Dual IRE 1 RN ase functions dictate glioblastoma development
2018; Springer Nature; Volume: 10; Issue: 3 Linguagem: Inglês
10.15252/emmm.201707929
ISSN1757-4684
AutoresStéphanie Lhomond, Tony Avril, Nicolas Dejeans, Konstantinos Voutetakis, Dimitrios Doultsinos, Mari McMahon, Raphaël Pineau, Joanna Obacz, Olga Papadodima, Florence Jouan, Héloïse Bourien, Marianthi Logotheti, Gwénaële Jégou, Néstor Pallares‐Lupon, Kathleen Schmit, Pierre‐Jean Le Reste, Amandine Etcheverry, Jean Mosser, Kim Barroso, Élodie Vauléon, Marion Maurel, Afshin Samali, John B. Patterson, Olivier Pluquet, Claudio Hetz, Véronique Quillien, Aristotelis Chatziioannou, Éric Chevet,
Tópico(s)RNA regulation and disease
ResumoResearch Article8 January 2018Open Access Transparent process Dual IRE1 RNase functions dictate glioblastoma development Stéphanie Lhomond Stéphanie Lhomond Université de Bordeaux, Bordeaux, France Search for more papers by this author Tony Avril Tony Avril INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Nicolas Dejeans Nicolas Dejeans Université de Bordeaux, Bordeaux, France Search for more papers by this author Konstantinos Voutetakis Konstantinos Voutetakis Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece Department of Biochemistry & Biotechnology, University of Thessaly, Larissa, Greece Search for more papers by this author Dimitrios Doultsinos Dimitrios Doultsinos INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Mari McMahon Mari McMahon INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author Raphaël Pineau Raphaël Pineau INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Joanna Obacz Joanna Obacz INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Olga Papadodima Olga Papadodima Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece Search for more papers by this author Florence Jouan Florence Jouan INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Heloise Bourien Heloise Bourien INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Marianthi Logotheti Marianthi Logotheti Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece e-NIOS PC, Kallithea-Athens, Greece Search for more papers by this author Gwénaële Jégou Gwénaële Jégou INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Néstor Pallares-Lupon Néstor Pallares-Lupon Université de Bordeaux, Bordeaux, France Search for more papers by this author Kathleen Schmit Kathleen Schmit Université de Bordeaux, Bordeaux, France Search for more papers by this author Pierre-Jean Le Reste Pierre-Jean Le Reste INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Department of Neurosurgery, University Hospital Pontchaillou, Rennes, France Search for more papers by this author Amandine Etcheverry Amandine Etcheverry Integrated Functional Genomics and Biomarkers Team, UMR6290, CNRS, Université de Rennes 1, Rennes, France Search for more papers by this author Jean Mosser Jean Mosser Integrated Functional Genomics and Biomarkers Team, UMR6290, CNRS, Université de Rennes 1, Rennes, France Search for more papers by this author Kim Barroso Kim Barroso INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Elodie Vauléon Elodie Vauléon INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Marion Maurel Marion Maurel INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author Afshin Samali Afshin Samali orcid.org/0000-0002-8610-8375 Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author John B Patterson John B Patterson Medinnovata Inc., Ventura, CA, USA Search for more papers by this author Olivier Pluquet Olivier Pluquet Institut Pasteur de Lille, CNRS UMR8161 “Mechanisms of Tumourigenesis and Targeted Therapies”, Université de Lille, Lille, France Search for more papers by this author Claudio Hetz Claudio Hetz Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Program of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Center for Geroscience, Brain Health and Metabolism, Santiago, Chile Buck Institute for Research on Aging, Novato, CA, USA Department of Immunology and Infectious diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Véronique Quillien Véronique Quillien INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Aristotelis Chatziioannou Aristotelis Chatziioannou Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece e-NIOS PC, Kallithea-Athens, Greece Search for more papers by this author Eric Chevet Corresponding Author Eric Chevet [email protected] orcid.org/0000-0001-5855-4522 INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Stéphanie Lhomond Stéphanie Lhomond Université de Bordeaux, Bordeaux, France Search for more papers by this author Tony Avril Tony Avril INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Nicolas Dejeans Nicolas Dejeans Université de Bordeaux, Bordeaux, France Search for more papers by this author Konstantinos Voutetakis Konstantinos Voutetakis Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece Department of Biochemistry & Biotechnology, University of Thessaly, Larissa, Greece Search for more papers by this author Dimitrios Doultsinos Dimitrios Doultsinos INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Mari McMahon Mari McMahon INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author Raphaël Pineau Raphaël Pineau INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Joanna Obacz Joanna Obacz INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Olga Papadodima Olga Papadodima Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece Search for more papers by this author Florence Jouan Florence Jouan INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Heloise Bourien Heloise Bourien INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Marianthi Logotheti Marianthi Logotheti Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece e-NIOS PC, Kallithea-Athens, Greece Search for more papers by this author Gwénaële Jégou Gwénaële Jégou INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Néstor Pallares-Lupon Néstor Pallares-Lupon Université de Bordeaux, Bordeaux, France Search for more papers by this author Kathleen Schmit Kathleen Schmit Université de Bordeaux, Bordeaux, France Search for more papers by this author Pierre-Jean Le Reste Pierre-Jean Le Reste INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Department of Neurosurgery, University Hospital Pontchaillou, Rennes, France Search for more papers by this author Amandine Etcheverry Amandine Etcheverry Integrated Functional Genomics and Biomarkers Team, UMR6290, CNRS, Université de Rennes 1, Rennes, France Search for more papers by this author Jean Mosser Jean Mosser Integrated Functional Genomics and Biomarkers Team, UMR6290, CNRS, Université de Rennes 1, Rennes, France Search for more papers by this author Kim Barroso Kim Barroso INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Elodie Vauléon Elodie Vauléon INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Marion Maurel Marion Maurel INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author Afshin Samali Afshin Samali orcid.org/0000-0002-8610-8375 Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author John B Patterson John B Patterson Medinnovata Inc., Ventura, CA, USA Search for more papers by this author Olivier Pluquet Olivier Pluquet Institut Pasteur de Lille, CNRS UMR8161 “Mechanisms of Tumourigenesis and Targeted Therapies”, Université de Lille, Lille, France Search for more papers by this author Claudio Hetz Claudio Hetz Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile Program of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile Center for Geroscience, Brain Health and Metabolism, Santiago, Chile Buck Institute for Research on Aging, Novato, CA, USA Department of Immunology and Infectious diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Véronique Quillien Véronique Quillien INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Aristotelis Chatziioannou Aristotelis Chatziioannou Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece e-NIOS PC, Kallithea-Athens, Greece Search for more papers by this author Eric Chevet Corresponding Author Eric Chevet [email protected] orcid.org/0000-0001-5855-4522 INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Author Information Stéphanie Lhomond1,†, Tony Avril2,3,†, Nicolas Dejeans1,‡, Konstantinos Voutetakis4,5,‡, Dimitrios Doultsinos2,3, Mari McMahon2,3,6, Raphaël Pineau2,3, Joanna Obacz2,3, Olga Papadodima4, Florence Jouan2,3, Heloise Bourien2,3, Marianthi Logotheti4,7, Gwénaële Jégou2,3, Néstor Pallares-Lupon1, Kathleen Schmit1, Pierre-Jean Le Reste2,8, Amandine Etcheverry9, Jean Mosser9, Kim Barroso2,3, Elodie Vauléon2,3, Marion Maurel2,3,6, Afshin Samali6, John B Patterson10, Olivier Pluquet11, Claudio Hetz12,13,14,15,16, Véronique Quillien2,3, Aristotelis Chatziioannou4,7 and Eric Chevet *,2,3 1Université de Bordeaux, Bordeaux, France 2INSERM U1242, “Chemistry, Oncogenesis, Stress, Signaling”, Université de Rennes 1, Rennes, France 3Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France 4Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece 5Department of Biochemistry & Biotechnology, University of Thessaly, Larissa, Greece 6Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland 7e-NIOS PC, Kallithea-Athens, Greece 8Department of Neurosurgery, University Hospital Pontchaillou, Rennes, France 9Integrated Functional Genomics and Biomarkers Team, UMR6290, CNRS, Université de Rennes 1, Rennes, France 10Medinnovata Inc., Ventura, CA, USA 11Institut Pasteur de Lille, CNRS UMR8161 “Mechanisms of Tumourigenesis and Targeted Therapies”, Université de Lille, Lille, France 12Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile 13Program of Cellular and Molecular Biology, Institute of Biomedical Sciences, University of Chile, Santiago, Chile 14Center for Geroscience, Brain Health and Metabolism, Santiago, Chile 15Buck Institute for Research on Aging, Novato, CA, USA 16Department of Immunology and Infectious diseases, Harvard School of Public Health, Boston, MA, USA † These authors contributed equally to this work as first authors ‡ These authors contributed equally to this work as second authors *Corresponding author. Tel: +33 223237258; E-mail: [email protected] EMBO Mol Med (2018)10:e7929https://doi.org/10.15252/emmm.201707929 Correction(s) for this article Dual IRE1 RNase functions dictate glioblastoma development08 February 2023 This article has the following note(s): Dual IRE1 RNase functions dictate glioblastoma development11 January 2022 Correction added on 9 February 2023, after first online publication: Panel A has been corrected. See the associated Corrigendum at https://doi.org/10.15252/emmm.202216731. Correction added on 9 February 2023, after first online publication: Legend for panels D and E has been corrected. See the associated Corrigendum at https://doi.org/10.15252/emmm.202216731. Correction added on 9 February 2023, after first online publication: The text has been updated. See the associated Corrigendum at https://doi.org/10.15252/emmm.202216731. Correction added on 9 February 2023, after first online publication: Panel A has been corrected. See the associated Corrigendum at https://doi.org/10.15252/emmm.202216731. Correction added on 9 February 2023, after first online publication: Legend for panel A has been corrected. See the associated Corrigendum at https://doi.org/10.15252/emmm.202216731. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1α (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1-dependent decay (RIDD) of RNA. Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1-dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression. Synopsis The IRE1 arm of the Unfolded Protein Response (UPR) plays a major role in cancer development. Dissecting IRE1 signals in human glioblastoma tumors, primary and established cell lines reveals the dual role of XBP1 mRNA splicing and RIDD in tumor aggressiveness. GBM tumors cluster into two groups exhibiting low or high IRE1 activity. XBP1s elicits pro-tumorigenic signals and promotes angiogenesis and macrophage recruitment to the tumor. RIDD dampens angiogenesis and tumor cell migration. Patients bearing tumors with high XBP1s low RIDD features show lower survival than those with low XBP1s high RIDD, thereby providing potential therapeutic avenues. Introduction Glioblastoma multiforme (GBM) is one of the most lethal adult cancers, as the majority of patients die within 15 months after diagnosis (Anton et al, 2007). GBM is an aggressive, incurable glioma (grade IV astrocytoma, WHO classification) due to great heterogeneity of cell subtypes within the tumor and to the presence of invasive spots that cannot be easily cured by surgical resection or targeted radiation. To limit tumor recurrences from invasive cells, chemotherapy [temozolomide (TMZ)] was added to surgery and radiation (Stupp et al, 2005). Although this combined therapy has demonstrated some efficiency, it only increases patient's median survival from 12.1 to 14.6 months. Thus, understanding biological processes of GBM progression and treatment resistance represents a major challenge to develop more effective therapies. The ER is the major subcellular compartment involved in protein folding and secretion. Accumulating evidence supports an emerging role of ER proteostasis alterations in cancer development, having been implicated in most hallmarks of cancer (Urra et al, 2016). ER stress triggers an adaptive reaction known as the unfolded protein response (UPR), which aims to recover proteostasis or to induce apoptosis of irreversibly damaged cells (Walter & Ron, 2011). Several studies in animal models of cancer using genetic or pharmacological manipulation of the UPR have demonstrated a functional role of this pathway in cancer (Hetz et al, 2013). The UPR is initiated by the activation of three ER transmembrane proteins known as PERK, ATF6, and IRE1 (Hetz et al, 2015). IRE1α (referred to as IRE1 hereafter) is a serine/threonine kinase and endoribonuclease that represents the most conserved UPR signaling branch in evolution, controlling cell fate under ER stress (Hetz et al, 2015). Once activated, IRE1 oligomerizes thus engaging three major downstream outputs including the activation of JNK (Urano et al, 2000; Han et al, 2009), the splicing of XBP1 mRNA (XBP1s) (Yoshida et al, 2001; Calfon et al, 2002), and the degradation of targeted mRNA and miRNA, a process referred to as RNA regulated IRE1-dependent decay (RIDD) (Maurel et al, 2014). Importantly, the universe of RIDD targets may depend on the tissue context and the nature of the stress stimuli, impacting different biological processes including apoptosis, cell migration, and inflammatory responses (Dejeans et al, 2014). Several functional studies have shown that targeting the expression or the RNase activity of IRE1 reduces the progression of various forms of cancer mostly due to ablating the prosurvival effects of XBP1 on tumor growth (Chevet et al, 2015; Obacz et al, 2017), and we have previously demonstrated its functional implication in various models of experimental glioblastoma (Drogat et al, 2007; Auf et al, 2010; Dejeans et al, 2012; Pluquet et al, 2013; Jabouille et al, 2015). Moreover, large-scale sequencing studies on human cancer tissue samples performed by The Cancer Genome Atlas (TCGA) initiative (Cancer Genome Atlas Research Network, 2008; Parsons et al, 2008) revealed the presence of three somatic mutations on the IRE1 gene in GBM leading to the S769F, Q780* (Greenman et al, 2007), and P336L (Parsons et al, 2008) variants. Although a previous report aimed at understanding the structural impact of some of those mutations in IRE1 function (Xue et al, 2011), little is known on how their differential contribution to RIDD and XBP1 mRNA splicing impacts on GBM development and progression. Our previous findings indicated that IRE1 also contributes to mRNA degradation in cancer, having unexpected roles in tissue invasion in GBM, in addition to affecting growth and vascularization (Dejeans et al, 2012; Pluquet et al, 2013). Here, we took advantage of the selective signaling properties of different IRE1 GBM somatic mutants and we demonstrate that the modulation of IRE1 signaling characteristics in GBM cells controls tumor aggressiveness, not only by providing selective advantages to the tumor cells themselves, but also by remodeling the tumor stroma to the benefit of growth. Furthermore, we provide evidence supporting a novel concept where IRE1-downstream signals play antagonistic roles in cancer development, where XBP1s provides pro-tumoral signals, whereas RIDD of mRNA and miR17 rather elicits anti-tumoral features. Our data, obtained using established cell lines, patient tumor samples, and primary GBM lines, depict a complex scenario where IRE1 signaling orchestrates distinct aspects of GBM biology, thereby offering novel targets for therapeutic intervention. Results IRE1 activity and human GBM tumor properties We previously identified an IRE1-dependent gene expression signature in U87 cells using IRE1 dominant-negative-expressing cells, an approach that fully blocks all RNase outputs of this ER stress sensor (Pluquet et al, 2013). Functional annotation of the genes comprised in the IRE1-dependent gene expression signature revealed the enrichment in biological functions associated with stress responses, cell adhesion/migration, and with the inflammatory and immune response (Fig 1A). This gene expression signature was processed through the Bioinfominer pipeline (Appendix Fig S1) to increase its functional relevance, and this led to the identification of 38 IRE1 signaling hub genes (Appendix Fig S1). This 38 genes signature was then confronted to the transcriptomes of the GBM TCGA (Cancer Genome Atlas Research Network, 2008) and GBMmark (in-house) cohorts (Fig 1B). This analysis revealed the existence of two populations of patients displaying either high or low IRE1 activity, respectively (Fig 1C and Appendix Fig S2). Tumors exhibiting high IRE1 activity also correlated with shorter survival of the corresponding patients (Fig 1D). We then tested the impact of IRE1 signaling on the expression levels of IBA1, CD14, and CD163 as markers of the inflammatory/immune response in the tumors (Fig 1E), the levels of CD31 and vWF to monitor angiogenesis (Fig 1F), or RHOA, CYR61, and CTGF expression as indicators of tumoral invasion (Fig 1G). This revealed that tumors exhibiting high IRE1 activity also presented markers of massive infiltration of macrophages, with high vascularization and invasive properties. Similar observations were also obtained when analyzing the GBMmark dataset (Appendix Fig S2B–D). Activation of the IRE1/XBP1 axis was confirmed in those tumors through the analysis of the expression of XBP1 target genes ERDJ4 and EDEM1 (Appendix Fig S2E). To confirm these observations at the protein level in GBM, fresh tumors presenting high or low IRE1 activity were dissociated and analyzed for CD45 and CD11b expression by FACS. This analysis revealed that high IRE1 signaling correlated with strong macrophage infiltration (Fig 1H). Moreover, the presence of endothelial cells in tumors was detected by FACS after CD31 labeling and was increased in GBM tumors exhibiting high IRE1 activity (Fig 1I). Finally, tumors exhibiting high IRE1 signaling were mainly classified as belonging to the mesenchymal type of GBM whereas those with low IRE1 activity mostly included pro-neural and classical tumors (Fig 1J). These data demonstrate that IRE1 activation is found in human tumors and correlate with more aggressive cancers with shorter patient survival. Figure 1. IRE1 signaling signatures in glioblastoma multiform Functional annotations of the IRE1 gene expression signature identified in U87 cells (Pluquet et al, 2013). Schematic representation of the analysis workflow. Hierarchical clustering of GBM patients (TCGA cohort) based on high or low IRE1 activity as assessed with the correlation index of their median z-score with the expression pattern of the IRE1 gene signature of 38 hub genes (see Materials and Methods and Appendix Fig S1). Pearson correlation was used to measure the similarity between different genes and tumor cases, as well. The correlation index refers to the gene expression median z-score with (+) or (−) sign for identical or reverse expression pattern with that of WT vs. DN, respectively. The expression pattern of WT vs. DN has been described in detail in Pluquet et al (2013). Blue: low correlation index, red: high correlation index. Survival analysis of the GBM patients exhibiting high (red) or low (green) IRE1 activity. Student's t-test was used with Welch's correction when SD different. Expression of microglial/monocyte/macrophage (IBA1, CD14, CD163) (E), angiogenesis (CD31, vWF) (F), and migration/invasion (RHOA, CYR61, CTGF) (G) markers mRNA in the IRE1high (red) and IRE1low (green) populations. Probe analysis was carried out in data from 258 and 265 tumors in IRE1high and IRE1low groups, respectively. Horizontal lines indicate median; box lines indicate first & third quartiles; whiskers indicate min & max. Student's t-test was used with Welch's correction when SD different. FACS analysis of CD45/CD11b in freshly dissociated GBM tumors exhibiting high or low IRE1 activity. FACS analysis of CD31 in freshly dissociated GBM tumors exhibiting high or low IRE1 activity. In both cases, 7AAD was used to exclude dead cells. Relative distribution of the different classes of GBM—pro-neural (blue), neural (orange), classic (green), and mesenchymal (red) according to the tumor status, namely IRE1high or IRE1low. Download figure Download PowerPoint Identification of a novel somatic mutation on IRE1 in human GBM IRE1 activation in tumors could be due to exposure to stressful environments (nutrient/oxygen deprivation, pH, immune response) but also to the presence of somatic mutations in the IRE1 coding gene. Previous tumor sequencing studies identified IRE1 mutations that were defined as driver in various cancers among which three were found in GBM (Greenman et al, 2007; Parsons et al, 2008). Here, we sequenced the IRE1 gene (ERN1) exons in 23 additional GBM samples and identified a fourth IRE1 mutation in one GBM human sample (Appendix Fig S3A). This somatic A414T mutation came from an aggressive, mesenchymal-like GBM developed in a 70-year-old female. Immunohistochemistry staining revealed that this tumor was also highly vascularized (CD31 staining) and showed strong XBP1s staining (Appendix Fig S3B). Sequence alignment indicates that whereas the mutations P336L, S769F, and Q780* affect conserved amino acids in various species, the mutation identified in our sequencing study altered an apparently less conserved amino acid, which was only conserved in dog, chimpanzee, and human but not in rodents (Appendix Fig S3C). This property could explain why the A414T mutation, previously described in GBM samples, has been excluded from further analyses, as it was considered as a SNP or a secondary acquired mutation (Cancer Genome Atlas Research Network, 2008; Parsons et al, 2008). Interestingly since the first discovery of IRE1 somatic mutations in cancers in 2007, a number of cancer exome or whole-genome sequencing studies have also reported around 50 mutations but none of them in GBM (Chevet et al, 2015). Different kinase and RNase activities of IRE1-related cancer variants IRE1 is a bifunctional protein that contains a kinase and a RNase domain involved in three downstream signaling pathways including (i) activation of stress pathways [i.e., JNK and NFKB (Hetz, 2012)], (ii) the degradation of targeted RNAs (RIDD), and (iii) the unconventional splicing of XBP1 mRNA. The localization of IRE1 mutations found in cancer revealed no apparent clustering of the mutations in the secondary structure, not even into IRE1 catalytic domains. However, the “cytosolic” mutations S769F and Q780* are located in the kinase domain of the protein whereas the “luminal” mutations P336L and A414T are located in putative alpha-helical domains (Appendix Fig S3D). To measure the potential impact of the four mutations found in GBM, we overexpressed either the wild-type (WT) or the mutated forms of IRE1 in U87 cells, in a normal endogenous IRE1 background (Appendix Fig S4A). The four variants were overexpressed in U87 cells using a lentivirus system, and as anticipated, the stop mutation Q780* leads to overexpression of a shorter IRE1 protein (80 kDa instead
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