Epigenetic changes in pediatric solid tumors: promising new targets

Abstract

Cancer is being reinterpreted in the light of recent discoveries related to the histone code and the dynamic nature of epigenetic regulation and control of gene programs during development, as well as insights gained from whole cancer genome sequencing. Somatic mutations in or deregulated expression of genes that encode chromatin-modifying enzymes are being identified with high frequency. Nowhere is this more relevant than in pediatric embryonal solid tumors. A picture is emerging that shows that classic genetic alterations associated with these tumors ultimately converge on the epigenome to dysregulate developmental programs. In this review, we relate how alterations in components of the transcriptional machinery and chromatin modifier genes contribute to the initiation and progression of pediatric solid tumors. We also discuss how dramatic progress in our understanding of the fundamental mechanisms that contribute to epigenetic deregulation in cancer is providing novel avenues for targeted cancer therapy.

Figure 1. Higher order complexity of DNA

To achieve required nuclear compactness, eukaryotic DNA is wrapped around core histone proteins (histone octamers) and packaged into compact chromatin structures termed nucleosomes. Epigenetic regulation of gene expression is predominantly controlled by covalent modifications to histones (on histone tails). These post-translational modifications signal the recruitment of protein complexes that: 1) more tightly package the nucleosomes causing condensed chromatin referred to as heterochromatin. Heterochromatin is devoid of gene transcription; 2) remodel the nucleosomes leading to more loosely or irregularly spaced nucleosomes referred to as euchromatin. Regulated gene transcription takes place in euchromatin regions; and 3) recruit proteins responsible for DNA methylation. Insert Box: Modifications to Histone Tails. In particular, methylation of lysine residues 9 and 27 on histone 3 (H3K9me2, H3K9me3 and H3K27me3) and ubiquitination of histone 2A (H2AUb) are associated with a more compact heterochromatin structure and gene silencing (6). The activity of methyltransferases is countered by proteins with demethylase activity. Lysines may also be acetylated by acetyltransferases including GCN5/PCAF or CBP/p300 and typically acetylated lysines favor gene transcription. A series of histone deacetylases (HDAC1–11) deacetylated lysines and increased activity or levels of these proteins is associated with gene silencing. Proteins involved in these processes are explained in more detail in Figure 2. Histone modifications associated with silencing and proteins mediating them are denoted in red while those associated with gene activation are denoted in green.

Figure 2. Protein modifications and complexes that regulate higher order chromatin conformation

A. PcG-protein complexes. The PRC2 protein EZH2 is the key effector of PRC2 action, catalyzing trimethylation of H3K27 (H3K27me3) (7, 8). Histone deacetylases (HDACs) also bind the PRC2 complex decreasing acetylation of H3K27 and favoring its methylation and inhibiting gene transcription. In contrast, inhibitors of histone deacetylases (HDACi) such as Vorinistat or Romidepsin would be expected to counteract this activity resulting in increased acetylation at these loci, favoring gene expression. For example, at steady-state EZH2 mediates increased H3K27me3 at the CASZ1 tumor suppressor gene and loss of gene transcription in neuroblastoma and Romidepsin (depsipeptide) treatment leads to increased H3K27Ac and increased gene transcription at this locus (57). PRC2 is targeted to DNA by JARID2, which binds GC containing DNA regions. PRC1 in turn mono-ubiquitinates H2A, a task that is achieved by the PRC1 protein BMI-1 in cooperation with the E3 ubiquitin ligase RING1B (7, 8). In contrast, acetylation of lysine residues on histones 3 and 4 (H3K, H4K) and methylation of H3K residue 4 (H3K4me3) promote an open euchromatin state and active transcription. B. Nucleosome remodeling The ATP-dependent chromatin remodeling complexes are a family of proteins SWI/SNF, ISWI, NURD/MI-2/CHD and INO80 characterized by common DExx and HELICc domains. These chromatin-remodeling complexes use energy-dependent mechanisms to move the DNA around the histone octamer and also alter histone octamer composition. Most significantly, the SWI/SNF complex has been shown to play essential roles in regulating nucleosome remodeling, contributing to both the activation (left panel denoted in green) and repression (right panel denoted in red) of gene expression programs in a context-dependent manner (17). During lineage-specific differentiation the SWI/SNF complex cooperates with transcription factors to promote activation of differentiation genes and silencing of proliferation genes. C. DNA methylation. Methylation of CpG islands located in proximity to transcription initiation is associated with heritable silencing (denoted in red) of gene expression (gene off) and is responsible for physiologic processes that depend on permanent gene silencing such as X-chromosome inactivation and gene imprinting (11). DNA methylation may be mediated by transcription factor or PcG –mediated recruitment of DNMTs to specific gene loci. D. DNA demethylation. More recent studies have identified DNA demethylases (TET1,2) which hydroxylate 5-Methylcytosine(5mC) in CpG dinucleotides to 5-Hydroxymethylcytosine(5-hC). Evidence proposes that TET enzymes are capable of iterative oxidation on substrates to 5-formylmethylcytosine (5-fC) or 5-carboxymethylcytosine(5-caC) (23, 24). This leads to substrates upon which base excision repair mechanisms mediated by thymine-DNA glycosylase (TDG) excise a modified C and replace with an unmodified C, allowing for rapid re-activation (gene ON) of previously silenced genes (24).

Figure 3. Chromatin Regulation of Developmental Gene Expression and Oncogenic Dysregulation

A. Genome wide analyses of chromatin marks indicate that in embryonic stem cells expression of lineage specific transcriptional programs is suppressed while pluripotency genes such as OCT4, MYC, SOX2 and BMI-1 are expressed (–4). Key developmental genes appear to be enriched for H3K4me3 gene activation and H3K27me3 gene suppression modifications at their enhancers or promoters. These “bivalent” marks are thought to identify key developmental genes “poised” to be dynamically regulated upon appropriate activation of lineage specific pathways (15). In a stem cell these genes may be viewed as being “reversibly silenced”. During normal differentiation, lineage specific programs silenced in the stem cell are activated while pluripotency genes are silenced. During cell specification, non-specified lineage gene programs are suppressed. B. Oncogenic alterations may occur at any point during stem cell differentiation leading to blocks in developmental programs. Moreover, failure to suppress alternate lineage programs could lead to tumors with mixed lineage phenotypes. In models of EFT initiation, ectopic expression of EWS-FLI1 in mesenchymal (MSC) or neural crest (NCSC) stem cells modulates the expression of numerous transcripts and proteins involved in epigenetic regulation (shown in red; functional significance of proteins in box – BMI-1, EZH2, NKX2.2 – has been experimentally validated – See Refs. , , , and 39). Over-expression of DNA methyltransferases (DNMTs) in these cells is also observed and contributes to preferential silencing of transcription factors that normally instruct terminal differentiation of neuro-mesenchymal cells (E.R. Lawlor Lab, unpublished data). The precise molecular mechanisms that dictate persistent PcG- and DNA methylation-mediated repression of these developmental pathways remain to be elucidated but may involve EWS-FLI1-mediated deregulation of non-coding RNAs (miRNAs and lncRNAs).

Figure 4. Inhibition of polycomb proteins promotes tumor cell differentiation

A. BMI-1 knockdown (shBMI1) EFT cells have reduced tumorigenic capacity in immune deficient mice (42). As shown here, TC-71 xenografts established from stably transduced BMI-1 knockdown cells express higher levels of peripherin, a cytoskeletal marker of differentiated peripheral neurons, than tumors established from non-silenced control cells (shNS). Top panel: Q-RT-PCR - expression of PRPH is normalized relative to expression of GAPDH (Taqman assays from Applied Biosystems). Bottom panel: Peripherin immunostaining of fresh-frozen sections (peripherin antibody 1:100 dilution; Cat# AB1530, Millipore, Temecula, CA.). B. Deazaneplanocin A (DZnep) inhibits EZH2 as well as histone methylation more broadly and induces apoptosis or differentiation in pre-clinical models of a number of tumors (102, 103). Treatment of the NB cell line SKN-BE2 for 4-days with DZnep inhibits growth and induces morphologic differentiation.