ABSTRACT
Therapies targeting the tyrosine kinase receptor HER2 have significantly improved survival of patients with HER2+ cancer. However, both de novo and acquired resistance remain a challenge, particularly in the brain metastatic setting. Here we report that, unlike other HER tyrosine kinase receptors, HER2 possesses a binding motif in its cytosolic juxtamembrane region that allows interaction with members of the Ezrin/Radixin/Moesin (ERM) family. Under physiologic conditions, this interaction controls the localization of HER2 in ERM-enriched domains and stabilizes HER2 in a catalytically repressed state. In HER2+ breast cancers, low expression of Moesin correlated with increased HER2 expression. Restoring expression of ERM proteins in HER2+ breast cancer cells was sufficient to revert HER2 activation and inhibit HER2-dependent proliferation. A high-throughput assay recapitulating the HER2-ERM interaction allowed for screening of about 1,500 approved drugs. From this screen, we found Zuclopenthixol, an antipsychotic drug that behaved as a Moesin-mimicking compound, because it directly binds the juxtamembrane region of HER2 and specifically inhibits HER2 activation in HER2+ cancers, as well as activation of oncogenic mutated and truncated forms of HER2. Zuclopenthixol efficiently inhibited HER2+ breast tumor progression in vitro and in vivo and, more importantly, showed significant activity on HER2+ brain tumor progression. Collectively, these data reveal a novel class of allosteric HER2 inhibitors, increasing the number of approaches to consider for intervention on HER2+ breast cancers and brain metastases. SIGNIFICANCE: This study demonstrates the functional role of Moesin in maintaining HER2 in a catalytically repressed state and provides novel therapeutic approaches targeting HER2+ breast cancers and brain metastasis using Moesin-mimicking compounds.
Subject(s)
Biomimetics/methods , Brain Neoplasms/drug therapy , Breast Neoplasms/drug therapy , Clopenthixol/pharmacology , Gene Expression Regulation, Neoplastic , Microfilament Proteins/metabolism , Receptor, ErbB-2/antagonists & inhibitors , Allosteric Regulation , Animals , Apoptosis , Brain Neoplasms/metabolism , Brain Neoplasms/secondary , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation , Dopamine Antagonists/pharmacology , Female , Humans , Mice , Mice, Nude , Microfilament Proteins/genetics , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.
Subject(s)
HIV Infections/therapy , HIV Integrase/metabolism , Protein Serine-Threonine Kinases/metabolism , Small Molecule Libraries/chemistry , Virus Replication/drug effects , Catalytic Domain , Drug Evaluation, Preclinical , HIV , HIV Integrase/genetics , High-Throughput Screening Assays , Humans , Models, Molecular , Protein Binding , Protein Serine-Threonine Kinases/genetics , Retroviridae , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Virus Replication/geneticsABSTRACT
Phylogeny is often used to compare entire families of genes/proteins. We previously showed that classification of Caenorhabditis elegans Rho GTPases on the basis of their enzymatic properties was significantly different from sequence alignments. To further develop this concept, we have developed an integrated approach to classify C. elegans small GTPases based on functional data comprising affinity for GTP, sub-cellular localization, tissue distribution and silencing impact. This analysis led to establish a novel functional classification for small GTPases. To test the relevance of this classification in mammals, we focused our attention on the human orthologs of small GTPases from a specific group comprising arf-1.2, evl-20, arl-1, Y54E10BR.2, unc-108 and rab-7. We then tested their involvement in protein secretion and membrane traffic in mammalian systems. Using this approach we identify a novel network containing 18 GTPases, and 23 functionally interacting proteins, conserved between C. elegans and mammals, which is involved in membrane traffic and protein secretion.
Subject(s)
Cell Membrane/metabolism , Protein Transport/physiology , ras Proteins/metabolism , Animals , Caenorhabditis elegans/metabolism , Humans , Monomeric GTP-Binding Proteins/metabolism , Protein Transport/genetics , Proteomics/methodsABSTRACT
AlphaScreen(®) is a technology particularly suitable for bi-molecular inhibitor screening assays, e.g. using protein-protein interactions with purified recombinant proteins. Each binding partner of the bi-molecular interaction is coupled either to donor or to acceptor beads. The technology is based on the quantifiable transfer of oxygen singlets from donor to acceptor microbeads brought together by a specific interaction between the partners. We identified the conserved interaction between WW domains of cellular ubiquitin ligases of the Nedd4 family and a short peptide motif (PPxY) present in several structural and non-structural viral proteins as a potential drug target. Using an AlphaScreen assay recapitulating the interaction between Nedd4.2 and the PPxY motif of the adenoviral capsid protein VI, we screened a library of small molecules and identified specific inhibitors of this interaction.
Subject(s)
Host-Pathogen Interactions/physiology , Protein Binding/physiology , Adenoviridae/genetics , Host-Pathogen Interactions/genetics , Nedd4 Ubiquitin Protein Ligases/metabolism , Protein Binding/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Singlet Oxygen/metabolismABSTRACT
An imbalance of protein homeostasis caused by external or internal stress in the endoplasmic reticulum triggers the initiation of signalling pathways downstream of the IRE1, ATF6 and PERK sensors to a translational or transcriptional adaptive response known as UPR (Unfolded Protein Response). According to the intensity and duration of stress, the dual function of the UPR leads to either cell adaptation or cell death. UPR pathways in cancer cells are often altered and generally lead to an adaptation to an hostile environment. As the UPR becomes an emerging therapeutic target due to its increasing contribution to various diseases, we describe in this review various strategies that have been developed to discover new compounds enabling to manipulate the magnitude of ER stress in the context of cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Endoplasmic Reticulum Stress/drug effects , Molecular Targeted Therapy/methods , Neoplasms/drug therapy , Animals , Endoribonucleases/antagonists & inhibitors , Endoribonucleases/metabolism , Humans , Neoplasms/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/drug effects , Unfolded Protein Response/drug effects , eIF-2 Kinase/antagonists & inhibitors , eIF-2 Kinase/metabolismABSTRACT
Aptamers are oligonucleotides displaying specific binding properties for a predetermined target. They can be easily immobilized on various surfaces such as nanoparticles. Functionalized particles can then be used to various aims. We took advantage of the AlphaScreen(®) technology for monitoring aptamer-mediated interactions. A particle bearing an aptamer contains a photosensitizer whereas another type of particle contains a chemiluminescer. Irradiation causes the formation of singlet oxygen species in the photosensitizer-containing bead that in turn activates the chemiluminescer. Luminescence emission can be observed if the two types of beads are in close proximity (<200 nm). This is achieved when the cognate ligand of the aptamer is grafted onto the chemiluminescer-containing bead. Using this technology we have screened oligonucleotide libraries and monitored aptamer-protein interactions. This constitutes the basis for aptamer-based analytical assays.
Subject(s)
Nanoparticles/chemistry , Nanotechnology/methods , SELEX Aptamer Technique/methods , Humans , Oligonucleotides/chemistry , Oligonucleotides/geneticsABSTRACT
The unfolded protein response (UPR) was originally identified as a signaling network coordinating adaptive and apoptotic responses to accumulation of unfolded proteins in the endoplasmic reticulum (ER). More recent work has shown that UPR signaling can be triggered by a multitude of cellular events and that the UPR plays a critical role in the prevention of cell transformation but also in tumor development. This has been particularly well illustrated with studies on one of the three major ER stress sensors, IRE1. This ER resident type I transmembrane protein senses luminal ER stress and transduce signals through its cytosolic RNase activity. IRE1 signaling has been shown to contribute to the progression of solid tumors through pro-angiogenic mechanisms. Herein, we expose the methodologies for investigating IRE1 signaling in tumor cells and in tumors. Moreover, we show that selective pharmacological inhibition of IRE1 RNase activity sensitizes tumor cells to ER stress.
Subject(s)
Endoribonucleases/metabolism , Neoplasms/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoribonucleases/genetics , Humans , Neoplasms/genetics , Protein Serine-Threonine Kinases/genetics , Regulatory Factor X Transcription Factors , Secretory Pathway/genetics , Secretory Pathway/physiology , Signal Transduction/genetics , Signal Transduction/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Unfolded Protein Response/genetics , Unfolded Protein Response/physiologyABSTRACT
The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.
Subject(s)
Adenosine Triphosphatases/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , Cell Cycle Proteins/metabolism , Endoplasmic Reticulum Stress/physiology , Signal Transduction/genetics , Transcription, Genetic/physiology , Unfolded Protein Response/physiology , Adenosine Triphosphatases/genetics , Animals , Caenorhabditis elegans Proteins/genetics , Cell Cycle Proteins/genetics , Endoplasmic Reticulum Stress/genetics , Proteomics/methods , RNA Interference , Repressor Proteins/metabolism , Valosin Containing ProteinABSTRACT
Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.
Subject(s)
Fibroblast Growth Factor 2/isolation & purification , Heparin/chemistry , Protein Multimerization , Binding Sites , Cell Line , Chromatography, Affinity , Fibroblast Growth Factor 2/chemistry , Fibroblast Growth Factor 2/metabolism , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Receptors, Fibroblast Growth Factor/chemistry , Receptors, Fibroblast Growth Factor/metabolismABSTRACT
Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen(®), and AMMP(®)) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253.
Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Cirrhosis/genetics , Liver Neoplasms/genetics , Neoplasm Proteins/genetics , Phosphoproteins/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Aged , Aged, 80 and over , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Calnexin/genetics , Calnexin/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Dipeptidases/genetics , Dipeptidases/metabolism , Endoplasmic Reticulum Chaperone BiP , Female , Gene Expression Profiling , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Liver/metabolism , Liver/pathology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Middle Aged , Molecular Sequence Annotation , Neoplasm Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/metabolism , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Proteomics/methods , Signal Transduction , Thioredoxins/genetics , Thioredoxins/metabolism , Valosin Containing ProteinABSTRACT
Neisseria meningitidis is a cause of meningitis epidemics worldwide and of rapidly progressing fatal septic shock. A crucial step in the pathogenesis of invasive meningococcal infections is the adhesion of bloodborne meningococci to both peripheral and brain endothelia, leading to major vascular dysfunction. Initial adhesion of pathogenic strains to endothelial cells relies on meningococcal type IV pili, but the endothelial receptor for bacterial adhesion remains unknown. Here, we report that the immunoglobulin superfamily member CD147 (also called extracellular matrix metalloproteinase inducer (EMMPRIN) or Basigin) is a critical host receptor for the meningococcal pilus components PilE and PilV. Interfering with this interaction potently inhibited the primary attachment of meningococci to human endothelial cells in vitro and prevented colonization of vessels in human brain tissue explants ex vivo and in humanized mice in vivo. These findings establish the molecular events by which meningococci target human endothelia, and they open new perspectives for treatment and prevention of meningococcus-induced vascular dysfunctions.
Subject(s)
Basigin/immunology , Blood Vessels/microbiology , Neisseria meningitidis/pathogenicity , Bacterial Adhesion , Fimbriae, Bacterial/physiology , Humans , Neisseria meningitidis/immunologyABSTRACT
Hepatocellular adenomas (HCA) are benign liver tumors predominantly developed in women using oral contraceptives. Here, exome sequencing identified recurrent somatic FRK mutations that induce constitutive kinase activity, STAT3 activation, and cell proliferation sensitive to Src inhibitors. We also found uncommon recurrent mutations activating JAK1, gp130, or ß-catenin. Chromosome copy number and methylation profiling revealed patterns that correlated with specific gene mutations and tumor phenotypes. Finally, integrative analysis of HCAs transformed to hepatocellular carcinoma revealed ß-catenin mutation as an early alteration and TERT promoter mutations as associated with the last step of the adenoma-carcinoma transition. In conclusion, we identified the genomic diversity in benign hepatocyte proliferation, several therapeutic targets, and the key genomic determinants of the adenoma-carcinoma transformation sequence.
Subject(s)
Adenoma, Liver Cell/genetics , Carcinoma, Hepatocellular/genetics , Cell Transformation, Neoplastic/genetics , Liver Neoplasms/genetics , Neoplasm Proteins/genetics , Protein-Tyrosine Kinases/genetics , Adenoma, Liver Cell/enzymology , Adenoma, Liver Cell/pathology , Animals , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Transformation, Neoplastic/pathology , DNA Methylation , Enzyme Activation , Gene Expression Profiling , Humans , Liver Neoplasms/enzymology , Liver Neoplasms/pathology , Mice , Mutation , NIH 3T3 Cells , Neoplasm Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Risk Factors , TransfectionABSTRACT
ATF6α, a membrane-anchored transcription factor from the endoplasmic reticulum (ER) that modulates the cellular response to stress as an effector of the unfolded-protein response (UPR), is a key player in the development of tumors of different origin. ATF6α activation has been linked to oncogenic transformation and tumor maintenance; however, the mechanism(s) underlying this phenomenon remains elusive. Here, using a phenotypic small interfering RNA (siRNA) screening, we identified a novel role for ATF6α in chemoresistance and defined the protein disulfide isomerase A5 (PDIA5) as necessary for ATF6α activation upon ER stress. PDIA5 contributed to disulfide bond rearrangement in ATF6α under stress conditions, thereby leading to ATF6α export from the ER and activation of its target genes. Further analysis of the mechanism demonstrated that PDIA5 promotes ATF6α packaging into coat protein complex II (COPII) vesicles and that the PDIA5/ATF6α activation loop is essential to confer chemoresistance on cancer cells. Genetic and pharmacological inhibition of the PDIA5/ATF6α axis restored sensitivity to the drug treatment. This work defines the mechanisms underlying the role of ATF6α activation in carcinogenesis and chemoresistance; furthermore, it identifies PDIA5 as a key regulator ATF6α-mediated cellular functions in cancer.
Subject(s)
Activating Transcription Factor 6/metabolism , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Piperazines/pharmacology , Protein Disulfide-Isomerases/metabolism , Pyrimidines/pharmacology , COP-Coated Vesicles/metabolism , Cell Survival/drug effects , Cystine/metabolism , Drug Resistance, Neoplasm , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress , HeLa Cells , Heat-Shock Proteins/metabolism , Humans , Imatinib Mesylate , Protein Transport , Unfolded Protein ResponseABSTRACT
Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1α. Analysis of the mechanism shows that IRE1α endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1α signaling using either siRNA-mediated silencing or a dominant-negative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-α substrate, thereby pointing toward an increased IRE1α activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development.
Subject(s)
Endoribonucleases/metabolism , Gene Expression Regulation, Neoplastic/physiology , Glioblastoma/metabolism , Period Circadian Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Unfolded Protein Response/physiology , Animals , Base Sequence , Endoribonucleases/genetics , Glioblastoma/genetics , Humans , Mice , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Period Circadian Proteins/genetics , Protein Serine-Threonine Kinases/genetics , RNA Interference , RNA Processing, Post-Transcriptional , RNA, Messenger , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Xenograft Model Antitumor AssaysABSTRACT
P97/CDC-48 is a prominent member of a highly evolutionary conserved Walker cassette - containing AAA+ATPases. It has been involved in numerous cellular processes ranging from the control of protein homeostasis to membrane trafficking through the intervention of specific accessory proteins. Expression of p97/CDC-48 in cancers has been correlated with tumor aggressiveness and prognosis, however the precise underlying molecular mechanisms remain to be characterized. Moreover p97/CDC-48 inhibitors were developed and are currently under intense investigation as anticancer drugs. Herein, we discuss the role of p97/CDC-48 in cancer development and its therapeutic potential in tumor cell biology.
Subject(s)
Adenosine Triphosphatases/physiology , Cell Cycle Proteins/physiology , Neoplasms/etiology , Adenosine Triphosphatases/antagonists & inhibitors , Animals , Autophagy , Cell Cycle Proteins/antagonists & inhibitors , DNA Damage , Endoplasmic Reticulum/metabolism , Homeostasis , Humans , Lysosomes/metabolism , NF-kappa B/physiology , Neoplasms/therapy , Valosin Containing ProteinABSTRACT
Oligomerization of protein complexes has been involved in various mechanisms that play a major role in endoplasmic reticulum (ER) functions. In particular, in response to the accumulation of misfolded proteins in the ER, an adaptive response is activated and named the unfolded protein response (UPR). To facilitate recovery of ER homeostasis, both the inositol-requiring enzyme-1 (IRE1) and the protein kinase RNA-like ER kinase, two transmembrane ER stress transducers, oligomerize and activate UPR-specific transcription factors to adjust the folding and productive capacity of the ER, to direct misfolded proteins to ER-associated degradation or autophagy. Recent advances in the molecular characterization of how ER protein sensors transduce signals to orchestrate the adaptive cellular response have greatly unlocked the development of tools to dissect their functions in health and disease. Here, we focus on the advances concerning oligomerization of ER stress transducers and, in particular IRE1, describe the oligomerization-dependent mechanisms for modulating UPR signals on and off.
Subject(s)
Endoplasmic Reticulum Stress , Protein Multimerization , Signal Transduction , Animals , Humans , Models, Molecular , Protein Serine-Threonine Kinases/metabolism , Unfolded Protein ResponseABSTRACT
In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr(164) by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr(164) to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies.
Subject(s)
Cell Survival , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Protein Processing, Post-Translational , Serine C-Palmitoyltransferase/metabolism , Amino Acid Substitution , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzamides/pharmacology , Cell Nucleus/enzymology , Ceramides/metabolism , Dogs , Drug Resistance, Neoplasm , Endoplasmic Reticulum/metabolism , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/metabolism , Gene Knockdown Techniques , Golgi Apparatus/enzymology , Humans , Imatinib Mesylate , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Microsomes/metabolism , Peptide Fragments/chemistry , Phosphorylation , Phosphotyrosine/metabolism , Piperazines/pharmacology , Protein Transport , Proteome/metabolism , Pyrimidines/pharmacology , RNA, Small Interfering/genetics , Serine C-Palmitoyltransferase/chemistry , Serine C-Palmitoyltransferase/genetics , Transcription, Genetic/drug effectsABSTRACT
MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3' untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5' UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3' UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.
Subject(s)
Endoribonucleases/metabolism , Gene Silencing , Glypicans/metabolism , MicroRNAs/metabolism , Protein Serine-Threonine Kinases/metabolism , 3' Untranslated Regions , Cell Line, Tumor , Computational Biology/methods , Endoplasmic Reticulum Stress , Endoribonucleases/genetics , Glypicans/genetics , Green Fluorescent Proteins/metabolism , Humans , MicroRNAs/genetics , Protein Serine-Threonine Kinases/genetics , RNA Cleavage , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transcription, Genetic , Transfection , TransgenesABSTRACT
Immunohistochemistry is a valid method to classify hepatocellular adenoma (HCA). The aim was to test the performance of routine histology combined to glutamine synthetase (GS) staining to identify the 2 major HCA subtypes: HNF1 α inactivated (H-HCA) and inflammatory HCA (IHCA). 114 surgical cases, previously classified by immunohistochemistry, were analysed. Group A comprised 45 H-HCAs, 44 IHCAs, and 9 ß -catenin-activated IHCAs (b-IHCA), and group B, 16 b-HCA and unclassified HCA (UHCA). Steatosis was the hallmark of H-HCA. IHCA and b-IHCA were mainly characterized by inflammation, thick arteries, and sinusoidal dilatation; b-IHCA could not be differentiated from IHCA by routine histology. Group B was identified by default. A control set (91 cases) was analyzed using routine and GS stainings (without knowing immunohistochemical results). Among the 45 H-HCAs and 27 IHCAs, 40 and 24 were correctly classified, respectively. Among the 10 b-IHCAs, 4 were identified as such using additional GS. Eight of the 9 HCAs that were neither H-HCA nor IHCA were correctly classified. Conclusion. Routine histology allows to diagnose >85% of the 2 major HCA subtypes. GS is essential to identify b-HCA. This study demonstrates that a "palliative" diagnostic approach can be proposed, when the panel of specific antibodies is not available.
ABSTRACT
LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) is a homolog of the hyaluronan receptor CD44, and one of the most widely used markers of lymphatic endothelial cells in normal and tumor tissues. However, the physiologic role of LYVE-1 in the lymphatic system still remains unclear. It is well established that fibroblast growth factor 2 (FGF2) induces lymphangiogenesis. Based on the known interaction between FGF2 and CD44 and based on the structural similarity of CD44 and LYVE-1, we investigated whether FGF2 might interact with LYVE-1. We found that FGF2 is able to bind LYVE-1 using AlphaScreen, or after surface-immobilization or in solution. FGF2 binds to LYVE-1 with a higher affinity than any other known LYVE-1binding molecules, such as hyaluronan or PDGF-BB. Glycosylation of LYVE-1 is important for FGF2 binding. Furthermore, FGF2 interacts with LYVE-1 when overexpressed in CHO cells. Soluble LYVE-1 and knockdown of LYVE-1 in lymphatic endothelial cells impaired FGF2 signaling and functions. In addition, FGF2 but not VEGF-C-induced in vivo lymphangiogenesis, was also inhibited. Conversely, FGF2 also modulates LYVE-1 expression in cells and ex vivo. Thus, our data demonstrate a functional relationship to the interaction between FGF2 and LYVE-1.