Abstract
Oropharyngeal cancer is a subtype of head and neck squamous cell carcinoma that is associated with unique risk exposures like consumption of smokeless tobacco and areca nut and is highly prevalent in the northeastern region of India, especially Meghalaya. However, the underlying epigenetic and transcriptomic changes in this cancer type is yet to be delineated. We have undertaken a study on genome wide somatic alterations in the DNA methylation and transcriptome in oropharyngeal cancer patients from this region using genome wide techniques in paired tumors and adjacent normal tissues. By using integrative approaches, we have identified 194 epigenetically silenced and 241 epigenetically overexpressed genes in the tumor tissue of these patients. Pathways that are significantly enriched by these genes include the pathways of immune systems, such as the interleukin signaling pathways and Toll-like receptor signaling pathway. Also, osteoclast differentiation pathway was found to be epigenetically upregulated. The pathways enriched by the epigenetically downregulated genes were found to be predominantly those involved in xenobiotic metabolism and keratinization. Two major transcription factors – SPI1 and RUNX1 were identified as epigenetically dysregulated, which further modulates 129 downstream genes. Comparison of our observations with the head and neck cancer data from TCGA revealed distinct DNA methylation and gene expression landscapes which might be specific for oropharyngeal cancer. HPV DNA sequences were not detected in any of the tumor samples in RNA-Seq data. The results obtained in this study might provide improved understanding of the disease.
Highlights
MATERIALS AND METHODSOropharyngeal cancer is a type of head and neck cancer (HNC) that develops in the oropharynx, which includes the soft palate, the tonsils, lingual tonsils, the base of the tongue and the posterior pharyngeal wall
We found a strong correlation between the β values of these 25,494 differentially methylated CpG loci (DMP) in the tumors of the discovery set and the same in tumors of the validation set (Spearman rho = 0.86 and P-value < 2.2e-16) (Supplementary Figure 1)
The methylation patterns observed in the CpG islands (CGI) including associated region and those observed in non-CGI open sea region revealed distinct differences
Summary
Oropharyngeal cancer is a type of head and neck cancer (HNC) that develops in the oropharynx, which includes the soft palate, the tonsils, lingual tonsils, the base of the tongue and the posterior pharyngeal wall. After Bisulfite conversion, whole genome DNA methylation assay of tumor and paired adjacent normal samples from 16 oropharyngeal cancer patients and tumor samples from another 10 independent oropharyngeal cancer patients was performed using an Illumina Infinium HumanMethylation450 BeadChip (Bibikova et al, 2011), that interrogates 485,577 CpG sites per sample, following manufacturer’s protocol. Wilcoxon signed-rank test was performed to detect genomic regions with significantly altered DNA methylation between tumor and adjacent normal samples. Cuffnorm was applied on the alignment file produced by TopHat, using the merged transcript file produced by Cuffmerge as annotation and normalized expression values (in FPKM- Fragments Per Kilobase of transcript per Million mapped reads) of all the expressed genes across all the samples (both tumor and adjacent normal) were obtained. Transcription factors having high out-degree value i.e., transcription factors targeting higher number of differentially expressed genes, were considered as significant transcription factors
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