Comparative epigenetic analysis of tumour initiating cells and syngeneic EPSC-derived neural stem cells in glioblastoma.

Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM.

Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates.

Most proteins in cell and tissue lysates are soluble. We show here that in lysate from human neurons, more than 1,300 proteins are maintained in a soluble and functional state by association with endogenous RNA, as degradation of RNA invariably leads to protein aggregation. The majority of these proteins lack conventional RNA-binding domains. Using synthetic oligonucleotides, we identify the importance of nucleic acid structure, with single-stranded pyrimidine-rich bulges or loops surrounded by double-stranded regions being particularly efficient in the maintenance of protein solubility. These experiments also identify an apparent one-to-one protein-nucleic acid stoichiometry. Furthermore, we show that protein aggregates isolated from brain tissue from Amyotrophic Lateral Sclerosis patients can be rendered soluble after refolding by both RNA and synthetic oligonucleotides. Together, these findings open new avenues for understanding the mechanism behind protein aggregation and shed light on how certain proteins remain soluble.

Identification of a subset of trace amine-associated receptors and ligands as potential modulators of insulin secretion.

The worldwide prevalence of diabetes has reached 8.5% among adults, and this is characterised by elevated glucose concentrations and failing insulin secretion. Furthermore, most people with type 2 diabetes are either obese or overweight, with the associated dyslipidaemia contributing to the development of insulin resistance and increased cardiovascular risk. Here we incubated INS-1 pancreatic ß-cells for 72 h in RPMI-1640 media, or media supplemented with 28 mM glucose, 200 µM palmitic acid, and 200 µM oleic acid as a cellular model of diabetic glucolipotoxicity. Illumina HiSeq gene expression analysis showed the trace amine-associated receptor (TAAR) family to be among the most highly downregulated by glucolipotoxicity. Importantly, MetaCore integrated knowledge database, from Clarivate Analytics, indicated potential TAAR impact on insulin secretion through adenylyl cyclase signalling pathways. We therefore investigated the effect of TAAR ligands on cAMP signalling and insulin secretion, and found that only the branch of the TAAR family tree that is activated by isopentylamine, 2-phenylethylamine, p-tyramine, and agmatine significantly increased intracellular cAMP and resulted in increased insulin secretion from INS-1 cells and primary mouse islets under normal conditions. Crucially however, this enhancement was not evident when the receptor family was downregulated by glucolipotoxic conditions. This data indicates that a subset of TAARs are regulators of insulin secretion in pancreatic ß-cells, and that their downregulation contributes to glucolipotoxic inhibition of insulin secretion. As such they may be potential targets for treatment of type 2 diabetes.

Glucolipotoxicity initiates pancreatic ß-cell death through TNFR5/CD40-mediated STAT1 and NF-κB activation.

Type 2 diabetes is a chronic metabolic disorder, where failure to maintain normal glucose homoeostasis is associated with, and exacerbated by, obesity and the concomitant-elevated free fatty acid concentrations typically found in these patients. Hyperglycaemia and hyperlipidaemia together contribute to a decline in insulin-producing ß-cell mass through activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)-1. There are however a large number of molecules potentially able to modulate NF-κB and STAT1 activity, and the mechanism(s) by which glucolipotoxicity initially induces NF-κB and STAT1 activation is currently poorly defined. Using high-density microarray analysis of the ß-cell transcritptome, we have identified those genes and proteins most sensitive to glucose and fatty acid environment. Our data show that of those potentially able to activate STAT1 or NF-κB pathways, tumour necrosis factor receptor (TNFR)-5 is the most highly upregulated by glucolipotoxicity. Importantly, our data also show that the physiological ligand for TNFR5, CD40L, elicits NF-κB activity in ß-cells, whereas selective knockdown of TNFR5 ameliorates glucolipotoxic induction of STAT1 expression and NF-κB activity. This data indicate for the first time that TNFR5 signalling has a major role in triggering glucolipotoxic islet cell death.

DNA methylation analysis of paediatric low-grade astrocytomas identifies a tumour-specific hypomethylation signature in pilocytic astrocytomas.

Low-grade gliomas (LGGs) account for about a third of all brain tumours in children. We conducted a detailed study of DNA methylation and gene expression to improve our understanding of the biology of pilocytic and diffuse astrocytomas. Pilocytic astrocytomas were found to have a distinctive signature at 315 CpG sites, of which 312 were hypomethylated and 3 were hypermethylated. Genomic analysis revealed that 182 of these sites are within annotated enhancers. The signature was not present in diffuse astrocytomas, or in published profiles of other brain tumours and normal brain tissue. The AP-1 transcription factor was predicted to bind within 200 bp of a subset of the 315 differentially methylated CpG sites; the AP-1 factors, FOS and FOSL1 were found to be up-regulated in pilocytic astrocytomas. We also analysed splice variants of the AP-1 target gene, CCND1, which encodes cell cycle regulator cyclin D1. CCND1a was found to be highly expressed in both pilocytic and diffuse astrocytomas, but diffuse astrocytomas have far higher expression of the oncogenic variant, CCND1b. These findings highlight novel genetic and epigenetic differences between pilocytic and diffuse astrocytoma, in addition to well-described alterations involving BRAF, MYB and FGFR1.

Molecular analysis of pediatric brain tumors identifies microRNAs in pilocytic astrocytomas that target the MAPK and NF-κB pathways.

INTRODUCTION: Pilocytic astrocytomas are slow-growing tumors that usually occur in the cerebellum or in the midline along the hypothalamic/optic pathways. The most common genetic alterations in pilocytic astrocytomas activate the ERK/MAPK signal transduction pathway, which is a major driver of proliferation but is also believed to induce senescence in these tumors. Here, we have conducted a detailed investigation of microRNA and gene expression, together with pathway analysis, to improve our understanding of the regulatory mechanisms in pilocytic astrocytomas. RESULTS: Pilocytic astrocytomas were found to have distinctive microRNA and gene expression profiles compared to normal brain tissue and a selection of other pediatric brain tumors. Several microRNAs found to be up-regulated in pilocytic astrocytomas are predicted to target the ERK/MAPK and NF-κB signaling pathways as well as genes involved in senescence-associated inflammation and cell cycle control. Furthermore, IGFBP7 and CEBPB, which are transcriptional inducers of the senescence-associated secretory phenotype (SASP), were also up-regulated together with the markers of senescence and inflammation, CDKN1A (p21), CDKN2A (p16) and IL1B. CONCLUSION: These findings provide further evidence of a senescent phenotype in pilocytic astrocytomas. In addition, they suggest that the ERK/MAPK pathway, which is considered the major driver of these tumors, is regulated not only by genetic aberrations but also by microRNAs.

BORIS/CTCFL is an RNA-binding protein that associates with polysomes.

BACKGROUND: BORIS (CTCFL), a paralogue of the multifunctional and ubiquitously expressed transcription factor CTCF, is best known for its role in transcriptional regulation. In the nucleus, BORIS is particularly enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism. However, little is known about cytoplasmic BORIS, which represents the major pool of BORIS protein. RESULTS: We show, firstly, that BORIS has a putative nuclear export signal in the C-terminal domain. Furthermore, BORIS associates with mRNA in both neural stem cells and young neurons. The majority of the BORIS-associated transcripts are different in the two cell types. Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes. CONCLUSION: We have demonstrated the RNA binding properties of cellular BORIS and its association with actively translating ribosomes. We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.

CTCF binds to sites in the major histocompatibility complex that are rapidly reconfigured in response to interferon-gamma.

Activation of the major histocompatibility complex (MHC) by interferon-gamma (IFN-γ) is a fundamental step in the adaptive immune response to pathogens. Here, we show that reorganization of chromatin loop domains in the MHC is evident within the first 30 min of IFN-γ treatment of fibroblasts, and that further dynamic alterations occur up to 6 h. These very rapid changes occur at genomic sites which are occupied by CTCF and are close to IFN-γ-inducible MHC genes. Early responses to IFN-γ are thus initiated independently of CIITA, the master regulator of MHC class II genes and prepare the MHC for subsequent induction of transcription.

Widespread expression of BORIS/CTCFL in normal and cancer cells.

BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a "cancer-testis" antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function.

RAF gene fusion breakpoints in pediatric brain tumors are characterized by significant enrichment of sequence microhomology.

Gene fusions involving members of the RAF family of protein kinases have recently been identified as characteristic aberrations of low-grade astrocytomas, the most common tumors of the central nervous system in children. While it has been shown that these fusions cause constitutive activation of the ERK/MAPK pathway, very little is known about their formation. Here, we present a detailed analysis of RAF gene fusion breakpoints from a well-characterized cohort of 43 low-grade astrocytomas. Our findings show that the rearrangements that generate these RAF gene fusions may be simple or complex and that both inserted nucleotides and microhomology are common at the DNA breakpoints. Furthermore, we identify novel enrichment of microhomologous sequences in the regions immediately flanking the breakpoints. We thus provide evidence that the tandem duplications responsible for these fusions are generated by microhomology-mediated break-induced replication (MMBIR). Although MMBIR has previously been implicated in the pathogenesis of other diseases and the evolution of eukaryotic genomes, we demonstrate here that the proposed details of MMBIR are consistent with a recurrent rearrangement in cancer. Our analysis of repetitive elements, Z-DNA and sequence motifs in the fusion partners identified significant enrichment of the human minisatellite conserved sequence/χ-like element at one side of the breakpoint. Therefore, in addition to furthering our understanding of low-grade astrocytomas, this study provides insights into the molecular mechanistic details of MMBIR and the sequence of events that occur in the formation of genomic rearrangements.

Tumour angiogenesis is reduced in the Tc1 mouse model of Down's syndrome.

Down's syndrome (DS) is a genetic disorder caused by full or partial trisomy of human chromosome 21 and presents with many clinical phenotypes including a reduced incidence of solid tumours. Recent work with the Ts65Dn model of DS, which has orthologues of about 50% of the genes on chromosome 21 (Hsa21), has indicated that three copies of the ETS2 (ref. 3) or DS candidate region 1 (DSCR1) genes (a previously known suppressor of angiogenesis) is sufficient to inhibit tumour growth. Here we use the Tc1 transchromosomic mouse model of DS to dissect the contribution of extra copies of genes on Hsa21 to tumour angiogenesis. This mouse expresses roughly 81% of Hsa21 genes but not the human DSCR1 region. We transplanted B16F0 and Lewis lung carcinoma tumour cells into Tc1 mice and showed that growth of these tumours was substantially reduced compared with wild-type littermate controls. Furthermore, tumour angiogenesis was significantly repressed in Tc1 mice. In particular, in vitro and in vivo angiogenic responses to vascular endothelial growth factor (VEGF) were inhibited. Examination of the genes on the segment of Hsa21 in Tc1 mice identified putative anti-angiogenic genes (ADAMTS1and ERG) and novel endothelial cell-specific genes, never previously shown to be involved in angiogenesis (JAM-B and PTTG1IP), that, when overexpressed, are responsible for inhibiting angiogenic responses to VEGF. Three copies of these genes within the stromal compartment reduced tumour angiogenesis, explaining the reduced tumour growth in DS. Furthermore, we expect that, in addition to the candidate genes that we show to be involved in the repression of angiogenesis, the Tc1 mouse model of DS will permit the identification of other endothelium-specific anti-angiogenic targets relevant to a broad spectrum of cancer patients.

Activation of the ERK/MAPK pathway: a signature genetic defect in posterior fossa pilocytic astrocytomas.

We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549-BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto-inhibitory domains of BRAF and RAF1, which are replaced in-frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low-grade gliomas.

Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development.

BACKGROUND: Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines. PRINCIPAL FINDINGS: All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response. SIGNIFICANCE: These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.

Genomic profiling identifies discrete deletions associated with translocations in glioblastoma multiforme.

Glioblastoma multiforme is the most common tumor arising in the central nervous system. Patients with these tumors have limited treatment options and their disease is invariably fatal. Molecularly targeted agents offer the potential to improve patient treatment, however the use of these will require a fuller understanding of the genetic changes in these complex tumors. In this study, we identify copy number changes in a series of glioblastoma multiforme tumors and cell lines by applying high-resolution microarray comparative genomic hybridization. Molecular cytogenetic characterization of the cell lines revealed that copy number changes define translocation breakpoints. We focused on chromosome 6 and further characterized three regions of copy number change associated with translocations including a discrete deletion involving IGF2R, PARK2, PACRG and QKI and an unbalanced translocation involving POLH, GTPBP2 and PTPRZ1.