Enhancer methylation dynamics contribute to cancer plasticity and patient mortality

Abstract

During development, enhancers play pivotal roles in regulating gene expression programs; however, their involvement in cancer progression has not been fully characterized. We performed an integrative analysis of DNA methylation, RNA-seq, and small RNA-seq profiles from thousands of patients, including 25 diverse primary malignances and seven body sites of metastatic melanoma. We found that enhancers are consistently the most differentially methylated regions (DMR) as cancer progresses from normal to primary tumors and then to metastases, compared to other genomic features. Remarkably, identification of enhancer DMRs (eDMRs) enabled classification of primary tumors according to physiological organ systems, and in metastasis eDMRs are the most correlated with patient outcome. To further understand the eDMR role in cancer progression, we developed a model to predict genes and microRNAs that are regulated by enhancer and not promotor methylation, which shows high accuracy with chromatin architecture methods and was experimentally validated. Interestingly, among all metastatic melanoma eDMRs, the most correlated with patient survival were eDMRs that "switched" their methylation patterns back and forth between normal, primary, and metastases and target cancer drivers, e.g., KIT. We further demonstrated that eDMR target genes were modulated in melanoma by the bone metastasis microenvironment, suggesting that eDMRs respond to microenvironmental cues in metastatic niches. Our findings that aberrant methylation in cancer cells mostly affects enhancers, which contribute to tumor progression and cancer cell plasticity, will facilitate development of epigenetic anticancer approaches.

Identification of activated enhancers and linked transcription factors in breast, prostate, and kidney tumors by tracing enhancer networks using epigenetic traits

“…To prioritize enhancers which may possibly regulate gene expression among all enhancers marked by active epigenomic marks, we first identified ~50,000 probes (located within nucleosome-depleted subregions of enhancers) that are differentially methylated in breast, prostate, and kidney tumors, as compared to normal tissues; these probes correspond to ~25,000 different enhancer regions that have gained or lost activity in the tumor tissues. Because previous studies have shown a significant association between the DNA methylation level of an enhancer and the expression level of a direct target gene of the enhancer [27, 28], we then developed an approach (TENET) that identifies statistically significantly associated relationships (links) between DNA methylation and gene expression genome-wide using raw p values by calculating z scores, empirical p values, and Wilcoxon rank sum test p values. Although the number of enhancer to gene links can vary depending on the settings of cut-offs, in general, when we linked gene expression levels with enhancer activity, we found that only ~20% of the enhancer probes show a positive relationship between activity state and expression of a gene.…”

Epigenetic plasticity and the hallmarks of cancer

“…Microenvironmental hypoxia has been shown to suppress DNA demethylase activity in breast cancer (97). Altered cellular contexts can also increase the rate of DNA methylation changes that affect enhancer activity and increase transcriptional plasticity in melanoma (98). Finally, the observation that non-malignant cells in the tumor ecosystem can also undergo striking phenotypic changes suggests that microenvironmental effects on epigenetic states and plasticity may extend beyond the malignant compartment (99).…”

“…Evasion of growth suppressors, such as p16/INK4a, can be mediated by promoter hyper-methylation or EZH2 hyperactivity (3, 35). Invasion and metastasis depend on cell fate transitions, such as epithelial-mesenchymal transition (EMT), with epigenetic etiology (98, 102–104). Replicative immortality may be driven by mutations to histone H3.3 or its chaperone proteins that promote alternative telomere lengthening (65, 105), or by non-genetic mechanisms that simulate self-renewing stem cell states (9, 106).…”

Aberrant DNA methylation in melanoma: biomarker and therapeutic opportunities

“…A significant correlation was found between specific enhancer methylation patterns and patient survival. Specifically, differential methylation of KIT-, CYTL1-, and KIF14-associated enhancers played a role in regulating proliferation [121]. …”