Epigenetic Effects in Head and Neck Cancer and di-2-ethylhexyl phthalate (DEHP) Exposure

Abstract

Epigenetics refers to the study of heritable changes in gene expression that do not involve changes in the underlying DNA sequence. Typical investigated epigenetic marks include DNA methylation, modifications to histone proteins, and more recently DNA 5-hydroxymethylation (5hmC). Epigenetic profiles in carcinogenesis have been a topic of increasing interest, with several studies showing that epigenetic silencing of tumor suppressor genes and de-repression of heterochromatic intergenic regions via DNA methylation dysregulation play essential roles. Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide, and accumulated scientific evidence suggests that epigenetic alterations, especially DNA methylation, are frequently involved in oral carcinogenesis, tumor progression, and resistance to therapy. In addition to cancer, other diseases and exposures can also modify the epigenome. For instance, environmental contaminants and toxicants such as the chemical DEHP, have been shown to alter epigenetic regulatory features such as DNA methylation and hydroxymethylation. Infection with a high-risk strain of human papillomavirus (HPV) has been shown to be a risk factor for HNSCC development. Previous studies have identified DNA methylation differences between these two classes of HNSCC, but we are the first to characterize 5hmC in HNSCC by HPV status (18 HPV(+); 18 HPV(-)). In Chapter 2, we showed significant hyper-hydroxymethylation in HPV(-) tumors, characterized the heterogeneity in 5hmC in promoters and enhancers relative to tumor subtypes and clinical variables, and detected important cancer genes with differential 5hmC by HPV status and between subtypes. In Chapter 3, we tested the transcriptomic and epigenomic effects of IRX-2 regimen in a HNSCC Phase 2b clinical trial. IRX-2 is a human cell-derived biologic with multiple active cytokine components. By comparing the IRX-2 versus control arms after 21 days of trial, we were able to characterize the significant change of more than 100 genes using immune gene expression data and investigate the correlation between response (clinical and immunological) and immune signatures. The exploration of DNA methylation showed a slight overall increase in methylation after treatment in both regimens, especially for immune responders in the IRX-2 study arm. Although a small number of genes were identified distinguishing the IRX-2 from the control arm, the most beneficial changes were common to both, suggesting that much of the benefit of IRX-2 treatment regimen is due to components other than the cytokine cocktail. In Chapter 4, we focused on the epigenetic effect of early-life di-2-ethylhexyl phthalate (DEHP) exposure. We studied the effect of perinatal DEHP exposure on the DNA methylation profile in liver (a primary target tissue of DEHP) and blood (a common surrogate tissue) of both juvenile and adult mice. Despite exposure ceasing at 3 weeks of age (PND21), we identified thousands of sex-specific differential DNA methylation events in 5-month-old mice, more than identified at PND21, both in blood and liver. However, only a small number of these differentially methylated cytosines overlapped between the time points, or between tissues (i.e. liver and blood), indicating changes by age and that blood may not be an appropriate surrogate tissue to estimate the effects of DEHP exposure on liver. In summary, we facilitated the interpretation of epigenetic effects by exploring the 5hmC profile in head and neck cancer, characterizing the immune response gene expression and DNA methylation in oral cavity cancer immunotherapy, and capturing the effects of DEHP exposure on DNA methylation in mouse models.