Aberrant Transcriptional Regulation of Super-enhancers by RET Finger Protein-histone Deacetylase 1 Complex in Glioblastoma: Chemoresistance to Temozolomide.

Glioblastoma (GBM), the most common primary brain tumor, is the most aggressive human cancers, with a median survival rate of only 14.6 months. Temozolomide (TMZ) is the frontline chemotherapeutic drug in GBM. Drug resistance is the predominant obstacle in TMZ therapy. Drug resistance occurs via multiple pathways such as DNA mismatch repair and base excision repair systems, by which glioma cells acquire chemoresistance to some extent (5% and 95%, respectively). Histone3 Lysin27 residue-acetylation (H3K27ac) status regulates cis-regulatory elements, which increases the likelihood of gene transcription. Histone deacetylase (HDAC) complex deacetylate lysine residues on core histones, leading to a decrease in gene transcription. In cis-regulatory element regions, complexes with HDAC repress histones by H3K27ac deacetylation. The cis-regulating and three-dimensional transcriptional mechanism is called "super-enhancer". RET finger protein (RFP) is a protein that is expressed in many kinds of cancer. RFP forms a protein complex with HDAC1. The disruption of the RFP-HDAC1 complex has resulted in increased drug sensitivity in other cancers. We conclude that the downregulation of RFP or the disruption of the RFP/HDAC1 complex leads to an increase in TMZ efficacy in glioblastoma by changing histone modifications which lead to changes in cell division, cell cycle and apoptosis.

Introduction

Glioblastoma (GBM) is the most aggressive of all brain tumors. Its prognosis is bleak, with a median survival time of 14.6 months.[1)] Temozolomide (TMZ) is an oral chemotherapeutic alkylating agent that offers some promise. Nevertheless, it only confers a 5-year survival rate in <10% of cases.[1,2)] TMZ is a prodrug, administered orally, but activated in the more alkaline environment of the brain tumor tissue.[3–5)] TMZ induces tumor cell cytotoxicity by methylating genomic DNA. The major site of methylation is at the N3 position of adenine (∼20%), the N7 of guanine (∼80%) and the O6 of guanine (5%). Acquired resistance to TMZ is a common phenomenon in the patient population.[6,7)] DNA repair mechanisms such as DNA mismatch repair[8–10)] and base excision repair (BER)[11,12)] contribute to TMZ resistance. Temozolomide methylates guanine residues in glioma cells, which results in cell death due to the failure of the DNA mismatch repair system to find a complementary base for methylated guanine. However, MGMT creates a DNA repair system by demethylating the guanine residues methylated by TMZ. Thus, MGMT plays a vital role in TMZ resistance.[13)] Poly(ADP-ribose) polymerase 1 (PARP1) plays a role in TMZ resistance via BER. PARP1 binding protein (PARPBP) or C12orf48 binds directly to PARP1, leading to an increase in PARP1 activity. The expression of PARPBP is evidently increased in many types of cancer[14)] (Fig. 1). It has been observed that PARP1 inhibitors augment the therapeutic effect of TMZ in glioma.[15)]

Fig. 1

MGMT demethylation is a known factor in the resistance of temozolomide (TMZ), which is the mainstream drug. The PARP1–PARP binding protein (PARPBP) complex also contributes to TMZ resistance by the BER pathway. Depletion of RET finger protein (RFP) decreases the transcription of PARPBP, and destroys the formation of complex, leading to cytotoxicity.

In this review article, we describe that how the disruption of histone deacetylase (HDAC) complex affects the status of Histone3 Lysin27 residue-acetylation (H3K27ac)-mediated cis-regulatory elements (super-enhancer), leading to chemoresistance to TMZ.

Gene expression regulated by super-enhancer

The variety of DNA regulatory elements in the genome includes silencers, insulators, and enhancer regions. Those control and maintain gene expression that occurs during mammalian development. Enhancers are particularly important in their regulatory roles that help determine cell fates. They are key cis-regulatory elements that can influence transcription of genes that differ in orientation or are thousands to millions pairs away from promoters and transcriptional start sites.[16,17)] Recent studies have sought to identify enhancers globally by focusing on the histone marker H3-lysine4-monomethylated (H3K4me1), with the aim of identifying many cell type-specific enhancer sites.[18–21)] However, many of the enhancers that are enriched by H3K4me1 have proximal gene transcriptional activity[22)] and many H3K4me1 associated enhancer regions are inactive.[18,22)] Conversely, H3K27ac is able to distinguish between inactive (poised) and active enhancer elements.[23)]

Even though a single enhancer can activate the expression of a nearby gene, high levels of cell type-specific and/or signal-dependent gene expression require enhancers located heterogeneously, with some genes residing in enhancer-rich regions of the genome. Such enhancer-rich regions have recently been termed as “super-enhancers”.[24–26)]

Super-enhancers were initially defined as large genomic loci tens of kilobases in length with an unusually high density of enhancer-associated markers, such as binding of the mediator complex, relative to most other genomic loci.[24,25)] These regions can also feature high density and/or extended (>3 KB) depositions of the histone marker H3K27ac. Super-enhancers can be differentiated from regular enhancers on the basis of differences in the density of mediator complex-binding sites or of H3K27ac markers. These differences have revealed the presence of 300–500 super-enhancers in most types of cells.[24)] Many super-enhancers and nearby genes are cell type-specific, and the gene sets that are associated with super-enhancers in a given cell type are highly enriched for the biological processes that define the identities of the cell types (Fig. 2).

Fig. 2

Histone3 Lysin27 residue-acetylation (H3K27ac) status regulates cis-regulatory elements, which increases the likelihood of gene transcription. Histone deacetylase (HDAC) complex deacetylate lysine residues on core histones, leading to a decrease in gene transcription. In cis-regulatory element regions (super-enhancer), complexes with HDAC repress histones by H3K27ac deacetylation.

In gliomas, tumor malignancy is associated with DNA hypomethylation, including altered cis-regulatory elements and promoter hypomethylation that leads to transcriptional upregulation of genes.[27)] CpG sites are found in gene promoters as well as in gene bodies or cis-regulatory elements such as enhancers, silencers, and insulators.[28,29)] These regulatory elements contain binding sites for transcription factors and act to increase or decrease transcription. The protein–DNA complex folds a three-dimensional chromatin loop called a topology associated domain (TAD). TADs are regulated by insulators, namely CCCTC-binding factors (CTCFs), which can block interactions between enhancers and promoters in the intergenic regions (Fig. 3). In IDH mutant gliomas, hypermethylation at CTCF binding sites inhibits the binding of CTCF, leading to aberrant enhancer-gene interplays and the upregulation of oncogenes such as platelet-derived growth factor receptor-α.[30)] The glioma-CpG island methylator phenotype-low subgroup has been speculated to exhibit a loss of genome-wide DNA methylation, including at CTCF binding sites, thereby affecting the chromatin architecture.[31)]

Fig. 3

(A) The protein–DNA complex folds a three-dimensional chromatin loop called a topology associated domain (TAD). (B) TADs are regulated by insulators, namely CCCTC-binding factors (CTCFs), which can block interactions between enhancers and promoters in the intergenic regions. Hypermethylation at CTCF binding sites inhibits the binding of CTCF, thereby affecting the chromatin architecture.

RET finger protein

RET finger protein (RFP, also termed as tripartite-motif-containing 27, TRIM27) is a transcription factor that can become oncogenic when fused with RET tyrosine kinase.[32–34)] RFP is involved in cell growth[35)] and apoptosis[36)] and is expressed in diverse types of cancer cells.[33,37–41)] In these cells, RFP forms a tripartite complex with HDAC1 and nuclear transcription factor Y (NF-Y). The knockdown of RFP (RFP-KD) disrupts the complex formed between RFP, HDAC1, and NF-Y, which increases the expression of thioredoxin-binding protein-2 (TBP-2).[42)] These events increase the chemosensitivity of cancer cells to cisplatin (Fig. 4A).

Fig. 4

RET finger protein (RFP, also termed as tripartite-motif-containing 27, TRIM27) is a transcription factor that can become oncogenic when fused with RET tyrosine kinase. RFP forms a tripartite complex with histone deacetylase 1 (HDAC1) and nuclear transcription factor Y (NF-Y). The knockdown of RFP (RFP-KD) disrupts the complex formed between RFP, HDAC1, and NF-Y, which increases the expression of thioredoxin-binding protein-2 (TBP-2) that is related to reactive oxygen species (ROS) and chemoresistance.

RET finger protein/HDAC1 complex and super-enhancer in GBM in association with cell cycles

RET finger protein is able to form a complex with HDAC1 in GBM cells, similar to Hela cells.[42)] Destruction of the complex using RFP siRNA can increase the efficacy of TMZ against RFP-expressing glioma cells. To investigate the mode of action, Ranjit et al.[43)] studied aberrant H3K27ac-controlled active cis-regulatory elements using chromatin immunoprecipitation next-generation sequencing and RNA-seq. The analyses revealed that disruption of the RFP–HDAC1 complex in turn disrupted the functioning of the H3K27ac-controlled active cis-regulatory elements. At first, the authors expected that the depletion of RFP–HDAC1 would lead to the upregulation of H3K27ac in most cis-regulatory-element regions. However, increases in the amount of H3K27ac were detected in some regions while decreases were evident in other regions. The findings implied that the RFP–HDAC1 complex directly controls the “H3K27ac gain” and upregulated genes and indirectly controls the “H3K27ac loss” and downregulated genes.

Gene Ontology analyses indicated that the genes in the “H3K27ac loss” group, whose expression levels significantly decreased, included genes with functions in the cell cycle, cell division, and DNA replication.

RET finger protein/HDAC1 complex and super-enhancer in GBM in association with the BER pathway

RET finger protein binds to the promyelocytic leukemia (PML) gene,[44)] and PML interacts with the mSin3a-HDAC1 corepressor,[45)] which in turn inhibits the expression of MGMT.[46)] Furthermore, RFP is linked with p53 sumoylation,[47,48)] and p53 downregulates MGMT in astrocytes.[48)] The PARPBP–PARP1 complex is a key BER agent that causes TMZ resistance. Ranjit et al.[43)] observed that RFP-KD led to a significant decrease in PARPBP expression. In addition, PARP1 inhibitors sensitize other cancers to antitumor agents by inhibiting the production of thioredoxin and reactive oxygen species.[49,50)]

Knockdown of RFP disrupts the RFP–HDAC1 complex, which results in the upregulation of Forkhead box1 (FOXO1) and TBP-2 proteins and the subsequent generation of ROS and induction of apoptosis. HDAC1 inhibits FOXO1,[51)] which induces the expression of TBP-2. Overexpression of TBP-2 results in the inactivation of thioredoxin (TRX) and increased oxidative stress.[42)] The inhibition of HDAC1 by RFP-KD results in the increased expression of FOXO1 and TBP-2 (Thioredoxin-interacting protein). RFP depletion and TMZ treatment induces oxidative stress in GBM cells (Fig. 4B).

Conclusion

In conclusion, RFP-KD or the disruption of the RFP–HDAC1 complex increases TMZ chemosensitivity of GBM cells by altering the pattern of histone modification, which alters oxidative stress and cell division.