The present study was undertaken to explore and validate novel hypermethylated DNA regions in squamous cell carcinoma of the tongue (SCCT). Genome-wide methylation changes were identified by differential methylation hybridization (DMH) microarray and validated by bisulfite genome sequencing (BGS). The results were compared against datasets from The Cancer Genome Atlas head and neck squamous cell carcinoma (TCGA-HNSCC), Gene Expression Omnibus (GSE26549), and ArrayExpress (E-MTAB-1328). DMH identified 116 hypomethylated and 241 hypermethylated regions. Of the latter, 24 were localized to promoter or 5'-UTR regions. By BGS, promoter sequences of DAPK1, LRPPRC, RAB6C, and ZNF471 were significantly hypermethylated in tumors when compared with matched normal tissues (P < 0.0001). A TCGA-HNSCC dataset (516 cases of cancer and 50 normal tissue samples) further confirmed hypermethylation of DAPK1, RAB6C, and ZNF471. Sensitivity and specificity of methylation markers for a diagnosis of cancer were in the range of 70-100% in our study and from TCGA-HNSCC datasets, with an area under curve (AUC) of 0.83 and above. Kaplan-Meier survival analysis of TCGA-HNSCC expression data revealed that patients with low expressions of DAPK1, RAB6C, and ZNF471 showed poorer survival than patients with high expression (P = 0.02). Human papillomavirus (HPV) was found in 55% of cases, HPV16 being the predominant genotype. DAPK1 immunohistochemical staining was lower in SCCT than in normal buccal epithelial cells. This is the first study to report hypermethylation of LRPPRC, RAB6C, and ZNF471 in SCCT and its diagnostic and prognostic potentials in a specific head and neck squamous cell carcinoma.

BackgroundScreening for lesions in the oral cavity is critical for early diagnosis of oral squamous cell carcinoma (OSCC). Targeted next generation sequencing‐based (NGS) mutation analysis of cancer driver genes becomes a reality for personalized medicine and cancer therapeutics.Materials and methodsIn the present study, we have performed a targeted NGS‐based mutation analysis of 50 known oncogenes and tumor suppressor genes in clinically diagnosed potentially malignant lesions and tissues of OSCC. NGS‐based analysis of DNA obtained from biopsies of histopathologically confirmed cases of potentially malignant lesions and OSCC specimens were performed using Ion AmpliSeq™ Cancer Hotspot Panel V2 using the Ion Proton™ Sequencer System, followed by data analysis using Ion Reporter™ and Torrent Suite™ software.ResultsNGS analysis indicated a total of 69 mutations present in 25 genes in potentially malignant lesions and OSCC specimens. We identified recurrent mutations in known OSCC driver genes ATM (11%), TP53 (55%), HRAS (16%), SMAD4 (13%), PIK3CA (16%), and ERBB4 (11%) in potentially malignant lesions and OSCC specimens. Driver mutation analysis identified recurrent TP53 and HRAS driver mutations in our OSCC specimens.ConclusionData generated from our study may enable an application of targeted NGS analysis of driver mutations for better therapeutic choice and improved outcomes for OSCC subjects when combined with clinical diagnosis.

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Antibiotics are the first line of treatment against infections and have contributed immensely to reduce the morbidity and mortality rates. Recently, extensive use of antibiotics has led to alterations of the gut microbiome, predisposition to various diseases and most importantly, increase in the emergence of antibiotic-resistant bacteria, which poses a major threat to global public health. Another major issue faced worldwide due to unregulated use of antibiotics in children as well as in adults is the influence of metabolism and body weight homeostasis, leading to obesity. Apart from the involvement of biosocial causes influencing diet, physical activity, and antibiotic use, pathogenesis of obesity is linked to interconnected functional alterations in cells, tissues and organs due to biochemical, epigenetic and genetic factors. Mitochondrial dysfunction is one such factor, which is becoming the primary focus of various aspects of research on multifactorial complex diseases and is providing new perspectives on etiology, biomarker-based diagnosis, and drug sensitivity. Through this review, we have made an attempt to present the interplay between use of antibiotics, obesity, and associated mitochondrial dysfunction. This may provide insights into the molecular basis, genetic predisposition and environmental triggers, which in turn may have potential clinical applications in the management of antibiotic use.

Somatic mutations in histone encoding genes result in gross alterations in the epigenetic landscape. Diffuse intrinsic pontine glioma (DIPG) is a pediatric high-grade glioma (pHGG) and one of the most challenging cancers to treat, with only 1% surviving for 5 years. Due to the location in the brainstem, DIPGs are difficult to resect and rapidly turn into a fatal disease. Over 80% of DIPGs confer mutations in genes coding for histone 3 variants (H3.3 or H3.1/H3.2), with lysine to methionine substitution at position 27 (H3K27M). This results in a global decrease in H3K27 trimethylation, increased H3K27 acetylation, and widespread oncogenic changes in gene expression. Epigenetic modifying drugs emerge as promising candidates to treat DIPG, with histone deacetylase (HDAC) inhibitors taking the lead in preclinical and clinical studies. However, some data show the evolving resistance of DIPGs to the most studied HDAC inhibitor panobinostat and highlight the need to further investigate its mechanism of action. A new forceful line of research explores the simultaneous use of multiple inhibitors that could target epigenetically induced changes in DIPG chromatin and enhance the anticancer response of single agents. In this review, we summarize the therapeutic approaches against H3K27M-expressing pHGGs focused on targeting epigenetic dysregulation and highlight promising combinatorial drug treatments. We assessed the effectiveness of the epigenetic drugs that are already in clinical trials in pHGGs. The constantly expanding understanding of the epigenetic vulnerabilities of H3K27M-expressing pHGGs provides new tumor-specific targets, opens new possibilities of therapy, and gives hope to find a cure for this deadly disease.

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Diffuse intrinsic pontine gliomas (DIPG) are deadly paediatric brain tumours, non-resectable due to brainstem localisation and diffusive growth. Patients with DIPG have a dismal prognosis of 9-12 months of survival with no effective therapy. Over 80% of DIPGs harbour a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine to methionine substitution (H3K27M). H3K27M causes global epigenetic alterations (a loss of H3K27 trimethylation and an increase in H3K27 acetylation) resulting in aberrant gene expression. To date, no therapeutic strategy exists to suppress the levels of oncogenic H3K27M. We show that pan-HDAC inhibitors (HDACi) lead to the temporary but significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines expressing the H3.3K27M histone variant, without changes in the H3F3A mRNA expression. The H3.3K27M occupancy at the chromatin is greatly reduced upon HDACi (SB939) treatment, as shown by ChIPseq analysis. H3.3K27M loss is most striking at SB939-upregulated genes suggesting the role in repression of these genes. In addition, genes previously reported as H3K27M-dependent become downregulated in response to SB939 treatment. We discover that the SB939-mediated loss of H3.3K27M is partially blocked by a lysosomal inhibitor, chloroquine. Moreover, the loss of H3.3K27M is facilitated by co-occurrence of H2A.Z, as evidenced by the knock-down of H2A.Z histone isoforms. ChIPseq analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. Altogether, we provide new insight into disease-specific mechanism of HDAC inhibition and demonstrate pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings open a new possibility to directly target the H3.3K27M oncohistone, which may be exploited in future therapies.