University of Kentucky Lab Seeks to Decode Epigenetic Signals in Cancer

Yvonne N. Fondufe-Mittendorf, PhD

Associate Professor

Department of Molecular & Cellular Biochemistry

University of Kentucky College of Medicine in Lexington

Although many labs study cancer as an all-or-nothing process, putting all metastable hybrid stages in the same bin, our laboratory at the University of Kentucky is exploring carcinogenesis as a continuum, from initiation through progression to maintenance. This approach has prompted us to focus on the role of epigenetics in cancer development.

Epigenetics is the study of gene regulation without a change in the DNA sequence. The DNA in eukaryotic cells is found as chromatin; its most basic form is a nucleosome, consisting of 147 base pairs of DNA wrapped around an octamer of 8 histone proteins. These histones can be modified, which aids in nucleosome remodeling, allowing accessibility of the DNA to regulatory proteins. The DNA, as well as the histones, can be modified to regulate gene expression.

Epigenomic dysregulation is implicated in diseases including cancer, cardiovascular disorders, diabetes, and neurodegeneration. Epigenetics is the reason why 2 cells (brain vs eye cell), with the same DNA, differ.

The body is constantly exposed to environmental signals—endogenous and exogenous—making the chromatin constantly adapt to allow for differential gene expression. It is now greatly recognized that defects in epigenetic regulation lead to diseases.

Scientists are making great strides in understanding how epigenetic modifications contribute to both health and disease; however, a complete understanding of these modifications is still very much a work in progress. My lab is interested in epigenetic regulation and dysregulation in diseases. One of our main projects is epigenetic reprogramming in response to an environmental cue.

Millions of people worldwide are chronically exposed to inorganic arsenic (iAs), a ubiquitous environmental carcinogen, through drinking water and food, with consequences ranging from acute toxicities to malignant transformations. One important example is that the rates of lung cancer in the coal-mining regions of Kentucky are approximately twice the national rate, correlating with high iAs levels in drinking water. Because iAs is not a mutagen, we hypothesize that it may be driving carcinogenesis through epigenetic dysregulation.

After cancer initiation, cells undergo several hybrid reversible states between the epithelial and mesenchymal states before full transformation. In binning these metastable hybrid states, we miss understanding and possibly harnessing the reversibility of the in-between states for therapeutic use. Our goal is to decipher the epigenetic changes—which are reversible—that can be potentially be targeted.

Chromatin Modeling

As part of that work, we developed a cancer model, instigating carcinogenesis with chronic low-dose iAs. We continuously exposed cells to low-dose iAs, measuring the changes in gene expression of specific carcinogenic markers until full transformation. We show an elegant dance between writers of histone marks, readers of these marks, and chromatin remodelers; this results in opening/closing of the chromatin structure, allowing transcription factors to access the DNA, with consequences in gene expression/repression of genes driving carcinogenesis (Figure).

Interestingly, during these hybrid carcinogenic states, the chromatin structure is poised for gene expression. Depending on the signal, this poised state regulated by epigenetic marks can be either reversed or driven to full transformation.

Our results can thus be interpreted in human populations. Sometimes we are exposed to an environmental toxicant and not develop the disease. This could lead to a poised epigenomic state, requiring a secondary hit either by exposure to the same toxicant or a new one, to drive the epigenomic state toward a full-blown disease state. This could explain why, in the same neighborhood, one resident and not another may develop the disease. Finally, although epigenetic marks are heritable, they are also reversible; harnessing this reversibility potential makes them good targets for drug therapy.

Histone Variants

A second exciting area involves understanding the impact of histone variants that are expressed at different times and locations. These variants differ by maximally 3 amino acids, and their incorporation into chromatin could change the histone octamer’s stability; histone octamer—DNA association and/or the variants can be differentially modified.

C. David Allis, PhD, who won the 2018 Albert Lasker Basic Medical Research Award for his discovery of the histone code, said recently that every amino acid in the histone counts.¹ Even though these variants differ by very few amino acids, their expression is implicated in several cancers. Thus, their modulation of the chromatin structure might result in differential gene expression critical in cancer. Using high-resolution mass spectrometry, we were the first to show the differential expression of histone H2B variants and their incorporation into the chromatin structure during iAs-mediated carcinogenesis.² We hypothesize that this incorporation occurs at specific regions of the genome, resulting in upregulation of oncogenes and downregulation of tumor suppressor genes. Analyses of these variants in human cancer tissues using the The Cancer Genome Atlas database showed high expression of these H2B variants in cancer, with some of them specific for one cancer over another. We posit that these H2B histone variants are “oncohistones.”

We are now delving into this exciting field, studying where these histone variants get incorporated into the genome and their functional impact using high-resolution genome-wide chromatin immunoprecipitation sequencing (ChIP-seq). Additionally, we are reconstituting in vitro nucleosomes and using fluorescence resonance energy transfer; biophysical, single molecule techniques; and cell biology techniques to study the functional impact of these variants on nucleosome stability and dynamics.

We are now in an exciting phase for epigenetics. We think that a combination of the various changes to the epigenome areas is driving carcinogenesis.

Circular RNA

We recently showed that a novel and less understood epigenetic regulator, circular RNA (circRNA), is also differentially expressed in iAs-mediated carcinogenesis.³ CircRNAs are specific to cell type and differentiation and are dysregulated in cancers. Due to their circulation in peripheral blood and their stability, circRNAs present great potential for being noninvasive biomarkers.

These highly stable isoforms derived from precursor messenger RNAs are also evolutionarily conserved, although their function remains unclear. They can act as microRNA/RNA-binding protein sponges, as well as being translated. We propose to profile the functional landscape of circRNAs and their modifications (epitranscriptome) as cells go through iAs carconogenesis, with the goal of revealing novel biomarkers and therapeutic targets.

References

1. Maze I, Noh KM, Soshnev AA, Allis CD. Every amino acid matters: essential contributions of histone variants to mammalian development and disease. Nat Rev Genet. 2014;15(4):259-271. doi: 10.1038/nrg3673.
2. Rea M, Jiang T, Eleazer R, Eckstein M, Marshall AG, Fondufe-Mittendorf YN. Quantitative mass spectrometry reveals changes in histone H2B variants as cells undergo inorganic arsenic-mediated cellular transformation. Mol Cell Proteomics. 2016;15(7):2411-2422. doi: 10.1074/mcp.M116.058412.
3. Eleazer R, Andreeva K, Mittendorf Y. Changes in the circular RNA transcriptome during inorganic arsenic-induced epithelial-to-mesenchymal transition. FASEB J. 2019;33(suppl 1; abstr 623.1). fasebj.org/doi/abs/10.1096/fasebj.2019.33.1_supplement.623.1.