Hypoxia is an inherent condition of tumors and contributes to cancer development and progression. Hypoxia-inducible factors (HIFs) are the major transcription factors involved in response to low O₂ levels, orchestrating the expression of hundreds of genes involved in cancer hallmarks’ acquisition and modulation of epigenetic mechanisms. Epigenetics refers to inheritable mechanisms responsible for regulating gene expression, including genes involved in the hypoxia response, without altering the sequence of DNA bases. The main epigenetic mechanisms are DNA methylation, non-coding RNAs, and histone modifications. These mechanisms are highly influenced by cell microenvironment, such as O₂ levels. The balance and interaction between these pathways is essential for homeostasis and is directly linked to cellular metabolism. Some of the major players in the regulation of HIFs, such as prolyl hydroxylases, DNA methylation regulators, and histone modifiers require oxygen as a substrate, or have metabolic intermediates as cofactors, whose levels are altered during hypoxia. Furthermore, during pathological hypoxia, HIFs’ targets as well as alterations in epigenetic patterns impact several pathways linked to tumorigenesis, such as proliferation and apoptosis, among other hallmarks. Therefore, this review aims to elucidate the intricate relationship between hypoxia and epigenetic mechanisms, and its crucial impact on the acquisition of cancer hallmarks.

Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δβ) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2, and THSD4, had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization.Significance: This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers.

Background: Esophageal squamous cell carcinoma (ESCC) shows a 5-year survival rate below 10%, demonstrating the urgency in improving its treatment. Alterations in epidermal growth factor receptors are closely related to malignancy transformation in a number of tumors and recent successful targeted therapies have been directed to these molecules. Therefore, in this study, we analyzed the expression of EGFR and HER2 and evaluatedEGFRmutation profile as well as the presence of mutations in hotspots ofKRASandBRAFin ESCC patients.Methods: We performed RT-qPCR, immunohistochemistry and Fluorescentin situhybridization to determine EGFR and HER2 expression in ESCC patients, and direct sequencing and PCR-RFLP for mutations and polymorphism analysis.Results: Our results showed an increasedEGFRmRNA expression in tumors compared to surrounding tissue (p T) in 2.1% of the patients, and at codon 787 (G>A) in 79.2% of the cases. This last polymorphism was also evaluated in 304 healthy controls, which presented a similar frequency (73.7%) in comparison with ESCC patients. The absence of mutations ofEGFR,KRASandBRAFas well as the overexpression of EGFR and HER2 in less than 10% of the patients suggest that this signaling pathway is altered in only a small proportion of patients with ESCC.Conclusion: HER receptors target therapies may have the potential to be effective in only a minor fraction of patients with ESCC.

HPV oncoproteins can modulate DNMT1 expression and activity, and previous studies have reported both gene-specific and global DNA methylation alterations according to HPV status in head and neck cancer. However, validation of these findings and a more detailed analysis of the transposable elements (TEs) are still missing. Here we performed pyrosequencing to evaluate a 5-CpG methylation signature and Line1 methylation in an oropharyngeal squamous cell carcinoma (OPSCC) cohort. We further evaluated the methylation levels of the TEs, their correlation with gene expression and their impact on overall survival (OS) using the TCGA cohort. In our dataset, the 5-CpG signature distinguished HPV-positive and HPV-negative OPSCC with 66.67% sensitivity and 84.33% specificity. Line1 methylation levels were higher in HPV-positive cases. In the TCGA cohort, Line1, Alu and long terminal repeats (LTRs) showed hypermethylation in a frequency of 60.5%, 58.9% and 92.3%, respectively. ZNF541 and CCNL1 higher expression was observed in HPV-positive OPSCC, correlated with lower methylation levels of promoter-associated Alu and LTR, respectively, and independently associated with better OS. Based on our findings, we may conclude that a 5-CpG methylation signature can discriminate OPSCC according to HPV status with high accuracy and TEs are differentially methylated and may regulate gene expression in HPV-positive OPSCC.