Publications by authors named "Tony Kaoma"

29 Publications

  • Page 1 of 1

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

Nucleic Acids Res 2021 Feb 3. Epub 2021 Feb 3.

IGMM, Univ Montpellier, CNRS, Montpellier, France.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab053DOI Listing
February 2021

Adrenomedullin-CALCRL axis controls relapse-initiating drug tolerant acute myeloid leukemia cells.

Nat Commun 2021 01 18;12(1):422. Epub 2021 Jan 18.

Centre de Recherches en Cancérologie de Toulouse, UMR1037, Inserm, Université de Toulouse 3 Paul Sabatier, Equipe Labellisée LIGUE 2018, F-31037, Toulouse, France.

Drug tolerant/resistant leukemic stem cell (LSC) subpopulations may explain frequent relapses in acute myeloid leukemia (AML), suggesting that these relapse-initiating cells (RICs) persistent after chemotherapy represent bona fide targets to prevent drug resistance and relapse. We uncover that calcitonin receptor-like receptor (CALCRL) is expressed in RICs, and that the overexpression of CALCRL and/or of its ligand adrenomedullin (ADM), and not CGRP, correlates to adverse outcome in AML. CALCRL knockdown impairs leukemic growth, decreases LSC frequency, and sensitizes to cytarabine in patient-derived xenograft models. Mechanistically, the ADM-CALCRL axis drives cell cycle, DNA repair, and mitochondrial OxPHOS function of AML blasts dependent on E2F1 and BCL2. Finally, CALCRL depletion reduces LSC frequency of RICs post-chemotherapy in vivo. In summary, our data highlight a critical role of ADM-CALCRL in post-chemotherapy persistence of these cells, and disclose a promising therapeutic target to prevent relapse in AML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-20717-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7813857PMC
January 2021

Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology.

Acta Neuropathol 2020 12 3;140(6):919-949. Epub 2020 Oct 3.

NORLUX Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health, 84, Val Fleuri, 1526, Luxembourg, Luxembourg.

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00401-020-02226-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666297PMC
December 2020

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

Mol Neurobiol 2021 Feb 30;58(2):576-602. Epub 2020 Sep 30.

Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Belvaux, Luxembourg.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-020-02085-zDOI Listing
February 2021

Reference-free deconvolution, visualization and interpretation of complex DNA methylation data using DecompPipeline, MeDeCom and FactorViz.

Nat Protoc 2020 10 25;15(10):3240-3263. Epub 2020 Sep 25.

Division of Cancer Epigenomics, German Cancer Research Center (DKFZ), Heidelberg, Germany.

DNA methylation profiling offers unique insights into human development and diseases. Often the analysis of complex tissues and cell mixtures is the only feasible option to study methylation changes across large patient cohorts. Since DNA methylomes are highly cell type specific, deconvolution methods can be used to recover cell type-specific information in the form of latent methylation components (LMCs) from such 'bulk' samples. Reference-free deconvolution methods retrieve these components without the need for DNA methylation profiles of purified cell types. Currently no integrated and guided procedure is available for data preparation and subsequent interpretation of deconvolution results. Here, we describe a three-stage protocol for reference-free deconvolution of DNA methylation data comprising: (i) data preprocessing, confounder adjustment using independent component analysis (ICA) and feature selection using DecompPipeline, (ii) deconvolution with multiple parameters using MeDeCom, RefFreeCellMix or EDec and (iii) guided biological inference and validation of deconvolution results with the R/Shiny graphical user interface FactorViz. Our protocol simplifies the analysis and guides the initial interpretation of DNA methylation data derived from complex samples. The harmonized approach is particularly useful to dissect and evaluate cell heterogeneity in complex systems such as tumors. We apply the protocol to lung cancer methylomes from The Cancer Genome Atlas (TCGA) and show that our approach identifies the proportions of stromal cells and tumor-infiltrating immune cells, as well as associations of the detected components with clinical parameters. The protocol takes slightly >3 d to complete and requires basic R skills.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41596-020-0369-6DOI Listing
October 2020

Extracellular ATP and CD39 Activate cAMP-Mediated Mitochondrial Stress Response to Promote Cytarabine Resistance in Acute Myeloid Leukemia.

Cancer Discov 2020 Oct 8;10(10):1544-1565. Epub 2020 Jul 8.

Centre de Recherches en Cancérologie de Toulouse, UMR1037 Inserm/Université Toulouse III-Paul Sabatier, ERL5294 CNRS, Equipe Labellisée LIGUE 2018, Toulouse, France.

Relapses driven by chemoresistant leukemic cell populations are the main cause of mortality for patients with acute myeloid leukemia (AML). Here, we show that the ectonucleotidase CD39 (ENTPD1) is upregulated in cytarabine-resistant leukemic cells from both AML cell lines and patient samples and . CD39 cell-surface expression and activity is increased in patients with AML upon chemotherapy compared with diagnosis, and enrichment in CD39-expressing blasts is a marker of adverse prognosis in the clinics. High CD39 activity promotes cytarabine resistance by enhancing mitochondrial activity and biogenesis through activation of a cAMP-mediated adaptive mitochondrial stress response. Finally, genetic and pharmacologic inhibition of CD39 ecto-ATPase activity blocks the mitochondrial reprogramming triggered by cytarabine treatment and markedly enhances its cytotoxicity in AML cells and . Together, these results reveal CD39 as a new residual disease marker and a promising therapeutic target to improve chemotherapy response in AML. SIGNIFICANCE: Extracellular ATP and CD39-P2RY13-cAMP-OxPHOS axis are key regulators of cytarabine resistance, offering a new promising therapeutic strategy in AML..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-19-1008DOI Listing
October 2020

Machine learning-assisted neurotoxicity prediction in human midbrain organoids.

Parkinsonism Relat Disord 2020 06 8;75:105-109. Epub 2020 May 8.

Developmental and Cellular Biology, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg. Electronic address:

Introduction: Brain organoids are highly complex multi-cellular tissue proxies, which have recently risen as novel tools to study neurodegenerative diseases such as Parkinson's disease (PD). However, with increasing complexity of the system, usage of quantitative tools becomes challenging.

Objectives: The primary objective of this study was to develop a neurotoxin-induced PD organoid model and to assess the neurotoxic effect on dopaminergic neurons using microscopy-based phenotyping in a high-content fashion.

Methods: We describe a pipeline for a machine learning-based analytical method, allowing for detailed image-based cell profiling and toxicity prediction in brain organoids treated with the neurotoxic compound 6-hydroxydopamine (6-OHDA).

Results: We quantified features such as dopaminergic neuron count and neuronal complexity and built a machine learning classifier with the data to optimize data processing strategies and to discriminate between different treatment conditions. We validated the approach with high content imaging data from PD patient derived midbrain organoids.

Conclusions: The here described model is a valuable tool for advanced in vitro PD modeling and to test putative neurotoxic compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.parkreldis.2020.05.011DOI Listing
June 2020

Oncogenic KIT mutations induce STAT3-dependent autophagy to support cell proliferation in acute myeloid leukemia.

Oncogenesis 2019 Jul 16;8(8):39. Epub 2019 Jul 16.

Cancer Research Center of Toulouse (CRCT), UMR1037 INSERM, ERL5294 CNRS, Equipe Labellisée LIGUE, Toulouse, France.

Autophagy is associated with both survival and cell death in myeloid malignancies. Therefore, deciphering its role in different genetically defined subtypes of acute myeloid leukemia (AML) is critical. Activating mutations of the KIT receptor tyrosine kinase are frequently detected in core-binding factor AML and are associated with a greater risk of relapse. Herein, we report that basal autophagy was significantly increased by the KIT mutation in AML cells and contributed to support their cell proliferation and survival. Invalidation of the key autophagy protein Atg12 strongly reduced tumor burden and improved survival of immunocompromised NSG mice engrafted with KIT TF-1 cells. Downstream of KIT, STAT3, but not AKT or ERK pathways, was identified as a major regulator of autophagy. Accordingly, STAT3 pharmacological inhibition or downregulation inhibited autophagy and reduced tumor growth both in vitro and in vivo. Taken together, our results support the notion that targeting autophagy or STAT3 opens up an exploratory pathway for finding new therapeutic opportunities for patients with CBF-AML or others malignancies with KIT mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41389-019-0148-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635375PMC
July 2019

Hub genes in a pan-cancer co-expression network show potential for predicting drug responses.

F1000Res 2018 7;7:1906. Epub 2018 Dec 7.

Luxembourg Institute of Health (LIH), Strassen, Luxembourg.

: The topological analysis of networks extracted from different types of "omics" data is a useful strategy for characterizing biologically meaningful properties of the complex systems underlying these networks. In particular, the biological significance of highly connected genes in diverse molecular networks has been previously determined using data from several model organisms and phenotypes. Despite such insights, the predictive potential of candidate hubs in gene co-expression networks in the specific context of cancer-related drug experiments remains to be deeply investigated. The examination of such associations may offer opportunities for the accurate prediction of anticancer drug responses.  Here, we address this problem by: a) analyzing a co-expression network obtained from thousands of cancer cell lines, b) detecting significant network hubs, and c) assessing their capacity to predict drug sensitivity using data from thousands of drug experiments. We investigated the prediction capability of those genes using a multiple linear regression model, independent datasets, comparisons with other models and our own experiments. These analyses led to the identification of 47 hub genes, which are implicated in a diverse range of cancer-relevant processes and pathways. Overall, encouraging agreements between predicted and observed drug sensitivities were observed in public datasets, as well as in our validations for four glioblastoma cell lines and four drugs. To facilitate further research, we share our hub-based drug sensitivity prediction model as an online tool. : Our research shows that co-expression network hubs are biologically interesting and exhibit potential for predicting drug responses . These findings motivate further investigations about the relevance and application of our unbiased discovery approach in pre-clinical, translationally-oriented research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/f1000research.17149.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406180PMC
December 2018

Distribution and prognostic impact of microglia/macrophage subpopulations in gliomas.

Brain Pathol 2019 07 15;29(4):513-529. Epub 2019 Jan 15.

Edinger Institute, Institute of Neurology, Goethe University Frankfurt, Frankfurt am Main, Germany.

While the central nervous system is considered an immunoprivileged site and brain tumors display immunosuppressive features, both innate and adaptive immune responses affect glioblastoma (GBM) growth and treatment resistance. However, the impact of the major immune cell population in gliomas, represented by glioma-associated microglia/macrophages (GAMs), on patients' clinical course is still unclear. Thus, we aimed at assessing the immunohistochemical expression of selected microglia and macrophage markers in 344 gliomas (including gliomas from WHO grade I-IV). Furthermore, we analyzed a cohort of 241 IDH1R132H-non-mutant GBM patients for association of GAM subtypes and patient overall survival. Phenotypical properties of GAMs, isolated from high-grade astrocytomas by CD11b-based magnetic cell sorting, were analyzed by immunocytochemistry, mRNA microarray, qRT-PCR and bioinformatic analyses. A higher amount of CD68-, CD163- and CD206-positive GAMs in the vital tumor core was associated with beneficial patient survival. The mRNA expression profile of GAMs displayed an upregulation of factors that are considered as pro-inflammatory M1 (eg, CCL2, CCL3L3, CCL4, PTGS2) and anti-inflammatory M2 polarization markers (eg, MRC1, LGMN, CD163, IL10, MSR1), the latter rather being associated with phagocytic functions in the GBM microenvironment. In summary, we present evidence that human GBMs contain mixed M1/M2-like polarized GAMs and that the levels of different GAM subpopulations in the tumor core are positively associated with overall survival of patients with IDH1R132H-non-mutant GBMs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bpa.12690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849857PMC
July 2019

Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures.

EMBO Rep 2018 11 11;19(11). Epub 2018 Sep 11.

NORLUX Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg

Microglia are specialized parenchymal-resident phagocytes of the central nervous system (CNS) that actively support, defend and modulate the neural environment. Dysfunctional microglial responses are thought to worsen CNS diseases; nevertheless, their impact during neuroinflammatory processes remains largely obscure. Here, using a combination of single-cell RNA sequencing and multicolour flow cytometry, we comprehensively profile microglia in the brain of lipopolysaccharide (LPS)-injected mice. By excluding the contribution of other immune CNS-resident and peripheral cells, we show that microglia isolated from LPS-injected mice display a global downregulation of their homeostatic signature together with an upregulation of inflammatory genes. Notably, we identify distinct microglial activated profiles under inflammatory conditions, which greatly differ from neurodegenerative disease-associated profiles. These results provide insights into microglial heterogeneity and establish a resource for the identification of specific phenotypes in CNS disorders, such as neuroinflammatory and neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201846171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216255PMC
November 2018

Inhibition of HIF1α-Dependent Upregulation of Phospho-l-Plastin Resensitizes Multiple Myeloma Cells to Frontline Therapy.

Int J Mol Sci 2018 05 23;19(6). Epub 2018 May 23.

Laboratory of Experimental Cancer Research, Luxembourg Institute of Health (LIH), L-1526 Luxembourg City, Luxembourg.

The introduction of novel frontline agents in multiple myeloma (MM), like immunomodulatory drugs and proteasome inhibitors, has improved the overall survival of patients. Yet, MM is still not curable, and drug resistance (DR) remains the main challenge. To improve the understanding of DR in MM, we established a resistant cell line (MOLP8/R). The exploration of DR mechanisms yielded an overexpression of HIF1α, due to impaired proteasome activity of MOLP8/R. We show that MOLP8/R, like other tumor cells, overexpressing HIF1α, have an increased resistance to the immune system. By exploring the main target genes regulated by HIF1α, we could not show an overexpression of these targets in MOLP8/R. We, however, show that MOLP8/R cells display a very high overexpression of gene (l-Plastin) controlled by HIF1α, and that this overexpression also exists in MM patient samples. The l-Plastin activity is controlled by its phosphorylation in Ser5. We further show that the inhibition of l-Plastin phosphorylation restores the sensitivity of MOLP8/R to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). Our results reveal a new target gene of DR, controlled by HIF1α.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19061551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032243PMC
May 2018

Predicting clinical outcome of neuroblastoma patients using an integrative network-based approach.

Biol Direct 2018 06 7;13(1):12. Epub 2018 Jun 7.

Proteome and Genome Research Unit, Department of Oncology, Luxembourg Institute of Health, 1A-B, rue Thomas Edison, Strassen, L-1445, Luxembourg.

Background: One of the main current challenges in computational biology is to make sense of the huge amounts of multidimensional experimental data that are being produced. For instance, large cohorts of patients are often screened using different high-throughput technologies, effectively producing multiple patient-specific molecular profiles for hundreds or thousands of patients.

Results: We propose and implement a network-based method that integrates such patient omics data into Patient Similarity Networks. Topological features derived from these networks were then used to predict relevant clinical features. As part of the 2017 CAMDA challenge, we have successfully applied this strategy to a neuroblastoma dataset, consisting of genomic and transcriptomic data. In particular, we observe that models built on our network-based approach perform at least as well as state of the art models. We furthermore explore the effectiveness of various topological features and observe, for instance, that redundant centrality metrics can be combined to build more powerful models.

Conclusion: We demonstrate that the networks inferred from omics data contain clinically relevant information and that patient clinical outcomes can be predicted using only network topological data.

Reviewers: This article was reviewed by Yang-Yu Liu, Tomislav Smuc and Isabel Nepomuceno.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13062-018-0214-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992838PMC
June 2018

Carboxypeptidase E transmits its anti-migratory function in glioma cells via transcriptional regulation of cell architecture and motility regulating factors.

Int J Oncol 2017 Aug 21;51(2):702-714. Epub 2017 Jun 21.

Molecular Neuro-Oncology, Hertie Institute for Clinical Brain Research and Center Neurology, University of Tübingen, D-72076 Tübingen, Germany.

Glioblastoma (GBM), the most frequent and aggressive malignant primary brain tumor, is characterized by a highly invasive growth. In our previous study we showed that overexpression of Carboxypeptidase E (CPE) mitigated glioma cell migration. In the present study we aimed at deciphering the regulatory mechanisms of the secreted form of CPE (sCPE). By transcriptome analysis and inhibition of signaling pathways involved in the regulation of cell growth and motility, we discovered that overexpression of sCPE was accompanied by differential regulation of mRNAs connected to the motility-associated networks, among others FAK, PAK, Cdc42, integrin, STAT3 as well as TGF-β. Especially SLUG was downregulated in sCPE-overexpressing glioma cells, paralleled by reduced expression of matrix-metalloproteinases (MMP) and, in consequence, by decreased cell migration. Expression of SLUG was regulated by ERK since inhibition of ERK reverted sCPE-mediated SLUG downregulation and enhanced cell motility. In a mouse glioma model, overexpression of sCPE significantly prolonged survival. Our results implicate a novel role for sCPE that mainly affects the expression of motility-associated genes via several signal pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/ijo.2017.4051DOI Listing
August 2017

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

BMC Genomics 2017 06 6;18(1):443. Epub 2017 Jun 6.

Proteome and Genome Research Unit, Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-017-3819-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461714PMC
June 2017

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.

J Pathol 2017 08 5;242(4):421-434. Epub 2017 Jul 5.

INSERM U954, Faculty of Medicine, Université de Lorraine, Vandoeuvre-lès-Nancy, France.

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 ) 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 ) and IOMM-Lee (p = 4 × 10 ) cells. Transcriptome analyses revealed HuR knockdown in IOMM-Lee cells to deregulate the HIF1A signalling pathway (p = 1.5 × 10 ) 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.4916DOI Listing
August 2017

Chemotherapy-Resistant Human Acute Myeloid Leukemia Cells Are Not Enriched for Leukemic Stem Cells but Require Oxidative Metabolism.

Cancer Discov 2017 07 17;7(7):716-735. Epub 2017 Apr 17.

Inserm, Cancer Research Center of Toulouse, U1037, Toulouse, France.

Chemotherapy-resistant human acute myeloid leukemia (AML) cells are thought to be enriched in quiescent immature leukemic stem cells (LSC). To validate this hypothesis , we developed a clinically relevant chemotherapeutic approach treating patient-derived xenografts (PDX) with cytarabine (AraC). AraC residual AML cells are enriched in neither immature, quiescent cells nor LSCs. Strikingly, AraC-resistant preexisting and persisting cells displayed high levels of reactive oxygen species, showed increased mitochondrial mass, and retained active polarized mitochondria, consistent with a high oxidative phosphorylation (OXPHOS) status. AraC residual cells exhibited increased fatty-acid oxidation, upregulated CD36 expression, and a high OXPHOS gene signature predictive for treatment response in PDX and patients with AML. High OXPHOS but not low OXPHOS human AML cell lines were chemoresistant Targeting mitochondrial protein synthesis, electron transfer, or fatty-acid oxidation induced an energetic shift toward low OXPHOS and markedly enhanced antileukemic effects of AraC. Together, this study demonstrates that essential mitochondrial functions contribute to AraC resistance in AML and are a robust hallmark of AraC sensitivity and a promising therapeutic avenue to treat AML residual disease. AraC-resistant AML cells exhibit metabolic features and gene signatures consistent with a high OXPHOS status. In these cells, targeting mitochondrial metabolism through the CD36-FAO-OXPHOS axis induces an energetic shift toward low OXPHOS and strongly enhanced antileukemic effects of AraC, offering a promising avenue to design new therapeutic strategies and fight AraC resistance in AML. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-16-0441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501738PMC
July 2017

Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma.

Oncotarget 2016 May;7(22):31955-71

NORLUX Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health (L.I.H.) Luxembourg, Luxembourg.

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.7454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077988PMC
May 2016

Isocitrate dehydrogenase 1 mutations prime the all-trans retinoic acid myeloid differentiation pathway in acute myeloid leukemia.

J Exp Med 2016 Apr 7;213(4):483-97. Epub 2016 Mar 7.

Institut National de la Santé et de la Recherche Médicale (INSERM), Cancer Research Center of Toulouse, U1037, F-31024 Toulouse, France Université de Toulouse, F-31300 Toulouse, France

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs caused by recurrent mutations, such as the isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that dysregulates hematopoietic differentiation. In this study, we identified mutant R132H IDH1-specific gene signatures regulated by key transcription factors, particularly CEBPα, involved in myeloid differentiation and retinoid responsiveness. We show that treatment with all-trans retinoic acid (ATRA) at clinically achievable doses markedly enhanced terminal granulocytic differentiation in AML cell lines, primary patient samples, and a xenograft mouse model carrying mutant IDH1. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, whereas inhibition of 2-HG production significantly reduced ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD-Scid-IL2rγ(null)mice and markedly increased overall survival, revealing a potent antileukemic effect of ATRA in the presence of IDH1 mutation. This therapeutic strategy holds promise for this AML patient subgroup in future clinical studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20150736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821643PMC
April 2016

Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material.

Sci Rep 2016 Jan 20;6:19529. Epub 2016 Jan 20.

Department of Oncology, Luxembourg Institute of Health (LIH), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg.

MicroRNAs (miRNAs) have emerged as promising cancer biomarkers. However, exploiting their informative potential requires careful optimization of their detection. Here, we compared the efficiency of commonly used RNA extraction kits in miRNA recovery from cells, plasma and urine/plasma-derived exosomes, using single-gene RT-qPCR and miRNA profiling. We used increasing amounts of starting material to investigate the impact of the input material size on miRNA extraction. We showed that miRNA recovery was largely influenced by the isolation method and by the amount of input material. In particular, the miRCURY™ kit provided highly pure RNA. However, its columns poorly recovered miRNAs from limiting amounts of cells and plasma, and rapidly saturated by large RNA species and plasma components, thus impeding miRNA recovery from high input amounts. Overall, the miRNeasy® kit permitted a better miRNA detection despite a less pure extracted RNA. Nevertheless, some miRNAs were preferentially or exclusively isolated by either of the methods. Trizol® LS resulted in very low purity RNA which affected RT-qPCR efficiency. In general, miRCURY™ biofluids kit efficiently extracted miRNAs from plasma. A careful selection of the RNA isolation method and the consideration of the type and size of input material are highly recommended to avoid biased results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep19529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726450PMC
January 2016

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

FASEB J 2016 Mar 30;30(3):1218-33. Epub 2015 Nov 30.

*Laboratory of Cytoskeleton and Cell Plasticity and Systems Biology Group, Life Sciences Research Unit, University of Luxembourg, Luxembourg City, Luxembourg; and Genomics Research Unit, Luxembourg Institute of Health, Luxembourg City, Luxembourg

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.15-276311DOI Listing
March 2016

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

BMC Genomics 2015 Oct 19;16:809. Epub 2015 Oct 19.

Life Sciences Research Unit, University of Luxembourg, 162a, Avenue de la Faïencerie, L-1511, Luxembourg, Luxembourg.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-015-1984-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617894PMC
October 2015

Comprehensive analysis of glycolytic enzymes as therapeutic targets in the treatment of glioblastoma.

PLoS One 2015 1;10(5):e0123544. Epub 2015 May 1.

NorLux Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health (L.I.H.), Luxembourg, Luxembourg; KG Jebsen Brain Tumour Research Center, Department of Biomedicine, University of Bergen, Bergen, Norway.

Major efforts have been put in anti-angiogenic treatment for glioblastoma (GBM), an aggressive and highly vascularized brain tumor with dismal prognosis. However clinical outcome with anti-angiogenic agents has been disappointing and tumors quickly develop escape mechanisms. In preclinical GBM models we have recently shown that bevacizumab, a blocking antibody against vascular endothelial growth factor, induces hypoxia in treated tumors, which is accompanied by increased glycolytic activity and tumor invasiveness. Genome-wide transcriptomic analysis of patient derived GBM cells including stem cell lines revealed a strong up-regulation of glycolysis-related genes in response to severe hypoxia. We therefore investigated the importance of glycolytic enzymes in GBM adaptation and survival under hypoxia, both in vitro and in vivo. We found that shRNA-mediated attenuation of glycolytic enzyme expression interfered with GBM growth under normoxic and hypoxic conditions in all cellular models. Using intracranial GBM xenografts we identified seven glycolytic genes whose knockdown led to a dramatic survival benefit in mice. The most drastic effect was observed for PFKP (PFK1, +21.8%) and PDK1 (+20.9%), followed by PGAM1 and ENO1 (+14.5% each), HK2 (+11.8%), ALDOA (+10.9%) and ENO2 (+7.2%). The increase in mouse survival after genetic interference was confirmed using chemical inhibition of PFK1 with clotrimazole. We thus provide a comprehensive analysis on the importance of the glycolytic pathway for GBM growth in vivo and propose PFK1 and PDK1 as the most promising therapeutic targets to address the metabolic escape mechanisms of GBM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123544PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416792PMC
February 2016

An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells.

J Leukoc Biol 2015 Mar 29;97(3):557-71. Epub 2014 Dec 29.

*University of Luxembourg, Life Sciences Research Unit, Calcium Signaling and Inflammation, Luxembourg; and Centre de Recherche Public Santé, Luxembourg

Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1α, IL-1β, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1α, IL-1β, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1189/jlb.3A0514-254RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477892PMC
March 2015

Differential effects of antofine N-oxide on solid tumor and leukemia cells.

Anticancer Agents Med Chem 2014 ;14(10):1315-23

Laboratory of Cellular and Molecular Oncology, Public Research Center for Health (CRP-Sante), L-1526 Luxembourg.

We have studied the anti-cancer activities of antofine N-oxide isolated and purified from the medicinal plant Cynanchum vincetoxicum. Antofine N-oxide displayed a strong inhibitory effect on several solid tumor cell lines (glioblastoma, breast carcinoma and lung carcinoma) and on a T-cell leukemia cell line. Remarkably, its cytotoxic effect was considerably weaker in non-cancer cells. Antofine N-oxide was found to inhibit proliferation of the solid tumor cells whereas it caused apoptotic cell death in the leukemia cells. A microarray analysis after a short treatment revealed that the number of differentially expressed genes was considerably higher in solid tumor than in leukemia cells. Up-regulated genes in the three solid tumor cell lines include genes related to TNFα signaling, of which TNFα was among the most significantly induced. A functional analysis revealed that TNFR1 signaling was most likely activated in the solid tumor cells. The increased mRNA levels of several genes of this pathway (namely TNFα, TNFAIP3 and BIRC3) were confirmed by real-time quantitative PCR after different treatment durations. Finally a slight inhibition of NFκB-mediated transcription was observed in the same cells. Together our results suggest that inhibition of cell proliferation in solid tumor cells essentially occurs through TNFα signaling whereas this pathway is not activated in leukemia cells. Apoptotic cell death in the latter is induced by a distinct yet unknown pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1871520614666140624110458DOI Listing
July 2015

Neurodegeneration by activation of the microglial complement-phagosome pathway.

J Neurosci 2014 Jun;34(25):8546-56

Neural Regeneration Group, Institute of Reconstructive Neurobiology, University of Bonn, 53127 Bonn, Germany,

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.5002-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608212PMC
June 2014

The ROS/SUMO axis contributes to the response of acute myeloid leukemia cells to chemotherapeutic drugs.

Cell Rep 2014 Jun 5;7(6):1815-23. Epub 2014 Jun 5.

Equipe Labellisée Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier, UMR 5535 CNRS, Université Montpellier 1, Université Montpellier 2, 1919 Route de Mende, 34293 Montpellier, France. Electronic address:

Chemotherapeutic drugs used in the treatment of acute myeloid leukemias (AMLs) are thought to induce cancer cell death through the generation of DNA double-strand breaks. Here, we report that one of their early effects is the loss of conjugation of the ubiquitin-like protein SUMO from its targets via reactive oxygen species (ROS)-dependent inhibition of the SUMO-conjugating enzymes. Desumoylation regulates the expression of specific genes, such as the proapoptotic gene DDIT3, and helps induce apoptosis in chemosensitive AMLs. In contrast, chemotherapeutics do not activate the ROS/SUMO axis in chemoresistant cells. However, pro-oxidants or inhibition of the SUMO pathway by anacardic acid restores DDIT3 expression and apoptosis in chemoresistant cell lines and patient samples, including leukemic stem cells. Finally, inhibition of the SUMO pathway decreases tumor growth in mice xenografted with AML cells. Thus, targeting the ROS/SUMO axis might constitute a therapeutic strategy for AML patients resistant to conventional chemotherapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2014.05.016DOI Listing
June 2014

NK cell killer Ig-like receptor repertoire acquisition and maturation are strongly modulated by HLA class I molecules.

J Immunol 2014 Mar 19;192(6):2602-10. Epub 2014 Feb 19.

Laboratory of Immunogenetics and Allergology, Public Research Center for Health, L-1526 Luxembourg, Luxembourg;

The interaction between clonally distributed inhibitory receptors and their activating counterparts on NK cells and HLA class I molecules defines NK cell functions, but the role of HLA class I ligands in the acquisition of their receptors during NK development is still unclear. Although some studies demonstrated that HLA-C affects the expression of killer Ig-like receptors (KIR), other studies showed that NK cells acquire their KIR repertoire in a stochastic manner. Only when infected with human CMV is an expansion of self-specific KIR(+) NKG2C(+) NK cells detected. To gain more insight into this question, we compared the coexpression of different KIR molecules, NKG2A, CD8, and CD57, on NK cells in healthy donors and seven patients with deficient HLA class I expression due to mutations in one of the TAP genes. Our results show a correlation between the presence/absence of HLA class I molecules and the coexpression of their receptors. In an HLA class I low-expression context, an increase in KIR molecules' coexpression is detected on the NKG2A(+) CD8(+) subset. In functional assays, hyporesponsiveness was observed for TAP-deficient NK cells derived from four patients. In contrast, NK cells from patient five were functional, whereas CD107a(+) and IFN-γ(+) CD56(dim) NK cells presented a different pattern of HLA class I receptors compared with healthy donors. Taken together, our results provide strong evidence for the role of HLA class I molecules in NK cell maturation and KIR repertoire acquisition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1302843DOI Listing
March 2014

The acquisition of resistance to TNFα in breast cancer cells is associated with constitutive activation of autophagy as revealed by a transcriptome analysis using a custom microarray.

Autophagy 2011 Jul 1;7(7):760-70. Epub 2011 Jul 1.

Laboratory of Experimental Hemato-Oncology, Luxembourg City, Luxembourg.

While the autophagic process is mainly regulated at the post-translational level, a growing body of evidence suggests that autophagy might also be regulated at the transcriptional level. The identification of transcription factors involved in the regulation of autophagy genes has provided compelling evidence for such regulation. In this context, a powerful high throughput analysis tool to simultaneously monitor the expression level of autophagy genes is urgently needed. Here we describe setting up the first comprehensive human autophagy database (HADb, available at www.autophagy.lu) and the development of a companion Human Autophagy-dedicated cDNA Microarray which comprises 234 genes involved in or related to autophagy. The autophagy microarray tool used on breast adenocarcinoma MCF-7 cell line allowed the identification of 47 differentially expressed autophagy genes associated with the acquisition of resistance to the cytotoxic effect of TNFα. The autophagy-core machinery genes DRAM (Damage-Regulated Autophagy Modulator), BNIP3L (BCL2/adenovirus E1B 19 kDa interacting protein 3-like), BECN1 (Beclin 1), GABARAP (Gamma-AminoButyric Acid Receptor-Associated Protein) and UVRAG (UV radiation resistance associated gene) were found upregulated in TNF-resistant cells, suggesting a constitutive activation of the autophagy machinery in these cells. More interestingly, we identified NPC1 as the most upregulated genes in TNF-resistant compared to TNF-sensitive MCF-7 cells, suggesting a relation between the intracellular transport of cholesterol, the regulation of autophagy and NPC1 expression in TNF-resistant tumor cells. In conclusion, we describe here new tools that may help investigating autophagy gene regulation in various cellular models and diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/auto.7.7.15454DOI Listing
July 2011