The Role of Non-Coding RNAs in Epigenetic Dysregulation in Glioblastoma Development.

Glioblastoma (GBM) is a primary brain tumor arising from glial cells. The tumor is highly aggressive, the reason for which it has become the deadliest brain tumor type with the poorest prognosis. Like other cancers, it compromises molecular alteration on genetic and epigenetic levels. Epigenetics refers to changes in gene expression or cellular phenotype without the occurrence of any genetic mutations or DNA sequence alterations in the driver tumor-related genes. These epigenetic changes are reversible, making them convenient targets in cancer therapy. Therefore, we aim to review critical epigenetic dysregulation processes in glioblastoma. We will highlight the significant affected tumor-related pathways and their outcomes, such as regulation of cell cycle progression, cell growth, apoptosis, angiogenesis, cell invasiveness, immune evasion, or acquirement of drug resistance. Examples of molecular changes induced by epigenetic modifications, such as DNA epigenetic alterations, histone post-translational modifications (PTMs), and non-coding RNA (ncRNA) regulation, are highlighted. As understanding the role of epigenetic regulators and underlying molecular mechanisms in the overall pro-tumorigenic landscape of glioblastoma is essential, this literature study will provide valuable insights for establishing the prognostic or diagnostic value of various non-coding transcripts, including miRNAs.

H3K9 dimethylation safeguards cancer cells against activation of the interferon pathway.

Activation of interferon genes constitutes an important anticancer pathway able to restrict proliferation of cancer cells. Here, we demonstrate that the H3K9me3 histone methyltransferase (HMT) suppressor of variegation 3-9 homolog 1 (SUV39H1) is required for the proliferation of acute myeloid leukemia (AML) and find that its loss leads to activation of the interferon pathway. Mechanistically, we show that this occurs via destabilization of a complex composed of SUV39H1 and the two H3K9me2 HMTs, G9A and GLP. Indeed, loss of H3K9me2 correlated with the activation of key interferon pathway genes, and interference with the activities of G9A/GLP largely phenocopied loss of SUV39H1. Last, we demonstrate that inhibition of G9A/GLP synergized with DNA demethylating agents and that SUV39H1 constitutes a potential biomarker for the response to hypomethylation treatment. Collectively, we uncovered a clinically relevant role for H3K9me2 in safeguarding cancer cells against activation of the interferon pathway.

Human telomerase is directly regulated by non-telomeric TRF2-G-quadruplex interaction.

Human telomerase reverse transcriptase (hTERT) remains suppressed in most normal somatic cells. Resulting erosion of telomeres leads eventually to replicative senescence. Reactivation of hTERT maintains telomeres and triggers progression of >90% of cancers. However, any direct causal link between telomeres and telomerase regulation remains unclear. Here, we show that the telomere-repeat-binding-factor 2 (TRF2) binds hTERT promoter G-quadruplexes and recruits the polycomb-repressor EZH2/PRC2 complex. This is causal for H3K27 trimethylation at the hTERT promoter and represses hTERT in cancer as well as normal cells. Two highly recurrent hTERT promoter mutations found in many cancers, including ∼83% glioblastoma multiforme, that are known to destabilize hTERT promoter G-quadruplexes, showed loss of TRF2 binding in patient-derived primary glioblastoma multiforme cells. Ligand-induced G-quadruplex stabilization restored TRF2 binding, H3K27-trimethylation, and hTERT re-suppression. These results uncover a mechanism of hTERT regulation through a telomeric factor, implicating telomere-telomerase molecular links important in neoplastic transformation, aging, and regenerative therapy.

Vps11 and Vps18 of Vps-C membrane traffic complexes are E3 ubiquitin ligases and fine-tune signalling.

In response to extracellular signals, many signalling proteins associated with the plasma membrane are sorted into endosomes. This involves endosomal fusion, which depends on the complexes HOPS and CORVET. Whether and how their subunits themselves modulate signal transduction is unknown. We show that Vps11 and Vps18 (Vps11/18), two common subunits of the HOPS/CORVET complexes, are E3 ubiquitin ligases. Upon overexpression of Vps11/Vps18, we find perturbations of ubiquitination in signal transduction pathways. We specifically demonstrate that Vps11/18 regulate several signalling factors and pathways, including Wnt, estrogen receptor α (ERα), and NFκB. For ERα, we demonstrate that the Vps11/18-mediated ubiquitination of the scaffold protein PELP1 impairs the activation of ERα by c-Src. Thus, proteins involved in membrane traffic, in addition to performing their well-described role in endosomal fusion, fine-tune signalling in several different ways, including through ubiquitination.

Aggressiveness of non-EMT breast cancer cells relies on FBXO11 activity.

Tumorigenesis is increasingly considered to rely on subclones of cells poised to undergo an epithelial to mesenchymal transition (EMT) program. We and others have provided evidence, however, that the tumorigenesis of human breast cancer is not always restricted to typical EMT cells but is also somewhat paradoxically conveyed by subclones of apparently differentiated, non-EMT cells. Here we characterize such non-EMT-like and EMT-like subclones. Through a loss-of-function screen we found that a member of the E3 ubiquitin ligase complexes, FBXO11, specifically fuels tumor formation of a non-EMT-like clone by restraining the p53/p21 pathway. Interestingly, in the related EMT-like clone, FBXO11 operates through the BCL2 pathway with little or no impact on tumorigenesis. These data command caution in attempts to assess tumorigenesis prospectively based on EMT profiling, and they emphasize the importance of next generation subtyping of tumors, that is at the level of clonal composition.

EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16INK4A. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.

Monoubiquitination of Histone H2B Blocks Eviction of Histone Variant H2A.Z from Inducible Enhancers.

Covalent modifications of histones play a crucial role in the regulation of gene expression. Histone H2B monoubiquitination has mainly been described as a regulator of transcription elongation, but its role in transcription initiation is poorly documented. We investigated the role of this histone mark (H2Bub1) on different inducible enhancers, in particular those regulated by estrogen receptor α, by loss- and gain-of-function experiments with the specific E3-ubiquitin ligase complex of H2B: RNF20/RNF40. RNF20/RNF40 overexpression causes repression of the induced activity of these enhancers. Genome-wide profiles show that H2Bub1 levels are negatively correlated with the accessibility of enhancers to transcriptional activators. We found that the chromatin association of histone variant H2A.Z, which is evicted from enhancers for transcriptional activation, is stabilized by H2Bub1 by impairing access of the chromatin remodeler INO80. We propose that H2Bub1 acts as a gatekeeper of H2A.Z eviction and activation of inducible enhancers.

Histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and cancer cell proliferation.

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.

Detection of changes in gene regulatory patterns, elicited by perturbations of the Hsp90 molecular chaperone complex, by visualizing multiple experiments with an animation.

BACKGROUND: To make sense out of gene expression profiles, such analyses must be pushed beyond the mere listing of affected genes. For example, if a group of genes persistently display similar changes in expression levels under particular experimental conditions, and the proteins encoded by these genes interact and function in the same cellular compartments, this could be taken as very strong indicators for co-regulated protein complexes. One of the key requirements is having appropriate tools to detect such regulatory patterns. RESULTS: We have analyzed the global adaptations in gene expression patterns in the budding yeast when the Hsp90 molecular chaperone complex is perturbed either pharmacologically or genetically. We integrated these results with publicly accessible expression, protein-protein interaction and intracellular localization data. But most importantly, all experimental conditions were simultaneously and dynamically visualized with an animation. This critically facilitated the detection of patterns of gene expression changes that suggested underlying regulatory networks that a standard analysis by pairwise comparison and clustering could not have revealed. CONCLUSIONS: The results of the animation-assisted detection of changes in gene regulatory patterns make predictions about the potential roles of Hsp90 and its co-chaperone p23 in regulating whole sets of genes. The simultaneous dynamic visualization of microarray experiments, represented in networks built by integrating one's own experimental with publicly accessible data, represents a powerful discovery tool that allows the generation of new interpretations and hypotheses.

Multidirectional interplay between nuclear receptors and microRNAs.

Nuclear receptors (NRs) form one of the largest superfamilies of transcription factors in metazoans. MicroRNAs (miRNAs) are small non-coding RNAs that bind the 3' untranslated region (3'UTR) of target mRNAs to reduce their stability and/or translation. miRNAs can directly regulate the protein output of target NR mRNAs, and, conversely, the expression of miRNAs can be modulated by NRs at the transcriptional level. At least one NR also regulates the posttranscriptional maturation of miRNAs by interacting with miRNA processing factors via NR co-regulators. Moreover, miRNAs regulate NR signaling by targeting the mRNAs of NR co-regulators and target genes. This complex set of interactions also leads to an extensive network of feedback and feedforward regulatory loops.

miR-22 inhibits estrogen signaling by directly targeting the estrogen receptor alpha mRNA.

Estrogen receptor alpha (ER alpha) is a ligand-regulated transcription factor with a broad range of physiological functions and one of the most important classifiers in breast cancer. MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as important regulators of gene expression in a plethora of physiological and pathological processes. Upon binding the 3' untranslated region (UTR) of target mRNAs, miRNAs typically reduce their stability and/or translation. The ER alpha mRNA has a long 3' UTR of about 4.3 kb which has been reported to reduce mRNA stability and which bears evolutionarily conserved miRNA target sites, suggesting that it might be regulated by miRNAs. We have performed a comprehensive and systematic assessment of the regulatory role of all miRNAs that are predicted to target the 3' UTR of the ER alpha mRNA. We found that miR-22 represses ER alpha expression most strongly and by directly targeting the ER alpha mRNA 3' UTR. Of the three predicted miR-22 target sites in the 3' UTR, the evolutionarily conserved one is the primary target. miR-22 overexpression leads to a reduction of ER alpha levels, at least in part by inducing mRNA degradation, and compromises estrogen signaling, as exemplified by its inhibitory impact on the ER alpha-dependent proliferation of breast cancer cells.

Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF.

The steroid hormone oestrogen can signal through several receptors and pathways. Although the transcriptional responses mediated by the nuclear oestrogen receptors (ER) have been extensively characterized, the changes in gene expression elicited by signalling through the membrane-associated ER GPR30 have not been studied. We show here for ER-negative human breast cancer cells that the activation of GPR30 signalling by oestrogen or by hydroxytamoxifen (OHT), an ER antagonist but GPR30 agonist, induces a transcription factor network, which resembles that induced by serum in fibroblasts. The most strongly induced gene, CTGF, appears to be a target of these transcription factors. We found that the secreted factor connective tissue growth factor (CTGF) not only contributes to promote proliferation but also mediates the GPR30-induced stimulation of cell migration. These results provide a framework for understanding the physiological and pathological functions of GPR30. As the activation of GPR30 by OHT also induces CTGF in fibroblasts from breast tumour biopsies, these pathways may be involved in promoting aggressive behaviour of breast tumours in response to endogenous oestrogens or to OHT being used for endocrine therapy.

HicA of Escherichia coli defines a novel family of translation-independent mRNA interferases in bacteria and archaea.

Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the hicAB loci constitute a novel TA gene family. Using the hicAB locus of Escherichia coli K-12 as a model system, we present evidence that supports this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.

Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells.

Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.

Competitive inhibition of natural antisense Sok-RNA interactions activates Hok-mediated cell killing in Escherichia coli.

Short regulatory RNAs are widespread in bacteria, and many function through antisense recognition of mRNA. Among the best studied antisense transcripts are RNA antitoxins that repress toxin mRNA translation. The hok/sok locus of plasmid R1 from Escherichia coli is an established model for RNA antitoxin action. Base-pairing between hok mRNA and Sok-antisense-RNA increases plasmid maintenance through post-segregational-killing of plasmid-free progeny cells. To test the model and the idea that sequestration of Sok-RNA activity could provide a novel antimicrobial strategy, we designed anti Sok peptide nucleic acid (PNA) oligomers that, according to the model, would act as competitive inhibitors of hok mRNA::Sok-RNA interactions. In hok/sok-carrying cells, anti Sok PNAs were more bactericidal than rifampicin. Also, anti Sok PNAs induced ghost cell morphology and an accumulation of mature hok mRNA, consistent with cell killing through synthesis of Hok protein. The results support the sense/antisense model for hok mRNA repression by Sok-RNA and demonstrate that antisense agents can be used to out-compete RNA::RNA interactions in bacteria. Finally, BLAST analyses of approximately 200 prokaryotic genomes revealed that many enteric bacteria have multiple hok/sok homologous and analogous RNA-regulated toxin-antitoxin loci. Therefore, it is possible to activate suicide in bacteria by targeting antitoxins.

Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes.

Prokaryotic chromosomes code for toxin-antitoxin (TA) loci, often in multiple copies. In E.coli, experimental evidence indicates that TA loci are stress-response elements that help cells survive unfavorable growth conditions. The first gene in a TA operon codes for an antitoxin that combines with and neutralizes a regulatory 'toxin', encoded by the second gene. RelE and MazF toxins are regulators of translation that cleave mRNA and function, in interplay with tmRNA, in quality control of gene expression. Here, we present the results from an exhaustive search for TA loci in 126 completely sequenced prokaryotic genomes (16 archaea and 110 bacteria). We identified 671 TA loci belonging to the seven known TA gene families. Surprisingly, obligate intracellular organisms were devoid of TA loci, whereas free-living slowly growing prokaryotes had particularly many (38 in Mycobacterium tuberculosis and 43 in Nitrosomonas europaea). In many cases, TA loci were clustered and closely linked to mobile genetic elements. In the most extreme of these cases, all 13 TA loci of Vibrio cholerae were bona fide integron elements located in the V.cholerae mega-integron. These observations strongly suggest that TA loci are mobile cassettes that move frequently within and between chromosomes and also lend support to the hypothesis that TA loci function as stress-response elements beneficial to free-living prokaryotes.