DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.

Fra-1 regulates its target genes via binding to remote enhancers without exerting major control on chromatin architecture in triple negative breast cancers.

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.

Crosstalk between different family members: IL27 recapitulates IFNγ responses in HCC cells, but is inhibited by IL6-type cytokines.

Interleukin-27 (IL27) is a type-I-cytokine of the IL6/IL12 family predominantly secreted by activated macrophages and dendritic cells. In the liver, IL27 expression was observed to be upregulated in patients with hepatitis B, and sera of hepatocellular carcinoma (HCC) patients contain significantly elevated levels of IL27 compared to healthy controls or patients with hepatitis and/or liver cirrhosis. In this study, we show that IL27 induces STAT1 and STAT3 phosphorylation in 5 HCC lines and 3 different types of non-transformed liver cells. We were especially interested in the relevance of the IL27-induced STAT3 activation in liver cells. Thus, we compared the IL27 responses with those induced by IFNγ (STAT1-dominated response) or IL6-type cytokines (IL6, hyper-IL6 (hy-IL6) or OSM) (STAT3-dominated response) by microarray analysis and find that in HCC cells, IL27 induces an IFNγ-like, STAT1-dependent transcriptional response, but we do not find an effective STAT3-dependent response. Validation experiments corroborate the finding from the microarray evaluation. Interestingly, the availability of STAT1 seems critical in the shaping of the IL27 response, as the siRNA knock-down of STAT1 revealed the ability of IL27 to induce the acute-phase protein γ-fibrinogen, a typical IL6 family characteristic. Moreover, we describe a crosstalk between the signaling of IL6-type cytokines and IL27: responses to the gp130-engaging cytokine IL27 (but not those to IFNs) can be inhibited by IL6-type cytokine pre-stimulation, likely by a SOCS3-mediated mechanism. Thus, IL27 recapitulates IFNγ responses in liver cells, but differs from IFNγ by its sensitivity to SOCS3 inhibition.

Transcriptional variations in the wider peritumoral tissue environment of pancreatic cancer.

Transcriptional profiling was performed on 452 RNA preparations isolated from various types of pancreatic tissue from tumour patients and healthy donors, with a particular focus on peritumoral samples. Pancreatic ductal adenocarcinomas (PDAC) and cystic tumours were most different in these non-tumorous tissues surrounding them, whereas the actual tumours exhibited rather similar transcript patterns. The environment of cystic tumours was transcriptionally nearly identical to normal pancreas tissue. In contrast, the tissue around PDAC behaved a lot like the tumour, indicating some kind of field defect, while showing far less molecular resemblance to both chronic pancreatitis and healthy tissue. This suggests that the major pathogenic difference between cystic and ductal tumours may be due to their cellular environment rather than the few variations between the tumours. Lack of correlation between DNA methylation and transcript levels makes it unlikely that the observed field defect in the peritumoral tissue of PDAC is controlled to a large extent by such epigenetic regulation. Functionally, a strikingly large number of autophagy-related transcripts was changed in both PDAC and its peritumoral tissue, but not in other pancreatic tumours. A transcription signature of 15 autophagy-related genes was established that permits a prognosis of survival with high accuracy and indicates the role of autophagy in tumour biology.

Cytoplasmic overexpression of RNA-binding protein HuR is a marker of poor prognosis in meningioma, and HuR knockdown decreases meningioma cell growth and resistance to hypoxia.

HuR regulates cytoplasmic mRNA stability and translatability, and the HuR expression level has been shown to correlate with poor disease outcome in several cancer types; however, the prognostic value and potential pro-oncogenic properties of HuR in meningioma remain unclear. Thus, in the present study, we analysed 85 meningioma tissue samples to establish the relationship between HuR expression, tumour cell proliferation, and/or patient survival. In addition, we examined the anti-proliferative effects of HuR knockdown in two meningioma cell lines (IOMM-Lee and Ben-Men-1) and conducted transcriptome-wide analyses (IOMM-Lee cells) to elucidate the molecular consequences of HuR knockdown. The results of the present study showed HuR cytoplasmic expression to correlate positively with tumour grade (p = 1.2 × 10-8 ) and negatively with progression-free and overall survival (p = 0.01) time in human meningioma tissues. In vitro, siHuR-induced HuR knockdown was shown to reduce the growth of both Ben-Men-1 (p = 2 × 10-8 ) and IOMM-Lee (p = 4 × 10-9 ) cells. Transcriptome analyses revealed HuR knockdown in IOMM-Lee cells to deregulate the HIF1A signalling pathway (p = 1.5 × 10-6 ) and to up-regulate the expression of genes essential for the assembly of the cytoplasmic mRNA processing body, global genome nucleotide-excision repair, poly(A)-specific ribonuclease activity, the positive regulation of apoptosis and of cell cycle arrest, and the negative regulation of RNA splicing [p(FDR) < 0.001]. Interestingly, HuR knockdown under hypoxic culture conditions further potentiated the effects of HuR knockdown on cell growth, apoptosis, and HIF1A expression. We thus conclude that cytoplasmic HuR expression is a marker of poor prognosis in meningioma and that HuR is a promising potential therapeutic target for use in tumours refractory to standard therapies. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.

BACKGROUND: RNA sequencing (RNA-seq) and microarrays are two transcriptomics techniques aimed at the quantification of transcribed genes and their isoforms. Here we compare the latest Affymetrix HTA 2.0 microarray with Illumina 2000 RNA-seq for the analysis of patient samples - normal lung epithelium tissue and squamous cell carcinoma lung tumours. Protein coding mRNAs and long non-coding RNAs (lncRNAs) were included in the study. RESULTS: Both platforms performed equally well for protein-coding RNAs, however the stochastic variability was higher for the sequencing data than for microarrays. This reduced the number of differentially expressed genes and genes with predictive potential for RNA-seq compared to microarray data. Analysis of this variability revealed a lack of reads for short and low abundant genes; lncRNAs, being shorter and less abundant RNAs, were found especially susceptible to this issue. A major difference between the two platforms was uncovered by analysis of alternatively spliced genes. Investigation of differential exon abundance showed insufficient reads for many exons and exon junctions in RNA-seq while the detection on the array platform was more stable. Nevertheless, we identified 207 genes which undergo alternative splicing and were consistently detected by both techniques. CONCLUSIONS: Despite the fact that the results of gene expression analysis were highly consistent between Human Transcriptome Arrays and RNA-seq platforms, the analysis of alternative splicing produced discordant results. We concluded that modern microarrays can still outperform sequencing for standard analysis of gene expression in terms of reproducibility and cost.

Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A.

BACKGROUND: Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM. METHODS: Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells. RESULTS: We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion. CONCLUSIONS: This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors.

L-plastin Ser5 phosphorylation in breast cancer cells and in vitro is mediated by RSK downstream of the ERK/MAPK pathway.

Deregulated cell migration and invasion are hallmarks of metastatic cancer cells. Phosphorylation on residue Ser5 of the actin-bundling protein L-plastin activates L-plastin and has been reported to be crucial for invasion and metastasis. Here, we investigate signal transduction leading to L-plastin Ser5 phosphorylation using 4 human breast cancer cell lines. Whole-genome microarray analysis comparing cell lines with different invasive capacities and corresponding variations in L-plastin Ser5 phosphorylation level revealed that genes of the ERK/MAPK pathway are differentially expressed. It is noteworthy that in vitro kinase assays showed that ERK/MAPK pathway downstream ribosomal protein S6 kinases α-1 (RSK1) and α-3 (RSK2) are able to directly phosphorylate L-plastin on Ser5. Small interfering RNA- or short hairpin RNA-mediated knockdown and activation/inhibition studies followed by immunoblot analysis and computational modeling confirmed that ribosomal S6 kinase (RSK) is an essential activator of L-plastin. Migration and invasion assays showed that RSK knockdown led to a decrease of up to 30% of migration and invasion of MDA-MB-435S cells. Although the presence of L-plastin was not necessary for migration/invasion of these cells, immunofluorescence assays illustrated RSK-dependent recruitment of Ser5-phosphorylated L-plastin to migratory structures. Altogether, we provide evidence that the ERK/MAPK pathway is involved in L-plastin Ser5 phosphorylation in breast cancer cells with RSK1 and RSK2 kinases able to directly phosphorylate L-plastin residue Ser5.

Neurodegeneration by activation of the microglial complement-phagosome pathway.

Systemic inflammatory reactions have been postulated to exacerbate neurodegenerative diseases via microglial activation. We now demonstrate in vivo that repeated systemic challenge of mice over four consecutive days with bacterial LPS maintained an elevated microglial inflammatory phenotype and induced loss of dopaminergic neurons in the substantia nigra. The same total cumulative LPS dose given within a single application did not induce neurodegeneration. Whole-genome transcriptome analysis of the brain demonstrated that repeated systemic LPS application induced an activation pattern involving the classical complement system and its associated phagosome pathway. Loss of dopaminergic neurons induced by repeated systemic LPS application was rescued in complement C3-deficient mice, confirming the involvement of the complement system in neurodegeneration. Our data demonstrate that a phagosomal inflammatory response of microglia is leading to complement-mediated loss of dopaminergic neurons.

Integrated metabolic modelling reveals cell-type specific epigenetic control points of the macrophage metabolic network.

BACKGROUND: The reconstruction of context-specific metabolic models from easily and reliably measurable features such as transcriptomics data will be increasingly important in research and medicine. Current reconstruction methods suffer from high computational effort and arbitrary threshold setting. Moreover, understanding the underlying epigenetic regulation might allow the identification of putative intervention points within metabolic networks. Genes under high regulatory load from multiple enhancers or super-enhancers are known key genes for disease and cell identity. However, their role in regulation of metabolism and their placement within the metabolic networks has not been studied. METHODS: Here we present FASTCORMICS, a fast and robust workflow for the creation of high-quality metabolic models from transcriptomics data. FASTCORMICS is devoid of arbitrary parameter settings and due to its low computational demand allows cross-validation assays. Applying FASTCORMICS, we have generated models for 63 primary human cell types from microarray data, revealing significant differences in their metabolic networks. RESULTS: To understand the cell type-specific regulation of the alternative metabolic pathways we built multiple models during differentiation of primary human monocytes to macrophages and performed ChIP-Seq experiments for histone H3 K27 acetylation (H3K27ac) to map the active enhancers in macrophages. Focusing on the metabolic genes under high regulatory load from multiple enhancers or super-enhancers, we found these genes to show the most cell type-restricted and abundant expression profiles within their respective pathways. Importantly, the high regulatory load genes are associated to reactions enriched for transport reactions and other pathway entry points, suggesting that they are critical regulatory control points for cell type-specific metabolism. CONCLUSIONS: By integrating metabolic modelling and epigenomic analysis we have identified high regulatory load as a common feature of metabolic genes at pathway entry points such as transporters within the macrophage metabolic network. Analysis of these control points through further integration of metabolic and gene regulatory networks in various contexts could be beneficial in multiple fields from identification of disease intervention strategies to cellular reprogramming.

Constitutive activation of oncogenic PDGFRα-mutant proteins occurring in GIST patients induces receptor mislocalisation and alters PDGFRα signalling characteristics.

BACKGROUND: Gastrointestinal stromal tumours (GIST) are mainly characterised by the presence of activating mutations in either of the two receptor tyrosine kinases c-KIT or platelet-derived growth factor receptor-α (PDGFRα). Most mechanistic studies dealing with GIST mutations have focused on c-KIT and far less is known about the signalling characteristics of the mutated PDGFRα proteins. Here, we study the signalling capacities and corresponding transcriptional responses of the different PDGFRα proteins under comparable genomic conditions. RESULTS: We demonstrate that the constitutive signalling via the oncogenic PDGFRα mutants favours a mislocalisation of the receptors and that this modifies the signalling characteristics of the mutated receptors. We show that signalling via the oncogenic PDGFRα mutants is not solely characterised by a constitutive activation of the conventional PDGFRα signalling pathways. In contrast to wild-type PDGFRα signal transduction, the activation of STAT factors (STAT1, STAT3 and STAT5) is an integral part of signalling mediated via mutated PDGF-receptors. Furthermore, this unconventional STAT activation by mutated PDGFRα is already initiated in the endoplasmic reticulum whereas the conventional signalling pathways rather require cell surface expression of the receptor. Finally, we demonstrate that the activation of STAT factors also translates into a biologic response as highlighted by the induction of STAT target genes. CONCLUSION: We show that the overall oncogenic response is the result of different signatures emanating from different cellular compartments. Furthermore, STAT mediated responses are an integral part of mutated PDGFRα signalling.

Interplay of microRNAs, transcription factors and target genes: linking dynamic expression changes to function.

MicroRNAs (miRNAs) are ubiquitously expressed small non-coding RNAs that, in most cases, negatively regulate gene expression at the post-transcriptional level. miRNAs are involved in fine-tuning fundamental cellular processes such as proliferation, cell death and cell cycle control and are believed to confer robustness to biological responses. Here, we investigated simultaneously the transcriptional changes of miRNA and mRNA expression levels over time after activation of the Janus kinase/Signal transducer and activator of transcription (Jak/STAT) pathway by interferon-γ stimulation of melanoma cells. To examine global miRNA and mRNA expression patterns, time-series microarray data were analysed. We observed delayed responses of miRNAs (after 24-48 h) with respect to mRNAs (12-24 h) and identified biological functions involved at each step of the cellular response. Inference of the upstream regulators allowed for identification of transcriptional regulators involved in cellular reactions to interferon-γ stimulation. Linking expression profiles of transcriptional regulators and miRNAs with their annotated functions, we demonstrate the dynamic interplay of miRNAs and upstream regulators with biological functions. Finally, our data revealed network motifs in the form of feed-forward loops involving transcriptional regulators, mRNAs and miRNAs. Additional information obtained from integrating time-series mRNA and miRNA data may represent an important step towards understanding the regulatory principles of gene expression.

Glioblastomas are composed of genetically divergent clones with distinct tumourigenic potential and variable stem cell-associated phenotypes.

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36%, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM.

Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells.

The identification and significance of cancer stem-like cells in malignant gliomas remains controversial. It has been proposed that cancer stem-like cells display increased drug resistance, through the expression of ATP-binding cassette transporters that detoxify cells by effluxing exogenous compounds. Here, we investigated the 'side population' phenotype based on efflux properties of ATP-binding cassette transporters in freshly isolated human glioblastoma samples and intracranial xenografts derived thereof. Using fluorescence in situ hybridization analysis on sorted cells obtained from glioblastoma biopsies, as well as human tumour xenografts developed in immunodeficient enhanced green fluorescence protein-expressing mice that allow an unequivocal tumour-stroma discrimination, we show that side population cells in human glioblastoma are non-neoplastic and exclusively stroma-derived. Tumour cells were consistently devoid of efflux properties regardless of their genetic background, tumour ploidy or stem cell associated marker expression. Using multi-parameter flow cytometry we identified the stromal side population in human glioblastoma to be brain-derived endothelial cells with a minor contribution of astrocytes. In contrast with their foetal counterpart, neural stem/progenitor cells in the adult brain did not display the side population phenotype. Of note, we show that CD133-positive cells often associated with cancer stem-like cells in glioblastoma biopsies, do not represent a homogenous cell population and include CD31-positive endothelial cells. Interestingly, treatment of brain tumours with the anti-angiogenic agent bevacizumab reduced total vessel density, but did not affect the efflux properties of endothelial cells. In conclusion our findings contribute to an unbiased identification of cancer stem-like cells and stromal cells in brain neoplasms, and provide novel insight into the complex issue of drug delivery to the brain. Since efflux properties of endothelial cells are likely to compromise drug availability, transiently targeting ATP-binding cassette transporters may be a valuable therapeutic strategy to improve treatment effects in brain tumours.

MicroRNAs miR-16 and miR-519 control meningioma cell proliferation via overlapping transcriptomic programs shared with the RNA-binding protein HuR.

Introduction: Meningiomas are the most common type of primary central nervous system tumors. In about 80% cases, these tumors are benign and grow very slowly, but the remainder 20% can unlock higher proliferation rates and become malignant. In this study we examined two miRs, miR-16 and miR-519, and evaluated their role in tumorigenesis and cell growth in human meningioma. Methods: A cohort of 60 intracranial grade 1 and grade 2 human meningioma plus 20 healthy meningeal tissues was used to quantify miR-16 and miR-519 expressions. Cell growth and dose-response assays were performed in two human meningioma cell lines, Ben-Men-1 (benign) and IOMM-Lee (aggressive). Transcriptomes of IOMM-lee cells were measured after both miR-mimics transfection, followed by integrative bioinformatics to expand on available data. Results: In tumoral tissues, we detected decreased levels of miR-16 and miR-519 when compared with arachnoid cells of healthy patients (miR-16: P=8.7e-04; miR-519: P=3.5e-07). When individually overexpressing these miRs in Ben-Men-1 and IOMM-Lee, we observed that each showed reduced growth (P<0.001). In IOMM-Lee cell transcriptomes, downregulated genes, among which ELAVL1/HuR (miR-16: P=6.1e-06; miR-519:P=9.38e-03), were linked to biological processes such as mitotic cell cycle regulation, pre-replicative complex, and brain development (FDR<1e-05). Additionally, we uncovered a specific transcriptomic signature of miR-16/miR-519-dysregulated genes which was highly enriched in HuR targets (>6-fold; 79.6% of target genes). Discussion: These results were confirmed on several public transcriptomic and microRNA datasets of human meningiomas, hinting that the putative tumor suppressor effect of these miRs is mediated, at least in part, via HuR direct or indirect inhibition.

Cross-Talk between miRNAs from the Dlk1-Dio3 Locus and Histone Methylation to Protect Male Cerebellum from Methyl Donor Deficiency.

SCOPE: Disruption of the one carbon metabolism during development, i.e., following a gestational vitamin B9 and B12 deficiencies, is involved in birth defects and brain development delay. Using a rat nutritional model, consisting of pups born to dams fed a vitamin B9 and B12 deficient diet (MDD), the study previously reports molecular and cellular alterations in the brain, in a sex dependent manner, with females being more affected than males. The study hypothesizes that epigenetic modifications could participate in the sex differences is observed. METHODS AND RESULTS: The study investigates lysine methylation of histones and expression of microRNAs in the cerebellum of MDD male and female pups. The study reports a differential regulation of H3K36Me2 and H4K20Me3 between males and females, in response to MDD. Moreover, distinct regulation of Kmt5b and Kdm2a expression by miR-134-5p and miR-369-5p from the Dlk1-Dio3 locus, contributes to the maintenance of expression of genes involved in synaptic plasticity. CONCLUSION: These results could explain the neuroprotection to MDD that male pups display. The work will contribute to the understanding of the consequences of vitamin starvation on brain development, as well as how the epigenome is affected by one carbon metabolism disruption.

Dynamic regulation of microRNA expression following interferon-γ-induced gene transcription.

MicroRNAs are major players in post-transcriptional gene regulation. Even small changes in miRNA levels may have profound consequences for the expression levels of target genes. Hence, miRNAs themselves need to be tightly, albeit dynamically, regulated. Here, we investigated the dynamic behavior of miRNAs over a wide time range following stimulation of melanoma cells with interferon-γ (IFN-γ), which activates the transcription factor STAT1. By applying several bioinformatic and statistical software tools for visualization and identification of differentially expressed miRNAs derived from time-series microarray experiments, 8.9% of 1105 miRNAs appeared to be directly or indirectly regulated by STAT1. Focusing on distinct dynamic expression patterns, we found that the majority of robust miRNA expression changes occurred in the intermediate time range (24-48 h). Three miRNAs (miR-27a, miR-30a, miR-34a) had a delayed regulation occurring at 72 h while none showed significant expression changes at early time points between 30 min and 6 h. Expression patterns of individual miRNAs were altered gradually over time or abruptly increased or decreased between two time points. Furthermore, we observed coordinated dynamic transcription of most miRNA clusters while few were found to be regulated independently of their genetic cluster. Most interestingly, several "star" or passenger strand sequences were specifically regulated over time while their "guide" strands were not.

Transcriptional repression of microRNA genes by PML-RARA increases expression of key cancer proteins in acute promyelocytic leukemia.

Micro(mi)RNAs are small noncoding RNAs that orchestrate many key aspects of cell physiology and their deregulation is often linked to distinct diseases including cancer. Here, we studied the contribution of miRNAs in a well-characterized human myeloid leukemia, acute promyelocytic leukemia (APL), targeted by retinoic acid and trioxide arsenic therapy. We identified several miRNAs transcriptionally repressed by the APL-associated PML-RAR oncogene which are released after treatment with all-trans retinoic acid. These coregulated miRNAs were found to control, in a coordinated manner, crucial pathways linked to leukemogenesis, such as HOX proteins and cell adhesion molecules whose expressions are thereby repressed by the chemotherapy. Thus, APL appears linked to transcriptional perturbation of miRNA genes, and clinical protocols able to successfully eradicate cancer cells may do so by restoring miRNA expression. The identification of abnormal miRNA biogenesis in cancer may therefore provide novel biomarkers and therapeutic targets in myeloid leukemias.

A New Synuclein-Transgenic Mouse Model for Early Parkinson's Reveals Molecular Features of Preclinical Disease.

Understanding Parkinson's disease (PD), in particular in its earliest phases, is important for diagnosis and treatment. However, human brain samples are collected post-mortem, reflecting mainly end-stage disease. Because brain samples of mouse models can be collected at any stage of the disease process, they are useful in investigating PD progression. Here, we compare ventral midbrain transcriptomics profiles from α-synuclein transgenic mice with a progressive, early PD-like striatal neurodegeneration across different ages using pathway, gene set, and network analysis methods. Our study uncovers statistically significant altered genes across ages and between genotypes with known, suspected, or unknown function in PD pathogenesis and key pathways associated with disease progression. Among those are genotype-dependent alterations associated with synaptic plasticity and neurotransmission, as well as mitochondria-related genes and dysregulation of lipid metabolism. Age-dependent changes were among others observed in neuronal and synaptic activity, calcium homeostasis, and membrane receptor signaling pathways, many of which linked to G-protein coupled receptors. Most importantly, most changes occurred before neurodegeneration was detected in this model, which points to a sequence of gene expression events that may be relevant for disease initiation and progression. It is tempting to speculate that molecular changes similar to those changes observed in our model happen in midbrain dopaminergic neurons before they start to degenerate. In other words, we believe we have uncovered molecular changes that accompany the progression from preclinical to early PD.

The actin filament cross-linker L-plastin confers resistance to TNF-alpha in MCF-7 breast cancer cells in a phosphorylation-dependent manner.

We used a tumour necrosis factor (TNF)-alpha resistant breast adenocarcinoma MCF-7 cell line to investigate the involvement of the actin cytoskeleton in the mechanism of cell resistance to this cytokine. We found that TNF resistance correlates with the loss of cell epithelial properties and the gain of a mesenchymal phenotype, reminiscent of an epithelial-to-mesenchymal transition (EMT). Morphological changes were associated with a profound reorganization of the actin cytoskeleton and with a change in the repertoire of expressed actin cytoskeleton genes and EMT markers, as revealed by DNA microarray-based expression profiling. L-plastin, an F-actin cross-linking and stabilizing protein, was identified as one of the most significantly up-regulated genes in TNF-resistant cells. Knockdown of L-plastin in these cells revealed its crucial role in conferring TNF resistance. Importantly, overexpression of wild-type L-plastin in TNF-sensitive MCF-7 cells was sufficient to protect them against TNF-mediated cell death. Furthermore, we found that this effect is dependent on serine-5 phosphorylation of L-plastin and that non-conventional protein kinase C isoforms and the ceramide pathway may regulate its phosphorylation state. The protective role of L-plastin was not restricted to TNF-alpha resistant MCF-7 cells because a correlation between the expression of L-plastin and the resistance to TNF-alpha was observed in other breast cancer cell lines. Together, our study discloses a novel unexpected role of the actin bundling protein L-plastin as a cell protective protein against TNF-cytotoxicity.