combination of PCR, viral load determination, HPV-16 E7 mRNA expression, p16, p53, and pRB immuno-histochemistry (IHC). RESULTS: A total of 226 HNC patients were enrolled in the present study. Sixty-seven (29.7%) of HNC cases were found to be HPV DNA positive. Thirty-two (14%) cases were HPV-16 DNA positive and 20 (9%) cases expressed HPV-16 E7 mRNA. HPV-16 mRNA/p16 positive cases had significantly increased viral load and integrated HPV-16 DNA. In summary, of total HNC patients, 6% cases were positive for both HPV-16 DNA and p16, and 5% were positive for both E7 mRNA and p16 IHC. We observed similar HPV-16 DNA/E7mRNA prevalence in oropharynx and oral cavity sites, however, oropharynx SCC had significantly higher viral load. CONCLUSION: Our results show low prevalence of active HPV-16 in North Indian HNC patients. HPV-16 E7 mRNA expression correlated with p16 nuclear positivity and increased viral load. Therefore, E7 mRNA expression may be used as a good surrogate indicator for active form of HPV-16 infection.

Role of autophagy in head and neck cancer and therapeutic resistance.

Head and neck squamous cell carcinomas (HNSCC) are one of the most common cancers worldwide, accounting for almost 50% of all malignancies in developing nations. Autophagy is a catabolic process involving turnover of long-lived proteins and organelles and is an important mechanism for cell survival under stress conditions. Autophagy has been shown to play a pivotal role in etio-pathogenesis of several cancers. Autophagy and apoptosis may be triggered by common upstream signals, and sometimes this results in combined autophagy and apoptosis, or defective apoptosis rendering immortalized epithelial cells highly tumorigenic. Autophagy has been found to buffer metabolic stress and may help in cell survival; however, inhibiting autophagy under conditions of nutrient limitation can restore cell death to apoptosis-refractory tumors. Therefore, autophagy acts as a double-edged sword in cancer therapeutics. Role of autophagy in pathophysiology and as a potential cancer therapeutics is a subject of intensive research. This review will focus on the role of autophagy and how it contributes to the pathogenesis and overcoming therapeutic resistance in HNSCC.

Structural basis of the strong cell-cell junction formed by cadherin-23.

Cadherin-23, a giant atypical cadherin, forms homophilic interactions at the cell-cell junction of epithelial cells and heterophilic interactions with protocadherin-15 at the tip-links of neuroepithelial cells. While the molecular structure of the heterodimer is solved, the homodimer structure is yet to be resolved. The homodimers play an essential role in cell-cell adhesion as the downregulation of cadherin-23 in cancers loosen the intercellular junction resulting in faster-migration of cancer cells and a significant drop in patient survival. In vitro studies have measured a stronger aggregation-propensity of cadherin-23 compared to typical E-cadherin. Here, we deciphered the unique trans-homodimer structure of cadherin-23 in solution, and show that it consists of two electrostatic-based interfaces extended up to two terminal domains. The interface is robust, with a low off-rate of ~8x10-4 s-1 that supports its strong aggregation-propensity. We identified a point-mutation, E78K, that disrupts this binding. Interestingly, a mutation at the interface was reported in skin cancer. Overall, the structural basis of the strong cadherin-23 adhesion may have far-reaching applications in the fields of mechanobiology and cancer.

Role of Host miRNA Hsa-miR-139-3p in HPV-16-Induced Carcinomas.

Purpose: Human papillomavirus 16 (HPV-16) is an important risk factor in head and neck cancer (HNC). Studies suggest that miRNAs play an important role in cancer; however, their role in HPV-mediated oncogenesis remains largely unknown. We investigated the role of miRNAs with HPV-16 as putative target in HPV-16-mediated cancers.Experimental Design: Using in silico tools, we identified miRNAs with putative binding sequences on HPV-16 miRNAs. Hsa-miR-139-3p was identified as best candidate miRNA by luciferase reporter assay and was found to be significantly downregulated in HPV-16-positive tissues and cell lines. Overexpression/inhibition studies were performed to determine the role of miRNA in regulating oncogenic pathways.Results: Hsa-miR-139-3p was found to target high-risk HPV-16 oncogenic proteins and revive major tumor suppressor proteins (p53, p21, and p16). This resulted in inhibition of cell proliferation and cell migration, cell-cycle arrest at G2-M phase and increased cell death of HPV-16-positive cells. Analysis of The Cancer Genome Atlas (TCGA) data showed decreased expression of Hsa-miR-139-3p in HPV-16-positive HNC and cervical cancer cases, and its higher expression correlated with better survival outcome in both cases. Increased DNA methylation of Hsa-miR-139-3p harboring gene PDE2A at its promoter/CpG islands was observed in HPV-16-positive tissues and cell lines, which further correlated with Hsa-miR-139-3p expression, suggesting its role in regulating Hsa-miR-139-3p expression. Furthermore, we observed an increased sensitization of Hsa-miR-139-3p overexpressed HPV-16-positive cells to chemotherapeutic drugs (cisplatin and 5-fluorouracil).Conclusions: HPV-16-mediated downregulation of Hsa-miR-139-3p may promote oncogenesis in HNC and cervical cancer. Clin Cancer Res; 23(14); 3884-95. ©2017 AACR.